CN101932599A

CN101932599A - Molecules and chimeric molecules thereof

Info

Publication number: CN101932599A
Application number: CN2006800080885A
Authority: CN
Inventors: J·D·普里斯特; A·D·沃茨; J·S·惠特克; G·R·皮尔金顿; C·A·利德尔; I·贝姆
Original assignee: Apollo Life Science Ltd
Current assignee: Apollo Life Science Ltd
Priority date: 2005-11-10
Filing date: 2006-01-25
Publication date: 2010-12-29

Abstract

The present invention relates generally to the fields of proteomics, diagnostics, therapeutics and nutrition. In particular, the present invention provides an isolated protein molecule belonging to or associated with the short-chain 4-helix bundle superfamily, such as: GM-CSF, IL-3, IL-4 and IL-5 and chimeric molecules comprising at least a portion of the protein molecules such as: GM-CSF-Fc, IL-3-Fc, IL-4-Fc and IL-5-Fc. Wherein the protein or chimeric molecule thereof has a measurable physiochemical parameter profile, wherein the profile is indicative of, associated with or forms the basis of one or more pharmacological properties. The invention further contemplates the use of the isolated protein or chimeric molecule thereof in a range of diagnostic, prophylactic, therapeutic, nutritional and/or research applications.

Description

Molecule and its chimeric molecule

Technical field

The present invention broadly relates to proteinology, diagnostics, acology and nutrition.Especially, the invention provides the protein molecule of separation, the molecule belongs to the helical bundle superfamily of short chain 4 (4helix bundle superfamily) or related to the helical bundle superfamily of short chain 4, such as：GM-CSF, IL-3, IL-4 and IL-5 and at least one of chimeric molecule comprising the protein molecule are for example：GM-CSF-Fc, IL-3-Fc, IL-4-Fc and IL-5-Fc.Wherein protein or its chimeric molecule has measurable parameter attribute, wherein character representation, be associated with one or more pharmacological characteristics or form the basis of one or more pharmacological characteristics.It is still further contemplated that the protein of separation or its chimeric molecule are used to diagnosing, prevented, is treated, in nutrition and/or research application.

Background technology

Any prior art being related in this specification also not shall not be construed as forming prior art a kind of accreditation or any type of prompting of a part for universal general knowledge.

The a variety of effects of cell factor and growth factor in hematopoiesis.Propagation, differentiation and the development of haemocyte are all regulated and controled by them.In addition, a variety of factors also play main function in immunologic process, the phagocytosis and cytotoxic effect, the other cell factors of promotion for such as stimulating leucocyte are produced.Participating in the cell factor and growth factor of hematopoiesis includes GM-CSF, interleukin Ⅲ (IL-3), interleukin-4 (IL-4) and t cell growth factor (IL-5).These cell factors belong to the helical bundle superfamily member of short chain 4.

GM-CSF may participate in hematopoiesis, cell migration and immunologic process as a kind of glycoprotein.The growth and development of the factor pair granulocyte and macrophage progenitors has a significant impact.GM-CSF and EPO acts synergistically in the atomization of CFU-E and megakaiyocyte progenitor.GM-CSF can stimulate propagation, differentiation and the phagocytosis of neutrophil leucocyte, eosinophil and monocytic series, can also stimulate the cytotoxic effect of eosinophil.GM-CSF also by raise MHCII expression promote antigen presenting cell effect and stimulate the generation of pro-inflammatory cytokine.And the use of rhGM-CSF clinically at present is mainly based upon the effect of its myelosis.It is primarily used to a variety of leukaemia such as acute myelogenous leukemia and acute lymphoblast type leukaemia, is also shared with tumor chemotherapeutic drug in such as a variety of diseases such as leukopenia, neutrophilic granulocytopenia.

People IL-3 be it is a kind of exist with monomeric form, the glycoprotein containing two N sections of glycosylation sites.IL-3 is mainly produced by activating T cell, can be detected in monocyte, macrophage, NK cells, mast cell, endothelial cell, keratinocyte.IL-3 is cloned as mast cell growth factor earliest.However, subsequent research finds that IL-3 has a variety of effects such as promotion myeloid lineage, acidophic cell system and megakaryocytic series progenitor cell proliferation, ripe and survival.IL-3 is the Main Factors of the inside and outside effect of candidate stem cell body, may additionally facilitate the expression of other colony stimulating factor acceptors.It can promote phagocytosis by the secretion of stimulating expression of macrophage and increase IL-1, IL-6, TNF.IL-3 can stimulate mast cell to produce histamine, and induce basophil cellular expression complement C 3 acceptor.IL-3 is also found energy chemotactic eosinophil leucocyte, makes platelet levels and neutrophil level increase.The application prospects of IL-3 clinically are based primarily upon it and maintain the effects such as candidate stem cell survival, propagation.Therefore it can individually or with other growth factors, such as GM-CSF and G-CSF, be united and applied in the marrow function abnormality disease such as MDS and peripheral blood cells reduction, anaemia, leukopenia, thrombopenia etc..

People IL-4 is glycoprotein related to GM-CSF, M-CSF and growth hormone in a kind of structure.IL-4 can promote the expression of the propagation and ripe up-regulation T cell antigen (CD3, CD5 and TCR) of thymocyte.In addition, IL-4 can also adjust B cell differentiation and by the post-stimulatory allosteric of activation.In non-leucocyte, IL-4 stimulates the extracellular matrix proteins such as epidermin mother cell secretion collagen, fibronectin as a kind of chemical inducer of fibroblast.IL-4 also stimulating endothelial cell expression Vcam1s (VCAM-1), so as to increase adhesion of the endothelial cell to T cell, basophilic granulocyte and eosinophil.IL-4 can also directly suppress the growth of kinds of tumor cells, such as colon cancer, people's kidney, malignant mela noma and breast cancer in vitro.

People IL-5 is a kind of homodimeric glycoprotein, is mainly produced by T lymphocytes and mast cell, can also be by the less generation of eosinophil, NK and endothelial cell.Main function in IL-5 bodies is that promotion is the generation, survival, differentiation and activation of eosinophil, and stimulates its degranulation.These effects are taken place mostly in during immune response and allergy to parasitic infection.There are some researches show IL-5 has the specificity for promoting eosinophil, the research is that IL-5 can be neutralized the eosinophil inhibiting antibody of generation is stimulated after parasitic infection.In addition, IL-5 is also found, it can be disturbed by film, extended, particle positions and increased the modes such as oxidative metabolism and activates eosinophil.Also it has been reported that IL-5 has the chemotaxis to eosinophil, moreover it is possible to stimulate basophilic granulocyte to produce histamine.

Protein with their own protein-bonded interaction by playing biological effect factor function, this shows each member of spiral aggressiveness superfamily of short chain 4 and associated albumen, the significant potentiality with respective part and acceptor with the treatment factor as regulation physiological processes.However, the small change of molecule such as one-level, two grades, three or four structure and common translation or posttranslational modification pattern may produce significant impact to the activity of albumen, secretion, antigenicity and removing.Accordingly, it is possible to which albumen can produce structure or assembling after special one-level, two grades, three or four structure, or common translation or rear translation, it assigns albumen uniqueness or significant useful properties.Therefore, it is necessary to assess the physiology characteristic of the albumen under different Production conditions to determine whether that it has useful physiologic character or other pharmacological characteristics.

So far, problem be to be carried out in the cell originated with the species of the remote edge of people's evolution available commercially as the production of protein, cell such as bacterium, yeast, fungi and insect.These cell marking proteins lack glycosylation or presentation different from the glycosylation repertoire of people's cell and it substantially have impact on their applications clinically.For example, the protein expressed in yeast or fungal systems such as aspergillus has high density mannose, albumen (Herscovics et al. FASEB J 7 invalid in the treatment are caused：540-550,1993).

Even in non-human mammal expression system such as Chinese hamster ovary (CHO) cell, it has proved that there is marked difference compared with people's cell on glycosylation pattern.For example, most of mammals, including rodent, enzyme (α 1,3) galactosyl transferase is expressed, it produces Gal (α 1,3)-Gal (β Isosorbide-5-Nitraes)-GlcNAc oligosaccharides on glycoprotein.But in people, ape and Old World Monkeys, the expression of the enzyme is inactivated by the frameshift mutation of gene.(Larsen et al.JBiol Chem 265：7055-7061, although 1990) most of Chinese hamster ovary celI systems synthesize for recombinant protein, such as Dux-B11, (the α 1 of inactivation gene expression, 3) galactosyl transferase, they still lack feature (α 2,6) sialyltransferase for the synthesis of present in human cell (α 2,6)-connection terminal sialic acid.Further, there is sialic acid motif in the Chinese hamster ovary celI of expression glycoprotein to tend to decompose (Gramer etal.Biotechnology 13 (7) by the endogenous sialidase of Chinese hamster ovary celI：692-8,1995).As a result, the albumen produced by these inhuman expression systems shows the physics and chemistry different from people's cell derived protein and pharmacological characteristics such as half-life period, antigenicity, stability and feature effect.

The progress of nearest stem cells technology substantially enhances the potentiality that such as transplantation treatment, drug screening, toxicologic study and functional genomics are applied to using stem cell.However, stem cell routinely maintains in the culture medium including non-human protein's matter, due to the possibility that inhuman infectious substance pollutes, therefore clinical practice is not suitable for.Further, the stem cell culture in inhuman derivative medium may cause to introduce inhuman carbohydrate portions therefore endanger graft application.(Martin et al Nature Medicine 11(2)：228-232,2005).Therefore, use of the special people's derived protein in the maintenance and/or differentiation of stem cell will improve the introducing of allogene protein and increase the clinical practice of stem cell.

Correspondingly, it is necessary development protein and their acceptor, it has special desired physics and chemistry and pharmacological characteristic for diagnosis, prevention, treatment, nutrition and/or research application, and is used for clinical, business and research application the invention provides the protein for belonging to or being relevant to the helical bundle superfamily of short chain 4.

Summary of the invention

Throughout the specification, unless stated otherwise, term " containing " or its variant for example " have " or "comprising", it will accordingly be understood that to imply the group of the group or entirety that include the key element or whole or key element, but be not excluded for any other key element or whole.

Nucleotides and amino acid sequence are expressed as sequence identification number (SEQ ID NO：).These SEQ IDNO：Sequence identification number is corresponded in number<400>1(SEQ ID NO：1),<400>2(SEQ IDNO：2), the rest may be inferred.The summary info of sequence identification number is shown in table 1.Sequence list is provided after claim.

The present invention broadly relates to belonging to physical and chemical parameter feature or is relevant to the protein or its chimeric molecule of the separation of the helical bundle superfamily of short chain 4, wherein described character representation, the basis for being associated with the pharmacological characteristics of one or more characteristics or forming the pharmacological characteristics of one or more characteristics.The protein or its chimeric molecule of the particularly separation that the present invention is provided are selected from GM-CSF, GM-CSF-Fc, IL-3, IL-3-Fc, IL-4, IL-4-Fc, IL-5 and IL-5-Fc, and it, which has, includes the physicochemical characteristicses { [P of multiple measurable physical and chemical parameters_x]₁、[P_x]₂、...[P_x]_n, wherein P_xRepresent measurable physical and chemical parameter and " n " be >=1 integer, wherein between and be included in [P_x]₁To [P_x]_nBetween parameter be respectively different measurable physical and chemical parameters, the value of any of which or more than one measurable physicochemical property represents, is associated with the pharmacological characteristics T of a characteristic_yBasis or series of features pharmacological characteristics { [T_y]₁、[T_y]₂、....[T_y]_mOr formed a characteristic pharmacological characteristics T_yBasis or series of features pharmacological characteristics { [T_y]₁、[T_y]₂、....[T_y]_mBasis, wherein T_yThe pharmacological characteristics and m for representing a characteristic are >=1 integers, wherein [T_y]₁To [T_y]_mFor different pharmacological characteristics.

Here term " characteristic " is related to protein or the pharmacological characteristics of its chimeric molecule, refer in the present invention protein or its chimeric molecule one or more be different from further feature physicochemical property pharmacological characteristics.Specifically, the characteristic of same protein of one or more pharmacological characteristics of protein isolate or its chimeric molecule from being produced in prokaryotic or low eukaryotic or even inhuman higher eucaryotic cells is different or has relevant difference.In other specific embodiment, help to realize a certain function as the albumen of the separation of target or the pharmacological characteristic of its chimeric molecule.Here term " measurable physical and chemical parameter " or P_xRepresent protein isolate or one or more measurable characteristics of its chimeric molecule.In one embodiment of the invention, as the albumen of the separation of target or measurable physicochemical property of its chimeric molecule or contribute to or generate pharmacological characteristics T_y。

The protein or chimeric molecule of the separation of the present invention have physical and chemical parameter (P_x), the parameter is used to intactly define protein molecule protein molecule or chimeric molecule.The physical and chemical parameter can be selected from：Apparent molecular weight (P₁), isoelectric point (pI) (P₂), isoform number (P₃), the relative intensity (P of different isoform number₄), carbohydrate percetage by weight (P₅), the actual measurement molecular weight (P after N- connection oligosaccharides deglycosylations₆), N- connections and O- connections oligosaccharides deglycosylation after actual measurement molecular weight (P₇), the percentage (P of acid contents of monosaccharides₈), contents of monosaccharides (P₉), sialic acid content (P₁₀), sulfate and phosphate content (P₁₁)、Ser/Thr：GalNAc ratios (P₁₂), the neutral percentage (P of N- connection oligosaccharides₁₃), the acid percentage (P of N- connection oligosaccharides₁₄), the neutral percentage (P of O- connection oligosaccharides₁₅), the acid percentage (P of O- connection oligosaccharides₁₆), the ratio (P of N- connection oligosaccharides₁₇), the ratio (P of O- connection oligosaccharides₁₈), the structure (P of N- connection oligosaccharide ingredients₁₉), the structure (P of O- connection oligosaccharide ingredients₂₀), the position of N- connection oligosaccharides and composition (P₂₁), the position of O- connection oligosaccharides and composition (P₂₂), common translation modification (P₂₃), posttranslational modification (P₂₄), acylated (P₂₅), acetylation (P₂₆), amidatioon (P₂₇), deamidation (P₂₈), biotinylation (P₂₉), carbamylation (P₃₀), carboxylation (P₃₁), decarboxylation (P₃₂), disulfide formation (P₃₃), fatty-acylation (P₃₄), myristoylation (P₃₅), palmitoylation (P₃₆), octadecane be acylated (P₃₇), formylated (P₃₈), saccharification (P₃₉), glycosylation (P₄₀), glycophosphatidyl inositol grappling (P₄₁), hydroxylating (P₄₂), the combination (P of selenocysteine₄₃), lipid (P₄₄), the addition (P of lipoic acid₄₅), methylate (P₄₆), N or C-terminal closing (P₄₇), N or C-terminal remove (P₄₈), nitrification (P₄₉), methionine oxidized (P₅₀), phosphorylation (P₅₁), protease digestion (P₅₂), prenylation (P₅₃), farnesylation (P₅₄), Mang ox base (P₅₅), phosphopyridoxal pyridoxal phosphate addition (P₅₆), sialylated (P₅₇), asialoglycoprotein (P₅₈), sulfation (P₅₉), ubiquitination (P₆₀), the addition (P of ubiquitin sample molecule₆₁), primary structure (P₆₂), secondary structure (P₆₃), tertiary structure (P₆₄), quaternary structure (P₆₅), chemical stability (P₆₆), heat endurance (P₆₇).The feature of these parameters is provided in table 2.

In a specific embodiment, with physical and chemical parameter (P_x) and pharmacological characteristics (T_y) feature come characterize the present invention GM-CSF, it includes 5-60 apparent molecular weight (P₁), such as：5th, 6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,56,57,58,59,60, it is in a specific embodiment 16-40kDa.The GM-CSF of present invention isoelectric point pI (P₂) it is 1-10, such as 1,2,3,4,5,6,7,8,9,10, and be 2-7 in a specific embodiment；With about 1 to 36 isoforms (isoform), such as 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36 isoforms, in a specific embodiment, isoform number (P₃) it is 10-30.The GM-CSF carbohydrate percentage by weights (P of the present invention₅) be 1 to 99 between, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, it is 0-76% in a specific embodiment, it is 0-65% in other specific embodiments.Actual measurement molecular weight (P after N- connection oligosaccharides is removed₆) it is 15-30kD, it is 15-2kD in a specific embodiment.Remove the actual measurement molecular weight (P after N- connections and O- connection oligosaccharides₇) it is 14-25kD, it is 14-20kD in a specific embodiment.Acid monose percentage (P contained by the GM-CSF of the present invention₈) it is 2 to 2 0% such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20%, it is 6-11% in a specific embodiment.Using GalNAc as standard, GM-CSF of the invention monose (P₉) and sialic acid (P₁₀) percentage composition be 1: 0-3 trehalose, 1: 1-16 GlcNAc, 0.1: 0.1-9 galactolipin, 1: 0.1-9 mannose, and 1: 0-5 NeuNAc, it is in specific embodiments 1: 0.1-1.5 trehalose, 1: 2-12 GlcNAc, 1: 1.0-6.0 galactolipin, 1: 1.0-6.0 mannose and 1: 0-3.0 NeuNAc；It is standard with 3 times of mannoses, for 3: 0-5 trehalose, 3: 0.1-3 GalNAc, 3: 2-15 GlcNAc, 3: 1-6 mannose, and 3: 0-4NeuNAc, it is in specific embodiments 3: 0.1-2.5 trehalose, 3: 0.5-2.5 GalNAc, 3: 5.0-10.0 GlcNAc, 3: 2.0-5.0 mannose and 3: 0-3.0 NeuNAc.Neutral percentage (the P of N- connection oligosaccharides₁₃) it is about 40 to 90%, such as 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90%, it is 49 to 83% in specific embodiments, is in specific embodiments 54 to 78%, is in specific embodiments 59 to 73%.Acid percentage (the P of N- connection oligosaccharides₁₄) it is about 10% to 70%, it is in specific embodiments 17% to 51%, is 22% to 46% in further specific embodiment, is in specific embodiments 27 to 41%.Acid percentage (the P of O- connection oligosaccharides₁₅) it is about 5 to 90%, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90%, it is 9 to 82% in a specific embodiment, it is 29 to 62% in specific embodiments, it is 34 to 57% in specific embodiments.Acid percentage (the P of O- connection oligosaccharides₁₆) it is about 10 to 100%, it is in specific embodiments 18 to 91%, is in specific embodiments 38 to 71%, is in specific embodiments 43 to 66%.The GM-CSF of present invention N- connections glycosylation (P₂₁) include N-44 and N-54 (from signal sequence section start numbering), differentiated in after PNGase processing with PMF.Stability (Ts of the GM-CSF of the present invention in serum/plasma₁₀) it is different from expressed human GM-CSF in non-human cell.Especially compared with the human GM-CSF expressed in E.coli, after being incubated 24 hours with calf serum, stronger rush proliferation activity is shown in the TF-1 GM-CSF of the invention expressed.

The GM-CSF of present invention multiplication capacity (T₃₂) it is different from the human GM-CSF expressed in non-human cell, specifically, GM-CSF proliferation activities (T of the invention₃₂) it is better than human GM-CSF expressed in non-human cell.GM-CSF differentiation capabilities (the T of the present invention₃₃) it is different from the human GM-CSF expressed in non-human cell, compared with the GM-CSF expressed in E.coli, the former has the ability of stronger induction TF-1 cell clonal formations.

In specific embodiments, with one or more physical and chemical parameter (P_x) and pharmacological characteristics (T_y) feature come characterize the present invention IL-3 of the invention, it includes 1 to 250 kD apparent molecular weight (P₁), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 kDa, it is 15 to 35kDa in specific embodiments.PI (the P of IL-3 molecules₂) it is 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 3.5-7.5 in specific embodiments, with about 2 to 50, such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, be in specific embodiments 5-15 isoform (P₃).Carbohydrate percentage (the P of IL-3 molecules₅) it is 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, it is 0 to 60% in specific embodiments.After N- connections oligosaccharides is removed, IL-3 of the invention actual measurement molecular weight (P₆) it is 10 and 25kDa.When using GalNAc as standard, IL-3 oligosaccharide contents (P₉) it is 1: 0.1-8 trehalose, 1: 0.1-7 GlcNAc, 1: 0.1-3 galactolipin, 1: 0.1-3 mannose and 1: 0-5 NeuAc；It is 1: 2-6 trehalose, 1: 3-5 GlcNAc, 1: 0.5-2 galactolipin, 1: 0.5-2 mannose and 1: 0-2 NeuNAc in a specific embodiment；It is 3: 2-25 trehalose, 3: 0.1-6 GalNAc, 3: 4-21 GlcNAc, 3: 0.1-9 galactolipin, and 3: 0-5 NeuAc when being standard with 3 times of mannoses；It is 3: 5-16 trehalose, 3: 2-4GalNAc, 3: 9-14 GlcNAc, 3: 3-6 galactolipin and 3: 0.1-2 NeuAc in a specific embodiment.The IL-3 molecular salivary acid contents of the present invention are expressed as accounting for the percentage (P of monose₁₀), it is 0 to 50%, it is 0 to 20% in a specific embodiment such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50%.Neutral percentage (the P of the IL-3 of present invention N connections oligosaccharides₁₃) it is 70 to 100%, it is 75 to 95% in a specific embodiment, is in specific embodiments 80 to 90%.Acid percentage (the P of the IL-3 of present invention N connection IL-3 oligosaccharides₁₄) it is 0 to 30%, it is 5 to 25% in a specific embodiment, is in specific embodiments 10 to 20%.The IL-3 of present invention immune response characteristic (T₁₃) different from the people IL-3 expressed in non-human cell, when especially being detected with the people IL-3 expressed containing non-human cell ELISA kit, IL-3 of the invention protein concentration is underestimated.In addition, the IL-3 of the present invention (T₁₃) different from the people IL-3 expressed in insect cell.The IL-3 of present invention multiplication capacity (T₃₂) it is different from the people IL-3 that expresses in non-human cell.The IL-3 of present invention multiplication capacity (T₃₂) it is better than the people IL-3 expressed in non-human cell.

In specific embodiments, with one or more physical and chemical parameter (P_x) and pharmacological characteristics (T_y) GM-CSF of the invention is characterized, it includes apparent molecular weight (P₁), its value is 5 to 60, it is 16-40kDa in a specific embodiment such as 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60.The GM-CSF of present invention pI (P₂) it is about 1 to 10, such as 1,2,3,4,5,6,7,8,9,10, in a specific embodiment be 2-7, it is 10-30 isoform (P in a specific embodiment including at least 1 to 36 isoform such as 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36₃).The GM-CSF carbohydrate percentages (P of the present invention₅) it is 1 to 99, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, it is 0-76% in a specific embodiment, it is 0-65% in more particular embodiment.After N- connections oligosaccharides is removed, molecule actual measurement molecular weight is (P₆) 10-35kDa, it is 12-30kDa in a specific embodiment.Actual measurement molecular weight (P after N- connections and O- connections oligosaccharides are removed₇) it is 9-30kDa, it is 11 to 25kDa in a specific embodiment.The GM-CSF of the present invention acid monose percentage (P₈) it is 2 to 20% such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20%, it is 6-11% in a specific embodiment.When using GalNAc as standard, GM-CSF of the invention monose (P₉) and sialic acid content (P₁₀) it is 1: 0-3 trehalose, 1: 1-16GlcNAc, 0.1: 0.19 galactolipin, 1: 0.1-9 mannose and 1: 0-5 NeuNAc, it is 1: 0.1-1.5 trehalose in a specific embodiment, 1: 2-12 GlcNAc, 1: 1.0-6.0 galactolipin, 1: 1.0-6.0 mannose, and 1: 0-3.0 NeuNAc；When being standard with 3 times of mannoses, it is then 3: 0-5 trehalose, 3: 0.1-3 GalNAc, 3: 2-15 GlcNAc, 3: 1-6 galactolipin, 3: 0-4 NeuNAc, it is 3: 0.1-2.5 trehalose, 3: 0.5-2.5 GalNAc in a specific embodiment, 3: 5.0-10.0 GlcNAc, 3: 2.0-5.0 galactolipin, and 3: 0-3.0NeuNAc.N connection oligosaccharides (P₁₃) neutral percentage is 40 to 90%, such as 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90%, in a specific embodiment be 49 to 83%, it 54 is 78% to be in specific embodiments, is 59 to 73% in more particular embodiment.Acid percentage (the P of N- connections connection₁₄) it is 10% to 70%, it is 17% to 51% in a specific embodiment, is in specific embodiments 22% to 46%, is 27 to 41% in more particular embodiment.Neutral percentage (the P of the oligosaccharides of O connections₁₅) be 5 be 90%, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90%, it is 9 to 82% in a specific embodiment, it is 29 to 62% in specific embodiments, it is 34 to 57% in more particular embodiment.Acid percentage (the P of O- connection oligosaccharides₁₆) it is about 10 to 100%, it is 18 to 91% in a specific embodiment, is in specific embodiments 38 to 71%, is 43 to 66% in more particular embodiment.The GM-CSF N- connection glycosylation sites (P of the present invention₂₁) include N-44 and N-54 (by signal sequence section start open numbering), these sites by PNGase handle after PMF recognize.The GM-CSF of present invention serum/plasma stability (T₁₀) different from the human GM-CSF expressed in non-human cell, especially after TF-1 is incubated 24 hours with hyclone, the former shows stronger proliferation activity than the GM-CSF expressed in E.coli.GM-CSF proliferation activities (the T of the present invention₃₂) it is different from the GM-CSF, GM-CSF proliferation activities (T of the invention that express in non-human cell₃₂) it is better than about 5-12 times of the human GM-CSF expressed in E.coli.Its differentiation activity (T₃₃) also different from the GM-CSF expressed in non-human cell, compared with the GM-CSF that E.coli is expressed, the former induces the ability of TF-1 Clone formations strong 1.5-2 times.

In specific embodiments, IL-3 molecules of the invention are with one or more physical and chemical parameter (P_x) and pharmacological characteristics (T_y) be characterized, including apparent molecular weight (P₁), its value is by 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, it is 15 to 35kDa in specific embodiments.IL-3 pI (P₂) it is 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 3.5-7.5 in specific embodiments, with about 2 to 50, such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, be in specific embodiments 5-15 isoform (P₃).IL-3 molecule carbohydrate percentages (P₅) it is 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, it is 0 to 60% in specific embodiments.After N- connections oligosaccharides is removed, IL-3 of the invention actual measurement molecular weight is (P₆) it is 8-30kDa, it is in specific embodiments 10-25kDa.When using GalNAc as standard, IL-3 contents of monosaccharides (P₉) it is 1: 0.1-8 trehalose, 1: 0.1-7 GlcNAc, 1: 0.1-3 galactolipin, 1: 0.1-3 mannose and 1: 0-5 NeuAc；It is 1: 2-6 trehalose, 1: 3-5GlcNAc, 1: 0.5-2 galactolipin, 1: 0.5-2 mannose and 1: 0-2 NeuNAc in a specific embodiment；It is 3: 2-2 5 trehalose, 3: 0.1-6 GalNAc, 3: 4-21 GlcNAc, 3: 0.1-9 galactolipin, and 3: 0-5 NeuAc when being standard with 3 times of mannoses；It is 3: 5-16 trehalose, 3: 2-4 GalNAc, 3: 9-14 GlcNAc, 3: 3-6 galactolipin and 3: 0.1-2 NeuAc in a specific embodiment.The IL-3 molecular salivary acid of the present invention accounts for monose percentage (P₁₀) it is 0 to 50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50%, it is 0 to 20% in a specific embodiment.IL-3 percentages (P in N connection oligosaccharides₁₃) it is 70 to 100%, it is 75 to 95% in a specific embodiment, is in specific embodiments 80 to 90%.Acid percentage (the P of the N connections oligosaccharides of the IL-3 molecules of the present invention₁₄) it is 0 to 30%, it is 5 to 25% in a specific embodiment, is in specific embodiments 10 to 20%.The IL-3 of present invention immune response characteristic (T₁₃) different from the people IL-3 expressed in non-human cell, when especially being detected with the people IL-3 expressed containing useful non-human cell ELISA kit, IL-3 of the invention protein concentration is underestimated.In addition, the IL-3 of present invention immune response characteristic (T₁₃) different from the people IL-3 expressed in insect cell.The IL-3 of present invention multiplication capacity is different from the people IL-3 expressed in non-human cell, IL-3 of the invention multiplication capacity (T₃₂) strong 1.1-2.5 times compared with the people IL-3 that E.coli is expressed.

In specific embodiments, IL-4 molecules of the invention are with one or more physical and chemical parameter (P_x) and pharmacological characteristics (T_y) be characterized, including apparent molecular weight (P₁), its value is by 1 to 120, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 14, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120kDa, it is 12 to 24kDa in specific embodiments.IL-4 pI (P₂) it is 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 8-11 in specific embodiments, with about 2 to 50, such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, be in specific embodiments 1-3 isoform (P₃).IL-4 molecule carbohydrate percentages (P₅) it is 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, it is 0 to 25% in specific embodiments.After N- connections oligosaccharides is removed, IL-4 of the invention actual measurement molecular weight is (P₆) 8-24kDa, it is in specific embodiments 10 to 20kDa.And after O- connections and N- connection oligosaccharides are removed, the IL-4 molecular weight (P detected₇) it is 8-22kDa, it is 10-18kDa in a specific embodiment.Neutral percentage (the P of the oligosaccharides of the IL-4 of present invention N connections₁₃) it is 50%-100%, it is 65-100% in a specific embodiment, is in specific embodiments 70-100%.Acid percentage (the P of the IL-4N connections oligosaccharides of the present invention₁₄) it is 0-50%, it is in specific embodiments 0-45%, is in specific embodiments 0-30%.The IL-4N- connection glycosylation sites (P of the present invention₂₁) include N-62 (by signal sequence section start open numbering), recognized after being handled with PNGase by PMF.Curing skeleton structure (P₃₃) include Cys27-Cys151, Cys48-Cys89 and Cys70-Cys123 (by signal sequence section start open numbering cysteine residues).

In specific embodiments, IL-4 of the invention immune response characteristic (T₁₃) different from the people IL-4 expressed in non-human cell, when especially being detected with the people IL-4 expressed containing non-human cell's system ELISA kit, IL-4 of the invention protein concentration is underestimated.The IL-4 of present invention proliferation activity (T₃₂) it is different from the people IL-4 of non-human cell's system expression, compared with the people IL-4 that E.coli is expressed, IL-4 proliferation activities (T of the invention₃₂) high 25-54 times；Compared with the people IL-4 of expressing cho cell, IL-4 proliferation activities (T of the invention₃₂) high 1.75 times.After elevated temperature preincubate, IL-4 proliferation activities (T of the invention₃₂) also different from the people IL-4 that non-human cell's system is expressed.After preincubate TF-1 cells at 37 DEG C of culture medium 4 days, IL-4 of the invention (T₃₂) value is high compared with the GM-CSF that E.coli is expressed 13-30 times.

In specific embodiments, IL-5 molecules of the invention are with one or more physical and chemical parameter (P_x) and pharmacological characteristics (T_y) be characterized, including apparent molecular weight (P₁), its value is by 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, it is 15 to 25kDa in specific embodiments.IL-5 pI (P₂) it is 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 4-9 in specific embodiments, plus about 2-50, such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, be in specific embodiments 5-12 isoform (P₃).Carbohydrate percentage (the P of I L-5 molecules₅) it is 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, it is 10-50% in specific embodiments.After N- connections oligosaccharides is removed, IL-5 actual measurement molecular weight (P of the invention₆) it is 9-30kDa, it is in specific embodiments 11-25kDa.And after O- connections and N- connection oligosaccharides are removed, the IL-5 molecular weight (P detected₇) it is 8-27kDa, it is 10-22kDa in a specific embodiment.When using GalNAc as standard, IL-5 of the invention contents of monosaccharides (P₉) it is 1: 0.1-3 trehalose, 1: 0.5-7GlcNAc, 1: 0.05-3 galactolipin, 1: 0.1-3 mannose and 1: 0-5 NeuAc；It is 1: 0-0.5 trehalose, 1: 2-4.5 GlcNAc, 1: 1-2 galactolipin, 1: 1-2 mannose and 1: 0.1-1 NeuNAc in a specific embodiment；It is 3: 0.1-3 trehalose, 3: 0.1-4 GalNAc, 3: 1-17 GlcNAc, 3: 1-8 galactolipin, and 3: 0-5 NeuAc when being standard with 3 times of mannoses；It is 3: 0-1 trehalose, 3: 2-3 GalNAc, 3: 3-12GlcNAc, 3: 2-5 galactolipin and 3: 0.2-1 NeuNAc in a specific embodiment.The IL-5 molecular salivary acid of the present invention accounts for monose percentage (P₁₀) it is 0 to 50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50%, it is 2 to 10% in a specific embodiment.When using GalNAc as standard, IL-5 sulphates contents (P of the invention₁₁) it is 1: 2-14 sulfate；It is 1: 5-10 sulfate in a specific embodiment；It is 3: 7-36 sulfate when being standard with 3 times of mannoses, is 3: 12-24 sulfate in a specific embodiment.Sulfate accounts for IL-5 monose percentages (P₅₉) it is 5-35%, it is 10-25% in a specific embodiment.N connection IL-5 oligosaccharides neutrality percentage (P₁₃) it is 30 to 90%, it is 40 to 80% in a specific embodiment, is in specific embodiments 50 to 75%.N connection IL-5 oligosaccharides acidity percentage (P₁₄) it is 10 to 70%, it is 20 to 60% in a specific embodiment, is in specific embodiments 25 to 50%.Neutral percentage (the P of the IL-5 of present invention O- connections oligosaccharides₁₅) it is 40 to 100%, it is 50 to 100% in a specific embodiment, is in specific embodiments 60 to 100%.Acid percentage (the P of the IL-5 of present invention O- connections oligosaccharides₁₆) it is 0 to 60%, it is 0 to 50% in a specific embodiment, is in specific embodiments 0 to 40%.

In a specific embodiment, present invention contemplates the form of the separation for belonging to the helical bundle superfamily of short chain 4 or the protein or its chimeric molecule related to the helical bundle superfamily of short chain 4 selected from GM-CSF, GM-CSF-Fc, IL-3, IL-3-Fc, IL-4, IL-4-Fc, IL-5 and IL-5-Fc.Protein or its chimeric molecule in the present invention have a pharmacological characteristics of characteristic, including the following characteristics in part or are made up of following characteristics：Therapeutic effect (T₁), dose therapeutically effective (TCID₅₀)(T₂), bioavilability (T₃), from the time (T for being administered into maintaining treatment level₄), absorption rate (T₅), discharge rate (T₆), special activity (T₇), heat endurance (T₈), lyophilized stability (T₉), serum/plasma stability (T₁₀), serum half-life (T₁₁), the solubility (T in blood flow₁₂), immune response feature (T₁₃), immunogenicity (T₁₄), neutralizing antibody suppress (T_14A), side effect (T₁₅), receptor/ligand affinity (T₁₆), receptor/ligand activation (T₁₇), tissue or cell category specificity (T₁₈), penetration capacity (such as intestines, lung, blood-brain barrier, skin) (T of biomembrane or barrier₁₉), generation blood vessel ability (T_19A), tissue resorption (T₂₀), degraded stability (T₂₁), freeze-thaw stability (T₂₂), protease stability (T₂₃), ubiquitin stability (T₂₄), administration reduce (T₂₅), administering mode (T₂₆), the compatibility (T with other pharmaceutical excipients or carrier₂₇), the residual (T in organism or environment₂₈), preserve during stability (T₂₉), (T such as toxicity in organism or environment₃₀)。

In addition, the protein or chimeric molecule of the present invention can have different biological effect (T in different cell categories₃₁), including but not limited to people's primary cell, such as lymphocyte, red blood cell, retina cell, liver cell, neuron, horn cell, endothelial cell, endoderm cell, ectoderm cell, mesoblastema, epithelial cell, nephrocyte, liver cell, osteocyte, bone marrow cell, lymph node cells, dermal cell, fibroblast, T cell, B cell, thick liquid cell, NK, macrophage, bite neutrophil leucocyte, grain cell of Langerhan, BMDC, bite sour granulocyte, bite alkali granulocyte, mammary cell, small leaf cell, prostatic cell, pneumonocyte, esophageal cells, pancreatic cell, Beta cells (insulin secretory cell), angioblast, myocyte, elliptocyte (liver cell), mesenchymal cell, brain microvessel endothelial cells in vitro, astroglia, spongiocyte, a variety of stem cells include adult and embryonic stem cell, a variety of progenitor cells；With other people permanent, conversion or cancer cell systems.

Biological effect in cell includes multiplication effect (T₃₂), differentiation (T₃₃), apoptosis (T₃₄), the growth (T of cell size₃₅), cell factor adhesion (T₃₆), cell adhesion (T₃₇), cell amplification (T₃₈), cell mobility (T₃₉), migration and intrusion (T₄₀), chemotaxis (T₄₁), cell phagocytosis (T₄₂), signal transduction (T₄₃), rich protein to receptor/ligand (T₄₄), the activation (T of JAK/STAT approach₄₅), the activation (T of Ras-erk approach₄₆), the activation (T of AKT approach₄₇), the activation (T of PKC approach₄₈), the activation (T of PKA approach₄₉), src activation (T₅₀), fas activation (T₅₁), TNFR activation (T₅₂), NFkB activation (T₅₃), p38MAPK activation (T₅₄), c-fos activation (T₅₅), secretion (T₅₆), acceptor caves in (T₅₇), acceptor reciprocation (T₅₈), the up-regulation of surface markers or downward (T₅₉), before FACS/change (T of other scattering signatures₆₀), the change (T of subgroup ratio₆₁), differential gene expression (T₆₂), meronecrosis (T₆₃), cell agglutination (T₆₄), cellular rejection (T₆₅) and heparin sulfate combination (T₆₆) and glycosylation structure combination (T₆₇) and chondroitin sulfate combination (T₆₈) and extracellular matrix combination (such as collagen, fibronectin) (T₆₉) and artificial material combination (such as support) (T₇₀) and carrier combination (T₇₁) and confactor combination (T₇₂), individually or the effect (T in the mixture containing other protein to stem cells hyperplasia, differentiation and/or self-renewing₇₃) etc..The feature of these characteristics is provided in table 3.

Invention further provides the chimeric molecule for including the albumen of separation or its segment, such as constant region (Fc) or framework region that the ectodomain of one membrane bound protein passes through one or more protein connexon combination human immunoglobulin(HIg)s.Such chimeric molecule also illustrates that into albumen-Fc herein.Include GM-CSF-Fc, IL-3-Fc, IL-4-Fc and IL-5-Fc present invention contemplates the example of such albumen-Fc chimeras.

Such protein-Fc has the characteristic of measurable physical and chemical parameter, these personality presentations, the albumen-Fc of association separation one or more specific pharmacological characteristics.It is contemplated by the invention that other chimeric molecules include albumen or albumen-Fc or its segment, be connected with an aliphatic radical such as polyunsaturated fatty acid molecule.Here aliphatic radical can be with an amino acid residue in binding molecule skeleton a amino acid residue or side chain.

Invention further provides the chimeric molecule for including protein isolate or its segment, such as Fc or framework region that the ectodomain of one embrane-associated protein passes through one or more protein connexon combination human immunoglobulin(HIg)s.In other respects, the constant region (Fc) or framework region of mammalian immunoglobulin derive from and include primate, including the mankind, suede, chimpanzee and gorilla, domestic animal (such as ox, sheep, pig, horse, donkey), experimental animal (such as mouse, rat, cavy, hamster, rabbit), wild animal (such as rodent of pet (such as cat, dog) and capture, fox, deer, kangaroo).In a further embodiment, Fc or framework region are human immunoglobulin(HIg)s.Mammal is the mankind in a particular embodiment.Such chimeric molecule also illustrates that into albumen-Fc herein.Include albumen or albumen-Fc or its segment present invention contemplates other chimeric molecules, be connected with the aliphatic radical of a such as polyunsaturated fatty acid molecule.Here an amino acid residue in the possible binding molecule skeleton of aliphatic radical or an amino acid residue in side chain.Chimeric molecule in the present invention, including GM-CSF-Fc, IL-3-Fc, IL-4-Fc and IL-5-Fc, it has the characteristic of measurable physical and chemical parameter, these personality presentations, the albumen-Fc of association separation one or more specific pharmacological characteristics.

Therefore, the invention provides by nucleotide sequence coded isolated polypeptide, the sequence is selected from SEQ ID NO：25th, 27,29,31,35,37,39,41,43,45,47,51,53,55,57,61,63,65,67, either there are at least nucleotide sequence of 65% uniformity or the sequence that can hybridize with any one in above-mentioned sequence or its complementary type under low stringency condition with above-mentioned sequence any of which one.

Another aspect provides the polypeptide of separation, the polypeptide is as coded by respectively correspond toing sequences of the respective mRNA of following sequences by cell processing montage, and the sequence is selected from SEQID NO：69、70、71、72.

Another aspect provides the polypeptide of separation, its amino acid sequence included is selected from SEQ ID NO：26th, 28,30,32,36,38,40,42,44,46,48,52,54,56,58,62,64,66,68, or there is with above-mentioned sequence any of which one amino acid sequence of at least 65% similitude.

Present invention further contemplates the pharmaceutical composition combined including at least a portion protein or its chimera, pharmacologically acceptable carrier, confactor and/or diluent.

In terms of primary structure, the invention provides the protein of separation or its chimera or its segment, its nucleotide coding sequence is selected from SEQ ID NO：25th, 27,29,31,35,37,39,41,43,45,47,51,53,55,57,61,63,65,67, either there are at least nucleotide sequence of 60% uniformity or the sequence that can hybridize with any one in above-mentioned sequence or its complementary type in low stringency condition with above-mentioned sequence any of which one.

In addition, other aspects of the present invention provide the isolated nucleotide molecule of encoding proteins matter or its chimeric molecule or its functional area, comprising with SEQ ID NO：25th, 27,29,31,35,37,39,41,43,45,47,51,53,55,57,61,63,65,67 have at least 60% similitude, or after optimal comparison and/or can be with SEQ ID NO：25th, 27,29,31,35,37,39,41,43,45,47,51,53,55,57,61,63,65,67 or the nucleotide sequence that hybridizes under low stringency condition of their complementary type.

In a specific embodiment, present invention is generally directed to the nucleic acid molecule of separation, it has the nucleotide sequence for encoding following molecules, and affiliated molecule is belonging to the helical bundle superfamily of short chain 4 or the protein or its chimeric molecule relevant with the helical bundle superfamily of short chain 4, and it is selected from：GGM-CSF, GM-CSF-Fc, IL-3, IL-3-Fc, IL-4, IL-4-Fc, IL-5 and IL-5-Fc or its segment, substantially with SEQ ID NO：26th, one or more amino acid sequences shown in 28,30,32,36,38,40,42,44,46,48,52,54,56,58,62,64,66,68, or with SEQ ID NO：26th, one or more sequences after 28,30,32,36,38,40,42,44,46,48,52,54,56,58,62,64,66,68 optimal comparisons have the amino acid sequence of at least 60% similitude.

In other respects, the invention provides the nucleic acid molecule of separation, coding is selected from belonging to the helical bundle superfamily of short chain 4 or the protein or its chimeric molecule relevant with the helical bundle superfamily of short chain 4, aforementioned proteins are selected from GM-CSF, IL-3, IL-4 and IL-5 or its segment, comprising selected from SEQ ID NO：27th, 29,37,39,41,43,53,55,63 or 65 nucleotide sequence, it directs or through the nucleotide sequence of well known encoding proteins matter connexon on one or more this areas, it is connected with constant (Fc) or framework region of encoding human immunoglobulin nucleotide sequence, constant (Fc) or framework region of described encoding human immunoglobulin nucleotides sequence are substantially such as SEQ ID NO：1st, 3,5,7,9,11,13,15,17 or 19 it is one or more shown in.In a specific embodiment, protein connexon includes IP, GSSNT, TRA or VDGIQWIP.

In other respects, the invention provides the protein of separation, the protein is to belong to the helical bundle superfamily of short chain 4 or the protein relevant with the helical bundle superfamily of short chain 4, and it is selected from GM-CSF, IL-3, IL-4 and IL-5 or its segment, and the protein, which includes to be selected from, has SEQID NO：28th, 30,38,40,42,44,54,56,64 or 66 amino acid sequence, it directs or through well known protein connexon on one or more this areas, it is connected with constant (Fc) or framework region of human immunoglobulin(HIg) nucleotide sequence, constant (Fc) or framework region of described encoding human immunoglobulin nucleotide sequence are substantially such as SEQ ID NO：2nd, 4,6,8,10,12,14,16,18 or 20 it is one or more shown in.

Invention further provides the albumen of separation or its chimeric molecule or encode its nucleic acid molecule diagnosis, prevent, treatment, nutrition and/or research application in application.Especially it is noted that the invention provides treating or preventing disease on subject animal or alleviating the method for disease symptomses, described method includes giving the protein or its chimeric molecule of the separation of effective dose to the subject animal.

Moreover, present invention contemplates be screened to have different diagnosis with the albumen or its chimeric molecule of the present invention, preventing, treatment, the small molecule of nutrition and/or research application effect.

Present invention further contemplates can be using protein isolate or its chimeric molecule as immunogene, to produce the antibody for treating and diagnosing.

Present invention further contemplates the culture medium for cultivating stem cell or related methods for the treatment of can be added using protein isolate or its chimeric molecule.

Present invention provides the pharmaceutical formulation containing albumen or its chimeric molecule in diagnosis, prevent, treatment, the application in nutrition and/or research application.

Present invention provides the purposes that people's source protein or its chimeric molecule are used as immunoassays or the standard protein of its kit.Present invention provides the method for determining people's cell expression people's albumen or its chimeric molecule level in biological agent.

Table 1

Sequence identifier

Sequence identifier	Sequence
		SEQ ID NO：1	Human IgG1's Fc nucleotide sequences
SEQ ID NO：2	Human IgG1's Fc amino acid sequences
		SEQ ID NO：3	Human IgG1 Fc nucleotide sequences (variant)
SEQ ID NO：4	Human IgG1 Fc amino acid sequences (variant)
		SEQ ID NO：5	Human IgG2's Fc nucleotide sequences
SEQ ID NO：6	Human IgG2's Fc amino acid sequences
		SEQ ID NO：7	The Fc nucleotide sequences of human IgG 3
SEQ ID NO：8	The Fc amino acid sequences of human IgG 3

SEQ ID NO：9	The Fc nucleotide sequences of human IgG 4
		SEQ ID NO：10	The Fc amino acid sequences of human IgG 4
SEQ ID NO：11	People's IgA1 Fc nucleotide sequences
		SEQ ID NO：12	People's IgA1 Fc amino acid sequences
SEQ ID NO：13	People's IgA2 Fc nucleotide sequences
		SEQ ID NO：14	People's IgA2 Fc amino acid sequences
SEQ ID NO：15	People's IgM Fc nucleotide sequences
		SEQ ID NO：16	People's IgM Fc amino acid sequences
SEQ ID NO：17	People's IgE Fc nucleotide sequences
		SEQ ID NO：18	People's IgE Fc amino acid sequences
SEQ ID NO：19	People's IgD Fc nucleotide sequences
		SEQ ID NO：20	People's IgD Fc amino acid sequences
SEQ ID NO：21	Human IgG1 Fc Apollo forward primers (pIRESbleo GSSNT cloning sites) (nucleotide sequence)
		SEQ ID NO：22	Human IgG1 Fc Apollo reverse primers (pIRESbleo GSSNT cloning sites) (nucleotide sequence)
SEQ ID NO：23	GM-CSF forward primers (nucleotide sequence)
		SEQ ID NO：24	GM-CSF reverse primers (nucleotide sequence)
SEQ ID NO：25	GM-CSF nucleotide sequences (signal peptide)
		SEQ ID NO：26	GM-CSF amino acid sequences (signal peptide)
SEQ ID NO：27	GM-CSF nucleotide sequences (mature peptide)
		SEQ ID NO：28	GM-CSF amino acid sequences (mature peptide)
SEQ ID NO：29	GM-CSF nucleotide sequences (signal peptide+mature peptide)
		SEQ ID NO：30	GM-CSF amino acid sequences (signal peptide+mature peptide)
SEQ ID NO：31	The nucleotide sequence (signal peptide+coded polypeptide+connexon+IgG1 Fc) of GM-CSF-Fc entire constructs

SEQ ID NO：32	The amino acid sequence (signal peptide+coded polypeptide+connexon+IgG1 Fc) of GM-CSF-Fc entire constructs
		SEQ ID NO：33	IL-3 forward primers (nucleotide sequence)
SEQ ID NO：34	IL-3 reverse primers (nucleotide sequence)
		SEQ ID NO：35	IL-3 nucleotide sequences (signal peptide)
SEQ ID NO：36	IL-3 amino acid sequences (signal peptide)
		SEQ ID NO：37	IL-3 nucleotide sequences (mature peptide)
SEQ ID NO：38	IL-3 amino acid sequences (mature peptide)

Sequence identifier	Sequence
		SEQ ID NO：39	IL-3 nucleotide sequences (mature peptide) (variant)
SEQ ID NO：40	IL-3 amino acid sequences (mature peptide) (variant)
		SEQ ID NO：41	IL-3 nucleotide sequences (signal peptide+mature peptide)
SEQ ID NO：42	IL-3- amino acid sequences (signal peptide+mature peptide)
		SEQ ID NO：43	IL-3-Fc nucleotide sequences (signal peptide+mature peptide (variant)
SEQ ID NO：44	IL-3-Fc amino acid sequences (signal peptide+mature peptide (variant)
		SEQ ID NO：45	The nucleotide sequence (signal peptide+mature peptide+GSSNT connexon IgG1 Fc) of IL-3-Fc entire constructs
SEQ ID NO：46	The amino acid sequence (signal peptide+mature peptide+GSSNT connexon I gG1 Fc) of IL-3-Fc entire constructs
		SEQ ID NO：47	The nucleotide sequence (signal peptide+mature peptide (variant)+GSSNT connexon IgG1 Fc) of IL-3-Fc entire constructs
SEQ ID NO：48	The amino acid sequence (signal peptide+mature peptide (variant)+GSSNT connexon IgG1 Fc) of IL-3-Fc entire constructs
		SEQ ID NO：49	IL-4 forward primers (nucleotide sequence)
SEQ ID NO：50	IL-4 reverse primers (nucleotide sequence)
		SEQ ID NO：51	IL-4 nucleotide sequences (signal peptide)

SEQ ID NO：52	IL-4 amino acid sequences (signal peptide)
		SEQ ID NO：53	IL-4 nucleotide sequences (mature peptide)
SEQ ID NO：54	IL-4 amino acid sequences (mature peptide)
		SEQ ID NO：55	IL-4 nucleotide sequences (signal peptide+mature peptide)
SEQ ID NO：56	IL-4 amino acid sequences (signal peptide+mature peptide)
		SEQ ID NO：57	The nucleotide sequence (signal peptide+mature peptide+GSSNT connexon+I gG1 Fc) of IL-4-Fc entire constructs
SEQ ID NO：58	The amino acid sequence (signal peptide+mature peptide+GSSNT connexon+I gG1 Fc) of IL-4-Fc entire constructs
		SEQ ID NO：59	IL-5 forward primers (nucleotide sequence)
SEQ ID NO：60	IL-5 reverse primers (nucleotide sequence)
		SEQ ID NO：61	IL-5 nucleotide sequences (signal peptide)
SEQ ID NO：62	IL-5 amino acid sequences (signal peptide)
		SEQ ID NO：63	IL-5 nucleotide sequences (mature peptide)
SEQ ID NO：64	IL-5 amino acid sequences (mature peptide)
		SEQ ID NO：65	IL-5 nucleotide sequences (signal peptide+mature peptide)
SEQ ID NO：66	IL-5 amino acid sequences (signal peptide+mature peptide)
		SEQ ID NO：67	The nucleotide sequence (signal peptide+mature peptide+GSSNT connexon IgG1 Fc) of IL-5-Fc entire constructs
SEQ ID NO：68	The amino acid sequence (signal peptide+mature peptide+GSSNT connexon IgG1 Fc) of IL-5-Fc entire constructs
		SEQ ID NO：69	GM-CSF gene group nucleotide sequence
SEQ ID NO：70	IL-3 genome nucleotide sequences
		SEQ ID NO：71	IL-4 genome nucleotide sequences
SEQ ID NO：72	IL-5 genome nucleotide sequences

Table 2：Physical and chemical parameter list

P_x	Physical and chemical parameter	GM-CSF	IL-3	IL-4	IL-5
						P₁	Apparent molecular weight	16-40kDa	15-35kDa	12-24kDa	15-25kDa
P₂	Isoelectric point (pI)	2-7	3.5-7.5	8-11	4-9
						P₃	Isoform number	10-30	5-15	1-3	5-12
P₄	The relative intensity of different isoform numbers
						P₅	Carbohydrate percentage	0-65%	0-60%	0-25%	10-50%
P₆	Molecular weight is surveyed after N- connection oligosaccharides deglycosylations	12-30kDa	10-25kDa	10-20kDa	11-25kDa
						P₇	Molecular weight is surveyed after removing N- connections and O- connection oligosaccharides deglycosylations	11-25kDa		10-18kDa	10-22kDa
P₈	Acid monose percentage composition	6-11%

P₉

Contents of monosaccharides

Using GalNAc as standard, it is：1: 0.1-1.5 trehalose, 1: 2-12 GlcNAc, 1: 1.0-6.0 galactolipin, 1: 1.0-6.0 mannose, 1 to 0-3.0 NeuNAc；It is standard with 3 times of mannoses, is：3: 0.1-2.5 trehalose, 3: 0.5-2.5 GalNAc, 3: 5.0-10.0 GlcNAc, 3: 2.0-5.0 galactolipin, 3: 0-3.0 NeuNAc.

Using GalNAc as standard, it is：1: 2-6 trehalose, 1: 3-5 G1cNAc, 1: 0.5-2 galactolipin, 1: 0.5-2 mannose, 1: 0-2 NeuNAc；It is standard with 3 times of mannoses, is：3: 5-16 trehalose, 3: 2-4 GalNAc, 3: 9-14 GlcNAc, 3: 3-6 galactolipin, 3: 0.1-2 NeuAc.

Using GalNAc as standard, it is：1: 0-0.5 is trehalose, 1: 2-4.5 GlcNAc, 1: 1-2 galactolipin, 1: 1-2 mannose, 1: 0.1-1 NeuNAc；It is standard with 3 times of mannoses, is：3: 0-1 trehalose, 3: 2-3 GalNAc, 3: 3-12 GlcNAc, 3: 2-5 galactolipin, 3: 0.2-1 NeuNAc.

P₁₀	Sialic acid content	It is 0-20 %. when being expressed as the percentage of contents of monosaccharides	It is 2-10%. when being expressed as the percentage of contents of monosaccharides
				P₁₁	Sulfate and phosphate content				It is 1: 5-10 sulfate when using GalNAc as standard；It is 3: 12-24 sulfate when with 3 times of mannoses being standard.
P₁₂	Ser/Thr: GalNAc ratio
				P₁₃	N connections oligosaccharide ingredient neutrality percentage	59-73%	80-90%	70-100%	50-75%

P_x	Physical and chemical parameter	GM-CSF	IL-3	IL-4	IL-5
							Than
P₁₄	The acid percentage of N connection oligosaccharide ingredients	27-41%	10-20%	0-30%	25-50%
						P₁₅	O connections oligosaccharide ingredient neutrality percentage	34-57%		60-100%
P₁₆	The acid percentage of O connection oligosaccharide ingredients	43-66%			0-40%
						P₁₇	N connection oligosaccharide ratios
P₁₈	O connection oligosaccharide ratios
						P₁₉	The structure of N junction fragments
P₂₀	The structure of O junction fragments
						P₂₁	The position of N- connection oligosaccharides and composition	Including N-44 and N-54 (by signal sequence section start open numbering), recognized after PNGa se processing with PMF	Including N-62 (by signal sequence section start open numbering), recognized after PNGase processing with PMF
P₂₂	The position of O- connection oligosaccharides and composition
						P₂₃	Common translation is processed
P₂₄	Post translational processing
						P₂₅	Acetylation
P₂₆	Acetylation
						P₂₇	Amination
P₂₈	Deaminizing
						P₂₉	Biotinylation
P₃₀	Carbamoylation and carbamoylation

P₃₁	Carboxylated
			P₃₂	Remove carbonylation
P₃₃	Disulfide formation	Disulfide formation site (P₃₃) include Cys27-Cys151, Cys48-Cys89 and Cys 70-Cys123 (by signal sequence section start open numbering cysteine residues.
			P₃₄	Fatty acid acyl
P₃₅	Myristoylation
			P₃₆	Palmitoylation
P₃₇	Stearoylation
			P₃₈	Formyl
P₃₉	Saccharification
			P₄₀	Glycosylation
P₄₁	Phosphatidyl sugar

P_r	Physical and chemical parameter	GM-CSF	IL-3	IL-4	IL-5
						P₄₂	Hydroxylating
P₄₃	With reference to selenocysteine
						P₄₄	It is esterified
P₄₅	Lipoic acid is added

P₄₆	Methylate
		P₄₇	N or C-terminal are blocked
P₄₈	N or C-terminal are removed
		P₄₉	Nitration
P₅₀	It is methionine oxidized
		P₅₁	Phosphorylation
P₅₂	Proteolysis
		P₅₃	Prenylation
P₅₄	Farnesylation
		P₅₅	Geranylgeranylation
P₅₆	Pyridoxal phosphate
		P₅₇	It is sialylated
P₅₈	Asialoglycoprotein
		P₅₉	Sulfation	It is when being expressed as contents of monosaccharides percentage：10-25%.
P₆₀	Ubiquitination
		P₆₁	Plus ubiquitin sample molecule
P₆₂	Primary structure
		P₆₃	Secondary structure
P₆₄	Tertiary structure
		P₆₅	Quaternary structure
P₆₆	Chemical stability
		P₆₇	Heat endurance

Table 3：Pharmacological characteristics list

T_y	Pharmacological characteristics	GM-CSF	IL-3	IL-4	IL-5
						T₁	Therapeutic effect
T₂	Dose therapeutically effective (TCID₅₀)
						T₃	Bioavilability
T₄	Maintaining treatment dose time
						T₅	Absorptivity
T₆	Excretion rate
						T₇	Specificity
T₈	Heat endurance
						T₉	Lyophilized rear stability
T₁₀	Serum/plasma stability	Hyclone was incubated TF-1 cells after 24 hours, and multiplication capacity is better than the GM-CSF of E.coli expression
						T₁₁	Serum half-life
T₁₂	Blood is soluble
						T₁₃	Immune response characteristic	To non-human cell's expression when being detected for the ELISA of standard, protein concentration is underestimated；It is different from the people IL-3 of insect cell expression	To non-human cell's expression when being detected for the ELISA of standard, protein concentration is underestimated

T₁₄	Immunogenicity
		T_14A	The suppression situation of neutralizing antibody
T₁₅	Side effect
		T₁₆	Receptor/ligand binding affinity
T₁₇	Receptor/ligand activity
		T₁₈	Tissue or cell-specific
T₁₉	By the ability of biomembrane or barrier (such as enteron aisle, lung,

T_y	Pharmacological characteristics	GM-CSF	IL-3	IL-4	IL-5
							Blood-brain barrier, skin etc.)
T_19A	Angiogenesis ability
						T₂₀	Tissue intake
T₂₁	Stability to degradation
						T₂₂	Freeze-thaw stability
T₂₃	To protease stability
						T₂₄	To ubiquitin stability
T₂₅	Reduce dosage

T₂₆	Administering mode
					T₂₇	With the compatibility of other pharmacology excipient or carrier
T₂₈	Stability in organ or environment
					T₂₉	Bin stability
T₃₀	Toxicity in organ or environment
					T₃₁	The different biological effects that different cell types are produced
T₃₂	Multiplication capacity	It is strong to TF-1 ability of cell proliferation 5-12 times compared with the GM-CSF that E.coli is expressed	It is strong to M-NFS-60 ability of cell proliferation 1.1-2.5 times compared with the people IL-3 that E.coli is expressed	It is strong to TF-1 ability of cell proliferation 25-54 times compared with the IL-4 that E.coli is expressed；It is strong 1.75 times compared with the IL-4 multiplication capacities that CHO is expressed；It is high 13-30 times compared with the GM-CSF that E.coli is expressed after preincubate TF1 cells at 37 DEG C of culture medium 4 days
					T₃₃	Differentiation capability	Ability than the E.coli GM-CSF induction TF-1 Clone formations expressed is strong 1.5-2 times.
T₃₄	Apoptosis capacity

T₃₅	Cell volume increase
						T₃₆	Cell factor is sticked

T_y	Pharmacological characteristics	GM-CSF	IL-3	IL-4	IL-5
						T₃₇	Cell adherence
T₃₈	Cellular invasion
						T₃₉	Cell mobility
T₄₀	Divide a word with a hyphen at the end of a line and invade
						T₄₁	Chemotaxis
T₄₂	Cell swallows
						T₄₃	Signal transduction
T₄₄	The albumen of receptor/ligand is raised
						T₄₅	JAK/STAT pathway activities
T₄₆	Ras-erk pathway activities
						T₄₇	AKT pathway activities
T₄₈	PKC and PKA pathway activities
						T₄₉	PKA pathway activities
T₅₀	Src activity
						T₅₁	Fas activity

T₅₂	TNFR activity
		T₅₃	NFkB activity
T₅₄	P38MAPK activity
		T₅₅	C-fos activity
T₅₆	Secretory
		T₅₇	Receptor internalisation
T₅₈	Acceptor cross reaction
		T₅₉	The up-regulation or downward of surface molecular
T₆₀	Before FACS/the distribution Long-term change trend of side
		T₆₁	Subset proportions change
T₆₂	Differentiation gene is expressed
		T₆₃	Meronecrosis
T₆₄	Cell is gathered together
		T₆₅	Cellular rejection
T₆₆	Combined with heparin sulfate

T_y	Pharmacological characteristics	GM-CSF	IL-3	IL-4	IL-5
						T₆₇	Combined with glycosylation structure

T₆₈	Combined with chondroitin sulfate
		T₆₉	Combined with extracellular matrix (such as collagen, fibronectin)
T₇₀	Combined with man-made structures (such as support)
		T₇₁	Combined with carrier
T₇₂	Acting factor is combined together
		T₇₃	Combine individually or with other albumen the effect to stem cells hyperplasia, differentiation and/or self-renewing

The list of conventional abbreviation is provided in table 4 and table 5 herein.

Table 4：Abbreviation and alias

Referred to as	Description
		AAA	Amino acid analysis
AFC	Affinity chromatography
		bFGF	Basic Fibroblast Growth Factor, FGF2
BSA	Bovine serum albumin(BSA)
		cDLC	Composite fuel part is chromatographed
CRD	Carbohydrate cog region
		CSF	Colony stimulating factor
DCS	Donor calf serum

DeoxGlc	1,5-anhydroglucitol
		DLC	The false affinity chromatography of fuel part
DSC	Differential scanning calorimetry
		ECD	Extracellular domain
EGF	EGF
		ELISA	Enzyme Linked Immunoadsorbent Assay
EPO	Hematopoietin
		EST	The sequence label of expression
Fc	Crystalline fragments or constant region for immunoglobulin
		FCS	Hyclone
FGF2	Basic Fibroblast Growth Factor, bFGF
		FTIS	Fourier transform infrared spectroscopy
Fuc	Trehalose
		G-CSF	Granulocyte colony stimulating factor
GM-CSF	Granulocyte-macrophage colony stimutaing factor (GM-CSF)；Colony stimulating factor 2 (CSF2) (genetic marker of accreditation)；Embryo erythroid colonies stimulating activity (BPA)；Colony stimulating factor α or β (CSF-alpha CSF-beta)；Colony stimulating factor 2 (CSF-2)；Eosinophil colony stimulating factor (Eo-CSF)；Eosinophil existence enhancer；Sour ball stimulates and actuates the factor (ESP)；Granulocyte-macrophage colony stimulating activity (GM-CSA)；Hematopoietic cell growth factor (HCGF)；Histamine produces a cell stimulating factor (HCSF)；Embryo KM102 colony stimulating activities (KM102-BPA)；The SCIF (KTGF) in keratinocyte source；Leukaemia infringement growth factor (LBGF)；The macrophage fusion factor (MFF)；Macrophage granulocyte inducer (MGI-1GM)；The neutrophil leucocyte migration inhibition factor (NIF-T) in T lymphocyte T cells source；pluripoietin alpha；WEHI-3B points

Referred to as	Description
			Change inducible factor.
Gal	Galactolipin
		GalNAc, galactosamine	2- deoxidations, 2 amine-galactoses
GFC	Gel permeation chromatography
		GlcA	Glucuronic acid
GlcNAc, glucosamine	2- deoxidations, 2 Glucosamines
		Glc	Glucose
GM-CSF	Granulocyte-macrophage colony stimutaing factor
		HBS	Hepes buffer salts
hES	Human embryo stem cell
		HIC	Hydrophobic interaction chromatograph
HPAEC-PAD	Use the high pH anion-exchange chromatographies of pulsed amperometric current detecting
		HPLC	High pressure liquid chromatography or high performance liquid chromatography
HSA	Human serum albumins
		HTS	High flux screening
IdoA	Iduronic acid
		IEC	Ion-exchange chromatography
IEF	Isoelectric focusing
		IFN	Interferon
Ig	Immunoglobulin
		IL	Interleukin

IL-3	Interleukin Ⅲ (IL3)；Embryo erythroid colonies stimulating activity (BP)；Blood progenitor cell activity factor (BPA)；Embryo erythroid colonies stimulating activity (BPA)；Spleen colony forming units (CFU-S)；Colony forming unit stimulating activity (CFU-SA)；Colony stimulating factor 2- α (CSF-2-alpha)；Colony stimulating factor 2- β (CSF-2-beta)；Epidermal cell IL-3 (EC IL-3)；Erythroid colonies stimulating factor (ECSF)；The basophilic granulocyte in epidermal keratinocytes (EK) source actuates activity；Eosinophil colony stimulating factor (Eo-CSF)；Hematopoietic cell growth factor (HCGF)；Histamine produces a cell stimulating factor (HCSF)；Hematopoietin 2 (HP2)；Hematopoietic cell growth factor (HPGF)；Maturation induction activity；Mast cell growth factor (MCGF)；Many colony stimulating activities (MCSA)；Megakaryocyte colony stimulating activity (MEG-CSA)；Megakaryocyte colony stimulating factor (MEG-CSF)；Mast cell growth factor (MFG)；Mix colony stimulating factor；Multi-colony stimulating factor (multi-CSF)；Polyphyly hematopoietic cell growth factor (multi-HGF)；Many auxin；Natural toxicity (NC) Porcine HGF；Neutrophil leucocyte-granulocyte colony stimulating factor；P cell-stimulating activities (PGSA)；P LSFs (PCSF)；The P factors；Multipotential stem cell support factor；Persisting cell stimulating factor (PSF)；Progenitor cells stimulating factor (PSF)；Panspecfic hematopoietin (PSH)；Synergistic activity (SA)；Stem cell activator (SAF)；The inducible factor of thymidine 1；The growth factors of WEHI 3；The hemopoieticgrowth factors of WEHI 3；The growth factors of WEHI 3 (WGF).
		IL-4	Interleukin-4 (IL4)；MHC II Ia inducible factors (IaIF)；B cell differential factor ε (BCGF-epsilon)；B cell differential factor γ (BCDF-gamma)；B

Referred to as	Description
		Cell differentiation factor γ (BCGF-gamma)；B cell differential factor 1 (BCGF-1)；B-cell stimulating factor -1 (BSF-1)；B-cell stimulating factor p1 (BSF-p1)；EL4-BCGF (EL4 Bcell growth factors)；Hodgkin ' s Porcine HGFs (HCGF)；IgE enhancers (IgE-EF)；IgG1- enhancers；IgG1- inducible factors；Low molecule amount Bcell growth factor (LMW-BCGF)；Mast cell growth factor-2 (MCGF-2)；The macrophage fusion factor (MFF)；SCIF -2 (TCGF-2)；Binetrakin(Sch39400).
IL-5	T cell growth factor (IL5)；B151 T cells exchange factor (B151-TRF)；B cell differential factor (BCDF)；B cell differential factor μ (BCDF-mu)；B cell differential factor α (BCDF-alpha)；B differentiation factors -2 (BCGF-2)；B cell growth and differentiation factor (BGDF)；Eosinophil differentiation's factor (EDF)；Eosinophil colony stimulating factor (Eo-CSF)；IgA enhancers (IgA-EF)；Kill confactor (KHF)；T cell exchange factor -1 (TRF-1)；Sour ball stimulates and actuates the factor (ESP)

GM-CSF	Interleukin 2 (IL2)；The bud life factor (BF)；Eosinophil differentiation factor (EDF)；Killer cell cofactor (KHF)；Lymphocyte mitogenic factor (LMF)；Lymphocyte conditionity mediated factor (LCM factor)；Lymphocyteproliferation factor thymocyte stimulating factors (LPF)；The cytotoxicity I (MAF-C I) of macrophage activating factor (MAF)；Spot formation cell enhancer (PFC-EA)；Thymocyte stimulating factor (SCIF)；SCIF (TCGF)；T cell Clone formation activity (TCPA)；Thymocyte differentiation factor (TDF)；Thymocyte mitogenic factor (TMF)；Eosinophil differentiation factor (TMF)；T cell mitogenic factor (TMF)；T cell Permutation Factor -3 (TRF-3)；T thymocyte stimulating factors (TSF).
		lacNAc	N- acetyl lactosamines (N-acetyl lactosamine)
lacdiNAc	N, N '-diacetyl lactose amine (N, N '-diacetyllactosediamine)
		LC	Fluid is chromatographed
MALDI-TOF	The mastrix-assisted laser desorption ionization time of flight mass spectrum of Matrix-assisted
		Man	Mannose
MCC	Metal chelate chromatography
		MS	Mass spectrum
NacSial, NeuAc or NeuNAc	N-acetyl-neuraminate (N-acetyl neuraminic acid)
		NGlySial, NeuGc or NeuGly	NeuGc ALPHA2-3Gal (N-glycolyl neuraminic acid)
PBS	Phosphate buffer solution
		PCS	Photon correlation spectroscopy
PDGF-AA	Platelet-derived growth factor A homodimers
		PNGase	Tire-N4- (N- acetyl group-β-D-glucosaminyl) asparagine acid amides enzyme
RMLP	Receptor-mediated part chromatography
		RPC	Reversed phase chromatography
SDS PAGE	Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
		SEC	Exclusion chromatography

Referred to as	Description
		Sia	Aluminosilicate
TCA	Trichloroacetic acid
		TFF	Tangential flow filtration
TGF	TGF
		TNF	TNF
TNFR	Tumor Necrosis Factor Receptors
		Xyl	Xylose

Table 5：Amino acid is referred to as

Amino acid	Three-letter codes	Single letter code
			Alanine	Ala	A
Arginine	Arg	R
			Asparagine	Asn	N
Aspartic acid	Asp	D
			Cysteine	Cys	C
Glutamic acid	Glu	E
			Glutamine	Gln	Q
Glycine	Gly	G
			Histidine	His	H
Isoleucine	Ile	I

Leucine	Leu	L
			Lysine	Lys	K
Methionine	Met	M
			Phenylalanine	Phe	F
Proline	Pro	P
			Serine	Ser	S
Threonine	Thr	T
			Tryptophan	Trp	W
Tyrosine	Tyr	Y
			Valine	Val	V

Table 6：Stem cell list

Cell type
	Common cell types s
Embryonic stem cell
	Thick liquid cell stem cell
Dry cell of microorganism
	Human embryo stem cell
Human epidermal stem cell
	The stem cell of adipose-derived
Brain
	Adult neural stem cell
Human neure

People's astroglia
	Epithelium
Human keratinocyte stem cell
	Human keratinocyte's transient amplification cell
Human melanocytes stem cell
	Human melanocytes
Skin
	Human foreskin fibroblasts
Pancreas
	People's urine output solencyte
Human pancreas' island
	Human pancreas' beta cell

Kidney
	The ripe kidney stem cell of people
Human embryo kidney (HEK) epithelial stem cell
	People's kidney epithelia cell
Liver
	People's Hepatic oval cells
People's liver cell
	People's list ductal epithelial cell
Human embryo endodermal stem cells
	People's adult human liver stem cell (has dispute) to it
Breast

Human breast epithelial stem cell
	Lung
The stem cell of bone marrow derived
	Human lung cancer cell A549
Human bronchial epithelial cell
	The non-race II type pneumonocytes of people
Muscle

Cell type
	Human Skeletal Muscle stem cell (satellite cell)
Heart
	Human Cardiomyocytes
Bone marrow interstital stem cell
	Simple squamous cell
Descending main artery epithelial cell
	Main artery arch epithelial cell
Aortic smooth muscle cells
	Eyes
Limbal stem cells
	Corneal epithelial cell
CD34+ candidate stem cells
	Mescenchymal stem cell
Osteoblasts (precursor is mescenchymal stem cell)
	Peripheral blood mononuclear progenitor cells (candidate stem cell)

Interstitial (precursor is above-mentioned cell type)
	Interstitial cell
Spleen
	People's spleen precursor stem cell
Human spleen cell
	Immunocyte
People's CD4+T- cells

People's CD8+T- cells
	NK cells of human beings
Person monocytic cell
	Human macrophage
Human dendritic cell
	People's B- cells
Nose
	Goblet cells (the mucilage secretion cell of nose)
The pseudostrimatic stanchion cell (being located at below nose regio olfactoria) for having a cilium
	The pseudostratified epithelial cell for having a cilium (the cell ratio is for ductus nasopharyngeus)
Tracheae
	Lamination epidermal cell (cell compares and constitutes tracheae)
There is the columnar cell of cilium (cell compares and constitutes tracheae)
	Goblet cells (cell compares and constitutes tracheae)
Basal cell (cell compares and constitutes tracheae)
	Oesophagus

Cricopharyngeus cell
	Reproduction
Female originally vesica
	Male spermatogonium

Brief description of the drawings

Fig. 1 is the diagram of the cloning procedure of the cDNA insertion pIRESbleo3 or pIRESbleo3-Fc carriers of the protein of the coding present invention.

Comparison of the human GM-CSF that the GM-CSF of Fig. 2 display present invention reaches with non-human cell's diagram of system to the proliferation function of TF1 cells.

The comparison for the human GM-CSF serum stability that the GM-CSF of Fig. 3 display present invention reaches with non-human cell's diagram of system, the TF-1 cel l proliferations induced with both are measured.

The comparison of human GM-CSF differentiation and proliferation granulocyte and macrophage colony that the GM-CSF of Fig. 4 display present invention reaches with non-human cell's diagram of system.

Comparisons of the people IL-3 that the IL-3 of Fig. 5 display present invention reaches with non-human cell's diagram of system to M-NFS-60 cel l proliferations.

Comparisons of the people IL-4 that the IL-4 (filled circles) and E.coli (triangle) and Chinese hamster ovary cell (CHO) (open circles) of Fig. 6 (a) display present invention is expressed to the proliferation function of TF1 cells.

Fig. 6 (b) displays are based on 37 DEG C of preincubates after 4 days with IL-4 culture, the comparison of IL-4 (circle) and the people IL-4 of (square) expression of E.coli of the invention to the proliferation function of TF1 cells.

The comparison of the ion vitro immunization response characteristic for the people IL-3 that the IL-3 (square) and E.coli (rhombus) and insect cell (triangle) of Fig. 7 display present invention is expressed.

The IL-4 (triangle) and the people IL-4 of (square) expression of non-human cell's system ion vitro immunization response characteristic of Fig. 8 display present invention comparison.

Embodiment

Be interpreted as except as otherwise noted, the invention is not restricted to special composition, preparation method, diagnostic method, analysis experimental design, nutrition experimental record or research experiment record or and so on possible change.It is further appreciated that only to be description specific embodiment for term purpose as used herein and does not limit specially.

It should be noted that what this specification was used, the indefinite article and definite article of singulative include plural number, unless context is otherwise indicated.Thus, for example, on " a kind of protein ", " a kind of cell factor " or " a kind of chimeric molecule " or " a kind of acceptor ", including single parameter and also including two or more parameters.

Term " compound ", " active factors ", " chemokines ", " pharmacologically active agents ", " medicament ", " active matter " and " medicine " exchanges use herein, is related to a kind of compound and the particularly a kind of desired physics and chemistry of induction and/or the protein of pharmacological effect or its chimeric molecule.The term also includes the pharmaceutically acceptable and pharmacological active component of these active factorses, and salt, esters, amide-type, pro-drug, active metabolite, analog etc. are more particularly to included but is not limited to herein.During using term " compound ", " active factors ", " chemokines ", " pharmacologically active agents ", " medicament ", " activity " and " medicine ", it is interpreted as its include active factors and pharmaceutically acceptable, pharmacological activity salt, esters, amide-type, pro-drug, active metabolite, analog etc. in itself.

Include the composition of two or more active materials on " compound ", " active factors ", " chemokines ", " pharmacologically active agents ", " medicament ", " active matter " and " medicine ", such as two or more cell factors." composition " also includes many part such as two parts compositions, wherein before formula, the factor is separately provided and given or prepare respectively or mixes.

For example, many part packs (multi-part pharmaceutical pack) can have belongs to 2 or more protein or its chimeric molecule the helical bundle superfamily of short chain 4 or related to the helical bundle superfamily of short chain 4 selected from GM-CSF, GM-CSF-Fc, IL-3, IL-3-Fc, IL-4, IL-4-Fc, IL-5 and IL-5-Fc, dividually preserves.

The term " effective dose " of reagent as used herein and " therapeutically effective amount " represent protein or its chimeric molecule individually or in the composition for have other reagents to provide the sufficient amount of desired treatment or physiological effect or result.Undesirable effect, such as side effect, are proved with desired therapeutic effect sometimes；Therefore, doctor balances possible benefit with possible risk to determine that what is appropriate " effective dose ".According to the species of subject, age and comprehensive condition, mode of administration etc., required definite amount can change between subject and subject.Therefore, it is not possible to specify one accurate " effective dose ".However, appropriate " effective dose " to any individual case can be determined by those skilled in the art using unique routine test.

" pharmaceutically acceptable " carrier is used, excipient or diluent represent that pharmaceutical carriers include abiotic or non-undesirable substance, i.e. material and selected active factors are administered to subject without causing any side effect or substantial side effect together.Carrier can include auxiliary material and other additives, such as diluent, detergent, colouring agent, wetting agent or emulsifying agent, pH buffer, preservative.

Similarly, " pharmaceutically acceptable " salt, esters, amide-type, pro-drug or the derivative of composition refer to abiotic or non-bad salt, esters, amide-type, pro-drug or derivative provided herein.

Term " treatment " as used herein and " therapy " are related to severity and/or the mitigation of frequency of the symptom of disease being treated, prevention and the improvement of the infringement of adjoint disease or remedy or take a turn for the better that the symptom of the elimination of symptom and/or potential cause, disease and/or their potential cause occurs.

" treatment " subject can be included in the disease in susceptible individual or the prevention and the treatment individual to clinical symptoms by improving the symptom of disease of other bad physiologic results.

" subject " as used herein is related to animal, in specific specific embodiment, mammal, and in further specific embodiment, the people that can be benefited from the pharmaceutical preparation and method of the present invention.The species of animal at this to that can be benefited from presently described pharmaceutical preparation and method is not limited.Whether people or non-human animal can be referred to as individual, patient, animal, host or acceptor to subject.The Compounds and methods for of the present invention is applied to physianthropy, veterinary science and general, animal breeding raise and train or wild.

Indicated above, in specific specific embodiment, animal is people or other primates such as orangutan, gorilla, ape, livestock animals, laboratory test animal, pairing animal or the wild animal and birds being captured.

Laboratory test animal citing includes mouse, mouse, rabbit, cavy and hamster.There is provided convenient pilot system or animal model for rabbit and rodent, such as mouse and mouse.Livestock animals include sheep, ox, pig, goat, horse and donkey.Nonmammalian such as birds, fish and amphibian include Xenopus, procaryon and non-lactation eucaryote.

Term " cytokine " " is used for its most universal meaning and including any various protein secreted by cell, and it adjusts the functional activity of immune system, regulation individual cells and/or tissue, and/or induces a series of physiological reactions.Term " cytokine " as used herein " is construed as being related to the cell factor of " complete " and includes the increase of one or more amino acid; missing is substituted; and it is kept substantially its fragment of the biological activity of the intact cell factor, derivative or homologue or chimeric molecule.

" cytokine receptor " is that cell membrane is associated or solvable albuminous cell factor acceptor, relevant with cytokine signaling system or regulation.Term " cytokine " acceptor as used herein " is construed as being related to the cytokine receptor of " complete " and includes the increase of one or more amino acid; missing is substituted, and is kept substantially its fragment, derivative or the homologue or chimeric molecule of the biological activity of intact cell factor acceptor.

Term " protein " is used for its most universal meaning and including cell factor and cytokine receptor.It is as used herein, term " protein " should be understood to be related to the protein of " complete " and include the increase of one or more amino acid, missing is substituted, and is kept substantially its fragment, derivative or the homologue or chimeric molecule of the biological activity of whole protein.

Present invention contemplates the protein of separation or its chimeric molecule, it has measurable physical and chemical parameter (P_x) feature, wherein this feature represents, is associated with the pharmacological characteristics of one or more characteristics or form the pharmacological characteristics (T of one or more characteristics_y) basis.The protein of separation or its chimeric molecule are to belong to the helical bundle superfamily of short chain 4 or the protein related to the helical bundle superfamily of short chain 4 selected from GM-CSF, GM-CSF-Fc, IL-3, IL-3-Fc, IL-4, IL-4-Fc, IL-5 and IL-5-Fc.Herein, GM-CSF, GM-CSF-Fc, IL-3, IL-3-Fc, IL-4, IL-4-Fc, IL-5 and IL-5-Fc include whole polypeptide and its fragment being related to.

More particularly, the invention provides a kind of protein of separation or its chimeric molecule, it, which has, includes a series of physicochemical characteristicses of measurable physical and chemical parameters, { [P_x]₁, [P_x]₂、··[P_x]_n, wherein P_xRepresent measurable physical and chemical parameter and " n " be >=1 integer, wherein [P_x]₁To [P_x]_nIndividually one different measurable physical and chemical parameter, the numerical value of the one or more measurable physicochemical characteristicses of any of which represents, is associated with the pharmacological characteristics T of a characteristic_y, or series of features pharmacological characteristics ([T_y]₁、[T_y]₂、....[T_y]_mOr formed a characteristic pharmacological characteristics T_y, or series of features pharmacological characteristics ([T_y]₁、[T_y]₂、....[T_y]_mBasis, wherein T_yThe pharmacological characteristic and " m " for representing a characteristic are >=1 integer, and [T_y]₁To [T_y]_mIndividually one different pharmacological characteristics.

Term " measurable physical and chemical parameter " (P as used herein_x) it is related to the protein of one or more measurable separation or the feature of its chimeric molecule.Representational " special measurable physical and chemical parameter " includes, but are not limited to：Apparent molecular weight (P₁), isoelectric point (pI) (P₂), isoform number (P₃), the relative intensity (P of different isoform number₄), carbohydrate percetage by weight (P₅), the actual measurement molecular weight (P after N- connection oligosaccharides deglycosylations₆), N- connections and O- connections oligosaccharides deglycosylation after actual measurement molecular weight (P₇), the percentage (P of acid contents of monosaccharides₈), contents of monosaccharides (P₉), sialic acid content (P₁₀), sulfate and phosphate content (P₁₁)、Ser/Thr:GalNAc ratios (P₁₂), the neutral percentage (P of N- connection oligosaccharides₁₃), the acid percentage (P of N- connection oligosaccharides₁₄), the neutral percentage (P of O- connection oligosaccharides₁₅), the acid percentage (P of O- connection oligosaccharides₁₆), the ratio (P of N- connection oligosaccharides₁₇), the ratio (P of O- connection oligosaccharides₁₈), the structure (P of N- connection oligosaccharide ingredients₁₉), the structure (P of O- connection oligosaccharide ingredients₂₀), the position of N- connection oligosaccharides and composition (P₂₁), the position of O- connection oligosaccharides and composition (P₂₂), common translation modification (P₂₃), posttranslational modification (P₂₄), acylated (P₂₅), acetylation (P₂₆), amidatioon (P₂₇), deamidation (P₂₈), biotinylation (P₂₉), carbamylation (P₃₀), carboxylation (P₃₁), decarboxylation (P₃₂), disulfide formation (P₃₃), fatty-acylation (P₃₄), myristoylation (P₃₅), palmitoylation (P₃₆), octadecane be acylated (P₃₇), formylated (P₃₈), saccharification (P₃₉), glycosylation (P₄₀), glycophosphatidyl inositol grappling (P₄₁), hydroxylating (P₄₂), the combination (P of selenocysteine₄₃), lipid (P₄₄), the addition (P of lipoic acid₄₅), methylate (P₄₆), N or C-terminal closing (P₄₇), N or C-terminal remove (P₄₈), nitrification (P₄₉), methionine oxidized (P₅₀), phosphorylation (P₅₁), protease digestion (P₅₂), prenylation (P₅₃), farnesylation (P₅₄), Mang ox base (P₅₅), phosphopyridoxal pyridoxal phosphate addition (P₅₆), sialylated (P₅₇), asialoglycoprotein (P₅₈), sulfation (P₅₉), ubiquitination (P₆₀), the addition (P of ubiquitin sample molecule₆₁), primary structure (P₆₂), secondary structure (P₆₃), tertiary structure (P₆₄), quaternary structure (P₆₅), chemical stability (P₆₆), heat endurance (P₆₇).The summary of these parameters is provided in table 2.

It is to include any pharmacological or clinically relevant characteristic of the protein or chimeric molecule of the present invention that term " (distinctive) pharmacological characteristics of characteristic ", which has been readily appreciated by one skilled in the art,.Representational " pharmacological characteristics " not only shall be limited only to the extent what invention included：Therapeutic effect (T₁), dose therapeutically effective (TCID₅₀)(T₂), bioavilability (T₃), from the time (T for being administered into maintaining treatment level₄), absorption rate (T₅), discharge rate (T₆), special activity (T₇), heat endurance (T₈), lyophilized stability (T₉), serum/plasma stability (T₁₀), serum half-life (T₁₁), the solubility (T in blood flow₁₂), immune response feature (T₁₃), immunogenicity (T₁₄), neutralizing antibody suppress (T_14A), side effect (T₁₅), receptor/ligand affinity (T₁₆), receptor/ligand activation (T₁₇), tissue or cell category specificity (T₁₈), penetration capacity (such as intestines, lung, blood-brain barrier, skin etc.) (T of biomembrane or barrier₁₉), generation blood vessel ability (T_19A), tissue resorption (T₂₀), degraded stability (T₂₁), freeze-thaw stability (T₂₂), protease stability (T₂₃), ubiquitin stability (T₂₄), administration reduce (T₂₅), mode of administration (T₂₆), the compatibility (T with other pharmaceutical excipients or carrier₂₇), the residual (T in organism or environment₂₈), preserve during stability (T₂₉), (T such as toxicity in organism or environment₃₀)。

In addition, the protein or chimeric molecule of the present invention can have different biological effect (T in different cell categories₃₁), including but not limited to people's primary cell, such as lymphocyte, red blood cell, retina cell, liver cell, neuron, horn cell, endothelial cell, endoderm cell, ectoderm cell, mesoblastema, epithelial cell, nephrocyte, liver cell, osteocyte, bone marrow cell, lymph node cells, dermal cell, fibroblast, T cell, B cell, thick liquid cell, NK, macrophage, bite neutrophil leucocyte, grain cell of Langerhan, BMDC, bite sour granulocyte, bite alkali granulocyte, mammary cell, small leaf cell, prostatic cell, pneumonocyte, esophageal cells, pancreatic cell, Beta cells (insulin secretory cell), angioblast, myocyte, elliptocyte (liver cell), mesenchymal cell, brain microvessel endothelial cells in vitro, astroglia, spongiocyte, a variety of stem cells include adult and embryonic stem cell, a variety of progenitor cells；With other people permanent, conversion or cancer cell systems.Biological effect in cell includes multiplication effect (T₃₂), differentiation (T₃₃), apoptosis (T₃₄), the growth (T of cell size₃₅), cell factor adhesion (T₃₆), cell adhesion (T₃₇), cellular invasion (T₃₈), cell mobility (T₃₉), migration and intrusion (T₄₀), chemotaxis (T₄₁), cell phagocytosis (T₄₂), signal transduction (T₄₃), raise albumen to receptor/ligand (T₄₄), the activation (T of JAK/STAT approach₄₅), the activation (T of Ras-erk approach₄₆), the activation (T of AKT approach₄₇), the activation (T of PKC approach₄₈), the activation (T of PKA approach₄₉), src activation (T₅₀), fas activation (T₅₁), TNFR activation (T₅₂), NFkB activation (T₅₃), p38MAPK activation (T₅₄), c-fos activation (T₅₅), secretion (T₅₆), acceptor caves in (T₅₇), acceptor reciprocation (T₅₈), the up-regulation of surface markers or downward (T₅₉), before FACS/change (T of other scattering signatures₆₀), the change (T of subgroup ratio₆₁), differential gene expression (T₆₂), meronecrosis (T₆₃), cell agglutination (T₆₄), cellular rejection (T₆₅) and heparin sulfate combination (T₆₆) and glycosylation structure combination (T₆₇) and chondroitin sulfate combination (T₆₈) and extracellular matrix combination (such as collagen, fibronectin) (T₆₉) and artificial material combination (such as support) (T₇₀) and carrier combination (T₇₁) and confactor combination (T₇₂), individually or the effect (T in the mixture containing other protein to stem cells hyperplasia, differentiation and/or self-renewing₇₃) etc..The summary of these characteristics is provided in table 3.

Term " characteristic " as used herein is relevant with the protein of the present invention or the pharmacological characteristics of chimeric molecule, is related to one or more protein or the pharmacological characteristics of its chimeric molecule, it is characteristic for special physicochemical characteristicses.In specific specific embodiment, one or more pharmacological characteristics of the protein of separation or its chimeric molecule are different from, or particularity relative to the same protein or the form of chimeric molecule produced in protokaryon or low eukaryotic or even high inhuman eukaryotic.In specific embodiment, the protein isolate matter or the pharmacological characteristics of its chimeric molecule tested are substantially similar to or function equivalence is in the protein of generation naturally.

Term " protokaryon " as used herein is related to any prokaryotic, and it includes any bacterial cell (including actinomycetes cells) or archeabacterial cell.Term " non-human eucaryote " as used herein means self evident.However, for clarity, the term especially includes any non-human eucaryote, it includes：Yeast such as saccharomyces or pichia；Other fungies；Insect, including Drosophila and insect cell culture；Fish, including chub mackerel category；Amphibian, including Xenopus；Plant and plant cell cultures.

It is related to " stem cell " including embryo or adult stem cell and is included in the stem cell listed in table 6.The protein or chimeric molecule of the present invention can be used alone or be used with the protein in cocktail, to induce one or more stem cells hyperplasias, differentiation or self-renewing.

The primary structure of protein or its chimeric molecule can be measured as amino acid sequence.Secondary structure can be measured as the quantity and/or relative position of one or more secondary protein structures, such as alpha-helix, parallel beta sheet, anti-parallel ss-sheet or corner.Tertiary structure describes the folding of polypeptide chain, and different Secondary structural elements are assembled into special comparison.Spiral and folding are secondary building units, and domain is tertiary structural elements.In multi-domain proteins, tertiary structure includes comparison domain each other.Accordingly, the presence that tertiary structure can be to one or more protein domains, missing, quantity and/or relative position are measured.Representational domain be not only the present invention limit include：Single-screw, helix turn helix domain, four-helix bundle, DNA binding domain, three helical bundles, Greece's key helical bundle, coiled-coil packaging structure domain, β-sandwich, β-tubbiness, anti-parallel ss-sheet up and down, Greece's key topological structure domain, jam volume topological structure domain, β-propeller, β-clover, β-spiral, Rossman is folded, α/β horseshoe, α/β bucket, alpha+beta topology, rich disulfide bond is folded, serine stretch protein enzyme level domain, actinocongestin domain, EGF spline structures domain, complement C- modular domains, wheat plant toxin domain, cobra (Cobra) neurotoxin domain, greenery cobra anticholinesterase domain, Kringle domains, mucoprotein sample area, spherical region, spacer region.The comparison of different polypeptide chains of the quaternary structure description with protein structure, each chain has unique one-level, two grades and tertiary structure elements.Citing include with-or miscellaneous-oligomer multimerization (for example dimer formation or tripolymer formed).

For the primary structure being related to, the invention provides the protein of separation or its chimeric molecule, or its fragment, by including SEQ ID NO selected from sequence table：25th, 27,29,31,35,37,39,41,43,45,47,51,53,55,57,61,63,65,67, or there is the nucleotide sequence of at least about 60% homogeneity with above-named any bar sequence, or coded by the nucleotide sequence that can hybridize with any of the above-described sequence or their complementary type under low stringency condition.

Another aspect of the present invention provides a kind of polypeptide of separation, and it is by the their own mRNA processed by cell spliced nucleotide sequence SEQ ID NO：69th, 70,71,72 coding.

The present invention is in yet a further aspect there is provided a kind of separation, encoding proteins matter or its chimeric molecule or its Functional portions nucleotide sequence molecule, and the nucleic acid molecule is included with including SEQ ID NO selected from sequence table：25th, the sequence similarity of the nucleotides at least 60% selected in 27,29,31,35,37,39,41,43,45,47,51,53,55,57,61,63,65,67, or after optimal comparison and/or can be with SEQ ID No：25th, 27,29,31,35,37,39,41,43,45,47,51,53,55,57,61,63,65,67 or their complementary type one or more nucleotide sequences hybridized under low stringency condition.

In a specific embodiment, present invention is generally directed to a kind of nucleic acid molecule of separation, the molecule includes encoding a kind of protein or its chimeric molecule, or its fragment nucleotide sequence, it has substantially such as SEQ ID NO：26th, a 28,30,32,36,38,40,42,44,46,48,52,54,56,58,62,64,66,68 shown amino acid sequence or multiple, or with SEQ ID NO：26th, 28,30,32,36,38,40,42,44,46,48,52,54,56,58,62,64,66,68 one or more amino acid sequences with least about 60% similitude after optimal comparison.

On the other hand, the invention provides a kind of nucleic acid molecule of separation, encoding proteins matter molecule, or its fragment, including selected from SEQ ID NO：27th, the nucleotide sequence in 29,37,39,41,43,53,55,63 or 65, its directly or the nucleotide sequences through one or more coding protein connexons known in the art with basic such as SEQ ID NO：1st, the nucleotide sequence connection of the constant region (Fc) of 3,5,7,9,11,13,15,17 or 19 one or more shown encoding human immunoglobulins or framework region.

On the other hand, the invention provides a kind of protein molecule of separation or its fragment, including selected from SEQ ID NO：28th, 30,38,40,42,44,54,56,64 or 66 amino acid sequence, it is directly or through one or more protein connexons known in the art and basic such as SEQ ID NO：2nd, the constant region (Fc) of 4,6,8,10,12,14,16,18 or 20 one or more shown human immunoglobulin(HIg)s or framework region connection.

Another aspect of the present invention provides the protein or its chimeric molecule or its fragment of a kind of separation, including the SEQ ID NO selected from sequence table：26th, 28,30,32,36,38,40,42,44,46,48,52,54,56,58,62,64,66,68 amino acid sequence, or one or more amino acid sequences with least about 65% similitude with above-mentioned sequence.

In a particular embodiment, protein similarities percentage or nucleotide identity level include at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% similitude or homogeneity.

Section or part of " derivative " of the polypeptide of the present invention also including total length parental polypeptide, it retains the part transcriptional activity of parental polypeptide and including variant.Such " biological active fragment " includes depletion mutant and small peptide, for example, having at least 10, in a particular embodiment, the at least 30 continuous amino acid with least 20 and in further specific embodiment, necessary to described continuous amino acid is displaying activity.This peptide can be obtained by the application of standard recombinant nucleotide technology or synthesized with conventional liquid phase or solid phase synthesis technique.For example, object of reference can be prepared with described solution synthesis or synthesis in solid state, e.g., including by Nicholson edit by Blackwell Scientific Publications publish entitled " the 9th chapter in Synthetic Vaccines " publication is named as " Peptide Synthesis " by Atherton and Shephard.Optionally, peptide can be produced by using protease such as endoLys-C, endoArg-C, endoGlu-C and the staphylococcus V8 protease digestions present invention amino acid sequence.Digestion fragment can be purified, for example, high performance liquid chromatography (HPLC) technology.Any such fragment, the method with generation is unrelated, is construed as being included in terminology used herein " derivative ".

Therefore, term " variant " is related to, and is shown as the substantially identical nucleotide sequence of sequence and reference nucleotide sequence or the polynucleotides hybridized under strict conditions with reference sequences being defined below.Term " nucleotide sequence ", " polynucleotides " and " nucleic acid molecule " can be exchanged herein to be used and including being added or lacking with one or more nucleotides, or the polynucleotides replaced with different nucleotides.In this respect, it is known in the art that some change including that can be mutated to reference nucleotide sequence, add, missing and replacement, the polynucleotides thus changed keep the biological function or activity of the polypeptide of reference polynucleotides or coding.Term " variant " also includes the variant of spontaneous allele.

The nucleic acid molecule of the present invention can be carrier or other constructs forms.

In a specific embodiment, carrier is DNA and comprising arbitrary selected marker.

The example of selected marker includes assigning the gene to compound such as antibiotic resistance, assigns the gene of the ability grown in Basic selective material, and coding produces the gene for the protein that can survey signal such as fluorescence.A variety of such genes are known and are available, including, such as antibiotics resistance gene such as neomycin resistance gene (neo) and hygromycin gene (hyg).Selected marker also includes assigning gene such as the tk genes (thymidine kinase) or hprt genes (hypoxanthine phosphoribosyltransferase) of the growth ability in some culture matrixes ability (hypoxanthine, ammonia petrin and thymidine) that its imparting grows in HAT culture mediums；With bacterial gpt gene (guanine/xanthine phosphoribosyl transferase), it allows to grow (mycophenolic acid, Ade and Xan) in MAX culture mediums.Other are used for selected markers of mammalian cell and carry the plasmid of multiple choices mark in Sambrook equimolecular Cloning-A Laboratory Manuals, Cold SpringHarbor, New York, USA, are described in 1990.

Selected marker can be obtained (the protokaryon marker gene in being used for example in purpose mammalian cell) by the expression of its own promoter and marker gene from the organism very different with purpose organism.However, replacing original promoter to be useful with the transcription structure of known function in recipient cell.Substantial amounts of transcription initiation region is useful to such purpose, for example, metallothionein promoter, thymidine kinase promoter, beta-actin promoter, immunoglobulin promoter, SV40 promoters and human cytomegalovirus promoter.Widely used example is pSV2-neo plasmids, its ability (a kind of related antibiotic of neomycin) for having the bacterial neomycin phosphoric acid transferase gene under the control of SV40 early promoters and being endowed the anti-G418 of mammalian cell.Other substantial amounts of mutation can be used for strengthening expression of the selected marker in zooblast, the addition of the translation initiation sequence of the addition and synthesis of such as poly (A) sequence.Composing type and inducible promoter can be used.

The genetic constructs of the present invention can also include 3 ' non-translated sequences.3 ' non-translated sequences are related to the part of gene, comprising containing polyadenylation signal and any other can influence mRNA process or gene expression Regulate signal DNA fragmentation.Polyadenylation signal has the feature for influenceing polyadenylic acid chain to be added to the end of mRNA precursor 3 '.Polyadenosine acid signal is generally identified by the presence with the homology of 5 ' AATAAA-3 ' normal forms, although variation is much.

Correspondingly, the genetic constructs of the nucleic acid molecule comprising the present invention, are effectively connected with promoter, can be cloned into suitable carrier to be delivered in regulation mistake, dysfunction or the cell or tissue of missing, to repair and/or provide appropriate regulation.Carrier containing suitable genetic constructs can be delivered in purpose eukaryotic by many distinct methods known to the technical staff of biology field.

Term " similitude " as used herein is included in accurate homogeneity between the sequence that nucleotides or amino acid levels compare.There is nonidentity in nucleotide level, " similitude " includes the difference between sequence, it causes the difference of amino acid, the difference of amino acid still with mutual structure, function, physics and chemistry and/or conformational levels are relevant.There is nonidentity in amino acid levels, " similitude " includes and mutual structure, function, physics and chemistry and/or conformational levels still relevant amino acid.In specific specific embodiment, the comparison of nucleotides and sequence is carried out rather than similitude in level of sequence identity.

The term of sequence relation for describing two or more polynucleotides or polypeptide include " canonical sequence ", " comparison block ", " sequence similarity ", " sequence identity ", " sequence similarity percentage ", " Percentage of sequence identity ", " substantially similar " and " substantially same " " canonical sequence " be at least with 12, but often 15 to 18 and usually at least 25 or more, such as 30 monomeric units including nucleotides and amino acid residue, in length.Because two polynucleotides can all include (1) sequence similar between two polynucleotides (such as the part for there was only complete polynucleotide sequence), (2) sequence different between two polynucleotides, the similitude that progress is typically relatively compared by the sequence of two polynucleotides, identification and comparative sequences regional area are removed by " comparison block " of sequence between two (or a plurality of) polynucleotides." comparison block " is related to notional fragment of general 12 consecutive residues, and it is contrasted with canonical sequence.For the optimal comparison of two sequences, comparison block can include about 20% or less addition or missing (such as gaps) compared with canonical sequence (wherein containing addition or missing).In order to compare comparison block, the optimal comparison of sequence can be by the computerization of algorithm or by checking and being realized by the optimal comparison (such as most high percentage homology is finally given between whole comparison block) of a variety of any generations of selected method.Control can also be obtained by the BLAST races of program, such as (the Nucl Acids Res 25 as disclosed in Altschul：389,1997) being discussed in detail for sequence analysis can find (In in Ausubel etc. Unit 19.3：Current Protocols in Molecular Biology、John Wiley & Sons Inc.1994-1998).

Term " sequence similarity " as used herein and " sequence identity " are related to sequence in comparison block, on the basis of nucleotides is than nucleotides or amino acid than amino acid on the basis of same or function or the similar scope of structure.Therefore, for example, the calculating of " percentage of sequence identity " is compared by the sequence of two optimal comparisons in comparison block, measure, which is present in two sequences, has identical nucleotide base (such as A, T, C, G, ) or identical amino acid residue (such as Ala I, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) site numerical value, to produce the numerical value with loci, with the sum in site in numerical value divided by comparison block with loci (for example, the size of frame), and result is multiplied by 100 to produce Percentage of sequence identity.For the purposes of the present invention, " sequence identity " will be understood to refer to by DNASIS computer programs (for windows of Version 2.5；Available from Hitachi SoftwareEngineering Co., Ltd., South San Francisco, California, USA) " the pairing percentage " that is calculated with the standard error that is used in the comparison handbook appended by software.Similar explanation application and sequence similarity.

It is as used herein it is low it is strict include and comprising being used to hybridize from least 0 at least about 15%v/v formamides and from least 1M at least about 2M salt, and at least about 1M is used for wash conditions at least about 2M salt.It is general, it is low strictly from about 25-30 DEG C to about 42 DEG C, such as 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41 and 42 DEG C.Temperature can change and higher temperature is used to replace formamide and/or provides optional stringent condition.Optional stringent condition can be used in the place of needs, for example moderate is strict, it is included and comprising from least 16%v/v at least about 30%v/v formamides, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30% and from least about 0.5M at least about 0.9M salt, such as 0.5, 0.6, 0.7, 0.8 or 0.9M is used for wash conditions, or it is high strict, it is included and comprising from least about 31%v/v at least about 50%v/v formamides, such as 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50% and from least about 0.01M at least about 0.15M salt, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 and 0.15M is used for wash conditions.General, wash in T_m=69.3+0.41 (G+C) % carries out (Marmur and Doty, J Mol Biol 5：109、1962).However, the T of double-stranded DNA_mThe quantity of 1 DEG C of base mismatch pair of often successively decreasing increases by 1% (Bonner and Laskey, EurJ Biochem 46：83、1974.Formamide is optional under these hybridization conditions.Accordingly, severity is defined below in specific embodiment：Low is strictly 6 × SSC buffer solutions, and 0.1%w/v SDS are at 25-42 DEG C；In strictly be 2 × SSC buffer solutions, 0.1%w/vSDS is at 20 DEG C to 65 DEG C of temperature range；High is strictly 0.1 × SSC buffer solutions, and 0.1%w/vSDS is at a temperature of at least 65 DEG C.

As used herein, term " common translation is modified or posttranslational modification " is related to when peptide chain is translated or occurred after translation covalent bond modification.Common translation is modified or posttranslational modification includes but is not limited to acylated (including acetylation), amidatioon or deamidation, biotinylation, carbamylation (or carbamylation), carboxylation or decarboxylation, two sulphur alkali are formed, fatty-acylation (including myristoylation, palmitoylation and octadecane are acylated), formylated, saccharification, glycosylation, hydroxylation, selenocysteine is combined, lipid, class resin acid addition, methylate, N- or C- endcappeds, N- or C- ends are removed, nitrification, it is methionine oxidized, phosphorylation, proteolytic cleavage, prenylation (including farnesylation, Mang ox base), phosphopyridoxal pyridoxal phosphate addition, sialylated or asialoglycoprotein, sulfation, ubiquitination (or ubiquitination) or Ubiquitin Like Proteins addition.

Acylation includes the hydrolysis of N- ends initial methionine and acetyl group is attached to new N-terminal amino acid.Acetyl group Co-A is the acetyl donor of acylation.

Amidatioon is the c-terminus of peptide and the covalent bond of amide groups and the stability for bioactivity and albumen is generally necessary.Go the hydrolysis removal that amidatioon is amide groups.Acid amides comprising amino acid residue goes amidatioon to be rare deformation, and it is by organism completion to recombinate 3D structures and change electric charge ratio/pI.

Biotinylation effect is that thus biotinyl is attached on molecule a technology; it is catalyzed or is carried out in vitro by holocarboxylase synthetase in the biosynthetic process of enzyme; it is intended to the visible special substrate of probe and the avidin hatching by using biotin labeling, or is intended to be connected to any streptavidin of many kinds of substance by physics and chemistry analytical control.

Carbamyl is transferred to acceptor portion such as amino by carbamylation (or carbamylation) from the molecule (such as carbamyl phosphate) containing carbamyl.

The carboxylation of glutaminic acid residue is the formation (Gla residues) that vitamin k-dependent reacts that it causes gamma-carboxyl glutamate.Gla residues are present in some protein of coagulation cascade, and it is required for the biological function of protein.Carboxylation can also betide asparatate residue.

Disulfide bond is the covalent bond of the disulphide formed when the sulfydryl of two cysteines is oxidized.Many mammalian proteins include disulfide bond, and it is for the generation and maintenance of tertiary protein structure, and such biological activity is conclusive.

Protein synthesis in bacterium includes the formylated of N- tenninal methionines and goes formylated.This formylated/go not occur in the formylated cytoplasm for circulating in eukaryotic and be the exclusive feature of bacterial cell.In addition to occurring a part of the hydroxylating in glycine residue as amidation process, under proline and lysyl hydroxylase catalysis (Kivirikko et al.FASEB Journal 3 can also occur on proline and lysine for hydroxylating：1609-1617,1989).

Saccharification is that glucose or other carbohydrates are uncontrolled, the amino acid backbone for being attached to protein of non-enzymatic.

Glycosylation is that sugar unit is attached to polypeptide backbone and will described further below.

Hydroxylation is the reaction of the vitamin C dependence as confactor.Hydroxylation is due to binding site of the hydroxyl lysine as glycosylation as the increase of the importance of posttranslational modification.

Selenoprotein is the protein of the selenium containing rare element, by adding unique amino acid, selenocysteine in translation process.TRNA for selenocysteine substitute serine and then enzyme seleno to produce selenocysteine-tRNA.Selenocystine-tRNA antisense codon and the terminator codon in mRNA (UGA) influence each other replacement serine codon.An element in selenoprotein mRNAs 3 ' non-translational regions (UTR) determines that UGA pronounces terminator codon or selenocysteine codon.

Lipidization is a covalently bound general name for including lipid on protein, and it includes fatty-acylation effect and prenylation.

Fatty-acylation effect includes covalent attachment thing such as 14 myristic acids (myristoylation) of aliphatic acid, 16 carbon palmitic acids (palmitoylation) and 18 carbon stearic acid (octadecane acylation).Aliphatic acid is connected to protein in preceding-Gorky separates and can be with targeting (the Blenis and Resh Curr Opin Cell Biol 5 (6) of regulatory protein confrontation film：984-9、1993).Therefore fatty-acylation effect is important (BernsteinMethods Mol Biol 237 in the functional activation of protein：195-204、2004).

Prenylation includes the combination of prenyl, i.e. 15 carbon farnesyls or 20 carbon Mang oxen-Mang ox base and receptor protein.Isoprenoid compounds, including farnesyl chloroquine or Mang ox benzylacetone chloroquine, are obtained in Biosynthesis of cholesterol approach.On the cysteine residues that isoprenoid base is attached in appropriate consensus sequence CAAX by thioether bond (wherein A is any aliphatic amino acid in addition to alanine), the c-terminus of protein is positioned at.Prenylation change protein and the united ability of lipid membrane and it is all known to GTP- combinations aminosal (G-protein) modify in this way so that prenylation is conclusive to signal transduction.(RandoBiochim Biophys Acta 1300(1)：5-16、1996；Gelb et al.Curr OpinChem Biol 2(1)j：40-8,1998).

Class resin acid is vitamin-like antioxidant, is used as the free radical of scavenger.Lipoic acid lysine is formed by lipoic acid protein ligase and class resin acid is attached on the lysine with reference to acid amides.

It is that a kind of common modification can be with the activity of regulatory protein matter or the new amino acid classes of generation that albumen, which methylates,.Protein methyltransferase by methyl from SAMe be transferred to albumen in nucleophilic oxygen, nitrogen or sulphur atom.The effect that methylates is divided into two kinds of general classification.First, the relative level of transmethylase and methyl esterase can control methylation on special carboxyl, the activity of its regulatory protein matter in turn.This methylate is reversible.Second group of protein methylation reaction includes the irreversible modification of sulphur or nitrogen-atoms in protein.This reaction produces the new amino acid with the physicochemical characteristicses changed, and it changes activity (the Clarke Curr Opin Cell Biol 5 of protein：977 983,1993).

Protein nitration is important posttranslational modification, and it is carried out in nitrous oxide signal transduction.The nitrification regulation catalytic activity of protein, cell signal and cytoskeleton organization.

Phosphorylation is related to the phosphate addition to protein kinase.Serine, threonine and tyrosine residue are the amino acid being phosphorylated.Phosphorylation is a kind of important mechanism, the bioactivity of its regulatory protein matter.

Most of protein is also modified by proteolytic cleavage.It can only include the removal of initial methionine.Other protein are synthesized in inactive precursor form, are activated by restricted or specific proteolysis.In order to secrete or signal sequence of the synthesis with the main hydrophobic amino acids of 12-36 with the albumen (preceding albumen) of film combination, it is after by being removed during ER films.

Phosphopyridoxal pyridoxal phosphate is the coenzyme derivative of vitamin B6 and participates in the transamination of amino acid side chain, decarboxylation, racemization, and many modifications.All phosphopyridoxal pyridoxal phosphates-desirability enzyme is worked by the formation of schiff bases between amino acid and coenzyme.The enzyme that most of dependence phosphopyridoxal pyridoxal phosphate bases are combined with lysine residue is self activation.

Sialylation is related to the terminal position that sialic acid is attached to glycoprotein by various sialyltransferases；And asialoglycoprotein is related to the excision of sialic acid.Sialic acid includes but is not limited to, N-acetyl-neuraminate (NeuAc) and NeuGc ALPHA2-3Gal (NeuGc).Sialic acid structure is caused by glycoprotein is sialylated, including sialic acid Lewis structures, for example, sialic acid Lewisa and sialic acid Lewis x, and sialic acid T structures, for example, sialic acid-TF and sialic acid Tn.

Sulfation occurs in tyrosine residue and is catalyzed by the enzyme tyrosine protein sulfurtransferase for being present in wire side on the outside of Gorky.Have determined that by the albumen within 1 to 20 that HepG2 cells are secreted and by least one tyrosine sulphate residue of the albumen within the 1 to 3 of fibroblasts to secrete.Sulfation is found to have an impact the bioactivity of albumen.It is particularly interesting that CCR5, main HIV co-receptors, optimal attachment and optimal HIV co- function of receptors of the discovery by the sulfation of one or more tyrosine residues in tyrosine sulfation and CCR5 N- terminal extracellular domains for MIP-1alpha/CCL3, MIP-1 beta/CCL4 and RANTES/CCL5 are necessary (Moore J Biol Chem278 (27)：24243-24246,2003).Sulfation can also occur on carbohydrate.In addition, the sulfation of the carbohydrate fraction of glycoprotein can occur by the activity of the sulfurtransferases of sugared sulfurtransferase such as GalNAc (β 1-4) GlcNAc (β 1-2) Man α 4.

Posttranslational modification can include protein-protein bonding.Ubiquitin is a kind of 76 aminoacid protein, and it both can also be covalently attached to other protein with itself combination in mammalian cell.Adhered to by the peptide bond between the amino of the lysine residue in the C-terminal of ubiquitin and other protein.The attachment of the chain and target protein of ubiquitin molecule, which is targeted, to be tended to by proteasome proteolytic and for the steady state levels of regulation regulatory protein matter, such as relevant with cell cycle protein, is a kind of important mechanism (Wilkinson Annu Rev Nutr 15：161-89、1995).On the contrary, single ubiquitination can be played an important role in the direct regulation of protein function.Ubiquitin Like Proteins can also be covalently attached on protein to influence their functional metabolism, including NEDD-8, SUMO-1 and Apg12.

Glycosylation is attachment of the saccharide residue on polypeptide backbone.Saccharide residue, such as monose, disaccharides and oligosaccharides include but is not limited to：Trehalose (Fuc), galactolipin (Gal), glucose (Glc), amine-galactose (GalNAc), gucosamine (GlcNAc), mannose (Man), N-acetyllactosamine (lacNAc), N, N '-diacetylamino lactose (lacdiNAc).These sugar units can be attached on polypeptide backbone at least seven kinds modes, i.e.

(1) consensus Asn-X-Ser is attached to by N- glycosidic bonds；Asn-X-Thr；Or the R- bases (N- glycosylations) of the day histidine residue in Asn-X-Cys.

(2) serine is attached to by O glycosidic bonds, threonine, hydroxy-proline, the R- bases (O- glycosylations) of tyrosine or oxylysine.

(3) the R- bases C- connection mannoses of tyrosine are passed through；

(4) glycophosphatidyl inositol grappling is used to some protein being fixed to cell membrane；

(5) GlcNAc of R- bases of serine or threonine is connected to as signal monose.The connection is usually reversible to adhere to (Yin-o-Yang) with inorganic phosphate；

(6) linear polysaccharide is to serine, the attachment (proteoglycans) of threonine or aspartic acid；

(7) the R- bases of cysteine are connected to by S-glycosides key.

Glycosylation structure can include one or more sugar antigens determinants following in table 7.

Table 7

Sugar antigens determinant list

Antigen title	O antigen polysaccharide o structure
		Blood group H (O), 1 type	Fuc(α1-2)Gal(β1-3)GlcNAc-R
Blood group H (O), 2 types	Fuc(α1-2)Gal(β1-4)GlcNAc-R
		Blood group A, 1 type	GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-3)GlcNAc-R
Blood group A, 2 types	GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc-R
		Blood group B, 1 type	Gal(α1-3)[Fuc(α1-2)]Gal(β1-3)GlcNAc-R

Blood group B, 2 types

Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc-R

Antigen title	O antigen polysaccharide o structure
		Blood group i	[Gal(β1-4)GlcNAc(β1-3)]_nGal(β1-R
Blood group I	Gal(β1-4)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)GlcNAc(β1-3)Gal(β1-R
		Lewisa(Le^a)	Gal(β1-3)[Fuc(α1-4)]GlcNAc-R
Sialic acid l Lewis a (sLe^a)	NeuAc(α2-3)Gal(β1-3)[Fuc(α1-4)]GlcNAc-R
		Lewis b(Le^b)	Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNAc-R
Lewis x(Le^x)	Gal(β1-4)[Fuc(α1-3)]GlcNAc-R
		Lewis x (the sLe of sialic acid 1^x)	NeuAc(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc-R
Lewisy(Le^y)	Fuc(α1-2)Gal(β1-4)[Fuc(α1-3)]GlcNAc-R
		Forssman	GalNAc(α1-3)GalNAc(β1-3)Gal-R
Thomsen-Friedenreich(TF or T)	Gal(β1-3)GalNAc(α1-0)-Ser/Thr
		Sialic acid 1-TF (sTF) or sialic acid 1-T (sT)	Gal(β1-3)[NeuAc(α2-6)]GalNAc(α1-0)-Ser/Thr
Tn	GalNAc(α1-0)-Ser/Thr
		The Tn of sialic acid 1 (sTn)	NeuAc(α2-6)Ga lNAc(α1-0)-Ser/Thr

Carbohydrate can also include some feeler structures, including list, double, three and four outboard structures.

Glycosylation can be by N linked glycosylations, O linked glycosylations, C connection mannose structures, and glycophosphatidyl inositol grappling presence, missing or pattern；Carbohydrate mass percent；Ser/Thr-GalNAc ratios；It is single, two, three and tetrose structure ratio or pass through agglutinin or antibody binding is determined.

The sialylation of protein can be determined by the immunoreactivity of protein and a kind of anti-antibody of specific sialic acid structure.For example, Lewis x distinct antibodies and the CEACAM1 reactions expressed by granulocyte but recombined human CEACAM1 reactions (the Luckaet al Glycobiology 15 (1) not expressed with 293 cells：87-100、2005).Optionally, presence of the sialic acid structure in protein can be by the mixture of glucosides ferment treatment through appropriate measurement process such as mass spectrum (MS), high performance liquid chromatography (HPLC) or sugared mass fingerprint (GMF) detection.

The apparent molecular weight of protein includes all constituents (confactor and non-covalent bond domain) and the modification of all common translations or the posttranslational modification of albumen composition (covalent groups cut off covalent groups to the attachment of peptide or on peptide).Apparent molecular weight is generally influenceed by common translation modification or posttranslational modification.The apparent molecular weight of protein can determine that it is also two dimension in its two-way analog, 2D-PAGE (two dimensional polyacrylamide gel electrophoresis) by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).However, the apparent molecular weight of protein can be by mass spectrum (MS)-by laser desorption Ionization-Time of Flight (MALDI-TOF) MS of the matrix-auxiliary for producing the electronic and ionic changed or can produce more sensitive electro-spray ionization (ESI) MS at multiple electrically charged peaks and more accurately determine.The apparent molecular weight of protein or its chimeric molecule can be in the range of 1 to 1000kDa.Accordingly,The protein or chimeric molecule of the separation of the present invention have 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,573,574,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592,593,594,595,596,597,598,599,600,601,602,603,604,605,606,607,608,609,610,611,612,613,614,615,616,617,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642,643,644,645,646,647,648,649,650,651,652,653,654,655,656,657,658,659,660,661,662,663,664,665,666,667,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713,714,715,716,717,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741,742,743,744,745,746,747,748,749,750,751,752,753,754,755,756,757,758,759,760,761,762,763,764,765,766,767,768,769,770,771,772,773,774,775,776,777,778,779,780,781,782,783,784,785,786,787,788,789,790,791,792,793,794,795,796,797,798,799,800,801,802,803,804,805,806,807,808,809,810,811,812,813,814,815,816,817,818,819,820,821,822,823,824,825,826,827,828,829,830,831,832,833,834,835,836,837,838,839,840,841,842,843,844,845,846,847,848,849,850,851,852,853,854,855,856,857,858,859,860,861,862,863,864,865,866,867,868,869,870,871,872,873,874,875,876,877,878,879,880,881,882,883,884,885,886,887,888,889,890,891,892,893,894,895,896,897,898,899,900,901,902,903,904,905,906,907,908,909,910,911,912,913,914,915,916,917,918,919,920,921,922,923,924,925,926,927,928,929,930,931,932,933,934,935,936,937,938,939,940,941,942,943,944,945,946,947,948,949,950,951,952,953,954,955,956,957,958,959,960,961,962,963,964,965,966,967,968,969,970,971,972,973,974,975,976,977,978,979,980,981,982,983,984,985,986,987,988,989,990,991,992,993,994,995,996,997,998,999,1000kDa apparent molecular weight.The molecular weight or molecular mass of protein can pass through any convenient method determination, such as electrophoresis, mass spectrum, gradient centrifugation.

The isoelectric point (or pI) of protein is pH when albumen does not carry net charge.The attribute can be determined by isoelectric focusing (IEF), and it is also the one-dimensional of 2D-PAGE.Experimental determination pI values can be up to 5 units by the difference between the pI and the pI of theory that the scope of common translation modification or posttranslational modification is influenceed and is therefore tested.Accordingly,The protein or chimeric molecule of the separation of the present invention can have 0,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5.0,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9.0,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10.0,10.1,10.2,10.3,10.4,10.5,10.6,10.7,10.8,10.9,11.0,11.1,11.2,11.3,11.4,11.5,11.6,11.7,11.8,11.9,12.0,12.1,12.2,12.3,12.4,12.5,12.6,12.7,12.8,12.9,13.0,13.1,13.2,13.3,13.4,13.5,13.6,13.7,13.8,13.9,Or 14.0 pI.

Term " isoform " as used herein represents a kind of different kinds of molecules form of given albumen, and is included in protein (1) primary structures (such as due to displacement RNA montages, or polymorphism) different in following level；(2) secondary structure (such as due to different common translation modifications or posttranslational modification)；And/or (3) three or four structure (such as because different subunits interacts, with-or iso- oligomer multimerization).Special, term " isoform " includes sugar-type, and it includes with continuous primary structure but modified or posttranslational modification in two grades or tertiary structure, or common translation, such as different glycosylation form, different protein or its chimeric molecule in level.

The chemical stability of protein can be measured in " half-life period " form of protein in special solvent or environment.Representational, the protein having less than 50kDa molecular weight has the half-life period of about 5 to 20 minutes.The protein or chimeric molecule of the present invention is focused on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, the half-life period of 99 or 100 hours.Another chemical stability is especially easily measured as the resistance that protein or its resistance molecule are acted on protease digestion, such as trypsase or pancreas milk reducing protease digesting effect.

Protein or its chimeric molecule can be measured its part or the affinity of acceptor in the form of equilibrium segregation coefficient (Kd) or function equivalence measurement.

The dissolubility of protein can be by being dissolved in the Tot Prot of given solvent and/or the wherein measurement of the ratio of proteolytic.Further, protein or its chimeric molecule are in heterogeneity such as polarity, pH, and the ratio and/or level dissolved in the solvent of temperature etc. can also provide protein or measurable physicochemical property of its chimeric molecule.

Any " measurable physical and chemical parameter " can determine that measurement quantifies or limited with known any method for a person skilled in the art.It is described below and is determined for, measures, quantify or limit the scope of the methodology of the protein of one or more separation or measurable physical and chemical parameter of its chimeric molecule.However, it should be understood to that the present invention is never simply defined as described special method, or it is measurable physical and chemical parameter to be defined to using these methods.

Glycoprotein can be described as having two interactions to produce one as overall molecule element-amino acid sequence and carbohydrate or sugared side chain.The carbohydrate part of molecule exists with the monose or oligosaccharide side chains form that are attached to by N- or O- keys on the hydroxyl side chains of Asn amino side chains or Ser/Thr residues respectively.Monose is the term on carbohydrate least unit, and it is considered as a sugar, with (CH₂O)_nBasic chemical formula and most of be usually formed 5 or six-membered cyclic structure (being respectively pentose and hexose).Oligosaccharides is the compound of the monose molding structure with Various Complex, and it can be linear or branch, but does not have the long-chain of tandem repeat unit generally (it is a kind of form of polysaccharide).The branch levels and the branch of end that oligosaccharides contains, which are replaced, significantly influences the feature of the glycoprotein as an entirety, and is played an important role on the biological function of molecule.Oligosaccharides is prepared and is attached in amino acid backbone in the endoplasmic reticulum (ER) and golgiosome of cell.Different organisms and the species of cell have the ratio of different glycosyl transferase and endoglycosidase and exoglycosidase and therefore produce different oligosaccharide structures.One of main defense mechanism of body is to find and destroy abnormal isoform, and same have correct glycosylated biopharmaceuticals to be neutralized being the discovery that for antibody important not only for heightening the effect of a treatment also for reducing.

Polysaccharide chains are generally expressed with branched form, even and if when it is linear, such chain is generally by a variety of modifications.Therefore, the complete sequence of oligosaccharides is difficult to be completed by single method and therefore need the combination repeatedly of physics and chemical method and finally obtain the details of studied structure.

Determining for the glycosylation pattern of protein can be carried out by using many different methods, for example, use SDS-PAGE.The fact that this technology is generally divided a word with a hyphen at the end of a line dependent on glycosylated protein in SDS-PAGE with different diffusion zones.The progress of differentiation between different isoforms is by using a series of agent treatment protein.For example, considering the glycosylated discrimination of N connections with the significant reduction of bandwidth after the digestion of peptide-N4- (N- acetyl-β-GLUCOSAMINE base) asparagine acid amides enzyme (PNGase) and the change for position of dividing a word with a hyphen at the end of a line.

In order to determine the glycosylated component of N connections, N- sugar chains from flavobacterium meningosepticum with cloning and cut off from protein in the PNGase of expression in escherichia coli.The N- sugar chains of excision can be from such as Packer et al Glycoconj J 5 (8)：737-47, the Alltech Carbograph SPE carbon posts (Deerfield, Illinois, USA) described in 1998 are reclaimed.Then, sample can carry out Monosaccharide analysis, saliva acid analysis or sulfate analysis with the Dionex systems with GP50 pumps ED50 pulsed amperometrics meter or electric conductivity detector and a variety of pH anion-exchange columns.

The glycosylated degree of O- connections can be determined by cutting off O- sugar chains from target protein first through β-elimination.The O- sugar chains of excision can be reclaimed as described in Packer et al. (1998, as above) from Alltech Carbograph SPE carbon posts (Deerfield, Illinois, USA).Then, sample can carry out Monosaccharide analysis, saliva acid analysis or sulfate analysis with the Dionex systems with GP50 pumps ED50 pulsed amperometrics meter or electric conductivity detector and a variety of pH anion-exchange columns.

The monose subunit of oligosaccharides has variable sensitivity to acid and therefore can discharged under the conditions of slight trifluoroacetic acid (TFA) condition, moderate TFA conditions, and strength hydrochloric acid (HCl) from destination protein.Then, mixture of monosaccharides is separated by using the high pH anion-exchange chromatographies (HPAEC) of a variety of post filled medias, and is detected with pulsed amperometric detection method (PAD).

High pH anion-exchange chromatographies have been widely used in measure monosaccharide component with Pulse amperometric detection method (HPAEC-PAD).Fluorescence-labeling method has been incorporated into and much used in a kit form.The obvious advantage of fluorescent method is sensitiveness enhancing (about 50 times).When one potential deficiency is coupling reaction, in hydrolysate and in external standard mixture, different monose can show different selectivity to different fluorophors.However, the enhancing of sensitiveness and differentiate the ability which monose is present from the sub-fraction of the total amount of actual glycoprotein, and the stronger sensitiveness with laser induced fluorescence potentiality so that this method is very attractive.Other electric conductivity detector can be used for determining sulfate and phosphate component.According to code is used, peak area can calculate the total amount of every kind of monose of presence.These data can represent the glycosylated level of N- and O- connections, sialylated degree, and amino acid composition in compound, glycosylate percentage by weight, acidoglycoprotein percentage by weight.

A small amount of monosaccharide composition analysis of protein is carried out preferably after the electric marking with PVDF (PSQ) film, or, less amount is analyzed with dot blot.PVDF is the preferable matrix of carbohydrate analysis, because being once all not joined to through peracid or enzyme hydrolysis, monose and oligosaccharides on film.

The measure of the oligosaccharide content of molecules of interest is carried out by many technologies.Sugar is cut off first from amino acid backbone by (such as being eliminated with hydroxide β) method of (such as being digested with PNGase) of enzyme or chemistry.Sugar can be by reducing stable or being made to be easy to detection with fluorescence labeling.Then, the free oligosaccharides of generation is separated, high pH anion-exchange chromatographies and pulsed amperometric detection method (HPAEC-PAD) can be passed through, it can be used in known standard to determine the ratio and sialylated level of various structures, or pass through fluorescence assisted carbohydrate electrophoresis (FACE), a kind of method separated similar to protein s DS-PAGE.In this process, oligosaccharides is marked with fluorophor, and it has impact on electrophoretic mobility.The banding pattern that they are separated and obtained in the polyacrylamide gel of high percentage provides the feature of the oligosaccharide content of molecules of interest.By using standard sample, obtaining some information or band of the practical structures existed can be cut and be analyzed with mass spectrum, can determine the structure of each of which.

Fluorescence assisted carbohydrate electrophoresis (FACE) is a kind of polyacrylamide gel electrophoresis system, designed for separating the single oligosaccharides discharged from glycoconjugate.Oligosaccharides by chemistry or enzyme method from sample protein matter be removed, reducing end is remained in this way.Then, oligosaccharides is digested as monose or keeps complete, and is fluorescently labeled (electrically charged or uncharged).High percentage polyacrylamide gel and a variety of buffer systems are used to migrate oligosaccharides/monose, and it is migrated relative to their size/component in the mode almost identical with protein.Carbohydrate is visualized by optical densitometric method and the relative amount of sugar can be measured by fluoroscopic examination.This process is consistent with MALDI-TOF MS, therefore this method can be used for illustrating practical structures.

Quartz crystal microbalance and surface plasma resonance (being respectively QCM and SPR) are the two methods of the physicochemical property acquisition biological information by molecule.Both measure protein-protein Indirect Interaction by the change of the physical features of fabricated chip caused by interaction.Single quartz crystal slice is handled with acceptor/antibody interacted with target ligand etc. in QCM.This chip is vibrated by microbalance and the frequency of chip is recorded.Target protein is allowed through chip and causes the frequency shift of thin slice with the interaction of molecules of combination.By the change of the condition of the interaction of part and chip, the binding characteristic of molecules of interest can be determined.

Apparent molecular weight is also a kind of physicochemical characteristicses, and it can be used for determining the similitude between the present invention and those protein produced with selectively mode or chimeric molecule.

As used herein, term " molecular weight " is defined as the summation of the atomic weight of composed atom in molecule, is directed to sometimes " molecular mass " (Mr).Molecular weight can add up to determine by the atomic mass to composed atom in molecule in theory.Term " apparent molecular weight " is defined as the molecular weight determined by one or more analytical technologies such as SDS page or ultracentrifugation, and dependent on the relation between molecule and detecting system.The apparent molecular weight of protein or its chimeric molecule can be measured with any of a series of experiments method.The analysis method of molecular weight for determining protein includes, and is not limited to, exclusion chromatography (SEC), gel electrophoresis, Rayleigh light scattering, analytical ultracentrifugation and, in a way, time-of-flight mass spectrometry (TOFMS).

Gel electrophoresis is the assay method of some physicochemical characteristicses (particularly apparent molecular weight and DI) of protein and on molecule to be separated into the dielectrophoresis of isoform, so as to provide the information of protein product posttranslational modification.Specifically, electrophoresis is to force charged molecule (such as protein or DNA) to be divided a word with a hyphen at the end of a line the method by gel-type vehicle (most of common polyacrylamides or agarose), passes through the use of the current potential through colloid.Most common electrophoretic type for protein is isoelectric focusing, non denatured, and sds polyacrylamide gel electrophoresis.Protein is placed in the polyacrylamide gel with pH gradient therebetween through in isoelectric focusing.Albumen will migrate to a position in gel, the net charge that it is zero that albumen, which has, in this place, so as to provide the isoelectric point of albumen.

Sugared quality fingerprinting (GMF) is a kind of method, through this method, and the oligosaccharides feature of one of protein or its isoform is accredited by electrophoresis and subsequent special mass-spectrometric technique.Sample protein matter is purified by the 1D SDS-PAGE that are determined for total protein or for the 2D gel electrophoresises of special isoform characteristic.Protein band/spot cuts off and decolourized to remove pollutant from gel.Carbohydrate and by chemistry or enzyme process release and using nanoflow LC systems desalination/separation then identify the oligosaccharides being present in sample.LC streams, which can directly be expelled to Electrospray Mass instrument, (is used for quality measurement and then discriminating amount is present in sample) its feature or fingerprint for providing each isoform, quantitative technique such as Dionex analyses can be combined, to determine total component of tested molecule.

Primary structure can be assessed by the physicochemical characteristicses for the protein or chimeric molecule for determining the present invention.

The primary structure of protein or its chimeric molecule can use one or more following systems to be analyzed.

The information of the primary structure of protein or its chimeric molecule can be constituted with mass spectrum (MS), DNA sequencing, amino acid, the combination of protein sequencing and peptide quality fingerprinting is measured.

In order to determine the sequence of amino acid backbone, N- terminator sequencing chemistries, tandem-mass spectrometry sequencing, or both combination can use.N- terminator sequencing chemistries utilize Edman chemistry (Edman P. " Sequence determination " Mol Biol Biochem Biophys 8：211-55,1970), peptide bond described in it between protein N-temiinal amino acid and the amino acid of 2 peptide bond more every other than in sequence is weak.By using intermediate acidity's condition, -terminal amino acid is removed, and derivative is the retention time with fluorophor (FTIC) and measure in reversed-phase HPLC post, and which kind of amino acid is compared with standard sample to determine is.This method can determine the actual primary structure of molecule but not be quantitative.Optional, nanoflow liquid chromatograies tandem-mass spectrometry can be used (LC-MS/MS).In this method, protein is hydrolyzed to peptide using special endo protease and the molecular weight of peptide is determined.Then it is broken peptide bond with energetic encounter gas such as nitrogen or argon and measures the quality of the peptide of acquisition.By the change for the quality for calculating peptide, the sequence of each peptide can be determined (every kind of amino acid has unique quality).Then by using different protease, peptide can overlap the order to determine them and thus determine the complete sequence of protein.

Clearly, enzymic digestion, chemically derived, liquid chromatogram (LC)/MS and series winding MS combination provide a kind of very effective instrument, for AA sequence analyses.For example, the detailed construction of recombinant soluble CD4 acceptor is characterized by the combination of method, have determined the primary structure of the 369AA glycoprotein more than 95% and have been found that the complete characteristic at N- and C- ends, correct configuration (the Carr et al.J BiolChem 264 (35) of the attachment position of polysaccharide, polysaccharide structure and disulfide bridge bond：21286-21295,1989).

Mass spectrum (MS) is a kind of method that deduction by the behavior of molecule in conductive environment under vacuum measures molecular mass.MS is highly useful in stability study and quality control.This method requirement first carries out (trypsase with proteolytic enzyme, V8 protease, chymotrypsin, subtilopeptidase A, and clostripain) treatments of the sample (Franks et al.Characterization of proteins, Humana Press, Clifton, NJ, 1988；Hearn et al.Methods in Enzymol 104：190-212,1984) then, pass through the separating digesting sample of RP chromatography (RPC).With Trypsin Induced combination LC-MS, peptide mapping can be used for detecting genetic stability, the homology of product batch, and fermentation, and purification, formulation prepares the stability with protein in storage process.

Before quality analysis, some modes are used to HPLC being connected to mass spectrograph：1) direct liquid injection；2) supercritical fluid；3) conveyer belt system；4) thermal spraying.Eluent in post is transported to the sample probe joint of mass spectrometric chamber for Caprioli ' the s HPLC/MS connections operated using Fused-silica capillary column.When sample solution appears in capillary tube connector, probe joint is continuously bombarded with the Xe atoms of high energy, causes sample solution to sputter.Then quality passes through Instrumental Analysis (Caprioli et al.Biochem Biophys Res Commun146：291-299,1987).

MS/MS and LC/MS connections have been expanded MS and potentially applied.MS/MS allows direct discriminating (the Carr et alAnal Chem 63 of part, deacylation amine site and the isomerization of more than 25AAs peptide complete sequence：2802-2824,1991).RPC or Capillary Electrophoresis (CE) are combined with MS can carry out high sensitive analysis (Figeys and Aebersold, Electrophoresis1 9 of protein：885-892、1998；Nguyen et al.J ChromatogrA 705：21-45,1995).LC/MS allows the LC methods of the isolated peptides before MS is entered, the constant current FAB being for example connected with micropore HPLC (Caprioli et al.1987, as above)." connection " below allows the sequencing of the single peptide from complex mixture：Selected peptide is broken by first time MS, is then passed through the ion cloud of collision cell：CID (collision induced dissociation).Collision produces the characteristic set of fragment, it is possible thereby to sequence is inferred to, without knowing other information, such as cDNA sequence.In single MS experiments, those of mixture (for example, from enzymic digestion) injection and the quality of leading ion with being expected by cDNA sequence that are not classified of peptide are compared.Fast atom bombardment (FAB)-MS analysis and proteolysis of the sequence of RhIL-2 by CNBr have been verified (Fukuhara et al.J Biol Chem260：10487-10494,1985).

Electro-spray ionization MS (ESI-MS) is inserted in pin under high voltages using proteolytic aerosol, produces a series of electric charge peak of identical molecules with a variety of electric charges.Because the peak for being each produced from different charge species produces the estimation of molecular weight, these estimations can combine to increase total accuracy of molecular weight estimation.Matrix assisted laser desorption ionization MS (MALDI-MS) uses high concentration chromophore.Higher-strength laser pulse by Matrix absorption and the energy evaporation section matrix absorbed and almost can bring protein example into gas phase completely.Then the MS flight time of the ion of acquisition is analyzed.The ionization of moderate can strengthen the ability that this method provides quaternary structure information.MALDI-MS can easily be run within 15.It does not need fragment chemoattractant molecule and when PAGE gel densitometric scan, as a result easily explains, for more than mass range 100kDa.

Amino acid sequence can be determined by determining the DNA of encoding proteins matter or its chimeric molecule.But, actual protein sequence is probably different once in a while.Generally, PCR reaction of the DNA sequencing reaction to replicating (DNA denaturation, duplication) DAN is similar.By DNA clone technology, the gene can be cloned and nucleotide sequence is determined.

The amino acid sequence of one or more following network analysis protein or chimeric molecule can be used.

Description to protein or the complete sequence of its chimeric molecule usually requires that the description product.Amino acid sequencing includes：Trypsin Induced is carried out on gel, fraction is carried out to the peptide of digestion with RPC-HPLC, the peptide peak with most symmetrical Absorption Characteristics is screened by MALDI-TOF MS, with the peptide of edman degradation first (N- ends).The primary sequence data that Ai Deman is chemically derived are the classical ways for determining protein on a molecular scale.MALDI-TOF MS can be used for N- terminal sequence analysis.But, all enzymic digestions and peptide sequencing for HPLC recommend to first pass through MS or MS/MS protein and differentiate to reduce time-consuming and reduce cost.After the endopeptidase digested protein separated from SDS-PAGE of the Lysyl by Trypsin Induced in situ or in matrix, separation is carried out with HPLC can obtain the amino acid sequence of inside.

Recommend that the inside of standard peptide is sequenced and is analyzed sample and runs together to make instrument maintain peak performance.Eukaryotic protein more than 80%, which is reported, closing amino-terminal end, hinders direct amino acid sequencing.When running into the eukaryotic protein of closing, the presence of internal standard can ensure that instrumentation is normal.

Edman degradation can be used to N-terminal direct Sequencing by chemical process, methods described derives N-terminal amino acid to discharge amino acid, exposes the amino terminal of next amino acid.Ai Deman sequencings include：1) N-terminal sequence analysis is repeated by Ai Deman chemical cycles by micropore HPLC in, and each Ai Deman chemical cycles can identify an amino acid.2) polypeptide generated in gels or after the digestion of PVDF associated proteins with HPLC separating digestings, internal sequential analysis of protein is carried out by edman degradation chemical method.

Peptide mixer is analyzed and purified using micropore HPLC and capillary HPLC and using RPC-HPLC methods.Use sample and PVDF samples in different pillar purifying gels.N-terminal analysis can be carried out using mastrix-assisted laser desorption ionization time of flight mass spectrum (MALDI-TOFMS) analysis of Matrix-assisted after HPLC segmentation separation.Selection standard is：1) apparent purity of HPLC fractions.2) quality of peptide and the length thus estimated.Peptide quality information is assigned with using to confirmation Ai Deman sequencing amino acid, and useful to the possible detection of translation or posttranslational modification.

(In-gel) digestion on gel is suitable for the purifying in high sensitivity HPLC system.Internal sequential analysis of protein carries out enzymic digestion by sds page (SDS-PAGE) first.Albumen in SDS-PAGE microgels can be reliably only by trypsase in digested in-gel.Purify fragments of peptides with RPC-HPLC, then with MALDI-TOF MS analyses, screening is adapted to the peptide of Ai Deman sequencing analysis.Albumen can only be analyzed with internal sequence analytic approach in gel, but can obtain very accurate peptide quality, and this, which can be provided, extra refers to the useful information of fixed sum data library searching to amino acid.

PVDF associated proteins are suitable to N-terminal and inside Ai Deman sequencing analysis.PVDF associated proteins for example hydrogenate Triton X-100 by suitable enzymic digestion (trypsase, interior protease Lys-C, interior protease Glu-C, clostripain, interior protease, Asp-N, thermolysin) and non-ionic detergent.In PVDF associated proteins, the mastrix-assisted laser desorption ionization time of flight mass spectral analysis for the detergent that the peptide of digestion discharges from film to can interfere with to Matrix-assisted.Before enzyme is added, cystine (Cys) is reduced with dithiothreitol (DTT) (DTT), and carboxyamidomethylation cystine is generated with iodoacetamide subsequently, can be identified in N-terminal sequencing analysis.

In order to determine that the amino acid of protein or its chimeric molecule is constituted, strength hydrochloric acid (HCl) the acid condition hydrolyzation sample being catalyzed in the gas phase with phenol.Once hydrolysis is completed, derive the amino acid of release with a kind of fluorophor compound, make reversed nature special on the molecular band of combination.Anti-phase high speed liquid chromatography (RP-HPLC) separates derivative amino, and is detected with fluorescence detector.Using outwardly and inwardly standard, the quantity of each amino acid in sample is calculated by the peak area observed.This information can be used for identification sample and to the protein quantification in sample.For example, the deviation of theoretical and actual result can be used for the possibility for initially determining that a desamidation position.It is combined with Monosaccharide analysis, it may determine that glycosylation percentage by weight composition and acidoglycoprotein percetage by weight.The limitation of this method is that it can provide the skeleton actual sequence information that inherent variation is produced because of the occasional breakage of environmental pollution and amino acid.Such as this method can not possibly detect the point mutation of sequence.

Peptide mass fingerprinting (PMF) is another method for being able to confirm that protein or its chimeric molecule.Its process is included initially with electrophoresis (1 dimension or 2 dimensions) separation sample, and point/band is cut from gel and is digested with special endo protease (typically with Porcine trypsin).Peptide is eluted from gel fragment and the quality of peptide existed is determined with mass spectral analysis.Then the Theoretical Mass debris database of the peptide quality of generation and the protein of all announcements is compared (or theoretical peptide quality of the implementation sequence built).The fact that this technology is unique dependent on " fingerprint " (i.e. its peptide mass combination) of protein.It can reliably identify that (degree of accuracy more than 90%) is small to 4 peptides and 30% sequential covering.Modification, such as fat part and desamidation can be identified in the PMF stages of analysis.Further analyzed by tandem mass spectrometry (MS-MS) with the peak that the protein of identification is not consistent, MS-MS technologies carry out colliding the energy of generation interrupting PTM weak bond using collision gas.Then quality is carried out to the molecule and original peptide that newly discharge to analyze to identify posttranslational modification and the fragments of peptides that it adheres to again.

It is different patterns to be divided HPLC according to the special component of size, electric charge, hydrophobicity, function or target biological molecules.Generally, a kind of protein is purified using two or more chromatographies.Above all to consider the characteristic of protein and sample solvent when selecting chromatogram mode

Their secondary structure can be evaluated by characterizing the protein of the present invention or the characteristic of its chimeric molecule.

Following one or more systems can be used to carry out the secondary structure of analysing protein or its chimeric molecule.

In order to study Secondary structure, it should use and relatively more the most frequently used several spectroscopic approach.Electromagnetic energy can be determined according to the size and shape of ripple with radiation continuous wave.Different spectroscopic approach uses different electromagnetic energy.

Wavelength is the length the distance between (two continuous wave maximum) of the single ripple of radiation.When radiant increase, wavelength shortens.Relation between frequency and wave number is：

Wave number (cm^-1)=frequency (s^-1)/the light velocity (cm/s)

Absorption of the molecule to electromagnetic radiation includes vibration and rotational transition and electron transition.The most popular method of measure molecular vibrational energy for identifying secondary structure is infrared (IR) and Raman spectrum.But their method is different with molecule absorption is determined.

Scatter emittance and be less than stokes line incident radiation.Scatter emittance and be more than anti-Stokes line incident radiation.Excite the base electronic state vibrational energy interval of the energy and molecule increased or decreased relevant.Therefore, Stokes and anti-Stokes line wave number are that the direct detection of molecular vibrational energy is measured.

Stokes shift (Stokes shift) is only observed in Raman spectrum.The wave number of stokes line is less than (or wavelength is more than) exciting light.High-energy excitaton source (such as laser) can be used to strengthen Raman scattering efficiency.Excitaton source should be monochromatic, because we are to exciting and the capacity volume variance (wave number) of stokes line is interested.

In order that vibration has infrared active (IR active), the dipole moment of molecule must change.Therefore, the symmetrical stretching, extension of carbonic anhydride is without infrared-active, because dipole moment does not change.It is that dipole moment is changed that asymmetry, which stretches the reason for having infrared active,.In order that vibration has Raman active (Raman active), the polarizability of molecule must occur vibration and change.The symmetry of carbonic anhydride, which stretches, Raman active, because the polarizability of molecule is changed.Therefore, Raman spectrum supplements infrared spectrum (Herzberg et al.Infrared andRaman Spectra of Polyatomic Molecules, Van Nostrand Reinhold, New York, NY, 1945).For example, in default of dipole moment motion, it is infrared to detect same core diatomic molecule, but Raman spectrum can be detected, because key stretches and shrinking is changed the polarizability of molecule, the interaction between this exoelectron and core is also changed.

For the polyatomic molecule (such as benzene) of the high degree of symmetry with upset center, it is possible to have bands of a spectrum activity in infrared spectrum and there is no bands of a spectrum activity in Raman spectrum, vice versa.Symmetry is low or chiral molecular in, it is possible to it is all active in infrared and Raman spectrum.

Infrared spectrum Detection wavelength and sample infrared absorption intensity.Infrared energy can make molecular vibration be energized into higher energy level.Infrared and Raman spectrum all detects the vibration of bond distance and bond angle.

It is infrared to characterize molecular vibration by detecting the light absorbs corresponding to molecule particular energy of the vibrational excitation of (or higher) state from v=0 to v=1.There are some to determine the selection of the ability of infrared spectrum detection molecules rule, infrared ray can not excite the vibration (Herzberget al.1945, as above) of all normal modes.

Infrared spectrum energy provides the qualitative and quantitative information of secondary protein structure, such as α spirals, β-pleated sheet, β-bend and irregular structure.Most useful infrared band in protein analysis is acid amides I (1620-1700cm^-1), acid amides II (1520-1580cm^-1) and acid amides III (1220-1350cm^-1).Acid amides I is the most strong absorption band of protein.Its stretching vibration comprising C=O (70-85%) and C-N groups (10-20%).Definite band position is determined by bone framework image and hydrogen bonding pattern.Acid amides II is more complicated than acid amides I.Acid amides II N-H in plane bend (40-60%), C-N (18-40%) and C-C (10%) stretching vibration and determined.Acid amides III bands use less (Krimm andBandekar, Adv Protein Chem 38：181-364、1986).Most of FTIR amide I bands B foldable structures are usually located at 1629cm^-1Left and right, minimum 1615cm^-1, maximum 1637cm^-1, secondary part may be in 1696cm^-1Neighbouring (minimum 1685cm^-1) display peak, α spirals are mainly in 1652cm^-1。1680cm^-1It is nearby β-bend.

The principle of Raman scattering is different from infrared absorption.The wavelength and intensity of the inelastic scattering light of Raman spectrum detection molecules.Raman scattering, molecular vibration energy makes lambda1-wavelength change, and produces Raman diffused light.

In order to there is Raman active, vibrate for inelastic scattering, its key is that polarizability changes in vibration.In symmetrical stretch, electronics bond strength is different between minimum and maximum nuclear separation.Therefore, polarizability changes in vibration, and this vibration mode scattering Raman light, and this vibration has Raman active.In asymmetric stretching, extension, the electronics that the electronics in the key of stretching, extension is easier in polarization, the key of compression is more difficult to polarization.Polarizability does not change generally, and asymmetric stretching, extension is no Raman active (Herzberg et al.1945, as above).

Circular dichroism can be used to detect any unsymmetric structure, such as protein.The different amounts of dextrorotation of optically-active chromophore absorption and left-handed rotation, this different absorption cause positive and negative absorption spectrum (right avertence vibrational spectrum generally, is subtracted from left-hand polarization spectrum).Generally, far ultraviolet or acid amides region (190-250nm) are mainly contributed by peptide bond, the information of amido link carbonyl environment is provided, therefore the information of secondary protein structure is provided, α spirals generally show two negative peak (Holzwarth et al J Am Chem Soc 178 at 208,222nm：350,1965), β-pleated sheet shows a negative peak in 196nm, and random coil shows a negative peak in 218nm.The peak of near ultraviolet region (250-350nm) by fragrant chromophore (Phe, Tyr, Trp) ambient contribution.Disulfide bond makes the minimum CD band increases near 250nm.

The three-dimensional structure that circular dichroism is generally folded with the height of side-chain structure tightly.Most protein is denatured Free up Memory steric hindrance, and denaturation degrees increase, circular dichroism spectra weakens.For example, hGH side chain circular dichroism spectra is very sensitive to adding partial denaturation caused by denaturant.Some reversible molecular chemistry changes, such as disulfide bond reduction or alkalimetric titration will change side chain circular dichroism spectra.For hGH, the change of the response of circle two of chromophore or the specific chromophore of influence is removed completely can cause these spectral differences, but denaturation or conformational change do not cause spectral difference (Aloj et al.J BiolChem 247：1146-1151,1971).

Ultra-violet absorption spectrum is to detect one of most efficient method of protein characteristic.It can provide the information of protein concentration and chromophore direct environment.Protein function group, such as amino, alcohol (or phenol) hydroxyl, carbonyl, carboxyl or sulfydryl can be converted into strong chromophore.It can be seen that be used to monitor two kinds of chromophore with near-ultraviolet spectrum：Metalloprotein (being more than 400nm) and the protein (260-280nm) containing Phe, Trp, Tyr residue.Ultraviolet or fluorescence signal change can be negative or positive, depend on protein sequence and solution properties.

Fluorescent detection molecules are radiated the energy launched after excited state.The aromatic amino acid of many protein is excited in 250 to 300nm, the fluorescence in the range of transmitting 300 to 400nm.Only the protein with Phe, Trp, Tyr residue can be detected, and intensity sequence is Trp》Tyr》Phe.Fluorescence spectrum can reflect the micro-loop environment information influenceed by protein folding.For example, the Trp of embedment is generally in hydrophobic environment, fluorescence is launched in the range of 325 to 330nm, but the residue or free amino acid of exposure launch fluorescence at 350 to 355nm.A kind of common agents for detecting protein stretching, extension are Bis-ANS.Bis-ANS fluorescence is pH dependent forms.Although its signal in water is weak, the hydrophobic site of the exposure that it can be stretched by being attached in protein increases signal (James and Bottomley Arch BiochemBiophy 356：296-300,1998).

In unfolded protein Tyr and Trp be effectively quenched cause protein stretch after signal dramatically increase.A kind of simple solute can also cause this change.In order that detection sensitivity is maximized, signaling rate can be used.For example, in research rFXIII stretches, using ratio (the Kurochkin et al.J Mol Biol248 of 350nm and 330nm fluorescence intensities：414-430,1995).Can be by the technique study conformational change that excitation energy is changed between fluorogenic donor and absorption acceptor, because conversion efficiency relies on the distance between both chromophores (Honroe et al.Biochem J 258：199-204,1989).Antitrypsin conformation (K won and Yu, Biophim Biophys Acta1335 are detected with fluorescence：265-272,1997), to determine HAS Tm (Farruggia et al.Int J BiolMacromol 20：43-51,1997), and detect that MerP stretches interaction (Aronsson etal.FEBS Lett.411：359-364,1997).

Under neutral ph, fluorescence emission spectrum intensity sequence is Trp ＞ Tyr.At acidic, because conformational change destroys ability conversion, Tyr fluorescence is better than Trp.Fluorescence experiments also confirm the intermediate in protein stretching, extension transformation caused by guanidine.

The three-level and quaternary structure of the protein of the present invention or the physics and chemistry form of chimeric molecule are to finding out that its function is also important.

Protein or the three-level and quaternary structure of their chimeric molecule can be determined using one or more following systems.

NMR and X ray diffractive crystal analysis are the most common techniques of research protein 3D structures.The method for detecting tertiary protein structure of other summaries includes two grades of derivatives of CD, ultraviolet spectra (Ackland et al.J Chromatogr 540 of near ultraviolet region：187-198,1991) and fluorescence.

NMR is one of main method of research molecular structure and intermolecular interaction in structure biology.In addition to studying protein structure, NMR can be used for the protein in the research present invention or the carbohydrate structure in chimeric molecule.Chemist studies the structure of chemical substance usually using simple one-dimensional NMR spectral technology.Two dimensional technique can be used to determine the structure of more complicated molecule.Time domain NMR be used to detect molecules in solution dynamics.Solid state NMR be used to determine solid-state molecular structure.NMR can be used for the structure and dynamics for studying protein, the nucleotides low molecular weight compound related to medical science to a variety of biological, pharmacology.But, such as not every core all has the appropriate characteristic that can be read by NMR, and not every core all has a spin, required for spin is NMR.Spin causes core to produce NMR signal, plays the function of small magnetic field.

The crystalline texture of one or more following system measurement protein or their chimeric molecule can be used.

X ray diffractive crystal analysis is a kind of experimental technique, and X-ray energy is by crystal diffraction, and the X-ray with suitable wavelength (in angstrom scope ,~10-8cm) is by the electron cloud institute diffraction of the atom of suitable size.The molecule or the diffraction pattern of atom X-ray diffraction combined according to the cycle in crystal, electron density can be reconstructed.Additional phase information can be obtained from diffraction data or in mending diffraction experiment, to complete reconstruct.Experimental electron density model is progressively then set up, according to data-optimized, very accurate molecular structure is as a result obtained.

X ray diffractive crystal analysis has developed into the structure with the stateful material of any ray research institute, and such as ion, electronics, epithermal neutron and proton, the distance between wavelength and atom to be measured or molecular structure are similar.

Light scattering spectrum is based on such a simple principle, and big particle is more than the light that small particles are scattered.In 310-400nm regions, based on a big KPT Scatter just inclined baseline, such as in solution aggregation Schmid et al.Protein structure, a practicalapproach, Creighton Ed.IRI Press, Oxford, England, 1989).

Light scattering spectrum can be used to assess protein molecular weight, and it is a kind of simple tool for monitoring quaternary structure of protein or protein aggregate.Protein aggregation degree can be characterized with simple Turbidity measurement.The pharmaceutical solutions finally produced carries out transparency inspection, because cloud and lacteous is presented in most of collectins.Quasi-elastic light scattering spectrum (QELSS), sometimes referred to as photon correlation spectroscopy (PCS) or dynamic light scattering (DLS), are a kind of non-intrusion type exploration technologies of macromolecular (protein, polysaccharide, synthetic polymer, micelle, colloidal solid and aggregation) complex fluid diffusion.In most examples, light scattering spectrum directly produces scattering class interdiffustion coefficient.When being applied to the monodisperse liquor of dilution, the diffusion coefficient that QELSS is obtained can estimate size.In polydisperse system, it estimates the width of molecular weight distribution.For accurate measure, using 200-500mW laser energies, widely used conventional Ar+/Kr+ gas lasers (Phillies Anal Chem62：1049A-1057A, 1990).Protein aggregate (Liet al.Biochemistry 34 are have detected with mankind's relaxin：5162-5772,1995).

The stability of protein or its chimeric molecule is also an important determinant of function.The analysis method of this characteristic includes DSC, TGA and freeze-drying cryomicroscope, analysis freeze thaw resistance and protease resistant.

The protein or chimeric molecule of the present invention is freezed can be more stable after (freeze-drying).Lyophilized stability and/or the shelf life that be used to increase product, because product is stored in powder form, rather than liquid form.Its process includes starting frozen samples, then removes liquid by being dehydrated under vacuo.Final result is a kind of protein of drying and " pie " of auxiliary material (the other materials composition used).The uniformity of the pie finally given is crucial to successful reconstitution.Freeze-drying process can cause protein to change, especially by the aggregated forms of crosslinking, also desamidation and other modifications.These can lose, the immune response of activity or induction for aggregation be reduced, so as to reduce effect.It is using stabilizer (such as mannitol, trehalose, Tween 80, human serum albumins) that protein is lyophilized by formula in order to detect lyophilized stability.If the activity of protein can be determined by suitable bioassay method, the amount of the protein recovered is detected after freezing with ELISA.HPLC or western blot analysis detection protein aggregate can be used.

Before lyophilized, it should determine the Tg or Te (defining Tg or Te) of composition, come set dry first in product maximum permissible temperature.Equally, the crystallinity of composition or amorphicity information help more reasonably to design lyophilized circulation.These thermal parameters product informations can be obtained using differential scanning calorimetry (DSC), thermogravimetry (TGA) or lyophilized cryomicroscope

Differential scanning calorimetry (DSC) is a kind of physics heat analysis method, and detection, the thermal characteristic of sign and analysis of material simultaneously determine thermal capacitance, melt enthalpy and corresponding transition point.DSC scans a temperature range with linear ratio.According to " power back-off zero balance " principle, individual other thermal source in instrument is respectively sample and provides heat with reference to disk.During physical transformation, energy absorption or the imbalance for causing the amount of energy for being supplied to sample bomb is sent.The different thermal behavior of sample is relied on, energy will be removed or spread from sample, and temperature difference will be detected to be transmitted to computer telecommunication number.As a result, the adjust automatically of heater makes the temperature of sample bomb equal with reference to holder.Compensate the electric energy needed and calorimetric effect is equal.

The purity of organic matter can be estimated with DSC according to shape and the DSC temperature for melting heat absorption.Under the same conditions, power back-off DSC is compared with hot-fluid DSC very high resolution ratio is provided.Power back-off DSC produces certainty and accuracy preferably melts regional area, is obscured because melting regional area not as secondary resolution factor hot-fluid DSC in a narrow temperature interval.Power back-off DSC can inherently produce more preferable local melting region, therefore can carry out more preferable purity analysis.By StepScan DSC help, using traditional and time-proven method, power back-off DSC can provide direct thermal capacitance detection, it is not necessary to which deconvolution extracts sinusoidal amplitude.

Thermogravimetry (TGA) detects sample weight loss and rate of weight loss, is used as temperature or the function of time.

In DSC, lyophilized low-temperature device can be rapidly achieved a wide temperature range.At present, as preplanning and planning experimental tool, platform of the lyophilized circulation there is provided best small-scale research protein component thermal parameters is simulated in lyophilized low-temperature device.The influence of component and process factors during lyophilized microscopes prediction is freezed and dried.Low temperature test only needs to 2-3mL samples, this technology is turned into that a valuable research is rare, is difficult to the instrument of medicine that obtains.It is refrigerating effect, ratio, the index of aridity, a kind of good instrument of thawing rate in the lyophilized circulation of research.Lyophilized cryomicroscope experiment can help to annealing research.Because the extensive use of freeze drying technology, and to extremely expensive medicine (such as protein and gene therapy medicament) stabilized wilderness demand, it is desirable to pharmaceuticals industry is in the near future by the Microobservation in implementation process.

Resistance is melted in the freezing that protein or its chimeric molecule can be determined using the one or more in following systems.

In translation or posttranslational modification, for example, glycosylate, freeze/thaw cycle repeatedly can be subjected to protected protein matter.In order to detect it, the carrier-free homologue that can be produced with the chimeric molecule and Escherichia coli of comparison protein or the present invention.Protein or its chimeric molecule are diluted in suitable medium (such as cell growth medium, PBS or similar other materials), then freezed with a variety of methods, for example, freezed at once in liquid nitrogen, be positioned over -70 DEG C of slow freezings or the snap frozen in dry ice.Although fast melt or slow melting samples at 4 DEG C at room temperature.Then some samples are freezed again, this process repeats some circulations.The albumen quality existed is detected with ELISA, experienced staff selects suitable biological detecting method to determine activity.The value of activity/remaining protein is compared with original material, to determine the resistance after many freezing/thaw cycles.

Protein or the chimeric molecule of the present invention can change thermal stability in the solution.In vitro can be by the following thermal stability for determining the present invention.

Protein or the chimeric molecule of the present invention can be mixed into buffer solution, such as containing carrier protein (such as human serum albumins) phosphate buffer, and are incubated the specific time (such as 37 DEG C, 7 days) at a certain temperature.The remaining quantity of protein or its chimeric molecule after being handled with ELISA measure, and want to compare with the material for being stored in -70 DEG C.Association area experienced person carries out suitable biological detection and determines the protein of remaining or the bioactivity of its chimeric molecule.

The protease resistant of one or more of following system measurement protein or its chimeric molecule can be used.

In order to compare protease resistant, the protease that the solution of the solution of the chimeric molecule containing protein or the present invention and the homologue containing Bacillus coli expression can and be selected is incubated (such as unpurified haemocyanin enzyme, the protease of purifying, recombinant protease) different period.The protein content of remaining is determined using suitable ELISA (such as capturing and detect that the identification epitope of antibody is split by proteolytic cleavage site), the suitable biological detecting method selected using experienced person determines remaining protein or the activity of its chimeric molecule.

One or more following system measurement protein or the bioavilability of its chimeric molecule can be used.

Bioavilability is administration latter medicine or other materials by the available degree of destination organization.Bioavilability is dependent on the half-life period of medicine or the ability of its arrival destination organization.

The mixture of chimeric molecule containing protein or the present invention is injected using subcutaneous or intramuscular.Egg then determines the level of protein or its chimeric molecule in blood with ELISA or radiocounting.Or, the suitable biological detecting method selected with experienced person detects the protein active in blood sample, for example, stimulating the propagation of specific targeted cell population.Because sample comes from blood plasma or serum, it is thus possible to there is some other molecule to have an impact the activity detected.This can be controlled by using the neutralizing antibody of testing protein.Therefore, any remaining bioactivity is all caused by other serum compositions.

Stability or the half-life period of one or more following system measurement protein or its chimeric molecule can be used.

Protein or the chimeric molecule of the present invention may have the half-life period of change in serum and blood plasma.In vitro can be by the following half-life period for determining the present invention.The mixture of chimeric molecule containing protein or the present invention can be mixed into human serum/blood plasma and be incubated special time (such as 37 DEG C, 4 hours, 12 hours etc.) at a certain temperature.The amount of remaining protein or its chimeric molecule after being handled with ELISA measure.The suitable biological detecting method selected using association area experienced person determines remaining protein or the bioactivity of its chimeric molecule.The serum of selection may come from different human blood groups (such as A, B, AB, O).

The half-life period of protein or its chimeric molecule can also be determined in vivo.The mixture of chimeric molecule containing protein or the present invention, it can be marked with radioactive tracer or other methods, can be by intravenous, subcutaneous, rear eye socket, tail vein, intramuscular or intraperitoneal injection into the species of experimental selection, such as mouse, rat, pig, primate, people.Different time points obtain blood sample after injection, analyze the protein existed or its chimeric molecule (precipitating radiocounting with ELISA or with TCA).With the control of the mixture containing Escherichia coli or the CHO protein produced or its chimeric molecule as a comparison.

, in vivo, can be to injecting a kind of protein or its chimeric molecule in male Wag/Rij rats or other suitable animals ivs in order to determine the half-life period of protein or chimeric molecule of the invention.

Before material is given, 200 μ l EDTA blood of sampling are used as negative control.Different time points, 200 μ l EDTA blood are sampled using constructed from animal after injection.After last time blood sampling, animal is killed.Sample is centrifuged 15 minutes at room temperature in 30 minutes after collection.The concentration of protein or the chimeric molecule of the present invention in each plasma sample is determined with special ELISA.

Protein or the chimeric molecule of the present invention can pass through blood-brain barrier.

Whether human brain epithelial cell can be combined using following determination method vitro detection protein or the chimeric molecule of the present invention.

The ability of the protein of energy detection of radioactive labels or the chimeric molecule combination human brain capillary epithelium cell of the present invention.The protein of separation or the chimeric molecule of the present invention are manually combined into upper radioactive label using methods known in the art, certain specific activity is realized, such as chloramine-T method is marked¹²⁵I or³H。

The original cuiture of human brain epithelial cell can grow in flat 96 orifice plate, gently be fixed with acetone within 5 days after covering with.Cell is dissolved, and is transferred on glass fibre membrane.Liquid scintillation counter detection of radioactive labels albumen or the chimeric molecule of the present invention can be used.

The chimeric molecule combination human brain epithelial cell of protein or the present invention can be detected in vivo using following determination methods.

The combination of human brain tissue section detection human specific protein being cut into slices using (not by fixing) of fresh food frozen, in cryostat, being positioned on sheet glass and fixed with acetone or the chimeric molecule and human brain capillary of the present invention.Using in quantitative autoradiography detection brain section³The combination of H- protein or the chimeric molecule of the present invention.

The Tissue distribution and blood clearance of human specific protein or the chimeric molecule of the present invention can be detected in vivo in primate system.

In two male cynomologus monkeys or other suitable primates, the chimeric molecule of a protein or the present invention are used for determining¹⁴C- mark albumen or the present invention chimeric molecule Tissue distribution and blood remove, protein or the present invention chimeric molecule and³The reference protein matter of H marks is given the animal with intravenous catheter simultaneously.In experimentation, collect blood sample to determine protein from the removing in circulation.24 hours after injection, kill animal and select organ, collect representational tissue to determine the distribution of isotope and remove naturally.In addition, according to Triguero et al. (J of Neurochemistry 54：1882-1888 1990) method to from brain different zones sample carry out capillary reduce test.The method eliminates the vascular system more than 90% from brain tissue homogenate (Triguero etc. is quoted above).

It is consistent that time-dependent redistribution of the chimeric molecule of radiolabeled protein or the present invention from capillary portion to substantial portion migrates across blood-brain barrier with the time-dependent of protein or the chimeric molecule of the present invention.

Protein or the chimeric molecule of the present invention can promote or suppress angiogenesis.

Methods known in the art can be used to determine the latent effect of the angiogenesis of protein or the chimeric molecule of the present invention.Angiogenesis degree is determined for example, can be sprouted in angiogenesis model by capilary.In this detection, by Shepherd et al. (Arterioscler ThrombVase Biol 24：898-904,2004) method separation rat fat microvessel fragment (RFMFs), epididymal adipose tissues pad, chopping blend compounds protoenzymes digestion are collected from the animal of execution.RFMFs and separate cell are being isolated from fat and fat cell by the method for centrifugation, and are being suspended in 0.1%BSAPBS.It is continuous to filter RFMF suspensions to remove the fragment of tissue in fragment, unicellular and red blood cell.With 15,000 RFMF/ml suspended in the neutral Collagen Type VI of big rat-tail 1 of cold, pH (such as 0.25ml/ holes) in RFMFs, and the hole being layered in 48 orifice plates cultivated.After collagen polymerization, the DMEM containing 10%FBS of equal volume is added in each glue.After gel is formed, the blood vessel extended characteristic of angiogenesis progressively occurs on the 4th day in culture.In default of roughening, smooth muscle with form, it is easy to distinguish these newly-generated blood vessels and parent blood vessel fragment.Energy protein or the chimeric molecule of present invention processing RFMF 3-D cultures, culture determine newly-generated length of vessel on the 5th and 6 day.

Guedez et al. (Am J Pathol 162 can also be used：1431-1439,2003) description body vessel generation detection method determine protein or the present invention chimeric molecule angiogenesis latent effect.This detection method is included in subcutaneous transplantation semi-closed (semiclosed) silicone cylinder (vascular reaction device, angioreactor) in nude mice.The extracellular matrix of premixing or non-pre-mixed proteins matter or the chimeric molecule of the present invention is filled with vascular reaction device.Fluorescein isothiocynate (FITC)-glucan is injected intravenously before recovery to quantify the vascularization in vascular reaction device, then carries out fluorescence spectrophotometry detection.Fluorescence spectrophotometry detection is carried out to vascular reaction device can show the cell of different developmental phases and the new vessels of intrusion.

Protein or the chimeric molecule of the present invention can have the immunoreactivity feature that different available immunoassays are determined, including the interaction with one or more antibody directly against molecule.The example of immunoassay includes enzyme crosslinking immuno absorbence detection (ELISA), Dot blot and immunochromatography detection, such as flow test or strip test.

Immunoassay process can be used to determine protein or the level of its chimeric molecule, for example, a kind of ELISA kit of commercial distribution.Because the specificity of the antibody provided in immune reagent kit, the chimeric molecule of protein or the present invention can have different immunoreactivity features to the protein or its chimeric molecule of non-human cell's expression.For example, the capture of immunoassay and/or detection antibody can be the specific human protein expressed directly against non-human cell or the antibody of its chimeric molecule.

In addition, the incorrect folding of the human protein or its chimeric molecule of non-human cell's expression can cause the unexposed epitope exposure in the human protein or its chimeric molecule of the people's cell expression correctly folded.Incorrect folding can be by, for example, and foreign preteins is excessively produced in non-human cell's endochylema to produce, (Baneyx Current Opinion inBiotechnology, 70 for example, Escherichia coli：411-421,1999).Further, the human protein or its chimeric molecule of non-human cell's expression can have different protein or chimeric molecule posttranslational modification form of the invention.For example, the human protein or its chimeric molecule of non-human cell's expression can have the quantity of exception and/or sugared structure, phosphate radical, sulfate radical, fat or the other residues of type.This may cause the exposure of the unexposed epitope in protein or the chimeric molecule of the present invention.Opposite, the change of posttranslational modification pattern may cause to lack exposure in the epitope in the chimeric molecule of protein or the present invention, human protein or its chimeric molecule that the epitope is expressed in non-human cell.

Any one or the above-mentioned factor combined may cause such as following incorrect detection：

(a) human protein naturally-produced in laboratory sample or people's tissue, or

(b) in laboratory sample, recombinant human protein's matter that people is organized or people's cell is expressed in human embryo stem cell (hES) culture medium or its chimeric molecule

The human protein of people's cell expression or the immunoreactivity feature of its chimeric molecule determined using suitable immunoassay can provide the sign of the immunogenicity of protein in human body, as described below.

Most biological products cause the antibody response for them of certain level.Antibody response can cause possible serious side effects and/or loss effect in some cases.For example, some receive the recombinant protein of non-human cell's expression or the patient of its chimeric molecule treatment there may be neutralizing antibody, especially in long-term treatment in use, simultaneously therefore weakening the effect of protein and/or facilitating side effect.The human protein or chimeric molecule of people's cell expression are less likely to produce neutralizing antibody, therefore the human protein or its chimeric molecule expressed with non-human cell are compared and enhance curative effect.

The immunogenicity of one or more following system measurement protein or its chimeric molecule can be used.

Most biological products cause the antibody response for them of certain level.Antibody response can cause possible serious side effects and/or loss effect in some cases.For example, some receive recombinant epo treatment patient there may be neutralizing antibody, and with the EPO cross reactions of patient itself.In this example, they can occur simple erythroid aplasia, and produce resistance to EPO treatments, result in the need for frequent dialysis.

Immunogenicity is to cause the ability of immune response in organism.Immunogenic portion depends on the size of the material, and partly depends on the dissimilar degree of it and host molecule.Because with new physiochemical properties, protein or its chimeric molecule may have the immunogenicity changed.For example, the glycosylation structure of protein or its chimeric molecule may shield or weaken identification of the antibody to epitope, therefore prevent or weaken antibody binding to protein or its chimeric molecule.Or, some antibody can recognize the non-existent glycopeptide epitope in non-glycosylated protein matter.

The protein or the ability of its chimeric molecule of plysiochemical form of the patient's sample identification with characteristic can be determined by different method of immunity, as described here.The appropriate method of immunity of design includes consideration directly suitable detection, quantitative and sign antibody response.Some are listed in Mire-Sluis et al. J ImmunolMethods 289 (1-2) to immunoassays design and the suggestion optimized：1-16, in 2004, is incorporated herein by reference document.

The protein or its chimeric molecule that one or more following system measurements can be used to be used in therapeutic engraftment.

Present invention extension operates stem cell using protein or its chimeric molecule.One main stem-cell therapy application is tissue, cartilage or bone regeneration.In one embodiment, the cell in the 3-dimensional matrix of bio-compatible is possible to be introduced in human body.Transplanting will include cell mixture, skeleton, growth factor and necessary composition, such as biodegradable polymer, proteoglycans.Intend to mix protein or its chimeric molecule into these matrix in building process to adjust the behavior of cell.This transplanting can be used for bone formation, nerve from growth of progenitor cells and other application.Protein derived from people's cell can reduce the quantity and/or kind of the allogene protein under stem cell cultivation conditions, and therefore reduce the risk of inhuman pathogenic infection.

The present invention protein or chimeric molecule can be different with matrix generations for forming graft interaction, and adjust cell mix graft in.It is expected that the protein or chimeric molecule and graft composition of the present invention, which is used in combination, will cause one or more following pharmacology characters, such as high proliferation, promote differentiation, the maintenance of desired differentiation state, stronger differentiation lineagespecific, increase the secretion of matrix components, form more preferable three-dimensional structure, strengthen signal, more preferable structural behaviour, reduce toxicity, reduce side effect, reduce inflammation, reduce immunocyte infiltration, reduce injection, the graft duration is longer, graft function is more permanent, graft peripheral cell is preferably stimulated, more preferable regeneration, more preferable organ dysfunction, more preferable tissue remodeling.

The effect that one or more following system measurement protein or its chimeric molecule can be used to express different genes.

The gene expression difference of the cell exposed to protein or its chimeric molecule can be analyzed.

Microarray technology can determine the mRNA expression of almost all of gene in an organism genome simultaneously.The method uses gene " chip ", and the oligonucleotides of correspondence different genes sequence is attached on the solid carrier in " chip ".Using the obtained marks of the mRNA extracted from cell or tissue interested cDNA and chip be incubated, allow cDNA and attachment complementary sequence hybridization.Also need to, using control, compare both signals after then being hybridized and being cleaned.Which gene upregulation or downward are determined using special software or which expression does not change.Each chip can analyze thousands of genes.For example using Affymetrix technologies, human genome U133 (HG-U133) gathers, including two GeneChip (registration mark) arrays, containing about 45000 probe set, more than 39000 transcripton for representing from about 33, extracting in 000 human gene well confirmed.GeneChip (registration mark) mouse genome 4302.0 contains more than 39000 transcripton in individual array.

It is such to analyze the change for disclosing Global mRNA expression profiles, therefore it can be found that the expression change of the unknown gene regulated and controled by particular stimulation thing.Therefore this technology is suitable for the gene expression of the analysis induction related to the protein or chimeric molecule of the present invention.

It is determined that by particular stimulation thing regulate and control known to and new gene will be helpful to confirm important biochemical pathway in the protein or chimeric molecule bioactivity of the specific present invention.These information will be useful to determination novel therapeutic target spot.

The product that the system can be used for observing with can purchase compares the protein of the present invention or the changes in gene expression of chimeric molecule induction.

The binding ability effect of one or more following system measurement protein or its chimeric molecule can be used.

The protein of the present invention or the binding ability of chimeric molecule and different material can be studied, the material includes extracellular matrix, artificial material, heparin sulfate, carrier or co-factor.

Following determination methods can be used to determine the effect of the specified protein combination extracellular matrix in protein or its chimeric molecule.

The pan coating extracellular matrix protein in suitable buffer solution, including but not limited to collagen, vitronectin, fibronectin, laminin.Uncombined site can be coated with methods known in the art, for example, being incubated with BSA solution.Surface is cleaned, for example, with PBS solution, then adding the solution containing albumen to be detected (protein or chimeric molecule of the invention) on surface.After coating, cleaning surface and and identification of protein or its chimeric molecule antibody incubation.The antibody that then detection is combined, for example, using a kind of enzyme-linked secondary antibody for recognizing primary antibody.By the way that the combining antibody visualization of color change reactions is incubated and observed with suitable material.Not glycosylated protein is compared, and glycosylation albumen stronger can be adhered on extracellular matrix protein.

Or, by the protein or chimeric molecule of (being specified with ELISA concentration or biologicall test active unit) present invention of equal amount, or the protein of the invention or chimeric molecule homologue expressed with non-human cell, it is incubated together with the coated aperture of matrix, then cleaning aperture, combined amount is detected with ELISA.The decline indirect determination combined amount of ELISA reactivity after being incubated by sample and coating surface.

One or more following system measurement protein or the ability of its chimeric molecule combination artificial material can be used.

In order to determine the ability of protein or its chimeric molecule combination artificial material, surface is coated with artificial material, including but not limited to metal, support in suitable buffer solution.Surface is cleaned, for example, with PBS solution is used, then adding the solution containing albumen to be detected (protein or chimeric molecule of the invention) on surface.After coating, cleaning surface and and identification of protein or its chimeric molecule antibody incubation.The antibody that then detection is combined, for example, using a kind of enzyme-linked secondary antibody for recognizing primary antibody.By the way that the combining antibody visualization of color change reactions is incubated and observed with suitable material.

Or, by the protein or chimeric molecule of (being specified with ELISA concentration or biologicall test active unit) present invention of equal amount, with the protein of the invention or chimeric molecule homologue expressed with non-human cell, it is incubated together with the coated aperture of artificial material, then cleaning aperture, combined amount is detected with ELISA.The decline indirect determination combined amount of ELISA reactivity after being incubated by sample and coating surface.

There may be biology effect with reference to the ability of artificial surfaces, such as in support coating.Or, the support for being coated with a kind of protein of the invention or chimeric molecule is used for repopulating cell.The then growth and differentiation of monitoring cell, and compared with the not coated or different support of coating.

One or more following system measurement protein or the ability of its chimeric molecule combination heparin sulfate can be used.

Due to the protein or the physics and chemistry form of chimeric molecule of the present invention, it is contemplated that they and heparin sulfate have different interactions.It is expected that these difference are obvious in the experimental models such as cell propagation, differentiation, migration.Protein or its chimeric molecule and the pre- pharmacology character in respect of characteristic of heparin sulfate are used in combination in designated treatment.It is probably extended serum half lives, bioavilability increase, reduces immune related removing, stronger effect, the advantage for reducing dosage reduction side effect and correlation.

The ability of one or more following system measurement protein or its chimeric molecule combination carrier or co-factor can be used.

When protein or its chimeric molecule are present in blood plasma, they will combine other molecules.These molecules can be named as " carrier " or " co-factor ", and will influence the bioavilability or serum half-life of these factors.

It is incubated with the plasma proteins of purifying and analyzes the interaction that resulting solution can determine the protein or chimeric molecule and their binding partner of the present invention with molecular-exclusion chromatography.If protein or its chimeric molecule combine a kind of co-factor, obtained compound causes elution time to change by with bigger molecular weight.The bioactivity of compound, external or Half-life in vivo and bioavilability can be compared.

The effect of one or more following system measurement protein or its chimeric molecule to biologicall test can be used.

The protein of a variety of bioassary method detection present invention or the activity of chimeric molecule can be carried out, including determines cell propagation, cell differentiation, Apoptosis, cell size, cell factor/cytokine receptor adhesion, cell adherence, cell dispersion, cell movement, migration and infiltration, chemotaxis, ligand receptor combination, receptor activation, signal transduction and isoform rate of change.

One or more following system measurement protein or the effect of its chimeric molecule cell proliferation can be used.

Cell, in specific embodiments, the cell of exponential growth are incubated in the growth medium of the protein containing the present invention or chimeric molecule.This can be carried out in arrow-necked bottle or 96 orifice plates.Cell growth then counts cell with the method for direct (such as cell count) or indirect (MTT, MTS, tritiated thymidine) for a period of time.Control only containing culture medium, which compares, determines increasing or decreasing for propagation.The protein or its chimeric molecule of different concentration can be used to obtain dose response feature in parallel serial experiment.ED50 and ED100 (producing the dosage required for the maximum and maximum response effect of half) can be determined with it.

One or more following system measurement protein or its chimeric molecule can be used to maintain the effect of undifferentiated state to cell differentiation or cell.

Cell is incubated in the protein or the growth medium of chimeric molecule that there are the present invention.After suitable a period of time, cell analysis is carried out to differentiation indicant.It can be the expression of cell surface special sign thing, the expression of endochylema mark, the change of cell size, shape or kytoplasm feature.Mark potentially includes protein, sugared structure (such as glycosaminoglycan, such as heparin sulfate, chondroitin sulfate), fat (glycosphingolipid or lipid bilayer composition).Multiple technologies can be used to detect that these change, the technology includes microscope, protein blot, FACS dyeing or forward direction/lateral scattering feature.

The effect of one or more following system measurement protein or its chimeric molecule to Apoptosis can be used.

Apoptosis is defined as programmed cell death, different with other cell death ways such as necrosis.It is characterized by the cellular change determined, and the activation (such as Fas, TNFR) of such as signal path causes a protease subgroup for being referred to as caspase (caspases) to be activated.Starting the activation of caspase causes the activation of lethal caspase, and it cuts various kinds of cell albumen, causes nuclear fragmentation, and nuclear lamins is broken, and cytoplasm bubbles and destroys cell.Apoptosis can be induced by protein ligands, such as FasL, TNFa, lymphocytotoxin or by signal induction, for example, ultraviolet light and cause the material of DNA damage.

Cell is incubated in the growth medium of the reagent containing protein or its chimeric molecule or other suitable detections.For example, it may be desirable to which transcription (actinomycin D) can be blocked or the reagent of (cycloheximide) is translated.After suitable a period of time is incubated, cell quantity is determined with suitable method.Cell quantity, which declines, might mean that apoptosis.Other apoptosis can be obtained by cell dyeing to characterize, for example, annexin or the trapezoidal DNA form of observation characteristic.The expression of apoptosis mark can be prevented by being incubated with Premeabilisation of cells Caspase inhibitors (such as z-VAD FMK), then detection apoptosis mark, further to confirm apoptosis.

The protein or chimeric molecule of the present invention may provide survival signaling to prevent apoptosis by cells survival path (such as Bc12 or Akt paths).Can be expressed by cell Bc12 increases or detects that Akt activates form (phosphorylation) increased protein blot and confirms the activation of these paths using the phospho-specif iotac antibodies directly against Akt.

For these detections, cell culture is under the conditions of Survival Factor (such as IL-3 and certain immunocyte) is contained or not contain.The apoptosis under Extending culture of the part cell under the conditions of Survival Factor is lacked is cultivated, cultivating the cell under the conditions of sufficient amount Survival Factor will survive or breed.The activation for the cell pathway for being responsible for these effects can be determined by protein blot, immunocytochemistry and facs analysis.

One or more following system measurement protein or its chimeric molecule can be used to suppress the effect of apoptosis.

Have detected the present invention protein or chimeric molecule protect in vitro rat, mouse and people's cortical neural cell oxygen content it is low and lack glucose condition under there is the activity of cell death.Therefore, being extracted cell culture from rat embryo.In order to determine the protein of the present invention or the effect of chimeric molecule, cell is containing 30mM Glucose Medium without serum and 95% wetness air/5%CO₂(oxygen content is normal) or without Glucose Medium without serum and 95% wetness nitrogen/5%CO₂Under the conditions of (hypoxemia and glucose lack), under the conditions of the protein or chimeric molecule of the absence or presence present invention, maintained 48 hours at 37 DEG C in the module culture storehouse in water jacketed mcubator.Cell culture returns to less than 24 hours and then oxygen content normal condition lower 24 hours under the conditions of hypoxemia and glucose shortage.With the blue fluorescence analysis cytotoxicities of Alamar, this method determines cell viability by metabolic activity.

In another method, neuronal cell cultures thing is under oxygen content normal condition, under the conditions of the protein of the invention or chimeric molecule of absence or presence various concentrations, exposed to 1mM Pidolidones or alpha-amido -3- hydroxyl -5- methyl oxazole -4- propionic acid (AMPA) 24 hours.With the blue fluorescence analysis cytotoxicities of Alamar, this method determines cell viability by metabolic activity.

Protein or its chimeric molecule can influence growth, apoptosis, development or the differentiation of various kinds of cell.In other detectable parameters, these changes can be developed the cell size change caused by intracellular organelle and the change of kytoplasm complexity reflects.For example, the culture induction keratinocyte differentiation that suspends, shows as surface marker (such as beta 1 integrin, the integrins of β 1) downward, cell becomes big, the increase of kytoplasm complexity.One or more following system measurement protein or its chimeric molecule can be used to cell size or the effect of kytoplasm complexity.

FACS determines the amount of light scattered after beam of laser is incided on cell.The argon laser for being 488nm usually using offer wavelength.Cell is bigger, and it is more that forward direction light beam is blocked, therefore preceding levels of scatter is related to cell size.In order to determine the change of cell size, the cell handled with the protein or chimeric molecule of the present invention is diluted in sheath fluid (sheath fluid) and is injected into flow cytometer (FACSVantage SE, Becton Dickinson).Untreated cell is used as control.Cell is by light beam, and the forward scattering quantity of light is related to cell size.

The change (kytoplasm complexity) of intracellular organelle growth and development can also be determined with FACS.Intracellular organelle is to side-scattered light.Accordingly, it is capable to the change for the light side-scattered quantitative measurement kytoplasm complexity that cell is caused in aforementioned manners, and the light side-scattered level that can be sent by determining cell determines the level of complexity and cell granulations degree level of intracellular organelle.

FACS can be used to determine protein or its chimeric molecule to cell size or the effect of kytoplasm complexity, come the signal of the untreated cell transmitting of comparing signal characteristic and equal amount that the cells of such as 20000 processing are sent.By comparing the signal of different disposal cell mass, the relative change of cell size and kytoplasm complexity can be detected.

One or more following system measurement protein or its chimeric molecule cell growth, apoptosis, development or the effect of differentiation can be used.

It can be marked with dyestuff such as propidium iodide (propidium iodine) and handle the DNA of cell to determine the apoptosis and cell growth or cell cycle change that protein is induced, the excitation wavelength of propidium iodide is launched in 620nm regions in 488nm regions.The cell DNA for occurring apoptosis is condensing, and size and granularity are also different.These factors give positive size scattering signatures and fluorescence signal, and therefore distinguish the positive cell mass and normal cell for occurring apoptosis.DNA quantity in cell also reflects which that cell is in the cell cycle in stage.For example, G₂The DNA quantity of phase cell is G₀Twice of phase.This can pass through G₂The fluorescence signal that phase cell sends twice is reflected.The signal of the untreated cell transmitting of fluorescence signal and equal amount that the cell that FACS can be used to compare such as 20000 processing is sent, determines protein or the effect of its chimeric molecule.

The protein or chimeric molecule of the present invention can also change the expression of multiple proteins.The protein or chimeric molecule of one or more following system measurement present invention can be used to the effect of protein expression.

In order to determine the increase and reduction of protein expression in whole cell, it can fix and permeabilized cells, the antibody incubation then and using proteins of interest matter epitope being crosslinked as the fluorescein of target spot.A variety of fluorescence labelings can be used in argon laser system.Often using FITC, Alexa Fluor 488, Cyanine 2, Cyanine 3 these fluorescence molecules in this experiment.By marking on cell surface, only the epitope of exposure can be by the non-permeabilized cells of antibody labeling, and the method can also be used to determine surface marker and protein expression change.The signal of the untreated cell transmitting of fluorescence signal and equal amount that the cell that FACS can be used to compare such as 20000 processing is sent, determines protein or the effect of its chimeric molecule.

The effect that one or more following system measurement protein or its chimeric molecule can be used to adhere to ligand/receptor.

Compared with known physics and chemistry form before those, protein of the invention or chimeric molecule there may be lower or more high-adhesiveness.Interacting can be and protein acceptor, because sugared structure (such as selects albumen, such as L- selects albumen and CD62P) and extracellular matrix components (such as fibronectin, collagen, vitronectin and laminin) or with non-protein component such as glycan molecule (heparin sulfate, other glycosaminoglycans).

Protein or its chimeric molecule can also be different with non-biological material such as tissue culturing plastic, medicine equipment composition (such as support or other grafts) or dental material generation interaction.For medicine equipment, this may change graft immigration rate, graft and specific cell type or the interaction between body connection type

Any suitable protein adherence detection method can be used.For example, in certain embodiments, the binding partners on protein or the solution of chimeric molecule and an immobilization surface containing the present invention are incubated.After incubation, the quantity of the protein or chimeric molecule in solution is detected with ELISA, the remaining quantity variance between parent material is bonded to the amount on gametophyte.For example, the interaction between protein or chimeric molecule and extracellular matrix proteins can be determined with the aperture of coated 96 orifice plate of ECM protein (such as fibronectin) first layer.Then it is incubated with BSA solution and prevents non-specific binding.After cleaning, the protein or its chimeric molecule solution of a kind of concentration known are added up to the time of determination.The amount of remaining protein or its chimeric molecule in subsequent removal solution, measure solution.Determine the amount being attached on ECM protein by using the antibody incubation aperture for protein or its chimeric molecule, then with a kind of suitable system (or a kind of mark secondary antibody or with used in biotin-avidin multienzyme complex such as ELISA) detected.

Determining the method for the amount for being attached to other surfaces can include, from inertia graft surface hydrolysis protein or its chimeric molecule, then determining the amino acid in solution.

The effect of one or more following system measurement protein or its chimeric molecule to cell adherence can be used.

Cell adherence is at least partly mediated to matrix (such as extracellular matrix components such as fibronectin, vitronectin, collagen, laminin) by integrin molecule.Integrin molecule includes α and beta subunit, and α and the unique combination physical efficiency increase of beta subunit are directed to the binding specificity of particular ligand (such as a2b1 integrins incorporating collagen, a5b1 binding fiber associated proteins).There is integrin subunit big extracellular domain to be used for binding partner, and shorter endochylema region domain is used for and cytoskeleton interaction.When there is part, endochylema region domain is responsible for inducing signal transduction event (signal transduction in outer).Extracellular signal event can regulate and control compatibility of the integrin to their part, subsequently result in the change (internal/external signal transduction) of integrin cytoplasmic tail.

It is incubated with the protein or chimeric molecule of the present invention and can change cell adherence by certain methods.First, it can qualitatively change the expression of specific integrin subgroup, cause the change of binding ability.Secondly, thus it is possible to vary the expression quantity of specific integrin, the combination of cell and its target matrix is caused to change.3rd, the affinity (internal/external signal transduction) of integrin can be changed in the case where not changing the surface expression of specific integrin.All these changes can change combination of the cell to a pedigree part, or change the combination to certain specific part.

Cell-ECM adhesion detection method the detections generally carried out in 96 orifice plates protein or chimeric molecule of the invention can be used.Aperture is coated with matrix, is then coated with BSA in hole and is not associated with site.By coated hole and the cell incubation of quantification, uncombined cell is then washed away, the cell of hatching combination under conditions of protein or its chimeric molecule is contained or not contain.The quantity of cell is determined by indirect method such as MTT/MTS.Or, with radioactively labelled substance (for example⁵¹Cr cell) is marked, the radioactivity (i.e. cell) of dose known amounts is added in each hole.Measure combination radioactivity, calculates the ratio for accounting for loading quantity.

Cell is also adhered on other cells, such as on another cell of a group cell adherence to individual layer.In order to detect this situation, the labeled upper radioactivity of suspension cell is simultaneously added on cell monolayer.Subsequent cell is incubated under conditions of presence or absence of protein or its chimeric molecule.Uncombined cell is washed away, the mixing with cells colony of remaining is dissolved and detects radioactivity.

The effect of one or more following system measurement protein or its chimeric molecule to Cell expansions can be used.

The protein or chimeric molecule of the present invention has different effects to Cell expansions.Cell starts to spread the committed step for being cell mobility and infiltration behavior.Cell can start diffusion in many ways in vitro.Suspension cell, which is layered on ECM compositions, to cause the adhesion by integrin and ligand binding.This starts signal transduction event, causes the activation of the small GTPases families of Cdc42, Rac and Rho.The activation of these protein causes actin polymerization and piece foot (lamellipodium) extension, causes cell gradually to flatten and their acceptor touches more integrins.Final cell is put down flat and forms focal adhension (big structure containing integrin and signal protein) completely.The cell that can also be adhered to by using factors stimulated growth carrys out active cell extension, also causes the activation of Cdc42/Rac/Rho protein and the formation of focal adhension.

Can by after protein or its chimeric molecule are stimulated different time points detect a large amount of cells Cell expansions quantified.Image analysis software can be used to determine the region of each cell, it is possible to be compared Cell expansions percentage and Cell expansions degree and time.The stronger activation of Cdc42/Rac/Rho paths, which can start, faster to be extended, in addition, it is temporary, the qualitative and quantitative differences that they are activated can be determined when having the protein or chimeric molecule of the present invention.The change of this protein for reflecting the present invention successively or the signal event of chimeric molecule induction.

One or more following system measurement protein or its chimeric molecule can be used to cell mobility, migration and the effect infiltrated.

It is not that static holding is anchored on a site to adhere to the cell on tissue culture dishes, but lasting stretching, extension and shrinks their cell body.When being observed with time lapse photography, it is observed that cell is strolled about in culture plate, the individual cells or cell colony of separation are not always the case.This motion both can be " random walk " (i.e. not towards specific direction) or orientation.Two kinds of motion can be strengthened by adding growth factor.The total distance and general direction of time lapse photography quantitative cell covering in preset time section can be used.

In directional migration, cell will be moved by experiencing chemical gradient and orienting their migration machine towards the source of chemoattractant.In many cases, chemoattractant is growth factor.Can be by providing chemoattractant source (such as by long pipette) and then cell being imaged to the migration in source with time lapse photography, to quantitative determine directional migration.

Another system for determining directional migration is Boyden chamber detection.In this detection, cell is placed in by splitting in the upper chamber that aperture is connected with lower room in film.Growth medium is all added in two rooms, but only adds chemoattractant in lower room, causes there is diffusion gradient between two rooms.Cell is attracted by growth factor source and migrates across the aperture in segmentation film, enters the lower surface of film.After a few hours, take film away and counting measure is carried out to the cell for moving to film bottom.

Cellular infiltration process has used many and migration identical component.Cellular infiltration multi-layer cellular epimatrix can be used to be used as cellular infiltration model.For example, matrigel is the mixture (ECM compositions, growth factor etc.) of basement membrane components, it is liquid at 4 DEG C, but gel is quickly formed at 37 DEG C.The upper surface of Boyden chamber can be utilized to be coated with, chemoattractant is added in lower floor.For by the cell to film lower surface, they must use enzyme (such as clostridiopetidase A and matrix metalloproteinase (MMP)) to carry out matrix degradation glue and be oriented migration towards chemoattractant.This detection simulates the various procedures needed for cellular infiltration.

The effect of one or more following system measurement protein or its chimeric molecule to chemotaxis can be used.

Cell can be determined in Boyden chamber in vitro towards the migration in chemoattractant direction.The protein or chimeric molecule of the present invention is placed to lower room, and suitable target cell group is placed into upper chamber.The migration by one layer of cells can be detected, to simulate the extracorporeal procedures of migration of the immunocyte from blood to inflammation part.With the upper surface in the hole of the one layer of cells cladding Boyden chamber covered with, the cell such as epithelium, endothelium or fibroblast, this is by blocking immunity cell Direct Transfer by the hole in hole, and immunocyte will need to adhere on cell monolayer and migrate across it to reach at detected albumen.Only there is the chemotactic sexuality that cell indicates protein or chimeric molecule in the Boyden chamber lower surface or lower opening culture medium in the hole handled with protein or its chimeric molecule.In order to show that this effect is that protein or its chimeric molecule are specific, albumen and neutralizing antibody can be allowed to be incubated together in lower room.

In addition, in order to detect that a kind of material (chemical substance, protein, sugar) prevents the ability of chemotaxis, the solution by the material and containing known chemotactic sexuality (can be the conditioned medium of the cell of specific chemokine, cell derived or a series of chemotactic factor (CF)s of secretion) is incubated together in the lower room of Boyden chamber.Permissive cell group then is added in upper chamber, detection is proceeded as described above.

The effect of one or more following system measurement protein or its chimeric molecule to ligand receptor-combination can be used.

The protein or chimeric molecule of the present invention may have different ligand-receptor binding abilities.Ligand-receptor can be determined by many kinds of parameters to combine, for example, dissociation constant (Kd), dissociation rate constant (k^-), association rate constant (k⁺).The difference that ligand-receptor is combined may be relevant with activation with the different signal time limits, causes different biological results.

Ligand-receptor combination can be determined and analyzed by scatchard plot method or other methods such as Biacore methods.

For scatchard plot method, with such as radioactive label (for example¹²⁵I) come the protein or its chimeric molecule that mark, under conditions of the "dead" protein or the competitor of its chimeric molecule that there is varying number, and expression respective ligand or the cell of acceptor or its extract are incubated together.Determine the protein or the quantity of its chimeric molecule of the mark of specific binding, and calculations incorporated parameter.

For Biacore methods, the restructuring part corresponding with protein or its chimeric molecule or acceptor and detection unit match.Solution containing selected protein or its chimeric molecule then determines combination by detecting cell, and by detecting the change of unit character.By by the solution containing protein or its chimeric molecule by detect cell until recorded fixed reading (when all permissions site all it is occupied sometimes) determine association rate constant.The solution of protein or chimeric molecule is not contained by cell, protein is disintegrated down from respective ligand or acceptor, gives dissociation rate constant.

The effect of one or more following system measurement protein or its chimeric molecule to receptor activation can be used.

Interaction between protein or its chimeric molecule and corresponding part or acceptor can be contrasted by the difference in the protein induced signal event of cellular endogenous.Protein or its chimeric molecule can be used, the interaction time limit is accurately characterized with combination/dissociation rate constant or dissociation constant.

The acceptor of activation is taken the photograph in cell is frequent.Subsequent receptor/ligand complex dissociation (for example, the low pH in cell follicles bubble causes part to depart from), and acceptor recirculates to cell surface.In addition, the compound may also turn into the target spot of degraded.In the process, acceptor will effectively be lowered and can not produce further signal, but then can repeating signal process when they recirculate.Different acceptors is combined or activation may cause acceptor to be changed into the path that recirculates from degraded, causes stronger biological response.

The effect of one or more following system measurement protein or its chimeric molecule to signal transduction can be used.

Part or acceptor, which are attached on protein or its chimeric molecule, to send signal by a variety of plasmosins, wherein potentially including reverse signal.When the part of film combining form is by combining its solubility or film combining form acceptor come conducted signal, reverse signal occurs.Reverse signal may also occur in by after a kind of antibody binding film combination part.These signal events (including reverse signal event) cause the change of gene and protein expression.Therefore, the protein or chimeric molecule of the present invention can induce or suppress different signal transductions or other signal transduction events in different paths, such as JAK/STAT paths, Ras-erk paths, the activation of AKT paths, PKC, PKA, Src, Fas, TNFR, NFkB, p38MAPK, c-Fos activation, by protein recruitment to acceptor, receptor phosphorylation, is taken the photograph in vivo, receptor cross-talk or secretion.

It may be unique to the protein or chimeric molecule of the present invention to raise protein or part or acceptor on its chimeric molecule, because induction of the different conformations of part or acceptor.A method for determining these differences is come immunoprecipitation part or acceptor with a kind of antibody being linked on sepahrose globules.After immunoprecipitation and cleaning, by protein loading 2D gel power supplys, com-parison and analysis dot pattern.Discrepant point can be cut and Mass Spectrometric Identification is used.

The effect that the upper mediation of one or more following system measurement protein or its chimeric molecule to surface marker can be used to lower.

Cell may have a variety of responses to the protein or chimeric molecule of the present invention.There are a series of protein to be responsible for the communication between cell and extracellular matrix in cell surface.By regulating and controlling encytosis and exocytosis process, multiple proteins are transported to and transported out of cell surface.The Representative Western found in cell surface includes acceptor, associated proteins, modulin and signaling molecule.The change of protein expression and degradation rate also changes the level of cell cortex protein.Some protein are also stored in intracellular container, transport of the distinctive signal energy inducible protein between these containers and cell membrane.

Cell is incubated the suitable time in the culture medium of the protein containing the present invention or chimeric molecule, is compared with the cell not contained exposed to identical in the protein of the present invention or the culture medium of chimeric molecule.Protein on cell membrane can be dissolved and by centrifugation and cell separation.Specific protein expression level can be determined with Western blot.The antibody labeled cells that can also be crosslinked with fluorescein, are visualized with Laser Scanning Confocal Microscope system or are counted by Fluorescence-activated cell sorting (FACS).This will detect any change of protein expression and distribution on cell.The change of protein dependent interaction can also be studied using Laser Scanning Confocal Microscope and immunoprecipitation by using multiple antibody.Similar, these experiments can be expanded in internal animal model.Cell can be extracted from the animal privileged site handled with the protein or chimeric molecule of the present invention, be detected with identical method.

Different marks will be expressed by adding the protein of the present invention or the cell of chimeric molecule induction differentiation in vitro or in vivo, the mark is separated by these cells and for processing cellular regions.Some cells, for example, progenitor cells or stem cell, can be divided into a variety of isoforms, are distinguished by their surface marker.One protein or chimeric molecule of the invention may stimulate progenitor cells to be divided into a variety of isoforms according to a specific ratio.

The protein and its chimeric molecule of the present invention is possible to effective to cellular rejection.

Protein or its chimeric molecule are convenient cellular rejection detection methods in regulating cell and neure growth and the effect pointed to.

Interaction between destruction subunit and the other components of protein is a kind of method for suppressing protein or the biological effect of its chimeric molecule.The compound for suppressing this biological effect is identified by a variety of methods.

High flux screening project, which produces lead compound using small chemical entities storehouse (compound or peptide), is used for clinical development.Some detection methods can be used to screen the ability of the biological related end of the final point of the compounds affect in storehouse.Each potential compound in storehouse is detected in an independent aperture in one-time detection, the effect of compound is determined.The embodiment of some detections is provided below.

To this detection, cell is taped against in micro plate (96 orifice plates, 384 orifice plates etc.).The reading mechanism that cell will be activated with protein or its chimeric molecule.This can include detection cell growth, the stimulation (such as technology based on FRET) for detecting specific passageways, the induction (such as CAT, beta galactosidase, fluorescin) of examining report gene, detection apoptosis and detection differentiation.Subsequent cell is being exposed in the protein of the present invention or chimeric molecule under conditions of existing or lacking specific small molecule.Medicine can be added before, after, or during protein or its chimeric molecule is added.After the suitable period, using suitable method to indivedual hole readings (such as the cell quantity in the induction of the fluorescence or fluorescin of FRET, MTT, betagalactosidase activity etc.).The control wells of any medicine or cell factor are added without as comparing.Any molecule that can suppress acceptor/cytokine complex will provide the readings different with compareing.Further experiment is also needed to show the specificity of suppression.In addition, medicine may influence detection method (false positive) by non-cytokine, non-receptor mechanism.

The part or acceptor of protein or its chimeric molecule are immobilized in the surface of solids.It is subsequently added protein or its chimeric molecule and compound to be detected.Protein or its chimeric molecule can be first added, compound is subsequently added；Compound is first added, protein or its chimeric molecule is subsequently added；Or compound and protein or its chimeric molecule are added together.Then by suitably detecting protein or chimeric molecule that antibody test is combined.Antibody can be detected with a kind of enzyme (such as alkaline phosphatase or horseradish peroxidase for colorimetric determination) or a kind of fluorescent tag label for fluoroscopic examination.Furthermore, it is possible to a kind of suitable technical mark (such as biotin, radioactive label) protein or chimeric molecule and with a kind of suitable technology (for example, for biotin labeling, streptavidin and colorimetric assay system；For radioactive label, dissolve compound and count) detection.The suppression that protein is combined is determined by comparing the decline of reading with control wells.

The soluble ligand or acceptor of protein or its chimeric molecule are combined on globule.This association reaction both can be that a kind of adsorption process relates to they being chemically crosslinked onto plate.Globule and protein or chimeric molecule and a kind of candidate compound are incubated in a suitable aperture.Protein or chimeric molecule can be first added, compound is subsequently added；Compound is first added, protein or chimeric molecule is subsequently added；Or add compound and protein or chimeric molecule together.Be subsequently added fluorescence labeling recognizes a kind of protein or the detection antibody of its chimeric molecule.Remove uncombined antibody and globule is passed through into FACS.If compound suppresses the interaction of protein or its chimeric molecule and its acceptor, the amount of fluorescence of detection will decline.

For multiple interactions between screening protein and its respective ligand/acceptor and a kind of inhibiting compound, it is necessary to use the ability of FACS Instrumental Analysis scattering signatures.The ball of larger diameter will have the scattering signatures different with smaller ball, it is possible to be separated for analyzing (" gating ").

Some different protein, one of which is the protein or chimeric molecule of the present invention, is all linked on the ball with special diameter.The ligand/receptor mixture of above-mentioned protein is then added in the ball mixed liquor containing a kind of candidate compound.The ligand/receptor combined is then detected with the secondary antibody of a species specific fluorescence labeling.Antibody can all mark identical to detect fluorophor.The ability that compound prevents protein from combining its ligand/receptor is then determined by the Run sample on FACS instruments and to the ball gating of each known dimensions.Then analysis individually each combines result respectively.The key benefit of this analysis method is each compound that can be screened with some protein Parallel testings, consequently reduces screening time.

Protein or its chimeric molecule can also be characterized by its crystalline structure.The physics and chemistry form of protein or its chimeric molecule can provide unique 3-dimensional crystal structure.Further, it is also possible to produce the crystal structure of protein-ligand/receptor complex using the protein or chimeric molecule of the present invention.Because the invention provides the protein essentially similar with naive existence form or its chimeric molecule, the compound is likely to be the more suitably representative of the internal structure of naturally occurring protein-ligand/receptor complex.Once obtaining crystal structure, the interaction between protein or its chimeric molecule and the compound for potentially suppressing this interaction just can determine that.

Once determine potential compound by high flux screening or from protein-ligand/receptor complex crystal structure, it is possible to proceed by design and rational pharmaceutical procedures.

Carried out generally using some steps in analogies design according to the compound with design specified characteristic.First, it is determined that compound privileged site crucial and/or important in characteristic needed for determining.For peptide, above-mentioned work can be completed by the amino acid residue in systematic change peptide, for example, replace each residue successively.This peptide motif is often refined using Alanine-scanning.These parts or residue constitute the active site of compound, and the active site is referred to as its pharmacophoric group.

Once it is found that pharmacophoric group, its structure is modeled using a series of data in sources according to its physical characteristic, the physical characteristic such as spatial chemistry, bonding, size and/or electric charge, data such as spectroscopy techniques, X ray diffracting data and the nuclear magnetic resonance in the series source.In this modeling process calculating analysis, similitude can be used to do figure (electric charge and/or volume-based model of pharmacophoric group, rather than interatomic bonding) and other technologies.

In a variant of this method, the 3 d structure model of part and its binding partners is made.This changes conformation when combining for part and/or binding partners and is particularly useful, and modeling can be allowed to consider this problem in design simulation thing.Modeling can be used to produce the inhibitor interacted with linear order or a kind of 3-dimensional conformation.

The template molecule that then selection can will simulate the chemical group transplanting of pharmacophoric group up.Template molecule can easily be selected and chemical group up is transplanted, therefore analogies are easily synthesized, it may be possible to it is pharmacologically acceptable and non-degradable in vivo, remain the bioactivity of lead compound.Or, when analogies are to be based on peptide, further stability can be obtained by the way that peptide is cyclized, increase its rigidity.The analogies that find by this method can be then screened to observe whether they have target property, or they show the target property of much degree.Then further it can be optimized or be modified, one or more final analogies are entered into internal or clinical detection.

The target of rational drug design is generation biologically active polypeptide interested or the analogue of small molecule, they and these polypeptides or small molecular phase interaction (such as activator, antagonist, inhibitor or reinforcing agent), in order to form medicine, for example, more active or more stable polypeptide form, or, for example strengthen or disturb the function of polypeptide in vivo.See, such as Hodgson (Bio/Technology 9：19-21,1991).In a procedure, first by x ray crystallography, by microcomputer modelling or it is most typical, be combined by a variety of methods and to determine the three-dimensional structure of protein interested.The useful information on polypeptide structure can also be obtained by the structural modeling based on homologous protein.The example of one rational drug design is exploitation hiv protease inhibitor (Erickson et al.Science249：527-533,1990).Furthermore, it is possible to analyze target molecule (Wells, Methods Enzymol 202 by Alanine-scanning：2699-2705,1991).In this technology, an amino acid residue is replaced by Ala, and determines its influence to peptide activity.Each amino acid residue of analytical peptide determines the important area of peptide in this way.

It is also possible to isolate target specificity antibody, is selected by functional analysis, and then parses its crystal structure.In principle, this method produces a drug core, and follow-up drug design can be carried out based on it.Bak protein crystallography is omitted completely possibly through anti-idiotype is produced to a functional, the antibody for having pharmacological activity.The binding site of anti-idiotype therefore is expected for the analog of original acceptor as the mirror image of mirror image.Therefore anti-idiotype can be used to identify and isolated peptides chemically or in biogenic peptide storehouse.Then drug core is used as with the peptide of selection.

On the one hand, protein of the invention or chimeric molecule are used as immunogen to produce antibody.The protein of the present invention or the physics and chemistry form of chimeric molecule can increase the antibody for protein or chimeric molecule；The antibody of the glycopeptide of protein or chimeric molecule for the present invention；Directly against in protein or its chimeric molecule another translation in or posttranslational modification peptide antibody.

The protein or its chimeric molecule of the present invention may have in vivo the epitope of not accessible (it is possible that presence) under normal circumstances.For example, it is possible to there is a region under normal circumstances with the part contact of a heteropleural acceptor in receptor domain.The monoclonal antibody for intersecting response with endogenous recipient can be produced with these epitopes.This antibody can block a receptor element and another interaction, and therefore prevent signal transduction.This can be used in the overexpressing cells factor or acceptor as treatment.Antibody can also acceptor overexpression and do not need part just produce signal when used as treatment.

Antibody can be also used for the level (for example, serum levels to determine half-life period) of detection protein or its chimeric molecule in treatment disease.

In addition, antibody can be used for protein or chimeric molecule of the diagnostic assay with the presence or absence of the present invention in specific sample.

" antibody " being previously mentioned includes the form of ownership for the antibody being previously mentioned, and includes but is not limited to：It is complete anti-(such as containing complete Fc regions), including such as monoclonal antibody；Antigen binding antibody fragment, including such as Fv, Fab, Fab ' and F (ab ')₂Fragment；Humanized antibody；Human antibodies (for example, in transgenic animals or the antibody produced by display technique of bacteriophage)；With the immunoglobulin derived peptide produced by technique for gene engineering.With other differences refered in particular to, term " antibody " and as described here including complete anti-and its antigen-binding fragment.

Unless otherwise noted, the present invention represents that the antibody substantially only combines its target antigen, the appreciable combination without unrelated protein on the specificity of antibody.It is, however, possible to which an antibody will be designed or select to combine two or more related proteins.Related protein includes different same proteins or comes from the splice variant or fragment of the homologous protein of different plant species.This antibody is also believed to have specificity to those protein, and is included in the present invention.Term " substantially " means in this context to be not above non target antigen the detectable combination of substrate level, i.e., non-specific.

The antibody of the present invention can be prepared by well known method.See, e.g., MonoclonalAntibodies, Hybridomas：A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980)；And Antibodies：A Laboratory Manual, Harlow and Lane (eds.), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

Producing a method of the antibody of the present invention includes a protein or chimeric molecule or its immunogenic portion of the invention being produced with animal (for example, one include receptor binding domain peptide, antibody is by receptor binding domain come with reference to the polypeptide or immunogenic portion in a protein or its chimeric molecule) non-human animal, such as one mouse or a transgenic mice is immunized.There are a variety of methods to increase the antigenicity of specified protein or its chimeric molecule, for example, give adjuvant or conjugated antigen, including for antigen and another element that required antibody is responded, these methods are all known in the art and are to use.Typical immunization includes a series of initial immune and subsequent enhancings and is immunized.Blood can be taken to animal and the antibody titer in serum is analyzed.Animal can be strengthened, the plateau until reaching titre.With protein fusion conjugant can be prepared in recombinant cell culture thing.Equally, aggregating agent such as alum is applied to enhancing immune response.

Polyclonal and monoclonal antibody can be prepared by this method.The method for obtaining two types antibody is well known in the art.Polyclonal antibody uses less but relatively easy preparation, suitable animal is injected with the protein of the invention or chimeric molecule or its immunogenic portion of effective quantity, serum is collected from animal and protein or the antibody of its chimeric molecule is directed to using the separation of any of immunoadsorbent technics is specific.The antibody produced by the method is using relatively fewer, because product has potential inhomogeneity.

The reason for significantly being had a preference for using monoclonal antibody is that they can largely be produced, and product has homogeney.Monoclonal antibody can be produced by conventional method.

Term " monoclonal antibody " used herein refers to the antibody obtained from substantial amounts of homogeneous antibody, i.e. by the independent antibody that identical colony constitutes in addition to the possible naturally-produced mutation existed on a small quantity.Monoclonal antibody is high specific, directly against single antigen site.In addition, and the polyclonal antibody preparations that are typically include for the different antibodies of different antigenic determinants (epitope) it is different, each monoclonal antibody is directly against antigenic determinant single on antigen.Modifier " monoclonal " refers to that antibody is characterized in be obtained from abundant homologous antibody colony, rather than is construed to need to produce antibody by any specific method.For example, Kohler et al. Nature 256 can be passed through according to monoclonal antibody used in the present invention：Prepared by the hybridoma method that 495 (1975) are announced first, or can prepare (see such as U.S. Patent number 4816567) by recombinant DNA method." monoclonal antibody " can also be separated from phage antibody library, use such as Clackson et al. Nature 352：624-628,1991 and Marks et al. J Mol Biol 222：581-597, the method described in 1991.

A kind of method of hybridoma cell line is produced present invention contemplates a kind of, including a kind of non-human animal, such as mouse or transgenic mice is immunized with the protein or chimeric molecule of the present invention；Splenocyte is harvested from immune animal；The splenocyte of harvest and myeloma cell line fusion are produced into hybridoma；And identify that production can combine the hybridoma cell line of a kind of protein or the monoclonal antibody of its chimeric molecule.

This hybridoma cell line and the monoclonal antibody that they are produced are included in the present invention.The monoclonal antibody secreted by routine techniques purified hybrid oncocyte system.The monoclonal antibody of hybridoma or their generations can further be screened to identify the monoclonal antibody with required particular characteristics, for example, suppress the ability of its signal by cytokine receptor.

Can be used for the protein or its chimeric molecule or its immunogenic portion of immune animal in the initial period of the antibody of the production present invention should come from people's expression source.

The antigen-binding fragment of the antibody of the present invention can be produced by conventional method.The example of this fragment includes but is not limited to：Fab、Fab′、F(ab′)₂With Fv fragments, including Single-Chain Fv Fragment of Murine (being named as sFv or scFv).The stable Fv fragments (dsFv) of the antibody fragment and derivative produced by genetic engineering, such as curing, single-stranded Variable domain (Abs), according to the present invention, it is also contemplated that miniantibody and binary are used to using.

Characteristic needed for this fragment and derivative directly against protein or the monoclonal antibody of its chimeric molecule can be prepared by known technology and is screened, the technology includes detection described herein.These detections provide the method for the fragment and derivative of identification energy conjugated protein or the antibody of the invention of its chimeric molecule, and can identify those active fragments and derivative for also retaining the signal for suppressing protein or its chimeric molecule.These technologies necessarily include the DNA of the polypeptide chain (or its part) in separation coding mAb interested, and operate these DNA by recombinant DNA technology.Can be by DNA being fused on another DNA interested or other methods (such as by mutagenesis or other routine techniques) increase, delete or replaced one or more amino acid residues.

Can be from the DNA (such as weighing or light chain, only variable region or total length) of separation encoding antibody polypeptide in the B cell of the immune mouse of the protein or chimeric molecule of the present invention.Conventional program can be used to separate DNA.Phage display is the example of another known technology, and the derivative of antibody can be prepared by it.In one approach, expressed in any suitable recombinant expression system as a kind of polypeptide of a part for antibody interested, and the polypeptide expressed can be assembled to form antibody molecule.

Heavy chain and light chain variable district (Fv areas) fragment can be connected to form single-chain antibody by amino acid bridge (small peptide linker), form a single polypeptide chain.This scFv s (scFvs) is prepared by the DNA of the fusion coded polypeptide linker between the DNA of two variable domain polypeptides (VL and VH) is encoded.According to the length of flexible connection between two variable domains, gained antibody fragment can form dimer or tripolymer (Kortt et al. ProteinEngineering 10：423,1997).The technology for the production single-chain antibody that developed includes United States Patent (USP) 4946778；Bird(Science 242：423rd, 1988), Huston et al. (ProcNatl Acad Sci USA 85：5879,1988) and Ward et al. (Nature334：544,1989) technology.The single-chain antibody derived from antibody provided herein is included in the present invention.

In one embodiment, the invention provides can combine the present invention protein or chimeric molecule and suppress protein or the signal of its chimeric molecule antibody antibody fragment or chimeric molecule, recon or synthesized form.

Known technology can obtain the antibody of different subclass or isotype from antibody interested, i.e. subclass is changed.It may be thus possible, for example, to obtain IgG1 or IgG4 monoclonal antibodies from IgM monoclonal antibody, vice versa.This technology can produce the new antibodies for the antigenic binding property for possessing specified antibody (female antibody), but the biological characteristics that also the display antibody isotype different with female antibody or subclass have.

Recombinant DNA technology can be used.The clone DNA of encoding particular antibodies polypeptide can be used in the process, such as the DNA of the constant region of isotype antibody needed for encoding.

Above-mentioned monoclonal production process, such as mouse, to produce monoclonal antibody can be used in animal.The conventional antibodies obtained from these animals, such as mouse antibodies, are typically considered to not be suitable for human body, because they can cause immune response.Therefore, this antibody may need modification to be applied to human body.The process for preparing chimeric molecule and/or humanized antibody is known in the art, be will be described in further detail below.

Monoclonal antibody herein particularly including " chimeric " antibody, wherein heavy chain and/or light variable domains are identical or homologous with the corresponding sequence for the antibody for coming from non-human species (such as mouse), and the remainder in chain is identical or homologous with the corresponding sequence for the antibody for coming from human body, the fragment of this antibody is also that so, therefore they show required biological activity (United States Patent (USP) 4816567；With Morrison et al. .Proc Natl Acad Sci USA81：6851-6855,1984).

" humanization " form of inhuman (such as mouse) antibody is chimeric antibody, wherein containing a small amount of sequence for coming from non-human immunoglobulin.For most part, humanized antibody is human immunoglobulin(HIg) (recipient's antibody), wherein the complementary determining region (CDRs) of recipient's antibody is replaced by the corresponding CDRs of non-human species' (donor antibody), and the non-human species for example possess mouse, rat, rabbit or the non-human primate of required characteristic (such as specificity and compatibility).In some instances, the framework residues of people's immune globulin are replaced by corresponding non-human residues.Also, humanized antibody may be embodied in undiscovered residue in acceptor antibody or donor antibody.These modifications are carried out further to refine the performance of antibody.In a word, humanized antibody will basically comprise it is all at least one, and typical case is the variable domains of two, wherein all or essentially all of complementary determining region corresponds to non-human immunoglobulin, and all or generally all of framework residues are human immunoglobulin sequences.Humanized antibody will also optionally contain at least one of constant region for immunoglobulin (Fc), typically human immunoglobulin(HIg).Further details are referring to Jones et al. Nature 321：522-525,1986；Reichmann et al. Nature 332：323-329,1988；Presta, Curr Op Struct Biol2：593-596,1992；Liu et al. Proc Natl Acad Sci USA 84：3439,1987；Larrick et al. Bio/Technology 7：934,1989；With Winter and Harris, TIPS 14：139,1993.

In a further embodiment, the invention provides immunity detection reagent, the level of the human protein expressed by the people's cell in a kind of biological products can be detected, the biological products include the biological products for including naturally-produced people's albumen.

The biological products that the energy of the present invention is detected using immunity detection reagent include but is not limited to laboratory sample, cell, tissue, blood, serum, blood plasma, urine, excrement, saliva and phlegm.

The immunity detection reagent of the present invention includes a kind of solid phase support matrix, is not limited to but including a kind of film, dipstick, pearl, gel, pipe or porous, flat, round bottom or the microtest plate at v-shaped bottom, such as 96 orifice plates；Directly against the antibody preparation (capture antibody) of human protein interested；It is coated with pharmaceutical solutions (such as BSA or casein)；Secondary antibody preparation (detection antibody), is conjugated directly against human protein interested and with suitable detection molecules (such as alkaline phosphatase)；A kind of Chromogenic Substrate Solution (such as nitro blue tetrazolium)；One kind addition substrate solution (the chloro- 3- indolyl phosphates of the bromo- 4- of such as 5-)；A kind of substrate buffer solution mother liquor (such as 0.1M Tris-HCL (pH7.5) and 0.1M NaCl, 50mM MgCl₂)；The protein of the invention of concentration known (standard) or the preparation of chimeric molecule；And operation instructions.

Suitable detection molecules can be selected to be attached on including but not limited to staphylococcal protein A or streptococcal protein G equimolecular from the list including reagents such as enzyme, dyestuff, fluorescence molecule, chemiluminescence agent, isotope or collaurums.

In a specific embodiment, capture and detect that antibody is monoclonal antibody, it, which is prepared, includes the protein with the present invention or chimeric molecule immunizing non-human animals, such as mouse or transgenic mice, and standard method is then carried out as described above.Monoclonal antibody can be produced in the method with restructuring of alternative, as described above, it is possible to including people or chimeric antibody part or domain.

In another embodiment, capture and detect that antibody is polyclonal antibody, it, which is prepared, includes the protein with the present invention or chimeric molecule immunizing non-human animals, such as mouse or rabbit, sheep or horse, and standard method is then carried out as described above.

The component of detection kit is provided by predetermined ratio, and the change of the relative populations of different reagents suitably maintains the reagent concentration in solution, allows detection sensitivity substantially to maximize.Especially, reagent can be provided in the way of dry powder, typically freezed, including auxiliary material, the dissolving each reagent solution of relief is for biological products to be measured with suitable concentration

The application method of the immunity detection reagent of the present invention can be discussed in detail in operation instructions.For example, operation instructions can introduce the method that solid phase support matrix is coated with suitable concentration with the capture antibody-solutions prepared, such as 4 DEG C overnight.The method for being coated with nonspecific protein binding site with the coating solution prepared can be described in further detail in operation instructions；(such as 37 DEG C 1 hour or room temperature 2 hours) adds sample and the incubation of the protein containing the present invention being serially diluted or chimeric molecule under suitable conditions, then serial cleaning, such as 0.1M PBS (pH7.2) solution containing 0.05% polysorbas20 are carried out using suitable buffer solution known in the art.In addition, specification can be provided, using being incubated under suitable conditions after detection antibody preparation, for example, 37 DEG C of 1 hour or room temperatures 2 hours, then carry out a series of cleanings.The working solution of detection buffer solution is prepared using the detection substrate and substrate buffer solution of offer, is subsequently added in each hole, is kept under suitable conditions, from room temperature 1 hour 5 minutes to 37 DEG C.Can be by adding 1N NaOH or 2N H₂SO₄Terminate chromogenic reaction.

In another alternate embodiment, operation instructions can provide any combinations for any or all said components to be added that can be added simultaneously in reservation ratio, the change of the relative populations of different reagents suitably maintains the reagent concentration in solution, allow compound formed produced by detectable signal substantially maximize.

ELISA Plate Readers or spectrophotometer can be used, under suitable optical density (OD), or it is used as exciting light, using spectrophotometer, fluorescence photometer or flow cytometer, under suitable wavelength, or radioactive counter is used, under suitable power spectrum, or by opacimeter, or by carrying out visual comparison, detection color products or fluorescence or chemiluminescence or radioactivity or other levels by combining, being conjugated the signal that connecting detection reagent is produced with chart or index.With the serial dilutions of above-mentioned sample Parallel testing standard preparation.Standard curve or chart are produced, the level of protein or its chimeric molecule in sample is calculated according to standard curve or chart interpolation (interpolated).

Present invention also offers people's derived protein or its chimeric molecule, as the standard protein in immune detection.The present invention extends further a kind of method for determining the level of people's albumen of people's cell expression or its chimeric molecule in biological products, including the suitable determination method for determining people's albumen or chimeric molecule, the detection method includes (a) and is combined biological products and one or more antibody directly against people's albumen or its chimeric molecule, and (b) determines the level of people's albumen or chimeric molecule in the antibody or each antibody binding biological products；(c) it will be combined between standard people albumen or a kind of chimeric molecule sample and one or more for the antibody of people's albumen or chimeric molecule；(d) level of people's albumen or chimeric molecule in the combination biological products of the antibody or each antibody is determined；(e) level of people's albumen or chimeric molecule and the level of the antibody or each antibody binding standard people albumen or chimeric molecule sample in the antibody or each antibody binding biological products are compared；

Especially, standard people albumen or chimeric molecule sample are the preparations of a kind of protein comprising the present invention or chimeric molecule.

Biological products include but is not limited to laboratory sample, cell, tissue, blood, serum, blood plasma, urine, excrement, saliva and phlegm.As described above or by methods known in the art, biological products can combine one or more capture antibody.For example, being coated with solid phase support matrix (for example, 4 DEG C are stayed overnight) under suitable conditions with the capture antibody-solutions prepared first；The binding site of nonspecific protein is then closed with the coating solution prepared；It is subsequently added the series of diluted samples of the protein containing the present invention or chimeric molecule and is incubated (such as 37 DEG C 1 hour or room temperature 2 hours) under suitable conditions, then carries out series cleaning (such as 0.1M PBS (pH7.2) solution containing 0.05% polysorbas20) using suitable buffer solution known in the art.

Then, biological products and one or more detection antibody for being combined with suitable detection molecules as described here are combined.For example, be incubated under suitable conditions after application detection antibody preparation (for example, 37 DEG C 1 hour or room temperature 2 hours), a series of cleanings are then carried out.

Level can be combined as described above or by methods known in the art measure.For example, preparing the working solution for detecting buffer solution using detection substrate and substrate buffer solution, it is subsequently added in each hole, keeps under suitable conditions, from room temperature 1 hour 5 minutes to 37 DEG C.Can be by adding 1N NaOH or 2N H₂SO₄Terminate chromogenic reaction.

In a specific embodiment, the present invention contemplates the protein or chimeric molecule of a separation as described above.

In specific embodiments, GM-CSF of the invention is with multiple physical and chemical parameter (P_x) and pharmacological characteristics (T_y) be characterized, including apparent molecular weight (P₁), its value is 5 to 60, it is 16-40kDa in a specific embodiment such as 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,56,57,58,59,60.The GM-CSF of present invention pI (P₂) it is about 1 to 10, such as 1,2,3,4,5,6,7,8,9,10, it is 2-7 in a specific embodiment, including at least 1 to 36 isoform, such as 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36 be 10-30 isoform (P in a specific embodiment₃).The GM-CSF carbohydrate percentages (P of the present invention₅) it is 1 to 99, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, it is 0-76% in a specific embodiment, it is 0-65% in further specific embodiment.After N- connections oligosaccharides is removed, molecule actual measurement molecular weight (P₆) it is 10-35kDa, it is 12-30kDa in a specific embodiment.Actual measurement molecular weight (P after N- connections and O- connections oligosaccharides are removed₇) it is 9-30kDa, it is 11 to 25kDa in a specific embodiment.

The acid contents of monosaccharides percentage (P of the GM-CSF of the present invention₈) it is 2 to 20% such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20%, it is 6-11% in a specific embodiment.When using GalNAc as standard, GM-CSF monose (P of the invention₉) and sialic acid content (P₁₀) it is 1: 0-3 trehalose, 1: 1-16 GlcNAc, 0.1: 0.1-9 galactolipin, 1: 0.1-9 mannose and 1: 0-5 NeuNAc, it is 1: 0.1-1.5 trehalose, 1: 2-12 GlcNAc, 1: 1.0-6.0 galactolipin in a specific embodiment, 1: 1.0-6.0 mannose, and 1: 0-3.0 NeuNAc；When being standard with 3 times of mannoses, for 3: 0-5 trehalose, 3: 0.1-3 GalNAc, 3: 2-15 GlcNAc, 3: 1-6 galactolipin, 3: 0-4 NeuNAc, it is 3: 0.1-2.5 trehalose, 3: 0.5-2.5 GalNAc, 3: 5.0-10.0 GlcNAc in a specific embodiment, 3: 2.0-5.0 galactolipin, and 3: 0-3.0 NeuNAc.N connection oligosaccharides (P₁₃) neutral percentage is 40 to 90%, such as 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90%, it is 49 to 83% in a specific embodiment, it 54 is 78% to be in specific embodiments, is 59 to 73% in further specific embodiment.Acid percentage (the P of N- connections connection₁₄) it is 10% to 70%, it is 17% to 51% in a specific embodiment, is in specific embodiments 22% to 46%, is 27 to 41% in further specific embodiment.Neutral percentage (the P of the oligosaccharides of O connections₁₅) be 5 be 90%, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90%, it is 9 to 82% in a specific embodiment, it is 29 to 62% in specific embodiments, it is 34 to 57% in further specific embodiment.Acid percentage (the P of O- connection oligosaccharides₁₆) it is about 10 to 100%, it is 18 to 91% in a specific embodiment, is in specific embodiments 38 to 71%, is 43 to 66% in further specific embodiment.The GM-CSF N- connection glycosylation sites (P of the present invention₂₁) include N-44 and N-54 (by signal sequence section start open numbering), these sites by PNGase handle after PMF recognize.The GM-CSF of present invention serum/plasma stability (T₁₀) different from the human GM-CSF expressed in non-human cell, especially after TF-1 is incubated 24 hours with hyclone, the former shows stronger proliferation activity than the GM-CSF expressed in E.coli.The GM-CSF and proliferation activity (T of the present invention₃₂) it is different from the human GM-CSF that non-human system expresses.GM-CSF proliferation activities (the T of the present invention₃₂) it is better than about 5-12 times of the human GM-CSF expressed in E.coli.Its differentiation activity (T₃₃) also different from the GM-CSF expressed in non-human cell, compared with the GM-CSF that E.coli is expressed, the former induces the ability of TF-1 Clone formations strong 1.5-2 times.

In specific embodiments, IL-3 molecules of the invention are with one or more physical and chemical parameter (P_x) and pharmacological characteristics (T_y) be characterized, including apparent molecular weight (P₁), its value is by 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, it is 15 to 35kDa in specific embodiments.IL-3 pI (P₂) it is 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 3.5-7.5 in specific embodiments, with about 2 to 50, such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, be in specific embodiments 5-15 isoform (P₃).IL-3 molecule carbohydrate percentages (P₅) it is 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, it is 0 to 60% in specific embodiments.After N- connections oligosaccharides is removed, IL-3 of the invention actual measurement molecular weight is (P₆) it is that 8-30kDa is 10 to 25kDa in a specific embodiment.When using GalNAc as standard, IL-3 contents of monosaccharides (P of the invention₉) it is 1: 0.1-8 trehalose, 1: 0.1-7 GlcNAc, 1: 0.1-3 galactolipin, 1: 0.1-3 mannose and 1: 0-5 NeuAc；It is 1: 2-6 trehalose, 1: 3-5 GlcNAc, 1: 0.5-2 galactolipin, 1: 0.5-2 mannose and 1: 0-2NeuNAc in a specific embodiment；It is 3: 2-25 trehalose, 3: 0.1-6 GalNAc, 3: 4-21 GlcNAc, 3: 0.1-9 galactolipin, and 3: 0-5 NeuAc when being standard with 3 times of mannoses；It is 3: 5-16 trehalose, 3: 2-4 GalNAc, 3: 9-14 GlcNAc, 3: 3-6 galactolipin and 3: 0.1-2 NeuAc in a specific embodiment.The IL-3 molecular salivary acid of the present invention is expressed as contents of monosaccharides percentage (P₁₀), it is 0 to 50%, such as 0,1st, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50%, it is 0 to 20% in a specific embodiment.Neutral percentage (the P of the IL-3 of present invention N- connection IL-3 oligosaccharides₁₃) it is 70 to 100%, it is 75 to 95% in a specific embodiment, is in specific embodiments 80 to 90%.Acid percentage (the P of the IL-3 of present invention N- connection IL-3 oligosaccharides₁₄) it is 0 to 30%, it is 5 to 25% in a specific embodiment, is in specific embodiments 10 to 20%.The IL-3 of present invention immune response characteristic (T₁₃) different from the people IL-3 expressed in non-human cell, when especially being detected with the people IL-3 expressed containing useful non-human cell ELISA kit, IL-3 of the invention protein concentration is underestimated.In addition, IL-3 (the T of the present invention₁₃) different from the people IL-3 expressed in insect cell.The IL3 of present invention multiplication capacity (T₃₂) it is different from the IL-3 that is expressed in non-human system, IL-3 of the invention multiplication capacity (T₃₂) strong 1.1-2.5 times compared with the people IL-3 that E.coli is expressed.

In specific embodiments, IL-4 molecules of the invention are with one or more physical and chemical parameter (P_x) and pharmacological characteristics (T_y) be characterized, including apparent molecular weight (P₁), its value is by 1 to 120, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120kDa, it is 12 to 24 kDa in specific embodiments.IL-4 pI (P₂) it is 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 8-11 in specific embodiments, with about 2 to 50, such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, be in specific embodiments 1-3 isoform (P₃).IL-4 molecule carbohydrate percentages (P₅) it is 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, it is 0 to 25% in specific embodiments.After N- connections oligosaccharides is removed, IL-4 of the invention actual measurement molecular weight (P₆) it is 8-24kDa, it is in specific embodiments 10 to 20kDa.And after O- connections and N- connection oligosaccharides are removed, the IL-4 molecular weight (P detected₇) it is 8-22kDa, it is 10-18kDa in a specific embodiment.Neutral percentage (the P of the oligosaccharides of the IL-4 of present invention N- connections₁₃) it is 50%-100%, it is 65-100% in a specific embodiment, is in specific embodiments 70-100%.Acid percentage (the P of the IL-4 of present invention N connections oligosaccharides₁₄) it is 0-50%, it is in specific embodiments 0-45%, is in specific embodiments 0-30%.The IL-4 of present invention N- connection glycosylation sites (P₂₁) include N-62 (by signal sequence section start open numbering), recognized after being handled with PNGase by PMF.Curing skeleton structure (P₃₃) include Cys27-Cys151, Cys48-Cys89 and Cys70-Cys123 (by signal sequence section start open numbering cysteine residues).

In specific embodiments, IL-4 of the invention immune response characteristic (T₁₃) different from the people IL-4 expressed in non-human cell, when especially being detected with the ELISA kit containing the people IL-4 expressed in non-human cell's system, IL-4 of the invention protein concentration is underestimated.The IL-4 of present invention proliferation activity (T₃₂) it is different from the people IL-4 that non-human cell's system is expressed, compared with the people IL-4 that E.co l i are expressed, I L-4 proliferation activities (T of the invention₃₂) high 25-54 times；Compared with the people IL-4 of expressing cho cell, IL-4 proliferation activities (T of the invention₃₂) high 1.7 5 times.After the temperature preincubate improved, IL-4 of the invention proliferation activity (T₃₂) also different from the people IL-4 that non-human cell's system is expressed.After preincubate TF1 cells at 37 DEG C of culture medium 4 days, IL-4 of the invention (T₃₂) value is high compared with the GM-CSF that E.coli is expressed 13-30 times.

In specific embodiments, IL-5 molecules of the invention are with one or more physical and chemical parameter (P_x) be characterized, including apparent molecular weight (P₁), its value is by 1 to 250, and such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, is in specific embodiments 15 to 25kDa.IL-5 pI (P₂) it is 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 4-9 in specific embodiments, with about 2-50, such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, be in specific embodiments 5-12 isoform (P₃).Contents of saccharide percentage (the P of IL-5 molecules₅) it is 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, it is 10-50% in specific embodiments.After N- connections oligosaccharides is removed, IL-5 actual measurement molecular weight (P of the invention₆) it is 9-30kDa, it is in specific embodiments 11-25kDa.And after O- connections and N- connection oligosaccharides are removed, the IL-5 molecular weight (P detected₇) it is 8-27kDa, it is 10-22kDa in a specific embodiment.When using GalNAc as standard, IL-5 contents of monosaccharides (P₉) it is 1: 0.1-3 trehalose, 1: 0.5-7 GlcNAc, 1: 0.05-3 galactolipin, 1: 0.1-3 mannose and 1: 0-5 NeuNAc；It is 1: 0-0.5 trehalose, 1: 2-4.5 GlcNAc, 1: 1-2 galactolipin, 1: 1-2 mannose and 1: 0.1-1 NeuNAc in a specific embodiment；It is 3: 0.1-3 trehalose, 3: 0.1-4 GalNAc, 3: 1-17 GlcNAc, 3: 1-8 galactolipin, and 3: 0-5 NeuNAc when being standard with 3 times of mannoses；It is 3: 0-1 trehalose, 3: 2-3 GalNAc, 3: 3-12 GlcNAc, 3: 2-5 galactolipin and 3: 0.2-1 NeuNAc in a specific embodiment.The IL-5 molecular salivary acid of the present invention is expressed as contents of monosaccharides percentage (P₁₀), for 0 to 50%, it is 2 to 10% in a specific embodiment such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50%.When using GalNAc as standard, IL-5 sulphates contents (P of the invention₁₁) it is 1: 2-14 sulfate；It is 1: 5-10 sulfate in a specific embodiment；It is 3: 7-36 sulfate when being standard with 3 times of mannoses, is 3: 12-24 sulfate in a specific embodiment.The IL-5 of present invention sulfate accounts for monose percentage (P₅₉) it is 5-35%, it is 10-25% in a specific embodiment.Neutral percentage (the P of the IL-5 of present invention N connections oligosaccharides₁₃) it is 30 to 90%, it is 40 to 80% in a specific embodiment, is in specific embodiments 50 to 75%.Acid percentage (the P of the IL-5 of present invention N connections oligosaccharides₁₄) it is 10 to 70%, it is 20 to 60% in a specific embodiment, is in specific embodiments 25 to 50%.Neutral percentage (the P of the IL-5 of present invention O- connections oligosaccharides₁₅) it is 40 to 100%, it is 50 to 100% in a specific embodiment, is in specific embodiments 60 to 100%.Acid percentage (the P of the IL-5 of present invention O- connections oligosaccharides₁₆) it is 0 to 60%, it is 0 to 50% in a specific embodiment, is in specific embodiments 0 to 40%.

In one embodiment, protein of the invention or chimeric molecule are in N- crosslink parts (P₁₉) in contain at least one following structure.In these charts, " u " or "" different header structure is represented for a or b, and/or crosslinking sites are 2,3,4, and/or 6.

+3×Gal(1-)GlcNAc(1-)

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-3)[Gal

(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-6)][GlcNAc

(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc+″+3×Gal

(1-)GlcNAc(1-)″

+3×Gal(1-)GlcNAc(1-)+Fuc(1-)

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-3)[Gal

(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-6)][Gl cNAc

(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc+″+3×Gal

(1-)GlcNAc(1-)+Fuc(1-)″

+3×Gal(b1-4)GlcNAc(b1-3)

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-)[Gal

(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(a1-)]Man(

b1-4)GlcNAc(b1-4)GlcNAc+″+3×Gal(b1-4)GlcNAc(b1-3)″

+3×Gal(b1-4)GlcNAc(b1-3)

(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(a1-)]Man(

b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc+″+3×Gal(b1-4)GlcNAc

(b1-3)″

+3×Gal(b1-4)GlcNHc(b1-3)+Gal(b1-3)GlcNAc(b1-3)

(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(a1-)]Man(

b1-4)GlcNAc(b1-4)GlcNAc+″+3×Gal(b1-4)GlcNAc(b1-3)+

Gal(b1-3)GlcNAc(b1-3)″

+3×Gal(b1-4)GlcNAc(b1-3)+Gal(b1-3)GlcNAc(b1-3)

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-

)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(

a1-)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc+″+3×

Gal(b1-4)GlcNAc(b1-3)+Gal(b1-3)GlcNAc(b1-3)″

+4×Gal(1-)GlcNAc(1-)

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-

3)[Gal(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(

a1-6)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)

]GlcNAc+″+4×Gal(1-)GlcNAc(1-)″

+4×Gal(1-)GlcNAc(1-)+Fuc(1-)

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a

1-3)[Gal(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]

Man(a1-6)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc

(1-6)]GlcNAc+″+4×Gal(1-)GlcNAc(1-)+

Fuc(1-)″

+5×Gal(1-)GlcNAc(1-)

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a

1-3)[Gal(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]

Man(a1-6)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc

(1-6)]GlcNAc+″+5×Gal(1-)GlcNAc(1-)″

Where j+k=14 ＆ j, k ＞=1

Glycan structures Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } kGl cNAc (b1-2) Man (a1-3) [

Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(b1-2)Man(a1-6)]

Man (b1-4) GlcNAc (b1-4) GlcNAc+ " Where j+k=14 ＆ j, k ＞=1 "

Where j+k=14 ＆ j, k ＞=1

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(b1-2

)Man(a1-)[Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2

)Man(a1-)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ " Where j+k=14＆

J, k ＞=1 "

Wherej+k=14＆k, j ＞=1

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2

)Man(a1-3)[NeuAc(a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}j

GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″Wher

E j+k=14 ＆ k, j ＞=1 "

Where j+k=14 ＆ j, k ＞=1

Glycan structures Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } kGlcNAc (b1-2) Man (a1-3) [

Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(b1-2)Man(a1-6)]

Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ " Where j+k=14 ＆

J, k ＞=1 "

Where j+k=14 ＆ j, k ＞=1

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(b1-2

)Man(a1-)[Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2

)Man(a1-)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc+″Where

J+k=14 ＆ j, k ＞=1 "

Hherej+k=14 ＆ j, k ＞=1

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2

)Man(a1-3)[NeuAc(a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jG

lcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]Glc

NAc+ " Where j+k=14 ＆ j, k ＞=1 "

Wherej+k=14 ＆ j, k ＞=1

Glycan structures Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } kGlcNAc (b1-2) Man (a1

-3)[Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(b1-2)M

an(a1-6)][GlcNAc(b1-4)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+ " Where j+k=14 ＆ j, k ＞=1 "

Where j+k=14 ＆ j, k ＞=1

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(

b1-2)Man(a1-)[Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlc

NAc(b1-2)Man(a1-)][GlcNAc(b1-4)]Man(b1-4)GlcNAc(b1

- 4) GlcNAc+ " Wherej+k=14 ＆ j, k ＞=1 "

Where j+k=14 ＆ j, k ＞=1

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(

b1-2)Man(a1-3)[NeuAc(a2-)Gal(b1-4){GlcNAc(b1-3)Gal

(b1-4)}jGlcNAc(b1-2)Man(a1-6)][GlcNAc(b1-4)]Man(b1-

4) GlcNAc (b1-4) GlcNAc+ " Where j+k=14 ＆ j, k ＞=1 "

Wherej+k=14 ＆ j, k ＞=1

-3)[Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(b1-2)M

an(a1-6)][GlcNAc(b1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(a

1-6)] GlcNAc+ " Wherej+k=14 ＆ j, k ＞=1 "

Where j+k=14 ＆ j, k ＞=1

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNA

c(b1-2)Man(a1-)[Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}

kGlcNAc(b1-2)Man(a1-)][GlcNAc(b1-4)]Man(b1-4)Glc

NAc (b1-4) [Fuc (a1-6)] GlcNAc+ " Where j+k=14 ＆ j, k ＞=1 "

Where j+k=14 ＆ j, k ＞=1

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(

b1-2)Man(a1-3)[NeuAc(a2-)Gal(b1-4){GlcNAc(b1-3)Gal

(b1-4)}jGlcNAc(b1-2)Man(a1-6)][GlcNAc(b1-4)]Man(b1-

4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ " Where j+k=14 ＆ j,

K ＞=1 "

Glycan structures GlcNAc (b1-2) Man (a1-6) [Man (a1-3)] Man (b1-4) GlcNAc (b1-4)

GlcNAc

Glycan structures GlcNAc (b1-4) Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4)

GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4)

[Fuc(a1-6)]GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] Man (b1-4)

GlcNAc(b1-4)GlcNAc

Glycan structures Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] G

lcNAc

Glycan structures GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-4)] [Man (a1-6)] Man (b1-

4)GlcNAc(b1-4)GlcNAc

Glycan structures Fuc (a1-6) [GlcNAc (b1-4)] GlcNAc

Glycan structures Man (a1-6) Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-6) Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] Gl

cNAc

Man a1-3 Man a1-6 Man b1-4 GlcNAcb1-4 GlcNAc

Glycan structures Man (a1-3) Man (a1-6) Man (b1-4) GlcNAc (b1-4) GlcNAc

NeuAca2 -u Gal b1-4 GlcNAcb1-2 Man a1-3 Man b1-4 GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-3)Man(b1-4)GlcNA

c

Glycan structures HSO3 (- 4) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3) [Man (a1-6)] Man

(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] Man (b1-4) Gl

cNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4

)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures GlcNAc (b1-2) [GlcNAc (b1-4)] Man (a1-3) [GlcNAc (b1-4)] [Man (a1

-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures GlcNAc (b1-2) [GlcNAc (b1-4)] Man (a1-3) [GlcNAc (b1-2) Man (a1-

6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

6)][GlcNAc(b1-4)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures GlcNAc (b1-2) [GlcNAc (b1-4)] Man (a1-3) [GlcNAc (b1-2) [GlcNAc

(b1-6)]Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures HSO3 (- 4) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3) [HSO3 (- 4) GalNAc

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-3)[Man(a1-6)]Man

(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc

(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Man (a1-3)] Man (b1-4) GlcNAc

(b1-4)GlcNAc

(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Fuc (1-)[Gal(1-)]GlcNAc(1-)Man(a1-)[Man(a1-)]Man

(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] Man

(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Man (a1-3) Man (a1-6) [Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures NeuAc (a 2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Ma

n(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [GlcNAc (b1-2) Man (a1-3)] Man

(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Gal (1-)GlcNAc(1-)Man(a1-)[GlcNAc(1-)Man(a1-)]Man

(b1-4)GlcNAc(b1-4)[Fuc(1-)]GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] [Gl

cNAc(b1-4)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [GlcNAc (b1-2) Man (a1-3)] [Gl

cNAc(b1-4)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [GlcNAc (b1-2) [GlcNAc (b1-4

)]Man(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures NeuAc (a2-6) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) Gl

cNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures HSO3 (- 4) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3) [NeuAc (a2-3) Gal

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

cNAc(b1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

cNAc(b1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Gal (1-)GlcNAc(1-)Man(a1-)[GlcNAc(1-)Man(a1-)][Gl

cNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [NeuAc (a2-6) Gal

NAc(b1-4)GlcNAc(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)[Fuc

(a1-6)]GlcNAc

NAc(b1-4)GlcNAc(b1-2)Man(a1-3)][GlcNAc(b1-4)]Man(b1-4)Gl

cNAc(b1-4)GlcNAc

+2×Man

Glycan structures Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+2 ×

Man″

Glycan structures NeuAc (a2-)Ga l(b1-4)GlcNAc(b1-2)Man(a1-3)[Man(a1-3)Man(

a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [NeuAc (a2-3) Gal

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man

(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Fuc (a1-2) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc

(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Fuc (1-)[Gal(1-)]GlcNAc(1-)Man(a1-)[Gal(1-)GlcN

Ac(1-)Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Fuc (a1-2) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc

(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Fuc (a1-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [NeuAc (a2-6) Gal (

b1-4)GlcNAc(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures HSO3 (- 6) [NeuAc (a2-3)] Gal (b1-4) GlcNAc (b1-2) Man (a1-)[Neu

Ac(a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-)]Man(b1-4)GlcNAc(

b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc

(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc

(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Fuc (a1-3) [Gal

(b1-4)]GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(

a1-6)]GlcNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Fuc (a1-3) [Gal

(b1-4)]GlcNAc(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)[Fuc(

a1-6)]GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) Man (a1-3) [Fuc (a1-3) [Gal

(b1-4)]GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(

a1-6)]GlcNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc

(b1-2)Man(a1-6)][GlcNAc(b1-4)]Man(b1-4)GlcNAc(b1-4)GlcNAc

(b1-4)GlcNAc(b1-2)Man(a1-3)][GlcNAc(b1-4)]Man(b1-4)GlcNAc

(b1-4)GlcNAc

Glycan structures Fuc (a1-2) [GalNAc (a1-3)] Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Fuc (a1-2) [GalNAc (a1-3)] Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal

(b1-4)GlcNAc(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc

(b1-2)Man(a1-6)][GlcNAc(b1-4)]Man(b1-4)GlcNAc(b1-4)GlcNAc

(b1-2)Man(a1-3)][GlcNAc(b1-4)]Man(b1-4)GlcNAc(b1-4)GlcNAc

(b1-2)Man(a1-6)][GlcNAc(b1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc

(a1-6)]GlcNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [NeuAc (a2-6) Gal

(b1-4)GlcNAc(b1-2)Man(a1-6)][GlcNAc(b1-4)]Man(b1-4)GlcNAc

(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Fuc (a1-2) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Fuc (a1-2) Gal (b1

-4)GlcNAc(b1-2)Man(a1-6)][GlcNAc(b1-4)]Man(b1-4)GlcNAc(

b1-4)GlcNAc

Glycan structures Gal (1-)GlcNAc(1-)[GlcNAc(1-)]Man(a1-)[Gal(1-)GlcNA

c(1-)Man(a1-)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc

(1-6)]GlcNAc

+NeuAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc+″+NeuAc″

Glycan structures Gal (b1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [NeuAc (a2-6) Gal (

b1-4)GlcNAc(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+2×NeuAc

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc+″+2×NeuAc″

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)[NeuAc(a2-)Gal(b1-4)GlcNAc

(b1-4)]Man(a1-3)[NeuAc(a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]

Man(b1-4)GlcNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (

b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1

-6)]GlcNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (

b1-4)GlcNAc(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1

-6)]GlcNAc

+NeuAc(2-6)

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Fuc

(a1-6)[Gal(b1-4)]Gl cNAc(1-2)Man(1-6)]Man(1-4)[Fuc(a1

-3)Fuc(a1-3)]GlcNAc+″+NeuAc(2-6)″

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6) [Gal

(b1-4)GlcNAc(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal

(b1-4)GlcNAc(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Man (a1-3) [Man (a1-6)] Man (a1-6) [Man (a1-2) Man (a1-3)] Man (b1

-4)GlcNAc(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Man (a1-3) [Man (a1-6)] Man (

a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-3)[Man(a1-3)[Man

(a1-6)]Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+Fuc(a1-3)

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+″+Fuc(a1-3)″

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-6)[Gal(b1-4)GlcNAc

(b1-2)[Gal(b1-4)GlcNAc(b1-4)]Man(a1-3)]Man(b1-4)GlcNAc(

b1-4)GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-4)[Gal(b1-4)GlcNAc(b1-2)]

Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc

(b1-4)GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-4)]

Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc

(b1-4)GlcNAc

+NeuAc(a2-6)

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-)[Gal

(1-)GlcNAc(1-)Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+″+NeuAc(a2-6)″

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)]

Man(a1-3)[NeuAc(a2-6)Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man

(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) [NeuAc (a2-3) Gal (b1-4) GlcNAc

(b1-4)]Man(a1-3)[NeuAc(a2-6)Gal(b1-4)GlcNAc(b1-2)Man(a1

-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+3×NeuAc(a2-)

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+″+3×NeuAc(a2-)″

(b1-4)GlcNAc(b1-2)Man(a1-3)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1

-6)]GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-2)]

Man(a1-)[Gal(b1-4)GlcNAc(b1-2)Man(a1-)]Man(b1-4)GlcNAc

(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1

-6)]GlcNAc

Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc

(b1-4)GlcNAc

+Fuc(a1-2)

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+″+Fuc(a1-2)″

+Fuc(a1-3)

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1

-6)]GlcNAc+″+Fuc(a1-3)″

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-4) [NeuAc (a2-6) Gal (b1-4) GlcNAc

(b1-2)]Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-

4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Man(a1-3)[NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man

(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-2)]

Man(a1-3)[NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man

(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

+NeuAc(a2-3)+NeuAc(a2-6)

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1

-6)]GlcNAc+″+NeuAc(a2-3)+NeuAc(a2-6)″

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) [Fuc (a1-3) [Gal (b1-4)] GlcNA

c(b1-4)]Man(a1-3)[NeuAc(a2-6)Gal(b1-4)GlcNAc(b1-2)Man(a1

-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+Fuc+2×NeuAc(a2-)

Glycan structures Gal (b1-4) GlcNAc (1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-)[Gal

(b1-4)GlcNAc(1-)Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+″+Fuc+2×NeuAc(a2-)″

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-4) [NeuAc (a2-6) Gal (b1-4) GlcN

Ac(b1-2)]Man(a1-3)[NeuAc(a2-6)Gal(b1-4)GlcNAc(b1-2)Man(a1

-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-4) [NeuAc (a2-6)

Gal(b1-4)GlcNAc(b1-2)]Man(a1-3)[NeuAc(a2-6)Gal(b1-4)Glc

NAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc

(b1-4)GlcNAc+″+3×NeuAc(a2-)″

+HSO3(-6)+2×NeuAc(a2-3)+NeuAc(a2-6)

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1

-6)]GlcNAc+″+HSO3(-6)+2×NeuAc(a2-3)+NeuAc(a2-6)″

+2×HSO3(-6)+2×NeuAc(a2-3)+NeuAc(a2-6)

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1

-6)]GlcNAc+″+2×HSO3(-6)+2×NeuAc(a2-3)+NeuAc(a2

-6)″

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-2) Man

(a1-3)[Fuc(a1-3)[Gal(b1-4)]GlcNAc(b1-2)Man(a1-6)]Man(b1

-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

+Gal(b1-2)GlcNAc(b1-3)+3×NeuAc

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc+″+Gal(b1-2

)GlcNAc(b1-3)+3×NeuAc″

+NeuAc(a2-)

Glycan structures Gal (a1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc

(b1-2)[Gal(b1-4)GlcNAc(b1-4)]Man(a1-3)]Man(b1-4)GlcNAc(

b1-4)[Fuc(a1-6)]GlcNAc+″+NeuAc(a2-)″

+NeuAc(a2-3)+NeuAc(a2-6)

(b1-2)[Gal(b1-4)GlcNAc(b1-4)]Man(a1-3)]Man(b1-4)GlcNAc(

b1-4)[Fuc(a1-6)]GlcNAc+″+NeuAc(a2-3)+NeuAc(a2-6)″

+HSO3(-6)+2×NeuAc(a2-)

(b1-2)[Gal(b1-4)GlcNAc(b1-4)]Man(a1-3)]Man(b1-4)GlcNAc(

b1-4)[Fuc(a1-6)]GlcNAc+″+HSO3(-6)+2×NeuAc(a2-)″

(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(a1-6)]Man(

b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-6)]

Man(a1-3)[Fuc(a1-2)[Gal(b1-4)]GlcNAc(b1-2)[Gal(b1-4)GlcNA

c(b1-6)]Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+3×NeuAc(a2-)

(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(a1-6)]Man(

b1-4)GlcNAc(b1-4)GlcNAc+″+3×NeuAc(a2-)″

Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man

(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [Gal (b1-4) GlcNAc (b1-2)]

Man(a1-6)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-4)]Man

(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)]

Man(a1-6)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-4)]Man

(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+3×NeuAc(a2-)

Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man

(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+3×NeuAc(a2-)″

(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(a1-6)]Man(

b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc+″+3×NeuAc(a2-)″

(b1-6)]Man(a1-6)[NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-4)[NeuAc

(a2-6)Gal(b1-4)GlcNAc(b1-2)]Man(a1-3)]Man(b1-4)GlcNAc(b1

-4)[Fuc(a1-6)]GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)[NeuAc(a2-)Gal(b1-4)GlcNAc

(b1-4)]Man(a1-3)[NeuAc(a2-)Gal(b1-4)GlcNAc(b1-2)[NeuAc

(a2-)Gal(b1-4)GlcNAc(b1-6)]Man(a1-6)]Man(b1-4)GlcNAc(b1

-4)[Fuc(a1-6)]GlcNAc

+4× NeuAc(a2-)

Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man

(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+4×NeuAc(a2-)″

+2×Fuc

Glycan structures Gal (b1-4) GlcNAc (1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-3)[Gal

(b1-4)GlcNAc(1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-6)]Man(

b1-4)GlcNAc(b1-4)GlcNAc+″+2×Fuc″

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-3)[Gal

(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-6)][GlcNAc

(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc

+Fuc

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-3)[Gal

(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-6)][GlcNA

c(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc+″+Fuc″

Glycan structures Gal (b1-4) GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-3) [Gal

(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc

(b1-6)]Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc

(b1-6)]Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Gal (a1-3) Gal (b1-4) GlcNAc (b1-2) [NeuAc (a2-)Gal(b1-4)GlcNAc

(b1-4)]Man(a1-3)[Gal(a1-3)Gal(b1-4)GlcNAc(b1-2)Man(a1-6

)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Gal (a1-3) Gal (b1-4) GlcNAc (b1-4) [NeuAc (a2-)Gal(b1-4)GlcNAc

(b1-2)]Man(a1-3)[Gal(a1-3)Gal(b1-4)GlcNAc(b1-2)Man(a1-6

)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

+2×Gal(h1-4)GlcNAc(b1-3)+2×NeuAc

(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc+″+2×Gal(

b1-4)GlcNAc(b1-3)+2×NeuAc″

+Gal(b1-4)GlcNAc(1-)+4×NeuAc(a2-)

Glycan structures Gal (b1-4) GlcNAc (1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-3)[Ga

l(b1-4)GlcNAc(1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-6)]Man(

b1-4)GlcNAc(b1-4)GlcNAc+″+Gal(b1-4)GlcNAc(1-)+4×

NeuAc(a2-)″

+5×NeuAc(a2-)

Glycan structures Gal (b1-4) GlcNAc (b1-)Gal(b1-4)GlcNAc(b1-6)[Gal(b1-4)GlcN

Ac(b1-2)]Man(a1-6)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc

(b1-4)]Man(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+5×NeuAc

(a2-)″

+Gal(b1-4)GlcNAc(b1-3)

(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(a1-6)]Man(

b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc+″+Gal(b1-4)GlcNAc(b1

-3)″

+2×Fuc+Gal(b1-4)GlcNAc(1-)

Glycan structures Gal (b1-4) GlcNAc (1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-3)[Gal

(b1-4)GlcNAc(1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-6)]Man(

b1-4)GlcNAc(b1-4)GlcNAc+″+2×Fuc+Gal(b1-4)GlcNAc(1

-)″

+2×Gal(b1-4)GlcNAc(b1-3)+Gal(b1-3)GlcNAc(b1-3)

(b1-4)GlcNAc(b1-2)Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc

+″+2×Gal(b1-4)GlcNAc(b1-3)+Gal(b1-3)GlcNAc(b1-3)″

In one embodiment, protein of the invention or chimeric molecule comprise at least a following structure (P in O- crosslink parts₂₀).In these charts, " u " or "" different header structure is represented for a or b, and/or crosslinking sites are 2,3,4, and/or 6.

Fuc

Glycan structures Fuc

Glc u1-u Fuc

Glycan structures Glc (1-)Fuc

GlcNAc

Glycan structures GlcNAc

GalNAc

Glycan structures GalNAc

NeuAca2- 6GalNAc

Glycan structures NeuAc (a2-6) Ga lNAc

GlcNAcb1-3 GalNAc

Glycan structures GlcNAc (b1-3) GalNAc

Glycan structures GlcNAc (b1-3) [NeuAc (a2-6)] GalNAc

Gal b1-3 GalNAc

Glycan structures Gal (b1-3) GalNAc

Gal

Glycan structures Gal

NeuAc a2-3 Gal

Glycan structures NeuAc (a2-3) Gal

Xylu1-u Glc

Glycan structures Xyl (1-)Glc

NeuAca2-3 Gal b1-4xyl

Glycan structures NeuAc (a2-3) Ga l (b1-4) Xyl

Xyl u1-u Glc

Glycan structures Xyl (1-)Glc

Xyl u1-u Glc

+Xyl

Glycan structures Xyl (1-)Glc+″+Xyl″

NeuAca2-3Galb1-3GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-3) GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-3) [NeuAc (a2-6)] GalNAc

Glycan structures Gal (b1-3) [NeuAc (a2-6)] GalNAc

Fuc a1-2 Galb1-3GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) [NeuAc (a2-6)] GalNAc

Glycan structures NeuAc (2-)Gal(1-)[Fuc(a1-)]GalNAc

Delta4,5GlcA b1-3 GalNAc b1-4GlcA b1-3 Gal b1-3 Gal b1-4 Xyl

Glycan structures delta4,5GlcA (b1-3) GalNAc (b1-4) GlcA (b1-3) Gal (b1-3) G

al(b1-4)Xyl

Glycan structures delta4,5GlcA (b1-3) [HSO 3 (- 4)] GalNAc (b1-4) GlcA (b1-3)

Gal(b1-3)Gal(b1-4)Xyl

Glycan structures HSO3 (-)[NeuAc(a2-)]GlcNAc(b1-6)[NeuAc(a2-3)Gal(b

1-3)]GalNAc

Glycan structures Gal (b1-3) [GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-4) GlcNAc (b1-6) [GlcNAc (b1-6) Gal (b3)] GalNAc

Glycan structures Fuc (a1-4) GlcNAc (b1-6) Gal (b1-3) [Fuc (a1-4) GlcNAc (b1-

6)]Gal NAc

Glycan structures Gal (b1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Fuc a1-2Galb1-3GlcNAcb1-3 GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) GlcNAc (b1-3) GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-3) GalNAc

Glycan structures Fuc (a1-2) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-6) [GlcNAc (b1-3)] GalNAc

Glycan structures Gal (b1-3) GlcNAc (b1-3) [GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [GlcNAc (b1-3)] GalNAc

Gal b1-4 GlcNAcb1-3 Gal b1-3 GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-3) GalNAc

Glycan structures GalNAc (b1-4) [NeuAc (a2-3)] Gal (b1-3) GalNAc

Glycan structures GalNAc (b1-4) [NeuAc (a2-3)] Gal (b1-3) [NeuAc (a2-6)] GalNAc

NeuAc u2-uGal u1-u GalNAcu1-u GalNAc

Glycan structures NeuAc (2-)Gal(1-)GalNAc(1-)GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] Ga

lNAc

Glycan structures Gal (b1-)GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-)GlcNAc(b1-6)[Gal(b1-3)]GalNAc

NeuAc a2-u Gal b1-u GlcNAcb1-u Gal u1-u GalNAc

Glycan structures NeuAc (a2-)Gal(b1-)GlcNAc(b1-)Gal(1-)GalNAc

lNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) [Gal (b1-4) GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] Ga

lNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-3) [HSO3 (- 6) GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] Gal

NAc

Glycan structures NeuAc (a2-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [Gal (b1-3)] Gal

NAc

Glycan structures Fuc (a1-2) Gal (b1-3) [Fuc (a1-4)] GlcNAc (b1-6) [Gal (b1-3)]

GalNAc

Glycan structures Fuc (a1-2) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [Gal (b1-3)]

GalNAc

+Fuc(a1-2)

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [Gal (b1-3)] GalNAc+ "+Fuc

(a1-2)″

Glycan structures Fuc (a1-2) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [NeuAc (a2-3) Gal

(b1-3)]GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) [Gal (b1-4) GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)]

GalNAc

Glycan structures NeuAc (2-3)Gal(1-3)[Fuc(1-4)]GlcNAc(1-3)Gal(1-3)

GalNAc

Glycan structures Fuc (a1-2) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) Gal (b1-3) GalNAc

(b1-3)]GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [NeuAc (a2-3)

Gal(b1-3)]GalNAc

Glycan structures Gal (b1-3) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [Gal (b1-3)]

GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal

(b1-3)]GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) GlcNAc (b1-3) Gal (b1-3) [Gal (b1-4) GlcNAc

(b1-6)]GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [Gal

(b1-3)]GalNAc

Glycan structures Gal (b1-3) GlcNAc (b1-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [Gal

(b1-3)]GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc

(a2-3)Gal(b1-3)]GalNAc

Glycan structures Gal (b1-3) GlcNAc (b1-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [Neu

Ac(a2-3)Gal(b1-3)]GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) [Gal (b1-4) GlcNAc (b1-6)] Gal (b1-3) [Gal

(b1-4)GlcNAc(b1-6)]GalNAc

Glycan structures Gal (b1-3) GlcNAc (b1-3) [Gal (b1-4) GlcNAc (b1-6)] Gal (b1-4) GlcN

Ac(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [Neu

Ac(a2-3)Gal(b1-3)]GalNAc

+Fuc

Glycan structures NeuAc (2-3)Gal(1-)GlcNAc(1-3)Gal(1-3)[Gal(1-4)GlcNAc

(1-6)]GalNAc+″+Fuc″

Glycan structures Gal (b1-)GlcNAc(b1-)Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1-6)[Neu

Ac(a2-3)Gal(b1-3)]GalNAc

Glycan structures Fuc (a1-)[Gal(b1-)]GlcNAc(b1-)Gal(b1-4)GlcNAc(b1-6)[Neu

Ac(a2-3)Gal(b1-3)]GalNAc

Glycan structures Fuc (1-)Gal(1-)[Fuc(1-)]GlcNAc(1-)Gal(1-)GlcNAc

(1-)[NeuAc(2-)Gal(1-)]GalNAc

+Fuc

Glycan structures Gal (1-)GlcNAc(1-)Gal(1-)[Fuc(1-)]GlcNAc(1-)[Neu

Ac(2-)Gal(1-)]GalNAc+″+Fuc″

Glycan structures Fuc (1-)Gal(1-)[Fuc(1-)]GlcNAc(1-)Gal(1-)[Fuc(

1-)]GlcNAc(1-)[NeuAc(2-)Ga l(1-)]GalNAc

Glycan structures Gal (1-)GlcNAc(1-)Gal(1-)GlcNAc(1-)Gal(1-)GlcN

Ac(1-)[NeuAc(2-)Gal(1-)]GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [Gal (b1-3) GlcNAc (b1-3)]

GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-3) [Gal (b1-4) GlcNAc (b1-6)]

GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-)GlcNAc(b1-3)Gal(b1-3)[NeuAc

(a2-6)]GalNAc

Glycan structures Gal (b1-)GlcNAc(1-)[Gal(b1-)GlcNAc(1-)]Gal(b1-)GlcNA

c(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc

(b1-6)[Gal(b1-3)]GalNAc

Ac(a2-3)Gal(b1-3)]GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) Gal (b1-4) GlcNA

c(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan knot Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-)Gal(b1-4)GlcNAc(b1-6)[NeuAc

Structure (a2-3) Gal (b1-3)] GalNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-)Gal(b1-4)GlcNA

c(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-)Gal(b1-4)GlcNAc(b1-)Gal

(b1-4)GlcNAc(b1-6)[Gal(b1-3)]GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) GlcNAc (b1-3) [Gal (b1-4) GlcNAc (b1-6)] Gal

(b1-3)[Gal(b1-4)GlcNAc(b1-6)]GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) [Fuc (a1-4)] GlcNAc (b1-3) Gal (b1-4) GlcNAc

(b1-3)Gal(b1-4)GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures Fuc (a1-4) [Gal (b1-3)] GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Ga

l(b1-4)[Fuc(a1-3)]GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]Gal

NAc

Glycan structures Fuc (a1-2) Gal (b1-3) [Fuc (a1-4)] GlcNAc (b1-3) [Fuc (a1-3) [Gal

(b1-4)]GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-6)[NeuAc(a2-3)Ga

l(b1-3)]GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) [Fuc (a1-4)] GlcNAc (b1-3) [Gal (b1-4) GlcN

Ac(b1-6)]Ga l(b1-4)[Fuc(a1-3)]GlcNAc(b1-6)[NeuAc(a2-3)Ga

l(b1-3)]GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Ga

l(b1-4)GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-)GlcNAc(b1-3)[Fuc(a1-2)[Gal

(a1-3)]Gal(b1-)GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-3)Gal(b

1-3)Gal(b1-3)[Gal(b1-4)GlcNAc(b1-6)]GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-)GlcNAc(b1-3)[Fuc(a1-2)[Gal

(a1-3)]Gal(b1-)GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-3)Gal(b1

-3)Gal(b1-3)[Gal(b1-4)GlcNAc(b1-6)]GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-)GlcNAc(b1-3)[Fuc(a1-2)[Gal

(a1-3)]Gal(b1-)GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-3)Gal(b1

-3)Gal(b1-3)[NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-6)]GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-)GlcNAc(b1-3)[Fuc(a1-2)[Gal

(a1-3)]Gal(b1-)GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-3)Gal(b1

-3)[Fuc(a1-2)[Gal(a1-3)]Gal(b1-4)GlcNAc(b1-6)]GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) Gal (b1-4) [Fuc

(a1-3)]GlcNAc(b1-3)Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1-6)[Gal

(b1-3)]GalNAc

(b1-3)Gal(b1-4)GlcNAc(b1-6)[Gal(b1-3)]GalNAc

(b1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc(b1-6)[Gal(b1

-3)]GalNAc

+Fuc(a1-3)

(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc+″+Fuc(a1-3)″

+2×Fuc(a1-3)

(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc+″+2×Fuc(a1-3)″

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc

(b1-3)Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1

-3)]Gal NAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-)Gal(b1-4)GlcNAc(b1-)Ga

l(b1-4)GlcNAc(b1-6)[Gal(b1-3)]GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-)Gal(b1-4)GlcNAc(b1-)Gal

(b1-4)GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Host cell can be modified by a variety of methods known in the art to obtain the protein of the present invention or the physics and chemistry form of chimeric molecule, a kind of one or more transgenosis for encoding enzyme or a variety of enzymes, producing required physics and chemistry form are including but not limited to introduced into host cell.This transgenosis includes polytype sialyltransferase, such as ST3Gal1, ST3Gal2, ST3Gal3, ST3Gal4, ST3Gal5, ST3Gal6, ST6Gal1, ST6Gal2, ST6GalNAc1, ST6GalNAc2, ST6GalNAc3, ST6GalNAc4, ST6GalNAc5, ST8Sia1, ST8Sia2, ST8Sia3, ST8Sia4, ST8Sia5, ST8Sia6；Galactosyl transferase, such as GalT1, GalT2；Fucosyltransferase such as FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11；Sulfurtransferase；GlcNAc transferases such as GNT1, GNT2, GNT3, GNT4, GNT5；Antenna nickase (antenna-cleaving enzymes) and endoglycosidase.

For example, the invalid terminal sialic acidization effect of N- glycan structures causes the protein sera half-life period of expression to reduce, the protein such as recombined human AchE, can improve this situation (JBiochem 336 by adding rat beta galactose glycosides α -2,6- sialyltransferase transgenosis into the cells of HEK 293：647-658,1998；J Biochem 363：619-631,2002).

Similarly, the sialyl Lewis x structures of the invalid structure such as N- glycan structures of specific Lewis x groups cause the reduction of expressing protein ligand binding, the expressing protein such as recombined human PSGL-1, can improve this situation (Fritz et al. PNAS 95 by adding fucosyltransferase transgenosis into the cells of HEK 293：12283-12288,1998)

In one embodiment, α -2,3 or the human cell line of the transfer of α -2,6 sialic acids or α -2,3 and α -2,6 sialyltransferases (" sialylated albumen ") produce protein or its chimeric molecule using conversion.The example of sialylated albumen includes sialylated-GM-CSF, sialylated-GM-CSF-Fc, sialylated-IL-3, sialylated-IL-3-Fc, sialylated-IL-4, sialylated-IL-4-Fc, sialylated-IL-5 and sialylated-IL-5-Fc.

Especially, with physical and chemical parameter (P_x) feature characterize the sialylated albumen of the present invention, including monose (P₉) and sialic acid content (P₁₀), it is 1: 0.1-100NeuNAc when using Ga l NAc as standard；It is 3: 0.1-100 NeuNAc when using 3 times of mannoses as standard.Neutral percentage (the P of the N- connections oligosaccharides of the sialylated albumen of the present invention₁₃) it is 0 to 9 9%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%.Acid percentage (the P of the N- connections oligosaccharides of the sialylated albumen of the present invention₁₄) it is 1 to 100%, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.Neutral percentage (the P of the O- connections oligosaccharides of the sialylated albumen of the present invention₁₅) it is 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%.Acid percentage (the P of the O- connections oligosaccharides of the sialylated albumen of the present invention₁₆) it is 1 to 100%, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

Compared with the protein of the invention of no transgenosis or the half-life period of chimeric molecule, the Half-life in vivo (T of sialylated albumen₁₁) increase.

In one embodiment, sialylated albumen contains at least one structural formula described herein, or at least one structural formula described herein, and wherein one or more NeuNAc crosslinkings are that the α 2,6 in N- crosslink parts is crosslinked.

In one embodiment, sialylated albumen contains at least one structural formula described herein, or at least one structural formula described herein, and wherein one or more NeuNAc crosslinkings are that the α 2,6 in O- crosslink parts is crosslinked.

In one embodiment, the human cell line for having FUT3 (" marine alga glycated protein ", " fucosylated-protein ") using conversion produces its chimeric molecule in protein or invention.The example of marine alga glycated protein includes marine alga saccharification-GM-CSF, marine alga saccharification-GM-CSF-Fc, marine alga saccharification-IL-3, marine alga saccharification-IL-3-Fc, marine alga saccharification-IL-4, marine alga saccharification-IL-4-Fc, marine alga saccharification-IL-5 and marine alga saccharification-IL-5-Fc.

Especially, with physical and chemical parameter (P_x) feature characterize the sialylated albumen of the present invention, including monose (P₉) and sialic acid content (P₁₀), arrive 0.1-100 NeuNAc when being normalized with GalNAc for 1；When with 3 times of mannose normalization 0.1-100 NeuNAc are arrived for 3.

In one embodiment, marine alga glycated protein possesses more structure divisions containing Lewis structures (such as Lewis a, Lewis b, Lewis x or Lewis y) or sialyl Lewis structures (such as sialyl Lewis a or sialyl Lewisx).

In one embodiment, with the expression without transgenosis protein of the invention or the binding affinity of chimeric molecule is compared, and marine alga glycated protein possesses the ligand binding affinity of change.

Use the respective forward primer and reverse primer of the protein molecular selected from GM-CSF, IL-3, IL-4 and IL-5, by DNA of the polymerase chain reaction (PCR) using methods known in the art amplification coding GAP-associated protein GAP from EST, such as according to the PCRSuper Mix High Fidelity (Cat.No. of Invitrogen companies：10790-020).Digest amplification is simultaneously connected in the corresponding restriction enzyme sites of suitable carrier, such as pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA 3.1/GS, pCEP4, pIRESpuro3, pIRESpuro4, pcDNA3.1/Hygro (+), pcDNA3.1/Hygro (-), pEF6/V5-His.Connection carrier is transformed into suitable e. coli host cell, such as XLGold, supercompetent cells (Strategene), XL-Blue, DH5 α, DH10B or other.

For the generation of chimeric molecule, the DNA sequence dna of the Fc domains of immunoglobulin, such as IgG1, IgG2, IgG3, IgG4, IgGA1, IgGA2, IgGM, IgGE, IgGD are expanded by PCR using suitable forward and reverse primer from EST.Extension is cloned into the corresponding restriction enzyme sites of suitable carrier, for example, pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA3.1/GS, pCEP4, pIRESpuro3, pIRESpuro4, pcDNA3.1/Hygro (+), pcDNA3.1/Hygro (-), pEF6/V5-His.The DNA sequence dna of corresponding albumen is amplified and is cloned into the corresponding restriction enzyme sites of the respective Fc- carriers using Fc as framework.

In a specific embodiment, the complement activation area in Fc receptor binding domains or Fc regions can be by the insertion of one or more amino acid, deletion or the replacement of recombinant modified, including Fc region amino acid sequences.In addition, the complement activation area in Fc receptor binding domains or Fc regions can be modified by sulphation, its glycoforms are changed into, physically adds or removes carbohydrate moiety on amino acid backbone or after the common translation entity of any association or translation, add polyunsaturated fatty acid part or other parts based on fat.Fc regions can also be shortening form, be realized by a kind of enzyme digestion, and the enzyme includes the protease of papain, pepsin or other any locus specificities.Fc regions can promote spontaneous conformation to be formed by the stronger chimeric protein of the combination respective ligand or acceptor ability of the polymer of dimer, tripolymer or greater degree.

Carry out differentiating digestion using suitable Restriction Enzyme identifying/separating containing the bacterial clone with corresponding gene.Separate positive colony simultaneously -70 DEG C of glycerin storages.Clonal expansion to 750ml is then contained to 37 DEG C of shakes in ampicillin (100 μ g/ml) sterile LB fluid nutrient mediums to cultivate 16 hours.Plasmid is extracted according to methods known in the art, it is preferred that according to QiagenEndofree Plasmid Mega kits (Qiagen Mega Prep Kit #12381).

The human host cell for being adapted for introduction into the cloned dna sequence of the protein containing the present invention or chimeric molecule includes but is not limited to HEK 293 and its any redundant organism, the c18 of HEK 293, HEK293-T, the CEN4 of HEK 293, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene companies), 293A (Invitrogen companies), Hela cells and its any redundant organism, HepG2, PA-1 Jurkat, THP-1, HL-60, H9, HuT 78, Hep-2, Hep G2, MRC-5, PER.C6, SKO-007, U266, Y2 (Apollo companies), WI-38, WI-L2.

Host cell can be modified by a variety of methods known in the art to obtain the protein of the present invention or the physics and chemistry form of chimeric molecule, a kind of one or more transgenosis for encoding enzyme or a variety of enzymes, producing required physics and chemistry form are including but not limited to introduced into host cell.It can be incorporated into by being introduced into special DNA sequence dna to optimize cloned dna sequence in host cell gene group, different types of integrate includes but is not limited to locus specificity, targeting, the mediation of direct or enzyme integration.

The DNA of protein or its chimeric molecule can be incorporated into suitable host cell by a variety of transfection methods known in the art, for example, using chemical reagent for example diethyllaminoethyl glucan (DEAE-dextran), calcium phosphate, artificial liposome or by direct microinjection, electroporation, biologic grain transport infection or transfected virus structure, as described below.

DEAE-dextran is a kind of cationic polymer, and negatively charged nucleic acid is combined.Positive charge excessive on condensate can allow closer associate of compound to carry the cell membrane of negative electrical charge in DNA/ polymer composites.The absorption of compound relies on endocytosis by inference.The cationic polymer of the other synthesis used in transfection includes the poly- Methobromide of methylene (polybrene) of 1,5- dimethyl -1,5- phenodiazine 11, polyethyleneimine and dendrimers.

The instantaneous and stable transfection of various kinds of cell type can use coprecipitation of calcium phosphate.DNA and calcium chloride are mixed by certain controlled mode, be then added to the alkali metal salt of buffering/phosphate solution in, mixed liquor is incubated at room temperature.Produce precipitation and absorbed by cell by endocytosis or phagocytosis.

The most frequently used fat composition of liposome is the fat composition under physiology pH with net positive charge in liposome-mediated gene delivery.Generally cationic lipid and neutral fats are mixed, such as L-dioleoyl phosphatidyl-ethanolamines (DOPE).The negative electrical charge association of the cationic moiety and nucleic acid of fat molecule, causes to tighten in liposome/nucleic acid complexes amplifying nucleic acid.Compound is absorbed by endocytosis.

It will be a kind of effective but laborious technology in the cell or nucleus of DNA direct microinjections to culture, be not suitable for needing the situation of a large amount of transfectional cells.

Electroporation produces hole using a kind of electric pulse, allows nucleic acid to enter cell, is required for carrying out fine tuning and optimization to the duration of pulse and intensity for each type of cell this technology used.Commercially available electroporation device includes the Nucleofector kits (Amaxa Biosystems, Germany) of Amaxa Biosystems companies.The method is dependent on nucleic acid microparticles rapid transit into recipient cell.

With virus or retroviral structural infection or transfection including the use of retrovirus, such as lentivirus, or DNA virus, such as adenovirus.Its process is entered in host cell including the use of a kind of virus or retroviral vector to transmit alien gene.

In some embodiments, protein or its chimeric molecule are produced by transient approach or from stably transfected cell line.Transiently transfected using adhesion or suspension cell line.For adherent cell system, cell grows in the culture medium (2-10% serum) containing serum, the culture medium such as DMEM, DMEM/F12 (JRH).The serum used can be hyclone (FCS), donor calf serum (DCS), newborn calf serum (NBCS) etc..Plasmid vector is incorporated into by cell by standard method known in the art.In a specific embodiment, the DEAE dextran or DNA of calcium phosphate precipitation transfected proteins matter or its chimeric molecule are used.After transfection, cell is transformed into the suitable albumen or its chimeric molecule collected and expression is collected in culture medium (such as serum-free DMEM/F12).

Plasmid vector can be introduced into by using the method for being briefly described above and carries out protein or its chimeric molecule from the transient expression in suspension cell.Suspension cell can grow in serum-containing media or serum free medium (such as Freestyle culture mediums (Invitrogen), CD293 culture mediums (Invitrogen), Excell culture mediums (JRH) etc.).It can be transfected under serum-free condition, by being transfected in a kind of suitable culture medium using suitable transfection method, for example, lipofectamine is in OptiMEM culture mediums.

Transient expression typically results in 2-3 days peak expressions after transfection.Episomal vector is replicated and continuous expression in cell.Therefore, in order to obtain substantial amounts of product, free expression vector is transfected into cell and amplifying cells.Protein or its chimeric molecule are expressed into culture medium, are collected after cell amplification several weeks.Expression culture medium can be that, containing serum or serum-free, cell can be adhesion or suspension.

Cell is entered by transfected expression vector and obtains stable clone, is then selected with suitable reagent, such as phleomycin, homomycin, puromycin, neomycin G418, methopterin.Stable clone can survive in selecting, because also containing resistant gene in addition to the gene of encoding proteins matter or chimeric molecule in plasmid.Introduce after gene 1 to 2 days, start to whole cells (stabilization pond) or the cell according to Clonal density bed board is selected.Non-transfected cell group is also selected to kill cell effect determine selective reagent.For adherent cell, cell is allowed to grow the clone until obtaining visible separation in tissue culturing plate.Then they are removed from flat board with pancreatin digestion or physical method and is taped against in tissue-culture well (each one clone in hole in 96 orifice plates).For suspension cell, limited dilution cloning is carried out after selection, clone is then expanded, and is then characterized and/or carried out the limiting dilution analysis of next round.

The stable clone being grown in the culture medium containing serum adapts to gradually decrease serum levels, then comes off and in the growth of low serum low suspension.Serum levels are then further reduced until serum-free state.Some culture mediums can make adaptation faster (for example, directly replacing with serum free suspension growth from the condition containing adhesion), and an example is the CD293 culture mediums of Invitrogen companies.

After being grown in serum free medium, clone can proceed by medium optimization.The industry characteristics of clone are detected in many different culture mediums, for example, complete vibrant cell quantity, until obtaining most suitable composition.This is dependent on the production method produced.For example, cell may be expanded in a culture medium, the additive of Enhanced expressing is then added before collection of products.

The albumen or chimeric molecule of overexpression may be accumulated in host cell.The recovery of intracellular protein includes handling host cell with lysis buffer, and the lysis buffer includes but is not limited to the buffer solution containing following composition：NP40, Triton X-100, Triton X-114, lauryl sodium sulfate (SDS), natrii tauroglycocholas, deoxysodium cholate, CHAPS, CHAPSO, Brij-35, Brij-58, Tween-20, Tween-80, octyl glucoside and octylthioglucoside.Another cracking host cell method can include sonication, homogenate, the crushing of Freund cell and multigelation and handle cell with hypotonic solution.

Final product can be produced in a variety of different bioreactors, via non-limiting example, including stirring pool, gas-lifting type (airlift), packed bed perfusion, microcarrier, doughnut, bag technique, cell factory.Its method can be continuous culture, batch, stream plus culture (fed batch) or induce.Peptone class can be added in low blood serum medium to increase volume protein.

The purification strategy customized using protein or chimeric molecule specifically for the present invention purifies the protein or chimeric molecule of the present invention.Purification process includes but is not limited to：Tangential flow filtration (TFF)；Ammonium sulfate precipitation；Molecular sieve chromatography (SEC)；Gel filtration chromatography (GFC)；Affinity chromatography (AFC)；Purification that A albumen is affine；Receptor-mediated coordinate chromatograph (RMLC)；Dyestuff coordinate chromatograph (DLC)；Ion-exchange chromatography (IEC), including anion or cation-exchange chromatography (AEC or CEC)；Reverse-phase chromatography (RPC)；Hydrophobic interaction chromatography (HIC)；Metal chelate chromatography (MCC).

TFF is a kind of quickly and efficiently bio-molecular separation method, for concentrating, desalination or fractionation sample.TFF can concentrate hundreds of liters of samples for 10 milliliters.It is combined with suitable molecular weight mwco membrane, TFF can separate different size and the biomolecule of molecular weight (nominal molecular weight retention (NMWC) is 5KDa, 10KDa, 30KDa, 100KDa).Diafiltration process is then concentrated again including dilute sample, can be used for desalination or be replaced sample buffer.

Saltout or ammonium sulfate precipitation can be used for condensing protein dilute solution.It is additionally operable to fractionating proteins mixture.Similar charge repels decreased effectiveness between the ionic strength of solution of the increase containing protein causes protein molecule.It also reduces the power of Protein requirement surrounding molecules solvent shell.When these power decrease to degree, protein will be precipitated；Compared with hydrophilic protein, hydrophobin sinks to forming sediment compared with low salt concn.It is a kind of very effective partial purification method of protein by the classification separation for being stepped up ionic strength and carry out centrifugation progress protein compound.

SEC flows through porous matrix according to sample, passes through size separation protein.SEC with GFC principles are identical, and it be used to separate the molecule in liquid phase systems.In SEC, the molecule bigger than hole in packing material and solvent front are flowed out first together, are completely excluded.The molecule of middle size, excludes between reservation between complete, the hole in host material is passed through according to its size.The small molecule for freeing in and out hole is retained.Therefore, different size of protein has different elution volume and retention time.For the similar molecule of structure, molecular size is bigger, their flows it is more early.Before any sample is run, it should set up standard curve to determine working limit and refer to retention time.

When protein shape is identical, the molecular weight of post eluate can quickly be screened by UV absorption, fluorescence or light scattering according to the filler of different pore size in post.Photon correlation spectroscopy (PCS) is generally used in static sample and liquid chromatographic detection.Low angle laser light scattering is also coupled in chromatogram detection, direct detection molecules amount, and unrelated (Carr et al. the AnalBiochem 175 of the shape of protein：492-499,1988).SEC-HPLC is used for detection hGH degradeds and aggregation (Pikal et al. Pharm Res 8：427-436,1991).It is also used for pollution condition (the Yoshioka et al.Pharm Res 10 of the beta galactosidase of Estimation Study：103-108,1993).

AFC is according to the specific interaction between the chemical constitution between biomolecule and suitable affinity ligand purifying biological molecule.By complementary immobilization ligand specificity's and reversible adsorbed target molecule.Part can be a kind of inhibitor, substrate, analogies or co-factor, or it is a kind of can specific recognition target molecule antibody.Then, the molecule of absorption is washed away by competitiveness displacement, or conformational change is allowed by pH or ionic strength conversion.

A albumen affinity purifications are an affinity purification example of the affinity using certain bacterioprotein, the bacterioprotein energy broad incorporation antibody, regardless of specificity of the antibody to antigen.A albumen, G-protein, L albumen are provided with the clear antibody binding properties of solution.These three albumen recombinant production and conventionally used for from a variety of middle affinity purification key antibody types.A albumen, the genetic engineering recombinant forms of G-protein are referred to as albumin A/G, are also to use.These antibody binding proteins can be immobilized on supported matrix.The method has been modified for being connected with protein of interest in the recombinant protein in antibody A protein binding region (Fc regions).It is attached in physiological conditions on the A protein moleculars of immobilization, by changing pH or ionic strength elution.

RMLC is a kind of AFC of specific type, has used acceptor to the intrinsic compatibility of its related objective molecule.Acceptor molecule is immobilized on suitable chromatogram supported matrix by active amino, reactive hydrogen, carbonyl, carboxyl or mercapto groups.In a RMLC example, acceptor-Fc chimeric molecules molecule is fixed on A albumen Ago-Gel pearls by acceptor Fc parts to the affinity of A albumen.The method has directional at-tachment acceptor, and the advantage in its ligand binding site is exposed to its corresponding cell factor.Target molecule is allowed to be adsorbed onto on acceptor in physiological conditions, by changing pH or ionic strength elution.

DLC is a kind of ALC, has used reactive dye selectivity and reversible associated proteins ability.Dyestuff is typically Monochlorpheanmide compound.Active cl radical able person triasine dyes is readily immobilized on supported matrix, such as Ago-Gel (Sepharose) or agarose, and can be immobilized in recently on nylon membrane.

Being originally found for these dye-bond proteins comes from it was observed that the glucan blue (cibacron indigo plants FG-3A conjugated compound) as solvent resistant column void volume mark can delay the elution of some protein.Then some the specific researchs of dyestuff to specific protein have been carried out, majority uses the blue dyestuffs of prototype cibacron.These dyestuffs show maximally effective binding characteristic, albumen and the enzyme such as kinases and dehydrogenase, although other albumen such as serum albumin can also combine closely in terms of protein and enzyme of the nucleotides as co-factor is used in combination.It is believed that aromatic series triasine dyes structure is similar with the nucleotide structure of nicotinamide adenine (NAD), and the nicotinamide adenine in dyestuff and these albumen folds and occurs dependent interaction.In many cases, associated proteins can be eluted under competition model by substrate or nucleotide cofactors, and dyestuff has shown that the characteristic that substrate binding site is competed in free solution.Appearing these dyestuffs can be by electrostatic and hydrophobic interaction and " pseudo- affine " the interaction associated proteins in more specific and ligand binding site.It has been employed successfully in by modifying the specificity of increase dye ligand further to simulate part (plan ecofriendly dyes) in many dehydrogenases of purifying and protease (McGettrick et al.).

Ion-exchange chromatography (IEC) has used delay of the albumen because of the electrostatic interaction between ion exchange column matrix and protein in pillar to carry out purifying protein.When mobile phase pH exceedes the pI of target protein, target protein is negatively charged, and will be interacted with anion-exchange column (AEC).When mobile phase pH is less than the pI of target protein, target protein positively charged should use cation exchange column (CEC).By using with target molecule identical electric charge target protein is eluted to increase the concentration of ion balance.

RPC is according to the hydrophobic interaction separation of biomolecules between molecule and chromatogram supported matrix.By controlling the pH in separation, under the neutral form of ionizable compound, they are easiest to analysis.Mobile Phase Additives, such as trifluoroacetic acid, albumen hydrophobicity, strong adsorption to stationary phase are increased by forming ion pair.By changing the polarity of stationary phase, biomolecule is eluted from the supported matrix of chromatogram.

HIC is similar with RPC, but with bigger nominal pore.In HIC, eluting solvent uses aqueous saline solution, instead of the aqueous phase or organic phase mobile phase used in RPC.Also, it is opposite that sample elution order is compared with RPC.Protein surface includes hydrophilic residue and hydrophobic " piece ", and stable protein is carried out in the inside that the latter is usually located at folded protein.When hydrophobic flakes are changed into being exposed in aqueous environment, they will destroy the normal solvent properties of albumen, be changed into thermodynamically unfavourable.In aqueous phase mobile phase, inorganic salts (such as ammonium sulfate) concentration is higher, and surface tension is bigger, therefore the intensity of the hydrophobic interaction between increase HIC resin hydrophobics group and albumen, adsorbs.But, when gradient reduces salinity, the reduction in surface tension of aqueous phase mobile phase, therefore cause hydrophobic interaction to reduce, cause albumen from desorption on pillar hydrophobic grouping.

MCC is a kind of according to technology of the protein to the affinity protein isolate of chelated metal ions.Different metal ions includes but is not limited to be fixed on the Cu of chromatogram supporter stationary phase by covalently bound cheland (such as diglycinee)²⁺, Co²⁺, Zn²⁺, Mn²⁺, Mg²⁺Or Ni²⁺.The free coordination site of metal ion be used to combine different proteins and peptides.Eluted by using competitive molecular replacement albumen or by changing pH.For example, reduction pH of buffer causes the binding affinity of protein-metal ion complex to reduce, protein desorption.Or, reduce pH (using discontinuous gradient or linear gradient form) protein of elution of bound from pillar using gradient.

It can modify host cell by a variety of methods known in the art to obtain the present invention protein or the physics and chemistry form of chimeric molecule.

Present invention contemplates after protein or chimeric molecule expression and purifying, carbohydrate chemistry or enzyme are coupled on the peptide chain of protein or chimeric molecule.Chemistry and/or enzyme coupling process can be used to modify, increase or decrease quantity or feature that quantity or carbohydrate are obtained.Conjugation pattern used in relying on, sugar can be attached to (a) arginic amide groups, (b) free carboxyl group group, (c) mercapto groups, such as in cysteine those, (d) those in oh group such as serine, threonine, oxylysine, (e) those in aromatic residue such as phenylalanine, tyrosine or tryptophan, (f) those in the amide group of glutamine, or (g) amino such as histidine, arginine or lysine.It can be added by chemistry or Enzymology method.It is, for example, possible to use suitably recombinating glycosyl transferase continuous additional sugared unit on protein or its chimeric molecule.Glycosyl transferase increase can also be used to be covalently attached the sugar of substituted base.For example, the sialic acid with covalently additional polyethylene glycol (PEG) can be transferred to terminal galactose residues to increase molecular size and serum half-life by sialyltransferase.

A variety of functional groups, including phosphate radical, sulfate radical, hydroxyl, carboxylate radical, O- sulfate radicals and N- acetyl groups can be mixed with the carbohydrate side chain of chemistry or enzymatic modification protein or chimeric molecule.

The carbohydrate that can be gone with chemistry or enzyme process in isolating protein or its chimeric molecule.Trifluoromethanesulfonic acid or a kind of suitable compound can be used to carry out Chemical deglycosylation.This processing can cause it is most of or all sugared isolate, except combining sugar, and keep polypeptide complete.With other sugar or whole chain can be removed from protein or its chimeric molecule by a variety of endoglycosidases and exoglycosidase.

The chitosan component of modifying protein or chimeric molecule can be carried out by using sialidase, or remove with moderate acid treatment the sialic acid of residual；With it is circumscribed-inscribe-glycosidase come cut N- crosslinking oligosaccharide antenna or shorten O- connection oligosaccharides.Side base, such as trehalose and sulfate radical can also be removed with fucosidase or sulfatase processing.Pseudo- glycan structures such as polyethylene glycol or glucan, or the sugared subunit of increase that the glycerine transferase cocktail with sugar-dUDP precursors can be used to be synthesized to glycan can be added on chemical normal direction amino acid backbone.

Present invention contemplates on chemistry or enzyme process coupling protein matter or its chimeric molecule to radionuclide.This protein or chimeric molecule can be selected from the list containing GM-CSF, GM-CSF-Fc, IL-3, IL-3-Fc, IL-4, IL-4-Fc, IL-5 and IL-5-Fc.

Can use iodination to protein or its chimeric molecule the additional iodine isotope of peptide chain (for example¹²³I).Especially, isotope can be attached on the phenol ring of (a) tyrosine of protein or the peptide chain of its chimeric molecule, or on the imidazole ring of (b) histidine.Can use chloramine-T (Chloramine-T), iodine monochloride, teriodide, electrolyte, enzyme, with reference to, the method for metallization removal, iodogen or iodine pearl carry out iodination.

Mtc labeled method can be used to use methods known in the art will^99mTc is attached on the protein of the present invention or chimeric molecule, for example, reduced by using go back original reagent (such as stannous chloride)^99mTcO₄ ^-, then carried out by bifunctional chelating agent (such as diethylenetriamine pentaacetic acid (DTPA))^99mTc labelled proteins or chimeric molecule.

It is coupled to present invention contemplates protein or its chimeric molecule chemistry or enzyme process on chemotherapeutant.It can use methods known in the art that suitable reagent (such as zoledronic acid) is attached on protein or its chimeric molecule, for example, the coupling reaction mediated by the enhanced carbodiimides of N-hydroxysulfosuccinimide.

It is coupled to present invention contemplates protein or its chimeric molecule chemistry or enzyme process on toxin.It can use methods known in the art that suitable toxin (including melittin, vanous toxin, Pseudomonas exotoxin, ricin (WA), gelonin and the diptheria toxin shortened) is attached on protein or chimeric molecule, methods described is for example acted on by maleimide or carbodiimides conjugation chemistry.

Can by allow separation protein or chimeric molecule and patient in target recipient or the method that comes in contact of part the protein of separation described herein or its chimeric molecule are transported in patient's body.In a specific embodiment, protein or its chimeric molecule are as in " pharmaceutical composition " transported patient.

On the other hand, present invention contemplates a kind of one or more protein or chimeric molecule and pharmaceutically acceptable carrier containing separation described above or the pharmaceutical composition of diluent.

The composition forms used suitable for injecting, which include aseptic aqueous solution (water soluble) and aseptic powdery, to be used for extemporaneous preparation of sterile and injects solution.It must be stable under production and condition of storage, and must keep avoiding the pollution of microorganism (such as bacterium and fungi).Carrier can be that a kind of solvent or diluent include, for example, water, ethanol, polyalcohol (for example, glycerine, propane diols, liquid polyethylene glycol etc.), their suitable mixture and vegetable oil.Suitable mobility can be maintained, for example, by using surfactant.Can be by a variety of antibacteriums and antifungal agent, such as metagin, anesin, phenol, sorbic acid, thirmerosal, to prevent the activity of microorganism.In many cases it is preferred to isotonic reagent, for example, sugar or sodium chloride.Can be by extending the absorption of Injectable composition using delayed absorption reagent in the composition, for example, aluminum monostearate and gel.

By mixing reactive compound in the suitable solvent with required active component and optional other active components of requirement, to prepare aseptic injectable solution, subsequent filtration sterilization or other suitable sterilizing methods are used.For the aseptic powdery for preparing aseptic injectable solution, suitable preparation method includes vacuum drying and lyophilized, and methods described produces the required composition that powdered active ingredient adds any addition.

When activating agent is suitably protected, it can be taken orally, for example; with inactive diluent or with assimilable edible carrier; it can either be encapsulated in hard or soft shell capsule or can be with boil down to tablet, or can be directly incorporated into the food of diet or be administered by breast milk.For oral therapeutic administration, active component can mix auxiliary material and be used in forms such as absorbable tablet, lozenge, capsule, elixir, suspension, syrup, wafers.This composition and preparation should contain the active component of at least 1% weight.The percentage of composition and preparation can be with, certainly, is change and can be with suitable between 5% to about the 80% of Unit Weight.The quantity of active agent is by the suitable dosage of acquisition in the composition of this treatment.In a specific embodiment, the composition or preparation according to the present invention are prepared, oral dosage unit form contains the conditioning agent between about 0.1 μ g and 200mg.Other dosage are included from about 1 μ g to about 1000mg, from about 10 μ g to about 500mg.These dosage can be each individual or per kilogram of body weight.Can administration per hour, daily, weekly, monthly or every year.

Tablet, lozenge, pill, capsule etc. can also contain the composition of following lists.Adhesive such as resin, Arabic gum, cornstarch, gelatin can be added；Auxiliary material such as Dicalcium Phosphate；Disintegrant is such as cornstarch, farina, alginic acid；Lubricant such as magnesium stearate；Sweetening agents such as sucrose, lactose or saccharin, or add fumet such as peppermint, wintergreen or cherry flavoring.When a dosage unit form is a capsule, it can be containing the above-mentioned type material and liquid-carrier.A variety of other materials can be used for coating or the in addition physical form of modification dosage unit.For example, shellac, sucrose or both together coating tablet, pill or capsule can be used.Syrup or elixir can contain reactive compound, and sucrose is used as preservative, dyestuff and flavoring such as cherry or citrus flavoring as sweetener, methanol and nipasol.Certainly, any material used in any dosage unit form is prepared should be pharmaceutical purity and be substantially nontoxic for the quantity used.In addition, reactive compound can be mixed in sustained release preparation and formulation.

Present invention further contemplates Topical dosage forms.In Topical dosage forms, active agent can suspend in emulsifiable paste or lotion or waxed dose or other liquid solutions, therefore topical application emulsifiable paste or lotion or waxed dose or liquid solution cause active agent to be introduced into the biological surface of patient.Vocabulary " biological surface " used herein, it is contemplated on body or internal any surface.The example of the local mixture adaptable " biological surface " of the present invention includes any epithelial surface, such as skin, respiratory tract, intestines and stomach and genitourinary tract.

In addition to traditional emulsifiable paste, emulsion, paster or spray, reagent of the invention can also use a series of methods based on ionotherapy or electroporation (poration) by local and/or transdermal transport.

" ionotherapy " causes the ability of charging particle movement based on electric current.It is placed in a pair on skin close electrodes and a potential is established between skin and following capillary.In positive pole, positively charged drug molecule is dislodged skin surface to capillary.Opposite, skin will be passed through by promotion in the electronegative drug molecule of negative pole.Because electric current can be closed and changed, Iontophoretic device quickly can start and close, and medicament transport is highly controllable and is sequencing.

Electroporation technology has used high-frequency impulse energy, and (such as radio-frequency radiation, laser, heat or light) is of short duration in a variety of forms breaks cuticula, and cuticula is the skin layer for preventing most medicines from entering in blood flow.Be important to note that it is different with iontophoresis, the energy that electroporation technology is used be not used in transport medicine by skin, simply facilitate its movement.Electroporation provides one " window ", and drug substrate is compared with normal condition can be easier and quickly pass through.

Pharmaceutically acceptable carrier and/or diluent include any and all solvent, dispersant, and coating, antibacterium and antifungal agent wait and blend delayed absorption agent etc..The use of this medium and reagent is known in the art to pharmaceutically active substance, and is incompatible without what conventional media or reagent and conditioning agent, their applications in pharmaceutical composition have been conceived to.The reactive compound of supplement can also be incorporated into composition.

In one embodiment, the pharmaceutical composition of the present invention can be used in combination individually or with other medicines or therapy, the other medicines or therapy employ the mode as protein or its chimeric molecule that non-human cell lines express, such as Escherichia coli, yeast or the protein or chimeric molecule of CHO expression, for individually or with other medicines treating disease together, the disease includes：Abetalipoproteinemia,A-V,Beta-2-Microglobulin amyloidosis,A-T,A1AD,A1AT,Aagenaes,Aarskog's syndrome,Aarskog-Scott syndrome,Aase-Smith syndrome,Aase syndrome,AAT,Abderhalden-Kaufmann-Lignac syndrome,Abdominal muscle deletion syndrome,Abdominal-wall defect,Abdominal epilepsy,Abdominal migraine,Abductor dysphonia spastica,Abductor spastic dysphonia,Abercrombie's syndrome,Eyelid meloschisis syndrome,ABS,HPRT lacks,Corpus callosum Schinzel Typ lack,Four limbs scalp and cranium defect,Amenorrhea Primar,HGPRT lacks,Absorptive Hyperoxaluriaor Enteric,Abt-Letterer-Siwe diseases,ACADL,ACADM lacks,ACADM,ACADS,Acanthocytosis neuropathy,Acanthocytosis,Epidermolysis bullosa,Acanthosis nigricans (disease),Acanthosis bullosa,Acanthosis nigricans (disease) with A type insulin resistances,Acanthosis nigricans (disease) with Type B insulin resistance,Acanthotic nevus,Acatalasemia,Acatalasia,ACC,Accessory atrioventricular pathways,Acephalia,With heart defect ACF,Relaxation can not,A Chade-ladder syndrome,A Chade (all mutation of horse),A Shaer Cotards,Acholuric jaundice,Achondrogenesis,The achondrogenesis of type four,3rd class achondrogenesis,Achondroplasia,Achondroplasia is slow,Achondroplastic dwarf,Sneeze syndrome,Colour blindness,Monochromat,Monochromasia,Monochromasia,Colour killing mole,Acid ceramidase deficiency,Acid maltase deficiency,Acid β-glucosidase deficiency disease,Acidaemia,Acidaemia,The sporadic incoordination of malonic acid mass formed by blood stasis and weakness,Acid poisoning,Tarsoepiphyseal aclasis,ACM,Acoustic nerve neurilemmoma,Acoustic neurinoma,With referring to (toe) more the undergrown tip of lower limb and refer to (toe) deformity,Refer to (toe) more tip and refer to (toe) deformity II,Refer to (toe) more tip and refer to (toe) deformity IV,Refer to (toe) more tip and refer to (toe) deformity III,The day after tomorrow aphasia convulsions disease,The day after tomorrow brown's syndrome,Acquired epileptic aphasia,Factor XIII deficiency,The ACC that the day after tomorrow is formed is (caused by infection,And be still in intrauterine),Acquired hyperoxaluria,Acquired hypogammaglobulinemia,Immune Deficiency Syndrome (AIDS),Acquired iron overload,Acquired fat nutritional disorders,Acquired partial fat dystrophia,Acquired splenectopia,ACR,Acrodysostosis is with face and genital malformation,Acro Renal,Acrocallosal Syndrome Scninzel Type,Acrocephalosyndactylism (toe) (deformity),I types acrocephalosyndactylism (toe) (deformity),I types acrocephalosyndactylism (toe) (deformity) hypotype I,II types acrocephalosyndactylism (toe) (deformity),Type III acrocephalosyndactylism (toe) (deformity),IV types acrocephalosyndactylism (toe) (deformity),V-type acrocephalosyndactylism (toe) (deformity) (ACS 5 or ACS V) hypotype I,Tip skull is asymmetric and slight and refers to,Tip,acrochondrohyperplasia,Acrodermatitis enteropathica,Acrodysostosis,Acrodystrophic neuropathy,Acrofacial dysostosis Nager types,Acrofacial dysostosis Postaxial types,Acrofacial dysostosis Genee-Wiedep types,Acrogeria family,Acromegalia,Acromelalgia,Acromesomelic dysplasia,Acromesomelic dwarfism,Akromikrie dysostosis,Akromikrie depauperation,Acroosteolysis and skull and mandibular change with osteoporosis,Acroosteolysis,Acroparesthesia,ACSI,ACS II types,ACS type IIIs,ACS,ACS3,Corticotropin deficiency disease,Action myoclonia,Acute brachial plexus neuritis syndrome,Acute brachial plexus nerve radicular syndrome,Acute brain Gaucher disease,Acute cholangitis,It is acute to send out encephalomyeloradiculopathy,Acute disseminated histiocytosis X,Acute hemorrhagic polioencephalitis,Extra urgent property polyneuritis,Acute immune reconciles polyneuritis,Children's acute familial centrolobar sclerosis,Accute porphyrin,Acute porphyria,Acute sarcoidosis,Acute shoulder neuritis,Acute toxic epidermolysis bullosa,Acyl-coenzyme dehydrogenase deficiency long-chain,Acyl-coenzyme dehydrogenase deficiency short chain,Acyl coenzyme A dihydroxyacetone (DHA) acyltransferase,Acetylation of coenzyme A oxidase lacks,ADA,ADA lacks,AdamComplex,Adamantiades-Behcet syndrome,Adamantinoma,Adams Oliver syndromes,Adaptive colitis,ADD combined type,ADD,Ai Di diseases carry cerebrosclerosis,Ai Di disease anaemias,Addison's disease,A Disen (family name) anaemia,Adrenoleukodystrophy,A Disen (family name) pernicious anaemia,Interior receipts thumb-mental retardation,Adductor spastic dysphonia,Adductor spastic dysphonia,Manlike elderly woman adenoma,Colon and rectal gland epithelial hyperplasia,Colonic adenoma polyposis,Family's adenomatous polyp,Adenosine deaminase deficiency,Adenylosuccinase deficiency,Hyperactivity mainly moves impulsive style,Absent-minded type based on hyperactivity,Hyperactivity,Arachnoid adhesion,Adie's syndrome,Adie's syndrome,Ai Di myotonic pupils,Adie's pupil,Referring to adipogenital (drawing) retinitis pigmentosa more,Adipogenital (drawing) retinitis pigmentosa syndrome,Adiposa Dolorosa,Adiposis dolorosa,Adiposogenital dystrophy,Adolescent cystinosis,Polycystic kindey,Adrenal cortical adenoma,Adrenal gland diseases,Adrenal cortex function is hyperfunction,Cause pituitary adrenocorticotropic hormone superfluous,Adrenal aplasia,Adrenal insufficiency,Adrenal tumor,Adrenal gland is manlike,Adrenal gland-retinitis pigmentosa-many toes syndrome,Adrenal insufficiency,Hypoadrenocorticism,Corticotropin lacks single,(congenital) adrenogenital syndrome,Adrenoleukodystrophy,Adrenomyeloneuropathy,Adrenal gland-retinitis pigmentosa-many toes syndrome,Adult's cystinosis,Adult dermatomyositis,Adult hypophosphatasia,Into macular degeneration in humans,Adult Onset ALD,Adult onset ceroidosis,Adult Onset's medullary substance cystic disease,Adult Onset's pernicious anaemia,Adult Onset's Schindler Disease,Adult onset subacute necrotizing cerebrospinal cord disease,Adult polycystic kidney disease,Adult Onset's medullary substance cystic disease,Adynlosuccinate lyases are lacked,AE,AEC syndrome,AFD,Fibrinogenemia,African siderosis,AGA,Aganglionic megacolon,Age-related macular degeneration,Agenesis of corpus callus,Agenesis of corpus callus,Agenesis of corpus callus-infantile spasm eye is abnormal,Agenesis of corpus callus and chorioretinal are abnormal,Agenesis of corpus callus-choroidoretinitis,Mastocytosis,Agnosis Primary,AGR Triad,AGU,Agyria (deformity),Agyria-pachygyria frequency range,AHC,AHD,AHDS,AHF lacks,AHG lacks,AHO,Ahumada Del Castillo,Ai Kaerdi syndromes,AIED,AIMP,AIP,AIS,Akinetic seizure,ALA-D porphyrias,Alactasia (disease),Alagille syndrome,Oran island illness in eye (x is chain),alaninuria,Ai Erbai-Arnold Schoenberg disease,Albefaction,Albinism,Albinoidism,Albright constitutional bone disease,Alkaptonuria,The related inborn defect of alcohol,Alcohol embryopathy,Alcoholic cirrhosis,Ald,ALD,ALD,Aldosterone,With normotensive aldosteronism,Congenital cataract,Alexander disease,Alexandria disease (infantile leukodystrophy),Algodystrophy,Algoneurodystrophy,Melanuria,Alkaptonuric ochronosis,Alkyl DHAP synthase is lacked,Allan-Herndon-Dudley syndromes,A Lan-henry syndrome,A Lan-henry-Dudley baryencephalia,Allergic granulomatous angiitis,The Canadian allergic granulomatous angiitis of Cronkhit e-,Alobar holoprosencephaly,Alopecia areata,Alopecia circumscripta,Alopecia circumscripta,Alopecia circumscripta,Alopecia-white hair (disease-dermatoglyph uveitis-leucoderma-deafness skin uvea-O,Alopecia seminuniversalis,Whole alopecia,General alopecia,Gray-matter degeneration,The ectocinerea and hepatic sclerosis of alpers diffusivities denaturation,Children's poliodystrophia (brain) of alpers progress,Alpha-1-antitrypsin deficiency,The glucosides enzyme deficiency disease of Alpha -14,Alpha-galactoside enzyme defect,Alpha-galactoside boron deficiency,Alpha's HDL lacks,Sick 3 type of alpha-L-fucosidase deficiency fucoside (storing up),α-GalNAc defect Schindler types,Alpha lipoprotein,Mannosidosis,Alpha-N-Acetylgalactosaminidase defect Schindler types,α-NAGA lack Schindler types,α-nerve ammonia (sugar) neuraminidase defect,α-thalassemia/mental retardation syndrome non-deletion type,Alpha lipoprotein,Alport syndrome,ALS,Alstroem Cotards,alstroem,Alstrom syndromes,Alternating hemiplegia syndrome,Children's alternating hemiplegia,Alzheimer's,Family black horny idiots,Family black horny idiots are grown up,Family black horny idiots children,Vulva sex is failed to understand,AMC,AMD,Ameloblastoma,Amelogenesis imperfecta,Amenorrhea-galactorrhea nonpuerperal,Amenorrhea-galactorrhea-FSH reduces syndrome,Amenorrhoea,Amino acid is not normal,Amino acid-osteomalacia-hyperphospheremia syndrome,AMN,Amniocentesis,Amniotic sheets,Amniotic band syndrome,Amnion desmorrhexis is complicated,Amniotic sheets sequence,Amniorrhexis sequence,Congenital amputation,AMS,The short and small syndrome de Lange in Amsterdam,Amylo-1:4,1:6-transglucosidase,6- glucosides enzyme deficiency diseases,Long term hemodialysis starch joint,Amylaceous corneal dystrophy,Amyloid polyneuropathy,Amyloidosis,Starch familial Mediterranean fever,Amylopectinosis,Amyoplasia congenita,ALS,ALS,ALS-glucan,AN,AN1,AN2,Hedratresia,Anal plate,Anorectal malformation,The stricture of anus,The amyloidosis of Analine 60,Blood (interior) alpha-lipoprotein lacks,analrectal,analrectal,Glioblastoma multiforme,Andersen's disease,Anderson -- Fabry disease,Andersen's glycogenosis,Anderson-Hua Bao syndromes,An Delie syndromes,The type of An Delie syndromes two,Androgen insensitivity,Androgen-insensitivity syndrome is local,Androgen-insensitivity syndrome is local,Androgenic Steroids,Anaemia autoimmune hemolytic,Anemia Blackfan Diamond,Anaemia,It is geneogenous,Triphalangeal thumb syndrome,The cold antibody of hemolytic anemia,Hemolytic anemia is lacked with PGK,Pernicious anaemia,Anencephalia,Angelman syndromes,Angio-osteohypertrophy syndrome,Blood vessel folliculus lymph node hyperplasia,Angiohemophilia,Angioceratoma body,Unrestrained property body angioceratoma,Diffusivity angioceratoma,Retinal angiomatosis,Lymphangiohemangioma,Angioneurotic edema heredity,Anhidrotic ectodermal dysplasia,Anhidrotic x is chain ectodermal dysplasia,Irideremia,Irideremia-unclear the mental retardation of genitals sex,Iris and mental retardation,Irideremia-cerebellar ataxia intelligence is residual,Iris part cerebellar ataxia mental retardation,Iris part cerebellar ataxia oligrophrenia,Irideremia type I,Irideremia type II,Aniridia-Wilms tumor association,Aniridia-Wilms tumor-gonadoblastoma,Ankyloblepharon-ectoderm developmental defect harelip/cleft palate,Ankylosing spondylitis,Annular groves,Anodontia,Anodontia vera,Anomalous trichromatism,Anormogenesis exception dentine,Coronal dentin dysplasia,Anomic aphasia,Anophthalmia,Anal intestine,Anorectal malformation,Anosmia,Front arcuate leg is with nanism,Tooth film corneal dystrophy,Anticonvulsion disease,Anti- Epstein-Barr Virus Nuclear Antigens (EBNA) antibody deficiency,Antibody deficiency,Antibody deficiency is carried close to normal immunoglobulin,Antihemophilic factor deficiency disease,Antihemophilic globulin deficiency,Antiphospholipid syndrome,Antiphospholipid antibody syndrome,Antithrombin III lacks,Typical Antithrombin III lacks (I types),Antitrypsin is lacked,Antley-Bixler syndromes,Antoni is benumbed,Shin is uneasy,Aortic arch syndrome,Sustainer and mitral atresia merge left heart syndrome,Aortic stenosis,aparoschisis,APC,Apeced syndromes,Apert syndrome,aperts,Aphasia,Congenital cranium outer shaft sexual dyspenesis,Congenital skin defect,The horizontal physical impairment of congenital skin defect and end,Alpastic anemia,Alpastic anemia birth defect,APLS,Apnea,Appalachian type amyloidosis,Apple skin syndrome,Appraxia (disease),Buccal surface appraxia (disease),Structure appraxia (disease),Ideational appraxias (disease),Ideokinetic appraxias (disease),Ideomotor movement appraxia (disease),Motor appraxia (disease),Eye movement appraxia (disease),APS,Archnoiditis,Arachnodactyly contracture Beals types,Arachnodactyly,Arachnoid cyst,Spider web periosteal ossification,Archnoiditis,Aran-Duchenne,Aran-Duchenne muscular atrophy,Alpastic anemia,Arginine depletion,Argininemia (disease),Arginino succinase are lacked,Argininosuccinase deficiency,Argininosuccinase Defect,Argininosuccinic acid-ASL,Argininosuccinate synthetase lacks,Smart ammonia (base) amber uraturia,Argonz-Del Castillo syndromes,Arhinencephalia [deformity],Armenia's syndrome,Arnold-Chiari malformation,Arnold-plus syndrome,ARPKD,Myoclonic arrhythmia cordis,Right ventricle depauperation,Arteriohepatic dysplasia,Arteriovenous malformation,Venous malformation brain,Arteritis giant cell,Arthritis,Reiter syndromes,Arthro-dento-osteo dysplasia,Arthro-ophthalmopathy,Arthrochalasis multiplex congenita,Distally,IIA types,ARVD,Fragrant (perfume) base sulfatase B deficiency diseases,AS,ASA lacks,Ascending paralysis,ASD,Atrial septal defect,ASH,Asherman syndrome,Ashkenazi type amyloidosis,ASL deficiency,Aspartylglucosaminuria,Eisberg syndrome,Eisberg type self-closing disease,Asphyxiating thoracic dysplasia,Splenic syndrome,ASS deficiency,Asthma,I grades of astrocytoma (benign),II grades of astrocytoma (benign),Asymmetric phase heart defect of crying,Asymmetric septal hypertrophy,Asymptomatic callosity,AT,AT III defects,AT III variants IA,AT III variants Ib,AT 3,Incoordination,Ataxia telangiectasia,Incoordination and lactic acidosis II types,Incoordination brain paralysis,Ataxiadynamia,Ataxiophemia,ATD,Brothers' brain paralysis,Dermatitis and eczema,Atretolemia is with or without esophago-tracheal fistula,Atrial septal defect,Original atrial septal defect,Atrial septum and small ventricular septal defect,Auricular flutter,Auricular fibrillation,Atriodigital dysplasia,Atrial septal defect,Atrioventricular block,Atrioventricular anomaly,Atrioventricular septal defect,(family name) disease in promise,Atrophic type athlete's foot,Olive atrophy,Attention deficit disorder,Attention deficit hyperactivity disorder [with how dynamic],It is attenuated colonic adenoma,Atypical amyloidosis,Atypia mass formed by blood stasis,Internal auditory meatus locking,Auriculotemporal syndrome,Autism,Self-closing disease-Eisberg type,Self-closing disease dementia incoordination and purpose hand use defect,Autism children autism,LADA Addison's disease,Autoimmune hemolytic anemia,Oneself immunity hepatitis,LADA-many (kind) endocrine adenopathy-candidiasis,Autoimmune polyglandular disease I types,Autosomal dominant inheritance albinism,Autosomal dominant CompellingHelioophthalmic Outburst syndromes,Autosomal dominant inheritance desmin distal myopathy is simultaneously tardy,Autosomal dominant EDS,Autosomal dominant emery-dreifuss type muscular dystrophies,Autosomal dominant keratoconus,Autosomal dominant Pei-plum Er Shi cerebral sclerosises,Autosomal dominant polycystic kidney disease,Autosomal dominant inheritance spinal cerebellar degeneration,Autosomal recessive inheritance agammaglobulin disease,Autosomal recessive inheritance central nucleus myopathy,Autosomal recessive inheritance Kang-Xu Er 's syndrome,Autosomal recessive inheritance EDS,Autosomal recessive inheritance ecuador's syndrome is malnourished,The eye albefaction of autosomal recessive inheritance form,Autosomal recessive inheritance callosity hypoplasia,Autosomal recessive keratoconus,Recessive hereditary polycystic kidney disease,Autosomal recessive inheritance severe combined immunodeficiency,AV,AVM,AVSD,AWTA,Oxter abscess,Axonal neuropathy is huge,Azorean sacred diseases,B-K mole syndrome,Babinski-Froelich syndromes,BADS,Baillarger Cotards,Balkan disease,Baller-Gerold syndrome,Sacculus bicuspid valve,Balo disease concentric sclerosis,Baltic Sea lafora's disease,Bannayan- Zuo Nana syndromes (bzs),Bannayan- FilippoGammarelli-ruvalcaba syndromes,This Di Shi diseases,Bardet-biedl syndromes,Bare lymphocyte symdrome,Concentric sclerosis,Bar [clarke that]-west [illiteracy] Er Shi diseases,Barrett esophagus,Bei Ruite ulcer,Bartter syndrome,Bartter's syndrome,Basal cell naevus syndrome,Basedow disease,A-betalipoproteinemia,Batten disease,Batten-Mayou syndromes,Batten-Spielmeyer-Vogt disease,The honest Turner's synodrome of shellfish,Bei Dun Teners type congenital myopathy,Batten-Vogt syndromes,BBB syndromes,BBB syndromes (opitz),BBBG syndromes,BCKD is lacked,BD,BDLS,BE,Bei Yaersi syndromes,Bei Yaersi syndromes,Bei Yaersi-Hecht syndrome,Bean syndrome,BEB,Bechterew syndromes,Becker disease,Becker muscular dystrophy,Becker's nevus,Beckwith Wiedemann syndromes,Beckwith syndrome,Begnez-Cesar syndromes,Behcet syndromes,Behcet diseases,Behr1,Behr2,Bell's palsy,Benign acanthosis nigricans,Benign astrocytoma,Benign cranial neuroma,Benign cystinosis,Benign essential blepharospasm,Benign essential tremor,Benign familial hematuria,Benign focal amyotrophia,The benign focal amyotrophia of ALS,Benign hydrocephalus,Benign hypermobility syndrome,Angling benign acanthosis nigricans,Benign paroxysmal peritonitis,Benign recurrent hematuria,Benign recurrent intrahepatic cholestasis,Benign spinal muscular atrophy and calf plumpness,Benign symmetric lipomatosis,Benign central nerve neuroma,Berardinelli-Seip syndrome,Primary Jie Shi diseases,Tinea pedis,Shellfish Mann syndrome,The Nellie syndrome of Claude Bernard-suddenly,Bernard Soulier syndrome,Bass Nie's pruigo,Vitelliform macular degeneration,Beta-alanine-Pyruvic Transaminase,Beta-Galactosidase deficiency Morquio syndrome,Beta-Glucuronidase deficiency,Beta-oxidation defect,β major thalaseemias,β minor thalassemias,Beta lipoprotein is lacked,Bei Telun myopathies,Beuren syndromes,BH4 deficiency diseases,Biber-Haab-Dimmer corneal dystrophies,Bicuspid aortic valve,Biedl-Bardet,Bifid brainpan,Bifunctional enzyme defect,Bilateral listens multiple neurofibromatosis,Bilateral Acoustic Neroumas,Bilateral right-sidedness sequence,Potter syndrome,Bilateral temporal lobe obstacle,Bile breaks out,Glucosiduronate (base) shifts enzyme defect I types,Binder syndromes,Binswanger diseases,Binswanger encephalopathics,Biotinidase deficiency,Bird-headed dwarf of Seckel Seckel types,Inborn defect,Birthmark,Double temporos clamp trace syndrome,Myocardial fibrosis,Bjornstad syndromes,B-K mole syndrome,Black lock-albinism-deafness sensoneural types (BADS),Blackfan-Diamond anemia,Pyorrhea primary arthritis,Blepharophimosis,Ptosis,Epicanthus inversus syndrome,Blepharospasm,Blepharospasm is benign primary,Oral cavity blepharospasm,Blessig's cysts,BLFS,Blindness,Bloch-Siemens incontinentia pigmenti melanoblastosis skins Linearis,Bloch-Siemens-Sulzberger syndromes,Bloch-Sulzberger syndrome,Blood group,Blood group A,Blood group B,Blood group AB,Blood group O,Facial telangiectasis of dwarfs syndrome,Bloom-Torre-Mackacek syndromes,Blue rubber bleb nevus,Blue baby,Blue diaper,BMD,BOD,BOFS,Bone tumour epidermoid cyst polyp,Bonnet-Dechaume-Blanc syndrome,Bonnevie-Ulrich syndromes,Book syndrome,BOR syndrome,BORJ,Borjeson syndromes,Borjeson's syndrome,Cerebrohepatorenal syndrome,Bowen-Conradi syndromes,Bowen-Conradi Hutterite,Bowen-Conradi type Hutterite syndromes,Bowman’s Layer,BPEI,BPES,Brachial plexus neuritis,Brachial plexus neuritis syndrome,Brachial plexus neuritis,Brachial plexus neuropathy,Arm ischemic,Brachmann-de Lange syndrome,Brachycephaly (deformity),Short shape type is congenital,Bradycardia,Craniocerebral injury is because of perinatal asphyxia,Brain tumor,Brain tumor is benign,Brain tumor is pernicious,Branched-chain alpha-keto acid dehydrogenase deficiency disease,Side chain beta-oxybutyria I,Brancher deficiency,Branchio-Oculo-Facial syndromes,Branchio-oto renal aplasias,Branchio-oto kidney syndromes,Branchiooculofacial syndromes,Branchiooculofacial syndromes,Brandt syndrome,Brandywine type hypoplasias of dentin,Brandywine type hypoplasia of dentin,Breast cancer,Benign recurrent intrahepatic cholestasis syndrome,Brittle bone disease,Broad-beta disease,Wide thumb syndrome,The facial MR of wide thumb and big toe feature,Wide thumb,Broca aphasias,Brocq-Duhring diseases,Bronze diabetes,Bronze diffusivity sclerosis,Brown albinism,Brown glaze heredity,Blang-fork clip that (family name) syndrome,Brown's syndrome,BRRS,Bo Lugeer syndromes,Bruton agammaglobulinemias (disease),BS,BSS,Buchanan syndromes,Budd syndromes,Budd-Chiari syndrome,Buerger-Gruetz syndromes,Bulbospinal muscular atrophies x is chain,Overwork syndrome,Heredity bleb,Bleb CIE,Epidermolysis congenital ichthyosis sample erythroderma,Epidermolysis ichthyosis,Bullous pemphigoid,Burkitt lymthomas,Burkitt lymthomas Africa type,Burkitt lymthomas,African type,BWS,Byler diseases,C syndromes,C1 esterase inhibitors exception II type angioedemas,C1-INH,C1 esterase inhibitors lack I type angioedemas,C1NH,Cacchi-Ricci disease,CAD,CADASIL,CAH,Valgus calcaneus,calcaneovalgus,Calcium pyrophosphate dihydrate is calm,Agenesis of corpus callus and eye are abnormal,The plump myeloid muscular dystrophy of calf,Campomelic dysplasia,Trunk nanism,Trunk syndrome,Count on one's fingers-cleft palate -- clubfoot,Limited jaw of counting on one's fingers is offset,Camptomelic dwarfism,Camptomelic syndrome,The long limb type of camptomelic syndrome,Camurati-Engelmann disease,Canada-Cronkhite diseases,Canavan's disease,What canavan's disease included,Ka Nawan leukodystrophies,Cancer,Cancer family syndrome Lynch types,Cantrell syndromes,Cantrell-Haller-Ravich syndromes,Cantrell pentalogys,Carbamyl phosphate synthetase deficiency,Carbohydrate defect glycoprotein syndrome,Carbohydrate defect glycoprotein syndrome Ia types,The hyperlipidemia that carbohydrate induces,The carbohydrate of glucose lactose is not tolerated,Carbon dioxide acid poisoning,Multiple carboxylation enzyme defect,Heart limb syndrome,Heart comprehensive hearing is levied,Jervell and and Lange-Nielsen cardioauditory syndromes,Cardiocutaneous syndrome,The heart-face-skin syndrome,Cardiofacial syndrome Cayler types,II type glycogen storage diseases,Cardiomyopathies lentiginosis,Cardiomyopathy,Cardiomyopathy and desmin storage myopathy,Cardiomyopathy is due to desmin defect,Cardiomyopathy-neutrophilic granulocytopenia,The lethal myocardium in children disease of cardiomyopathy-neutrophilic granulocytopenia,Heart disease amyloidosis,Cardiospasm,Cardocardiac syndromes,Carnitine-acylcarnitines transposition azymia,Carnitine lacks and disorderly,Carntine deficiency is primary,Carntine deficiency is secondary,Carntine deficiency is secondary to be lacked to MCAD,Meat [poison] base deficit disease,Carnitine palmitoyltransferase I ＆ II (CPT I ＆ II),Carnitine palmitoyltransferase lacks,Carnitine palmitoyltransferase lacks I types,Carnitine palmitoyltransferase lacks I types includes infant's severe form including benign typical muscle form,Carnitine transport defect (primary carnitine deficiency disease),Carnosinase deficiency,Carnosinemia,Caroli disease,Carpenter's syndrome,Ka Pengte's,Cartilage-hair hypoplasia,Castleman diseases,Castleman disease hyaline-vascular types,Castleman disease thick liquid cell types,Castleman tumours,Cat's eye syndrome,Cat is cried syndrome,Catalayse is lacked,Cataract- dental composites are levied,The chain syphilitic teeths of Cataractx,Catecholamine hormones,Catel-manzke syndromes,Catel-manzke type palatodigital syndromes,Tail dysplasia,Caudal dysplasia sequence,Caudal dysplasia syndrome,Cusalgia syndrome is grown up,Cvernous hemangioma,Cvernous hemangioma,Cvernous hemangioma,Spongy lymphatic vessel,Spongy deformity,Cayler syndromes,Cazenave leucoderma,CBGD,CBPS,CCA,CCD,CCHS,CCM syndromes,CCMS,CCO,CD,CDG1a,CDG1A,CDGS Ia types,CDGS,CDI,CdLS,Celiaca,Sprue,Abdominal cavity sprue-dermatitis,Cellular immunity deficiency and purine nucleoside phosphorylase deficiency,Celsus leucoderma,Central choking,Central core disease,Central diabetes insipidus,Center forms multiple neurofibromatosis,Maincenter hypoventilation,Centric sleep apnea,Telecentricity fat nutritional disorders,Centronuclear myopathy,CEP,Naoning tablet,Head (with) thorax fat nutritional disorders,Ceramide trihexosidase deficiency,Cerebellar hypoplasia,Cerebellar hypoplasia,Cerebellum hemiageusia,Cerebellar hypoplasia,Vermis of cerebellum hypoplasia,The dynamic incoordination of vermis of cerebellum hypoplasia-hypernea-curtain formula eye is slow,Cerebellar syndrome,Cerebellarparenchymal diseases IV,Celand-Arnold-Chiari syndrome syndrome,Cerebellum Oculocutaneous telangiectasis,Cerebellarparenchymal disease IV familials,Cerebellopontine angle tumours,Cerebral arachnoiditis,Brain autosomal inheritance cerebrovascular disease is with infarct and leukodystrophy under cortex,Wernicke-Korsakoff syndromes,Cerebral diplegia,Cerebral gigantism,Cerebral ischemia,Cerebrovascular malformation,Cerebral paralysis,Brain oculorenal is malnutritive,Brain eye facial skeleton syndrome,Cerebro-costo-mandibular syndrome,Cerebrohepatorenal syndrome,Cerebromacular degeneration,Cerebromuscular malnutrition Fukuyama types,Brain (with) agenesis of eye,Brain (with) agenesis of eye leyden-Mobius myodystrophia,Cerebro-oculo-facio-skeletal syndrome,Cerebroretinal arteriovenous aneurysm,Cerebroside lipidosis,Brain glucoside (deposition) disease,Brain-tendon xanthomatosis,Cerebrovascular Ferrocalcinosis,Ceroid lipofuscinosis adult form,Cervical dystonia,Cervical dystonia,Neck eye listens distress syndrome,Cervical spinal stenosis,Cervical vertebral body is merged,CES,CF,CFC syndromes,CFIDS,CFND,CGD,CGF,General hair Chalasodermia,Chanarin Dorfman diseases,Chanarin Dorfman syndromes,Chanarin Dorfman fish scale ringworms,Chandler syndromes,Charcot diseases,Charcot-Marie-Tooth atrophy,Figure thinks (family name) disease,Charcot-Marie-Tooth disease,Figure thinks (family name) disease,Charcot-Marie-Tooth disease mutation,Charcot-Marie-Tooth-Roussy-Levy diseases,CHARGE association,Charge syndromes,CHARGE syndromes,Chaund is ectodermal dysplasia,Chédiak-Higashi syndrome,CSH syndromes,Granulomatous cheilitis,Harelip,Chemke syndromes,Cheney syndromes,Cherry red spot and myoclonic syndrome,CHF,CHH,Chiari diseases,Chiari deformities I,Chiari deformities,ChiariI types (chiari deformities I),Chiari II types (chiari deformities II),Chiari I pattern synthesis is levied,Chiari-Budd syndromes,Chiari-Arnold syndrome,Chiari deformities II,Child syndrome,Children's fish scale ringworm,Child syndrome ichthyosis,Children's adrenoleukodystrophy,Children with dermatomyositis,Children's hair style myodystony,Children's cyclic vomiting,Children's giant axonal neuropathy,Childhood hypophosphatasia,Childhood muscular dystrophy,CHN,Cholestasia,Cholestasia genotype Norway type,Intrahepatic cholestasis,Neonate's cholestasia,Oral contraceptive user's cholestasia,Cholestasia and peripheral pulmonary artery stenosis,Cholestasis of pregnancy,Cholesterol desmolase deficiency,Chondrodysplasia punctata,Heng Naman (family name) syndrome,Fetal cartilage nutritional disorders,Chondrodystrophic myotonia,Chondrodystrophy,Chondrodystrophy and clubfoot,Epiphyseal cartilage dystrophia,Hyperplasia form chondrodystrophy,Chondroectodermal dysplasia,Achondroplasia,chondrohystrophia,Chondro-osteodystrophy,Choreoacanthocytosis,Chorionic villi sampling,Choroidal abnormalities,Choroidal abnormalities and ACC,Chorireninal defect-Joubert syndromes,Choroidal sclerosis,Choroideremia,Tip-many and zygodactyly syndrome,Christ-Siement-Touraine syndrome,Christ-Siement-Touraine syndrome,Christmas Day disease,Christmas disease,No. 3 chromosome deficiency distal end 3p,No. 3 distal chromosome 3p monosomy,No. 3 distal chromosome 3q2 overlap,No. 3 bodies of distal chromosome 3q2 tri-,No. 3 chromosome monosomy 3p2,Chromosome 3q partly overlaps syndrome,Chromosome 3q,Partial trisomy syndrome,No. 3 chromosome-trisomy 3q2,No. 4 chromosome 4q31-qter deletion syndromes,No. 4 chromosome 4q32-qter deletion syndromes,No. 4 chromosome 4q33-qter deletion syndromes,No. 4 chromosome long arm missings,No. 4 chromosome long arm missings,No. 4 chromosome monosomy 4q,No. 4 chromosome monosomy 4q,No. 4 chromosome monosomy distal side 4q,No. 4 partial deletion of chromosome 4p,No. 4 chromosomes,Local deletion of short arm,No. 4 chromosomal section monomer distal side 4q,No. 4 chromosomal section monomer 4p,No. 4 bodies 4 (q25-qter) of chromosomal section three,No. 4 bodies 4 (q26 or q27-qter) of chromosomal section three,No. 4 bodies 4 (q31 or 32-qter) of chromosomal section three,No. 4 body 4p of chromosomal section three,No. 4 chromosomal sections three body 4q2 and 4q3,No. 4 body of chromosomal section three distal ends 4,No. 4 rings,No. 4 chromosome 4q terminal deletion syndromes,Chromosome 4q syndromes,Chromosome 4q syndromes,No. 4 chromosome trisomies 4,No. 4 chromosome trisomy 4p,No. 4 chromosome x Y/47 XXY (Mosiac),No. 5 chromosome monosomy 5p,No. 5 chromosomes,Local deletion of short arm syndrome,No. 5 chromosome trisomy 5p,No. 5 chromosome trisomy 5p are all (5p11-pter),The 5p parts (5p13 or 14-pter) of No. 5 chromosome trisomies,Chromosome 5p syndromes,No. 6 body 6q of chromosomal section three,No. 6 rings,No. 6 chromosome trisomy 6q2,Monomer 7p2 on No. 7 chromosomes,No. 7 chromosomal section deletion of short arm (7p2-),No. 7 chromosome terminal 7p lack [del (7) (p21-p22)],No. 8 chromosome monosomy 8p2,No. 8 chromosome monosomy 8p21-pter,No. 8 partial deletion of chromosome (galianconism),No. 8 chromosomal section monomer 8p2,The complete three bodies 9P of No. 9 chromosomes,No. 9 partial deletion of chromosome galianconism,No. 9 chromosomal section monomer 9p,No. 9 chromosomal section monomer 9p22,No. 9 chromosomal section monomer 9p22-pter,No. 9 body 9P of chromosomal section three include,No. 9 rings,No. 9 body 9p of chromosome four,No. 9 body 9p of chromosome four inlay,No. 9 chromosome trisomy 9p (multiple mutation),No. 9 chromosome trisomies 9 (pter-p21 to q32) include,No. 9 chromosome trisomies are inlayed,No. 9 chromosome trisomies are inlayed,No. 10 body 10q of distal chromosome three,No. 10 chromosome monosomies,No. 10 chromosome monosomy 10p,No. 10 chromosomes,Excalation (galianconism),No. 10 chromosomes,10p- is local,No. 10 chromosomal section chromosome 10q24-qter,No. 10 chromosome trisomy 10q2,No. 11 chromosome long arm partial monosomies,No. 11 chromosomal section monomer 11q,No. 11 bodies of chromosomal section three,No. 11 body 11q13-qter of chromosomal section three,No. 11 body 11q21-qter of chromosomal section three,No. 11 body 11q23-qter of chromosomal section three,Chromosome 11q,Partial trisomy,No. 12 chromosome isochromosome 12p inlay,No. 13 chromosomal section monomer 13q,No. 13 chromosomes,Arm portion monomer,No. 14 rings,No. 14 chromosome trisomies,No. 15 body 15q of distal chromosome three,Chromosome r15,No. 15 rings,No. 15 chromosome trisomy 15q2,Chromosome 15q,Partly overlap syndrome,No. 17 chromosome deletion 17p,Grouchy-Royer-Salmon-Lumy syndrome,No. 18 chromosome monosomy 18p,No. 18 chromosome monosomy 18Q,No. 18 rings,No. 18 body 18p of chromosome four,Chromosome 18q syndromes,No. 21 syndromes of chromosomal mosaic 21,No. 21 rings,No. 21 syndromes of chromosome translocation 21,No. 22 chromosomes are overlapping (22pter-22q11),No. 22 bodies of chromosomal section three (22pter-22q11),No. 22 rings,No. 22 chromosome trisomies are inlayed,The xxyy of chromosome 48,The xxxy of chromosome 48,Chromosome r15,Chromosome trisomy,Chromosome trisomy,Trisome syndrome,X chromosomes,Chromosome x XY,Chronic acholuric jaundice,Chronic arachnoid adhesion,Addison disease,Carotid cavernous body is scorching,Congenital chronic aplastic anemia,Chronic dysphagocytosis,Familial chronic granuloma,Chronic familial jaundice,Chronic fatigue immune dysfunction syndrome (CFIDS),Chronic granulo matosis,Chronic guillain-Barre syndrome,Chronic idiopathic jaundice,Chronic special hair polyneuritis (CIP),Chronic inflammation demyelinating polyneuropathy,Chronic inflammation demyelinating neuropathy,Chronic motor tic,Chronic mucocutaneous candidiasis,Chronic multiple is twitched,Chronic nonspecific ulcerative colitis,Chmnic. obstructive's cholangitis,Chronic peptic ulcer and esophagitis syndrome,Chronic progressive chorea,Chronic progressive ballet's disease syndrome,Chronic progressive ballet's disease and myopathy,Chronic progressive ballet's disease and ragged red fiber,Chronic recurrent neuropathy,Chronic sarcoid,Chronic spasm,Children chronic is vomitted,CHS,Churg-Strauss syndrome,Cicatricial pemphigoid,CIP,The congenital pigment of hepatic sclerosis,Hepatic sclerosis,cistinuria,Citrullinemia,CJD,Typical Schindler diseases,The general syndrome of classic form,Classic form Pfeiffer syndromes,Classical haemophilia,Canonical form Cockayne syndrome I types (A types),Typical Leigh disease,Typical PKU,The typical chain Pei of x-plum Er Shi cerebrosclerosis,CLE,Harelip/cleft palate mucinous cyst lower lip PP finger-like and genitals are abnormal,Harelip-cleft palate blepharophimosis lagophthalmos and broadening,Harelip/cleft palate thumb deformity and microcephaly's deformity,The cleft palate contracture of joint-stretcher walking aid deformity,Cleft palate and harelip,Clavicular skull hypoplasia w/ micrognathias (disease),Lack thumb ＆ distal ends aphalangia (toe) deformity,Agenesis of clavicle,Clavicular skull hypoplasia,Click murmur syndrome,CLN1,Palmospasmus,Cloustons syndromes,Clubfoot,CMDI,CMM,CMT,CMTC,CMTX,COA syndromes,Aortic coaractation,Coats disease,Cobblestone dysplasia,Cochin Jewish diseases,Cockayne syndrome,COD-MD syndromes,COD,Coffin Lowry syndromes,Coffin syndromes,Coffin Siris syndromes,COFS syndromes,Cogan corneal dystrophies,Cogan reese's syndromes,Cohen syndrome,Cold coagulation disease,Antibody disease,Cold antibody hemolytic anemia,Ulcerative colitis,Ulcerative colitis,Ulcerative colitis chronic nonspecific ulcerative colitis,Collodion baby,Defect heart defect locking nostril retarding of growing development urogenital system exception and abnormal ear,Defect,Colon neurosis,Colour blindness,Colour blindness,Colpocephaly,Columnar samples oesophagus,With reference to cone rod cell denaturation,Combined immunodeficiency and immunoglobulin,Joint mesoectodermal defect,Common variable hypogammaglobulinemia,Common variable immunodeficiency,Common ventricle,Communicating hydrocephalus,Complete lack of hypoxanthine-guanine phosphoribosyl transferase,Treatment of Complete Atrioventricular Defect,The inhibitor of complement component 1 lacks,Complement component c1 modifying ingredients is lacked,Complete cardiac conduction block,Complex carbohydrate is not tolerated,Complicated regional pain syndrome,Complicated V atp synthases missing,Composite I,Composite I nadh dehydrogenase defect,Complex II,Complex II succinate dehydrogenase,Succinate dehydrogenase deficiency disease,Complex II I,Complex II I Co-Q10 cytochrome c oxidoreductase defects,Complex IV,Complex IV cytochrome C oxidase defect,Complex IV is lacked,Compound V,Cerebral concussion,Bore rod cell denaturation,Bore rod cell denaturation progress,Cone dystrophy,Bore rod cell malnutritive,Converge reticulated papillomatosis,Congenital low pk dynamics,It is congenital without abdominal muscles,Congenital athymia and parathyroid gland,Albinism,Congenital Addison's disease,Adrenal,congenital hyperplasia,Adrenal,congenital hyperplasia,Congenital afibrinogenemia,Congenital alveolar hypoventilation,Neonatal Congenital anaemia,Congenital bilateral persylvian syndromes,Congenital brown's syndrome,Congenital heart defect,The low hypopnea syndrome of congenital central,Congenital cerebral palsy,Congenital cervical vertebra bony union,The tight simultaneously MR of Congenital Thumb,CCA (toe),Congenital multiple contracture and arachnodactyly,Congenital cyanosis,Congenital craniofacial and scalp defect,Stones in intrahepatic bile duct congenital dilatation,Congenital dysmyelination,Congenital dysphagocytosis,Congenital sexual abnormality blood vessel dilatation,CEP,Congenital factor XIII deficiency diseases,It is congenital can not autonomous control breathing,Nonhemolytic jaundice,congenital familial I types,Congenital family's delayed diarrhea,Congenital form Cockayne syndrome II types (Type B),Congenital generalized fibromatosis,Congenital German measles,Congenital giant axonal neuropathy,CHB,Congenital heart defect,Congenital Hemidysplasia and fish scale sample erythroderma and physical impairment,Acholuric familial jaundice,Congenital hemolytic anemia,Congenital hepatic fibrosis,Congenital hereditary corneal degeneration,Congenital hereditary lymphedema,Congenital hyperchondroplasia,Congenital hypomyelinating DPNs,Congenital hypomyelination neuropathy,Congenital hypomyelination,Congenital hypomyelination (Onion Bulb) DPN,Congenital ichthyosis sample erythroderma,Congenital keratoconus,Congenital hyperlactacidemia,Congenital lactose intolerance,Congenital lipodystrophia,Congenital cirrhosis,CLE,Congenital localized emphysema,Congenital macroglossia (disease),Congenital marrow is narrow,Congenital megacolon,Congenital mole,Congenital mesoderm dysmorphodystrophy,Congenital mesoderm is malnutritive,Congenital microvillus atrophy,Congenital multiple joint,Congenital muscular dystrophy,Congenital neuropathy caused by hypomyelination,Congenital whole blood trace elements,Congenital pernicious anemia,Congenital pernicious anemia is due to lacking intrinsic factor,Congenital pernicious anemia is due to lacking intrinsic factor,Congenital pigmentation,Congenital porphyria,Congenital peri position myopathy and desmin storage myopathy,Congenital emphysema,Congenital pure red cell anaemia,Congenital dyserythropoietic anemia,Congenital retinal is blinded,Congenital cyst of retina,Congenital retinitis pigmentosa,Congenital retinal is cleaved,Congenital Rod diseases,Congenital rubella syndrome (CRS),Congenital scalp defects and distal limb defect,Congenital sensory neuropathy,Congenital SMA and arthrogryposis,Congenital congenital hemolytic jaundice,Pediatric congenital myogenic torticollis,Congenital bolt neck marrow syndrome,Congenital tyrosinosis,Congenital varicella syndrome,Congenital cavernous malformations,Congenital vascular covers retina,Congenital word blindness,Congenital splenectopia (paediatrics),Congestive cardiomyopathy,Keratoconus,Conjugated hyperbilirubinemia,Conjunctivitis,Conjunctivitis Ligneous,Conjunctivo-urethro-synovial syndrome,Conn syndromes,CTD,Conradi disease,Conradihunermann syndromes,Systemic aplastic anemia,Constitutional red blood cell development is not complete,Constitutional eczema,Constitutional dyshepatia,Constitutional thrombopathy blood platelet,Restrict congenital rank,Constrictive pericarditis and nanism,Continuous muscle fibre activity syndrome,Contracted arachodactylia (toe) syndrome,The double-legged muscular atrophy contracture and utilization of dynamic eye (motion) can not,Chi Zong,Cooley anaemias,Copper transports disease,Fecal porphyria,Cor triatriatum,Cor triatriatum Sinistrum,Cor triloculare biatriatum,Cor biloculare,Cori disease,Corneal dystrophy,Cornea amyloidosis,The opacity of the cornea-skin Laxa- MRs,Corneal dystrophy,Cornelia de Lange syndromes,Corona hypoplasia of dentin,Coronary heart disease,Coronary heart disease,Agenesis of corpus callus,Corticobasal ganglionic is denatured,Crust deformity,Corticobasal ganglionic is denatured (CBGD),Corticobasal is denatured,Cortex methloxidase lacks I types,Cortex methyloxidase lacks II types,Cortisol,Costello syndromes,SIDS sudden infant death syndrome,COVESDEM syndromes,COX,COX is lacked,The Canadian type of COX defects France,COX defect babies mitochondrial myopathy includes deToni-Fanconi-Debre,The benign infant's mitochondrial myopathy of COX deficiencies,CP,CPEO,CPEO and myopathy,CPEO and ragged red fiber,CPPD familial forms,CPT defects,CPTD,Cranial arteritis,The meningoencephalocele of cranium,Cranio-Oro-Digital syndromes,Cranio-carpo-tarsal dystrophy,Naoning tablet,Cranium syndrome MR Scott types,Craniofacial dysostosis,Craniofacial dysostosis-PD arteries-hirsutism-atelocheilia,Craniofrontonasal dysplasia,Craniometaphyseal dysplasia,Cranioorodigital syndromes,Cranioorodigital syndrome i I types,Craniostenosis crouzon types,Craniostenosis,Craniosynostosis nasal atresia radius humerus bony union,Craniosynostosis hirsutism face and other exceptions,Craniosynostosis mid facial hypoplasia and foot deformity,Craniosynostosis is primary,Craniosynostosis-radial aplasia syndrome,Craniosynostosis and radial segmental defect,Cranioschisis,CREST syndromes,Creutzfeldt-Jakob disease,Cat's cry syndrome,Cot death,Crigler-Najjar syndrome I types,Clone disease,Cronkhite-Canada syndrome,Cross syndromes,Cross syndromes,Cross-McKusick-Breen syndrome,Crouzon,Crouzon syndromes,Crouzon craniofacial dysostosis,Cryoglobulinemia primary is mixed,Cryptophthalmus-syndactyly syndrome,Cryptorchidism-dwarf-feeblemindedness,Shi Naide (family name) crystalloid corneal dystrophy,CS,CSD,CSID,CSO,CST syndromes,Hair crimping ankyloblepharon nail dysplasia,Curschmann-Battern-Steinert,Curth Macklin type fish scales hystric,CurthMacklin types,Cushing’s,Cushing's syndrome,Cushing’s III,The heredity of malignant melanoma of skin,Cutaneous porphyria,Cutis laxa,Cutis laxa-growth deletion syndrome,Cutis marmorata telangiectatica congenita,CVI,CVID,CVS,Cyclical vomiting syndrome,Kidney medulla disease,Cystic hygroma,Cystic fibrosis,Cystic lymphangioma,Cystine-Lys-Arg ornithinuria,Cystinosis,Abderhalden-Kaufmann-Lignac syndrome,Cystinuria,Cystinuria and dibasic aminoaciduria,Cystinuria I types,Cystinuria II types,Cystinuria type III,Congenital kidney medulla tumour,Cytochrome c oxidase defect,D.C.,dacryosialoadenopathy,dacryosialoadenopathia,dalpro,Dalton,Colour blindness,Danbolt-Cross syndromes,Twitching of the eyelid is stamped one's foot syndrome,Dandy-Walker syndrome,Dan Di-Wo Ke tumours,Dan Di-Wo Ke deformities,Dan Di-Wo Ke deformities,Danish heart-type amyloidosis (type III),Darier disease,Davidson diseases,Davies diseases,DBA,DBS,DC,DD,De Barsy syndromes,De Barsy-Moens-Diercks syndromes,De Lange syndrome,De Morsier syndromes,De Santis Cacchione syndromes,De Toni Fanconi syndrome,Congenital deafness and functional cardiac disease,Deafness-dwarf-neurodeatrophia,Deafness-functional cardiac disease,Deafness refers to (toe) Onychodystrophy osteodystrophy and baryencephalia,Deaf and curly hair Bjornstad types,Nerve deafness and hedratresia and thumb hypoplasia,Debrancher deficiency,Deciduous skin,Enterocyte intrinsic factor receptor lacks,Constant killer cell lacks,Carnitine kidney reabsorption defect,Glycoprotein neuraminidase lacks,Mitochondrial respiratory chain complex IV defect,Lack platelet membrane glycoprotein ib,Lack von Willebrand factor acceptor,Short chain acyl coenzyme dehydrogenase deficiency (ACADS),Mesomelic dwarfism deformity,Degeneration chorea,Treatment of Degenerative Lumbar Spinal Canal Stenosis,Degos' disease,Degos-Kohlmeier diseases,Netted pigmentation disease,DEH,Dejerine-Roussay syndrome,Dejerine-Sottas disease,9p minus syndrome is local,11q deletion syndromes are local,13q minus syndrome is local,Delleman-oorthuys syndromes,Delleman syndromes,Dull-witted and lobe of the lung atrophy and Neuronal cytoplasmic inclusions,Demyelinating disease,DeMyer syndromes,Hypoplasia of dentin corona,Hypoplasia of dentin root of the tooth,Hypoplasia of dentin I types,Hypoplasia of dentin II types,Hypoplasia of dentin brandywine types,Hypoplasia of dentin shielded type,Hypoplasia of dentin type III,Tooth-eye-dysosteogenesis,Dentooculocutaneous syndromes,Denys-Drash syndromes,Depakene,DepakeneTM exposes,Divalproex sodium,Depakote Sprinkle,Discoloration-gingival fibroma-microphthalmia,Dercum disease,Atopic dermatitis,Exfoliative dermatitis,Dermatitis herpetiformis,Multiformity dermatitis,General hair property Dermatochalasia,General hair property Dermatochalasia,Dermectasia,Dermatomyositis sine myositiss,Dermatomyositis,Dermatosparaxis,Dermatostomatitis StevensJohnson types,Desbuquois overstates syndrome,Desmin stores myopathy,Neonate peels,Deuteranomalia,Developmental reading dyslexia,Develop Gerstmann syndrome,De Veulle Ji disease,Devic's disease,Neuromyelitis optica,Dextrocardia-bronchiectasis and nasosinusitis,Dextrocardia and visceral reversal,DGS,DGSX Golabi-Rosen syndromes include,DH,DHAP alkyl-transferases are lacked,DHBS is lacked,DHOF,DHPR is lacked,Diabetes insipidus,Diabetes insipidus diabetes optic atrophy and deafness,Diabetes insipidus is basophilic adenoma of pituitary,Diabetes insulin dependent form,Diabetes,Diabetes Addison's disease oedema,Diabetic ketoacidosis,Diabetic bearded woman syndrome,Diabetic neuropathy,Diamond-Blackfan anemia,Diaphram apnea,Diaphysial aclasis,Diastrophic dwarfism,Diastrophic dysplasia,Diastrophic nanism syndrome,Dicarboxylic aminoaciduria,The dicarboxylic aciduria that aliphatic acid beta oxidation defect is caused,The dicarboxylic aciduria that aliphatic acid beta oxidation defect is caused,The dicarboxylic aciduria that MCADH defects are caused,Dichromasia,Dicker-Opitz,DIDMOAD,Diencephalic syndrome,Children's diencephalic syndrome,Become thin diencephalic syndrome,Dienoy1- CoA reductase defects,Baby diffusivity cerebral degeneration,Diffusivity brain degenerative disease,Diffusivity idiopathic osteoproliferation,Diffusum-Glycopeptiduria,DiGeorge syndromes,Digital-Oro-Cranio syndromes,Digito-Oto-Palatal syndromes,Digito-Oto-Palatal syndrome I types,Digito-Oto-Palatal syndrome II types,Dihydrobiopterin synthetase deficiency,Dihydropteridine reductase deficiency,Dihydroxyacetone phosphate synthase,Expanding (hyperemia) cardiomyopathy,Dimitri disease,The diplegia of cerebral paralysis,Diplo-Y syndromes,Disaccharidase is lacked,Disaccharide intolerance I,Lupus erythematosus discoides,Lupus erythematosus discoides,DISH,Keratosis,Keratosis I types,Keratosis 4,Keratosis 6,Keratosis 8,The Netherton types of keratosis 9,The phytane acid type of keratosis 11,Keratosis 12 (neutral fats memory type),Keratosis 13,Keratosis 14,The hair sulphur dystrophia type of keratosis 14,Keratosis 15 (keratitis deafness type),Keratosis 16,The form variation crythrokeratodermia type of keratosis 18,Keratosis 19,Keratosis 20,Keratosis 24,Spleen is shifted,Lupus erythematosus disseminatus,Disseminated neurodermatitis,Multiple sclerosis,Distal end 11q monomers,Distal end 11q syndromes,Distal end AMC IIA types,Distal end AMC IIA types,Far end arthrosis bend IIA types,Far end arthrosis bend 2A types,Distal end repeats 6q,Distally overlapping 10q,Repeat (10q) syndrome,Distally overlapping 15q,Distal end monomer 9p,Distally three body 6q,Distally three body 10q syndromes,Distally three body 11q,Divalproex sodium,DJS,DKC,DLE,DLPIII,DM,DMC syndromes,DMC diseases,DMD,Hereditary DNS,Doc I,Doc 2,Doc 4,Doc 6 (Harlequin types),The Curth-Macklin types of Doc 8,The phytane acid types of Doc 11,Doc 12 (neutral fats memory type),Doc 13,Doc 14,The hair sulphur dystrophia types of Doc 14,Doc 15 (keratitis deafness type),Doc 16,The UnilateralHemidysplasia types of Doc 16,Doc 18,Doc 19,Doc 20,Doc 24,Sinus Le Shi bodies-myelopathy,Dolichospondylic dysplasia,Dolichostenomelia (toe),Dolichostenomelia (toe) syndrome,Phenotype Kenny-Caffe syndromes,Phenotype myotonia congenita,Donahue syndromes,Donath-Landsteiner hemolytic anemias,Donath-Landsteiner syndrome,It is deaf-to refer to (toe) first hypoplasia-dysosteogenesis-MR syndrome,It is deaf-to refer to (toe) first hypoplasia-dysosteogenesis-MR syndrome,DRD,DorfmanChanarin syndromes,Dowling-Meara syndromes,Down's syndrome,DR syndromes,Drash syndromes,DRD,Dreifuss-Emery types muscular dystrophy and contracture,Postmyocardial infarction syndrome,Drift about spleen,Drug-induced acanthosis nigricans,Drug-induced lupus erythematosus,Medicine correlation adrenal insufficiency,Drummond syndromes,Dry type athlete's foot,Xerophthalmia,DTD,Duane retraction syndromes,Duane's syndrome,Duane's syndrome IA 1B and 1C types,Duane's syndrome 2A 2B and 2C types,Duane's syndrome 3A 3B and 3C types,1867 johnsen syndromes,Dubin-Johnson syndrome,Dubowitz syndrome,Progressive Erb's atrophy,Duchenne paralyses,Duhring diseases,Duncan diseases,Duncan diseases,Duodenal atresia,Duodenal stenosis,Duodenitis,Overlapping 4p syndromes,Repeat 6q local,Dupuy syndromes,Dupuytren contractures,Dutch-Kennedy syndromes,Nanism,Nanism campomelic,Nanism tubular bone cortex is thickened and instantaneous low calcium,Nanism Levi types,Tropism between nanism,Nanism-onychodysplasia,Nanism-pericarditis,Nanism and nephrarctia and deafness,Nanism and rickets,DWM,Dyggve MelchiorClausen syndromes,Dysautonomia,Familial dysbetalipoproteinemia,Dyschondrodysplasia and hemangioma,Dyschondrosteosis,Dyschromatosis universalis hereditaria,Dysencephalia splanchnocystica,Congenital dyskeratosis,Congenital dyskeratosis autosomal recessive inheritance,Congenital dyskeratosis Scoggins type,Congenital dyskeratosis syndrome,Follicular dyskeratosis Vegetans,Dislexia,Dysmyelogenic leukodystrophy,Dysmyelogenic leukodystrophy-megalobare,Dysphonia spastica,Heng Naman (family name) syndrome,Epiphysis osteodysplasty Hemimelica,Nail dysplasia and metodontiasis,Clavicle atelocephaly,Fibre Development is abnormal,Dysplasia giant's syndrome X is chain,Osteodentin dysplasia,Dysplastic nevi syndrome,Dysplastic nevus type,Dyssynergia cerebellaris myoclonica,Oesophagus dyssynergia,Myodystony,Dystopia canthi,Adiposogenital dystrophy,Malnutritive endothelitis cornea,Malnutritive mesodermalis,Malnutrition bullous epidermolysis,It is malnutritive,Asphyxiating thorax,Myotonia dystrophy,E-D syndromes,Eagle-Barrett syndromes,Retina Eales,Periphlebitis of retina,Ear exception-contracture-osteodysplasty and scoliokyphosis,The short and small syndrome of ear kneecap,Early stage about harness defects,Early stage hypercalcemia syndrome and Elfin Facie,Early onset myodystony,Eaton Lambert syndromes,EB,Ebstein is abnormal,EBV neurological susceptibilities (EBVS),EBVS,ECD,ECPSG,It is ectodermal dysplasia,Ectodermal dysplasia and cleft lip and cleft palate,Ectodermal dysplasia-exocrine pancreas insufficiency,Ectodermal dysplasia Rapp-Hodgkin types,Ectoderm and mesoderm depauperation are congenital,Ectoderm and mesoderm depauperation are participated in bone,Ectodermosis erosiva pluriorificialis,Ectopia lentis,Ectopia vesicae,Ectopic ACTH syndrome,Ectopic adrenocorticotropic hormone syndrome,Hedratresia,Hand ectrodactyly,Adactylism (deformity),Adactylism (toe)-ectodermal dysplasia-cleft lip and palate syndrome,Adactylism (toe)-ectodermal dysplasia-cleft lip and palate syndrome,Adactylism (toe)-ectodermal dysplasia-harelip,Eczema,Eczema-decrease of platelet acquired immunodeficiency syndrome,EDA,EDMD,EDS,EDS artery ecchymosis types,EDS arthrochalasis,EDS typical case's severe forms,EDSdysfibronectinemic,EDS is heavy,EDS hyperkinesias,EDS scoliokyphosis,EDS scoliokyphosis,EDS mitigation types,EDS eyeball scoliosis,EDS Progeroid,EDS periodontosis,EDS blood vessels,EEC syndromes,EFE,EHBA,EHK,Hlers-Danlos syndrome,Hlers-Danlos syndrome,Ai Lesi-as Lip river IX,Eisenmenger complex,Eisenmenger complex,Ai Senmangeer diseases,Ai Senmangeer reacts,Eisenmenger's syndrome,Eisenmenger's syndrome,Ekman-lobstein diseases,Hand ektrodactyly,EKV,Elastosis,Extensive elastic fibrous tissue (fiber) rupture,Elastosis dystrophica's syndrome,Elective mutism (obsolete),Elective mutism,Electrocardiogram (ECG or EKG),Electron transfer flavoprotein (ETF) dehydrogenase deficiency：(GAII & MADD),Electrophysiologic study (EPS),Birth is as nail,Elephantiasis congenital angiomatosis,Distensibility of blood vessel is loose,Elfin facies and hypercalcinemia,Ellis-van Creveld syndrome,Ellis-van Creveld syndrome,Embryoma kidney,Embryonal adenomyosarcoma kidney,Embryo's renal cancer kidney,Embryo's mixed rumour kidney,EMC,Emery Dreyfus muscular dystrophies,Ecuador's syndrome is malnourished,Diamond dust-dreifuss pattern synthesis is levied,EMF,EMG syndromes,Empty sella syndrome,Encephalitis periaxialis,Encephalitis periaxialis concentrica,Naoning tablet,Cranium face angiomatosis,Encephalopathic,Encephalotrigeminal angiomatosis,The multiple cvernous hemangioma of chondrodysplasia,Endemic multiple neuritis,Endocardial cushions defect,Endocardial cushions defect,Endometrial hyperplasia,Endocardial fibroelastosis (EFE),Endogenous hypertriglyceridemia,Endolymphatic hydrops,Endometrial growth,Endometriosis,Myocardium internal membrane of heart fibrosis,The congenital malnutrition of corneal endothelium,Esoderma cornea epithelium is malnutritive,Endothelium,Engelmann disease,Macroglossia,Enterocolitis,Enterocyte vitamin B12 malabsorption,Eosinophia syndromes,Acidophilia cellulitis,Eosinophilic fasciitis,Eosinophilic granuloma,Eosinophil syndrome,Epidermal nevus syndrome,Epidermolysis bullosa,Acquired bleb epidermidolysis,Epidermolysis bullosa hereditaria,Epidermolysis bullosa is lethal,Epidermidolysis Hereditaria Tarda,Epidermolytic hyperkeratosis (disease),Epidermolytic hyperkeratosis (disease) (bleb CIE),Cursive epilepsy,Epilepsy,Adrenaline,Epiphysis changes and high myopia,Epiphyseal cartilage knurl is benign,Epiphysealis Hemimelica dysplasia,Accidental abnormal eye movement,Basement membrane of epithelium corneal dystrophy,Meesmann teenager's dystrophia epithelialis corneae,Multiple epitheliomatosis is with mole,Epithelium,Epival,EPS,Male's Epstein-Barr virus induction lymphoproliferative diseases,Eaton-Lambert myasthenic syndrome,Erdheim Chesters disease,Erythema multiforme oozes out,Multiform (property) erythema Stevens Johnson types,erythroblastophthisis,Fetal red blood cells,Neonate's erythremia,Children's erythroblastosis anaemia,Red blood cell phosphoglycerate kinase deficiency,Infull property red blood cell occurs,Progressive erythrokeratodermia is symmetrical,The symmetrical ichthyosis of progressive erythrokeratodermia,Erythrokeratodermia variabilis,Erythrokeratodermia variabilis type,Erythrokeratolysis hiemalis,Erythropoietic porphyria,Erythropoietic porphyria,Ai Sikewaer syndromes,Atresia oesophageal,Esophageal peristalsis stops,Esophagitis peptic ulcer,Atresia oesophageal and/or tracheoesophageal fistula,Familial hyperlipemia,essential,Essential fructosuria,Essential hematuria,Essential hemorrhagic thrombocythemia,Essential mixed cryoglobulinemia,Primary moschowitz diseases,Essential thrombocythemia,Essential thrombocytopenia is reduced,Piastrenemia,Essential tremor,Esterase inhibitor defect,Fanconi anemia Estren-Dameshek mutation,The cholestasia relevant with estrogen,ET,ETF,ethylmalonic adipicaciduria,Eulenburg disease,pc,EVCS,Exaggerate startle reaction,Exencephalia (deformity),Exogenous hypertriglyceridemia,Umbilical hernia-macroglossia-giant's syndrome,Anemic goiter,Expanded rubella syndrome,Ectopia vesicae,EXT,External osteochondromatosis syndrome,Extrahepatic biliary atresia,Extramedullary plasmacytoma,Exudative retinitis,Eyes retreat syndrome,FA1,FAA,Fabry disease,FAC,FACB,FACD,FACE,FACF,FACG,FACH,Facioplegia,Facioplegia,Face is ectodermal dysplasia,Face is ectodermal dysplasia,Facio-scapulo-humeral dystrophy,Facio-auriculo-vertebral spectrum,Face-the heart-skin syndrome,Face-volume-nose hypoplasia,Faciocutaneoskeletal syndromes,Face-refer to (toe)-genital disease,Faciogenital dysplasia,Faciogenitopopliteal syndromes,Faciopalatoosseous syndromes,Faciopalatoosseous syndrome i I types,Facioscapulohumeral muscular dystrophy,Artificial hypoglycemia,Factor VIII deficiency,Factors IX deficiency disease,Factor XI deficiency disease,Factor XII deficiency,FXIII defect,Fahr disease,Fahr disease,Stomachial secretion gastric anti-pernicious anemia factor exhaustion,Fairbank diseases,Fallot tetra logys,Family is acrogeria,Family's akromikrie,Familial Adenomatous polyp of colon,Familial adenomatosis polyposis and parenteral performance,Familial alobar holoprosencephaly,Familial alpha-lipoprotein deficiency disease,Familial amyotrophic chorea and acanthocytosis,Familial myoclonia arrhythmia cordis,Familial articular cartilage calcification,Familial atypical mole-malignant mela noma syndrome,Familial broad beta disease,Family's calcium gout,Family's calcium pyrophosphate arthropathy,Familial chronic obstructive pulmonary disease,Family persistently peels,Familial amyloidosis cutis,Familial paraproteinemia,Familial emphysema,Familial enteropathy microvillus,Familial central fixation retinoschisis,Family's hibernation syndrome,Familial high cholesterol,Familial hemochromatosis,Familial hypercholesterolemia,Familial high-density lipoprotein deficiency,Familial high anteserum cholesterol,Family's hyperlipidemia,Family's hypoproteinemia and angioleucitis enteropathy,Icterus gpavis familiaris,The juvenile eye with nephronophtisis of familial is abnormal,Familial lichen amyloidosis (IX types),Familial lumbar spinal stenosis,Familial primary lymphedema,Familial Mediterranean fever,Multiple familial polyposis,Familial neck blister,Familial paroxysmal polyserositis,Familial colon polyp,Familial primary pulmonary hypertension,Familial renal glucosuria,Familial splenic anemia,The astonished disease of familial,Familial visceral amyloidosis (VIII types),FAMMM,FANCA,FANCB,FANCC,FANCD,FANCE,Fanconi panmyelopathys,Fanconi pancytopenias,Fancon III,Fanconi anaemias,Fanconi anaemia I types,Fanconi anaemia complementation groups,Fanconi anaemia complementation groups A,Fanconi anaemia complementation groups B,Fanconi anaemia complementation groups C,Fanconi anaemia complementation groups D,Fanconi anaemia complementation groups E,Fanconi anaemia complementation groups G,Fanconi anaemia complementation groups H,Fanconi anaemia Estren-Dameshek mutation,FANF,FANG,FANH,FAP,FAPG,Farber diseases,Farber lipogranulomas,FAS,Fasting hypoglycemia,Hyperlipidemia caused by fatty,The fatal granulomatosis of children,Fat oxidation obstacle,Fatty liver and encephalopathic,FAV,FCH,FCMD,FCS syndromes,FD,FDH,Febrile mucocutaneous mucous membrane syndrome Stevens Johnson types,Heat generation neutrality acute dermatological,Febrile seizure,Feinberg syndromes,Model [Singh]-Le [Roy] two Cotard,Women puppet Turner's synodrome,The bilateral Robin of femoral hypoplasia is abnormal,Femoral hypoplasia is bilateral,Distal femoral surface syndrome,Femoral hypoplasia-unusual facies syndrome,Fetal alcohol syndrome,Fetus anticonvulsant disease,Fetus cystic hygroma,Alcohol fetus effect,Varicella fetus effect,Reaction stops fetus effect,Varicella virus fetus effect,Myocardium internal membrane of heart fibrosis,Fetus face syndrome,Fetal iritis syndrome,Placental transfusion syndrome,Fetal valproate syndrome,Fetus valproic acid exposes syndrome,Fetal infection varicella,Fetus varicella zoster syndrome,FFDD II types,FG syndromes,FGDY,FHS,Fibrin stabilizing factor deficiency,Fibrinase is lacked,Star (colloid) cell cellulose is denatured,Class (blood) fibrin leukodystrophy,Fibrinoligase is lacked,Desmocytoma,(ductus pancreaticus) mucoviscidosis,(drawing) progressive fibrodysplasia ossificans,Fibrous elasticity tissue endocarditis,Fibromyalgia,Fibromyalgia-fibromyositis,Fibromyositis,Fibrosis cholangitis,Fibrositis,Multi-joint fibrous ankylosis,Fibrous cavernitis,Fibrous dysplasia,Penis fibrous plaque,Penis fibrosclerosis,Fickler-Winkler types,Fiedler's disease,Fifth Digit syndromes,Filippi syndromes,Finnish type amyloidosis (V-type),First order CHB,First and second branchial arch syndromes,Fischer syndromes,Fishiness syndrome,Crack tongue,Adenoma syndrome,Flatau-Schilder disease,Flavin containing monooxygenase 2,Floating beta disease,Floating-Harbor syndromes,Splenectopia,Floppy infant syndrome,Floppy valve syndrome,Fluent aphasia,FMD,FMF,FMO Adult LiverForm,FMO2,FND,Focal cerebral ischemia,Focal dermal hypoplasia syndrome,Focal dermal hypoplasia,Focal skin phalanges hypoplasia,Focal dystonia,Focal seizure,The infull II types of focal dermal facial development,Focal neuromyotonia,FODH,Folling syndrome,Fong diseases,FOP,Glycogen storage disease type III,Forbes-Albright syndrome,Forestier disease,Aland's disease (X is chain),Fothergill disease,Fountain syndromes,Progressivity macular dystrophy,FPO syndrome i I types,FPO,Fraccaro types achondrogenesis (IB types),Fragile X syndrome,Franceschetti-Zwalen-Klein syndromes,Francois dyscephaly syndromes,Francois-Neetens spots are malnutritive,Mottled corneal nutrition is not,Fraser syndrome,FRAXA,FRDA,Fredrickson type I hyperlipoprotememias,Cranio-carpo-tarsal dysplasia,Freire-Maia syndromes,Not thunder Cotard,Friedreich incoordination,Friedreich diseases,Friedreich is weak,FRNS,Froelich syndromes,Frommel-Chiari syndrome,Frommel-Chiari syndrome lactation atrophy of uterus,Volume refers to (toe) syndrome,Frontofacionasal hypoplasia,Frontofacionasal dysplasia,Volume nose dysplasia,Volume nose dysplasia and coronal craniosynostosis,Fructose-1-phosphate aldolase deficiency,Fructosemia,Fructosuria,Fryns syndrome,FSH,FSHD,FSS,Fuchs' dystrophy,Sick 1 type of fucoside (storing up),Sick 2 type of fucoside (storing up),Sick 3 type of fucoside (storing up),Fukuhara syndrome,Fukuyama diseases,Fukuyama type muscular dystrophy,Fumarylacetoacetase is lacked,Ditch tongue,G syndromes,G6PD defects,G6PD,GA I,GA IIB,GA IIA,GA II,GAII & MADD,Overflow breast-amenorrhoea syndrome nonpuerperal,Breast-amenorrhoea overflow without pregnancy,Amine-galactose 6-sulfatase is lacked,Galactose-1-phosphate uridyl transferase is lacked,Galactosemia (disease),GALB is lacked,Galloway-Mowat syndromes,Galloway syndromes,GALT is lacked,Agammaglobulinaemia,GAN,Gangliosides nerve ammonia (sugar) neuraminidase defect,Ganglioside sialidase deficiency,The types of gangliosidosis GM1 1,The types of gangliosidosis GM1 2,Gangliosidosis β hexosaminidases B lacks,Gardner syndromes,Gargoylism,Garies-Mason syndromes,Gasser syndrome,Gastric anti-pernicious anemia factor fails secretion,Enterocyte vitamin B12,Gastrinoma,Gastritis,Stomach oesophagus lacerated wound bleeding,Pipe intestinal polyp and ectoderm change,Gastroduodenal ulcer,Abdomen splits (deformity),Gaucher disease,Gaucher-Schlagenhaufer,Gayet-Wernicke syndromes,GBS,GCA,GCM syndromes,GCPS,Gee-Herter diseases,Gee-Thaysen disease,Gehrig diseases,Southern net Cotard,Genee-Wiedemann syndromes,Systemic myodystony,General race's neuromyotonia of building up a family fortune,General hair fibromatosis,Generalized flexion epilepsy,Generalized glycogenosis,Generalized hyperhidrosis,General hair lipofuscinosis,General hair myasthenia gravis,General hair myotonia,It is systemic to distribute neuromyxoma,Genetic disease,Genitals defect,Reproductive system and urinary system defect,Gerstmann syndrome,Gerstmann Tetrad,GHBP,GHD,GHR,Giant axon disease,Giant axonal neuropathy,Huge benign lymphoma,Giant cell glioblastoma,Giant cell arteritis,Giant cell lesion liver,Giant cell hepatitis,Giant cell neonate's hepatic sclerosis,Retina giant cyst,Castleman disease,Bernard-Soulier syndrome heredity,Macroglossia,Gic macular dystrophies,Gilbert diseases,Gilbert syndrome,Gilbert-Dreyfus syndrome,Gilbert-Lereboullet syndromes,Gilford syndromes,Gilles de la Tourette syndromes,Gillespie syndromes,Gingival fibromatosis-exception finger nail nose ear splenomegaly,GLA lacks,GLA,GLB1,Glaucoma,Retinal glioma,Total aphasia,Globoid leukodystrophy,Glossoptosis micrognathia (disease) and cleft palate,Glucocerebrosidase is lacked,Glucocerebrosidosis,Glucose 6 phosphate dehydrogenase deficiency,G6P trafficking defect,G-6-P salt transfer missing,Glucose G phosphoric acid enzyme deficiency diseases,Glucose-galactose malabsorption,Ceramide lipidosis [disease],Glutaric aciduria I,Glutaric acidemia I,Glutaric acidemia II,Glutaric aciduria II,Glutaric aciduria type II,Glutaric aciduria type III,Glutaric acidemia I,Glutaric acidemia II,Glutaric aciduria I,Glutaric aciduria II,Glutaric aciduria IIA types,Glutaric aciduria IIB types,Glutaryl-CoA dehydrogenase deficiency disease,Glutaurate- aspartic acid trafficking defects,Glutelin sensitive enteropathy,Muscle glycogen disease type VII,Glycogenic thesaurismosis I,Glycogenic thesaurismosis III,Glycogenic thesaurismosis IV,Glycogenic thesaurismosis V-shaped,Glycogenic thesaurismosis VI,Glycogenic thesaurismosis VII,Glycogenic thesaurismosis VIII,Glycogenic thesaurismosis II types,Glycogenic thesaurismosis type II,Glycogenosis,Glycogenosis I types,Glycogenosis IA types,Glycogenosis IB types,Glycogenosis II types,Glycogenosis II types,Glycogenosis type III,Glycogenosis type IV,Glycogenosis V-shaped,Glycogenosis VI types,Glycogenosis type VII,Glycogenosis type VIII,Glycolic aciduria,Glycolipid lipidosis,The type of GM2 gangliosidosis 1,GNPTA,Goitrous autoimmune thyroiditises,Ge Erdengha (family name) syndrome,Goldenhar-Gorlin syndromes,Goldscheider diseases,Ge Erci syndromes,Goltz-Gorlin syndromes,Gonadal agenesis 45X,Gonadal agenesis XO,Goniodysgenesis-metodontiasis,Goodman syndromes,Goodman,Goodpasture's syndrome,Gordon's protein-losing enteropathy,Gorlin syndromes,Gorlin-Chaudhry-Moss syndrome,The congenital erythrokeratodermia of gottron progressive symmetry,Gottron Cotards,Gougerot-Carteaud syndrome,Grand mal,Granular pattern corneal dystrophy,Granulomatous arteritis,Granulomatous colitis,Granulomatous dermatitis and eosinophilia,Granulomatous ileitis,Graves disease,Robert Graves hyperthyroidism,Graves disease,Greig short rib-polydactyly syndromes,Groenouw I type corneal dystrophies,Groenouw II type corneal dystrophies,Graefe-Sjogren syndrome,Grotton syndromes,Growth hormone receptor lacks,Growth hormone binding protein is lacked,Growth hormone deficiency,Myhre Growth-Mental deletion syndromes,Hypoevolutism-Rieger is abnormal,GRS,Gruber syndrome,GS,GSD6,GSD8,GTS,GTP cyclohydrolase is lacked,GTP cyclohydrolase is lacked,Guenther porphyrias,Guerin-Stern syndrome,Guillain-Barré,Guillain-Barre syndrome,Congenital erythropoeitic porphyria,H diseases,H.Gottron syndromes,It is accustomed to spasm,HAE,Congenital factor XII deficiency diseases,Hageman factor (HF),Haim-Munk syndromes,Hajdu-Cheney syndrome syndrome,HajduCheney,HAL is lacked,Hall-Pallister syndrome,Hallermann-Streiff-Francois syndrome,Hallerman-Streiff syndrome,Hallervorden Spatz disease,Hallerman-Streiff syndrome,Hallopeau-Siemens diseases,Thumb repeats postaxial polydactyly and lacks callosity,Halushi-Behcet syndromes,Lymph hamartoma,Hand-Schueller-Christian syndromes,HANE,Hanhart syndromes,Angelman's syndrome,Harada's syndrome,HardE syndromes,HARD syndromes,Harelip,Harlequin fetus,Harlequin types Doc6,Harlequin type ichthyosis,Harley syndromes,Harrington syndromes,Hart syndromes,How Hart flutters disease,How Hart flutters illness,Hartnup syndrome/disease,Hashimoto diseases,Hashimoto-Pritzker syndromes,Hashimoto syndromes,Hashimoto thyroiditis,Hashimoto-Pritzker syndromes,Hay Well syndromes,Hay-Wells syndrome of ectodermal dysplasia,HCMM,HCP,HCTD,HD,Heart-hand syndrome (Holt-Oram types),Heart disease,Hecht syndrome,HED,Heerferdt-Waldenstrom and Lofgren syndromes,Hegglin diseases,Heinrichsbauer syndromes,Hemangioma,Familial hemangioma,Hemangioma-thrombocytopenia syndrome,Chondrodystrophic dwarf,Hemangioma gill slit lip pseudocleft syndromes,Demifacet (stature) is short and small,Hemimegalencephaly,Cerebral paralysis hemiparesis,Cerebral paralysis hemiplegia,Spinal cord half is cut off,Hematochromatosis (disease),Hemochromatosis syndrome,Dialysis associated amyloidosis,Hemoglobin touch-control syndrome,Hemolytic anemia neonate,Cold cut hemolytic hemolytic anemia antibody,Neonatal hemolytic disease,Hemolytic uremic syndrome,Hemophilia,Hemophilia A,Hemophilia B,Hemophilia B factors IX,Congenital XI factor deficiency,Hemorrhagic decrease of platelet is malnutritive,Hemorrhagica Aleukia,Iron xanthematin thesaurismosis,Hepatic fructokinase is lacked,Liver phosphokinase is lacked,Hepatic porphyria,Hepatic porphyria,HVOD,Hepatitis C,Hepatorenal syndrome,Hepatolenticular degeneration,Hepatophosphorylase is lacked,Hepatorenal glycogenosis,Hepatorenal syndrome,Liver kidney tyrosinemia,Hereditary acromelalgia,Hereditary Melanuria,Hereditary amyloidosis,Hereditary angioedema,Heredity Areflexic dysstasias,Heredopathia atactic polyneuritifor,Ataxia hereditaria,Ataxia hereditaria spinocebellar ataxia type,Hereditary benign acanthosis nigricans,Hereditary cerebellar ataxia,Huntington's chorea,Genetic chronic progressive chorea,Heredity CTD,Hereditary coproporphyrin,Hereditary coproporphyrin,Heredity skin malignant melanoma,Hereditary hearing impairment-retinal pigment degeneration,Heredity zinc-deficiency disease,Heredity DNS,Hereditary dystopic lipidosis,Hereditary emphysema,Hereditary fructose intolerance,Hereditary hemorrhagic telangiectasia,Hereditary hemorrhagic telangiectasia type I,Hereditary hemorrhagic telangiectasia type II,Hereditary hemorrhagic telangiectasia type III,Heredity hyperuricemia and choreoathetosis,Hereditary leptocytosis Major,Hereditary leptocytosis Minor,Hereditary lymphedema,Heredity lymphedema tarda,Heredity lymphedema tarda I types,Heredity lymphedema tarda II types,Hereditary motor and sensory neuropathy,Hereditary motor and sensory neuropathy I,Hereditary motor and sensory neuropathy type III,Hereditary nephritis,Hereditary nephritis and nerve deafness,Hereditary amyloidosis nephrosis,Heredity nephrosis and deafness,Hereditary nonpolyposis colorectal cancer,Hereditary nonpolyposis colorectal cancer,Hereditary nonspherocytic hemolytic anemia,Heredity onycho-osteodysplasia,Hereditary optic retinopathy,Hereditary polyposis coli,Hereditary sensory nerve and autonomic neuropathy I types,Hereditary sensory nerve and autonomic neuropathy II types,Hereditary sensory nerve and autonomic neuropathy type III,Hereditary sensorimotor neuropathy,Hereditary sensory neuropathy I types,Hereditary sensory neuropathy I types,Hereditary sensory neuropathy II types,Hereditary sensory neuropathy group iii,Hereditary sensory radicular neuropathy I types,Hereditary sensory radicular neuropathy I types,Hereditary sensory radicular neuropathy II types,Heredity locus specificity cancer,Heredity spherocyte hemolytic anemia,Hereditary spherocytosis,The type of hereditary tyrosinemia 1,Heredity CTD,Herlitz syndrome,Hermans-Herzberg phakomatosses,Hai-general two Cotard,Hermaphroditic,Herpes zoster,Herpes iris Stevens-Johnson types,Glycogenosis type VI,Heterozygous β-thalassemia,Hexoaminidase alpha subunits missing (variation B),Hexoaminidase alpha subunits missing (variation B),HFA,HFM,HGPS,HH,HHHO,HHRH,HHT,Ceasma hernia-microcephaly's deformity-nephrosis Galloway types,Suppurative hidradenitis,Hidrosadenitis axillaris,Hidrosadenitis suppurates,Perspiration is ectodermal dysplasia,HIE syndromes,High hedratresia,High potassium,High scapula,HIM,Congenital megacolon,Acquired congenital megacolon,Referring to ulnar side ＆ hallux toes and VSD congenital megacolon more,Congenital megacolon and D type brachydactylias,Hirsutism,HIS lacks,Histadine ammoniacal (HAL) is lacked,Histidase is lacked,Histidinemia,Histocytosis,Histiocytosis X,HLHS,HLP II types,HMG,HMI,HMSN I,HNHA,HOCM,Hodgkin's disease,Hodgkin's disease,He Jiejin lymphomas,Hollaender-Simons diseases,Holmes-Adie syndrome,Holocarboxylase synthetase deficiency,Holoprosencephaly,Holoprosencephaly is complicated,Holoprosencephaly sequence,Holt-Oram syndrome,Holt-Oram type heart-hand syndromes,Blood (interior) homocystine is excessive,Homocystinuria,Homogentisic acid oxidase deficiency,homogentisicacidura,Homozygosis alpha-1-antitrypsin deficiency,HOOD,Nellie syndrome suddenly,Horton diseases,HOS,HOS1,Houston-Harris types Achrondrogenesis (IA types),HPS,HRS,HS,HSAN I types,HSAN II types,HSAN-III,HSMN,HSMN type IIIs,HSNI,HSN-III,Huebner-Herter diseases,Hunner’s Patch,Hunner ulcer,Hunter syndrome,Hunter-Thompson type acromesomelic dysplasias,Huntington's chorea,Huntington's chorea,I type mucopolysaccharidosis,Hurley syndrome,Hurler-Scheie compound,HUS,Hao-Ji Er Shi early ageing syndromes,Gilford's syndrome,Hutchinson-Weber-Peutz syndrome,Hutterite syndrome Bowen-Conradi types,Hyaline Panneuropathy,Hydranencephaly (deformity,Hydrocephalus,Hydrocephalus agyria (deformity) and retinal development are abnormal,Dandy-Walker types inside hydrocephalus,Hydrocephalus hydrocephalus noncommunicating hydrocephalus Dandy-Walker types,Hydrocephalus,Uronephrosis and peculiar facial expression,Hydroxylase defect,Hygroma colli,Hyper-IgE syndromes,Hyper-IgM syndromes,Hyperaldosteronism,Hyperaldosteronism and hypopotassemia Alkatosis,Hyperaldosteronism is not accompanied by hypertension,Hyperammonemia,Hyperammonemia caused by ammonia [base] formyl phosphate synthase lacks,Hyperammonemia caused by ornithine carbamyltransferase lacks,Hyperammonemia II types,Super β carnosinemias,Physiologic liver dysfunction,Hyperbilirubinemia II,Familial hypercalcinemia and nephrocalcinosis and urocyanosis,Hypercalcinemia-supravalvar aortostenosises,Hypercalciuric rickets,Hypercapnic acidosis,Hypercatabolism protein losing enteropathy,Hypercholesterolemic acidosis,Hypercholesterolemia,Hypercholesterolemia type IV,The excessive disease of blood (interior) chylomicron,Homocystinemia,hyperekplexia,Can hyper-extended joint,Hyperglobulinemic purpura,Hyperglycinemia and DKA and lactic acidosis,The excessive non-ketosis of glycine in blood,Hypergonadotropic hypogonadism,Hyperimmunoglobulin E syndrome,Hyperimmune globulin E-recurrent infection syndrome,Blood (in clear) excessive disease E- staphylococcics of immunoglobulin,Hyperkalemia,Hyperkinetic syndrome,The Hyperlipemic retinitiss,Hyperlipidemia I,Hyperlipidemia IV,Hyperlipoprotememia type I,Hyperlipoprotememia type III,Hyperlipoprotememia type IV,Hyperoxaluria,Hyperphalangy- forefingers refer to bending and micrognathia-glossoptosis syndrome,Hyperphenylalaninemia,Hyperplastic epidermolysis bullosa,Hyperpnea,Potassemia,Hyperprebetalipoproteinemia,Hyperprolinemia (disease) I types,Hyperprolinemia (disease) II types,Hypersplenism,Away from broadening and esophagus malformation and hypospadia,Hypertelorism-hypospadias syndrome,Hypertrophic cardiomyopathy,Hypertrophic interstitial neuropathy,Hypertrophic interstitial neuritis,Stromal hyperplasia nerve root neuropathy,The hypertrophica neuropathy of refsum,Hypertrophic obstructive cardiomyopathy,Hyperuricemia choreoathetosis autotomy syndrome,Hyperuricemia-oligrophrenia,Hypervalinemia,Hypocalcification (hypomineralization) type,Hypochondrogenesis,hypochrondroplasia,Blood (interior) gamma globulin is very few,The temporary blood of baby (interior) gamma globulin is very few,The malnutritive simultaneously potential diabeteses of hypogenital,Lack tongue-adactylism (toe) syndrome,Hypoglycemia,Exogenous hypoglycemia,Hypoglycemia and macroglossia (disease),Hypoglycosylation syndrome 1a types,Hypoglycosylation syndrome 1a types,Hypogonadism and anosmia,Low gonadotropic hormone hypofunction and anosmia,Ectodermal dysplasia,hypohidrotic,Sweat ectodermal dysplasia disease autosomal dominant inheritance type,Sweat ectodermal dysplasia disease autosomal dominant inheritance type autosomal dominant type,Hypokalemia,Hypokalemic alkalosis and hypercalciuria,Hypokalemic syndrome,Lactase lacks,Hypomaturation types (milky white teeth),Ito hypomelanosises,Hypomelia-hypotrichosis-facial hemangioma syndrome,Hypomyelination neuropathy,Hypoparathyroidism,Hypophosphatasia,Phosphate hypophosphatemic rickets and hypercalcinemia,Hypopigmentation,Pigment reduces ARM,Fallen scalp hypoplasia and heart defect,Hypoplastic anemia,Congenital aplasia anaemia,Chondrodystrophia hypoplastica,Hypoplasia enamel-onycholysis-hypohidrosis,Hypoplasia (hypoplasia-explastic) type,Hypoplastic left heart syndrome,Hypoplasia-(thumb) triphalangia,Hypokalemia,Hypospadia-dysphagia syndrome,Hyposphresia,Referring to hypothalamus hamartoblastoma hypopituitarism hedratresia more,Thalamus-childhood obesity,Hypothyroidism,H3O syndrome,Hypoxanthine-guanine phosphoribosyl transferase defect (missing completely),I- cytopathies,Iatrogenic hypoglycemosis,IBGC,IBIDS syndromes,IBM,IBS,IC,I- cytopathies,ICD,ICE syndrome Cogan-Reese types,Iceland type amyloidosis venereal disease (type VI),I- cytopathies,Ichthyosiform erythroderma cornea is participated in and deaf,The red skin trichosis of ichthyosiform and male,Ichthyosiform erythroderma and leucocyte vacuolization,Ichthyosis,Ichthyosis congenita,Congenital fish scale and trichothiodystrophy,Ichthyosis hystrix,Ichthyosis hystrix,Ichthyosis linearis circumflexa,Ichthyosis simplex,Ichthyosis Tay syndromes,Ichthyosis vulgaris,Ichthyotic neutral lipid storage disease,Leptospira icterogenes disease,Leptospira icterogenes disease,Jaundice (chronic familial),Icterus gravis neonatorum,Jaundice intermittens juvenalis,Primary alveolar hypoventilation,Primary amyloidosis,Primary Takayasu arteritis,Primary basal ganglion calcification (IBGC),Primary brachial plexus neuropathy,Primary cervical dystonia,Primary pulmonary arterial dilatation,Primary peripheral facial paralysis,Essential familial hypelipemia,The hypertrophica aortic stenosis of primary,Primary hypoproteinemia,Primary immunoglobulin deficiency,Primary neonatal hepatitis,Primary nonspecific ulcerative colitis,It is scorching around primary PeV,Idiopathic pulmonary fibrosis,Intractable primary iron granule erythrocyte anemia,Primary renal hematuria,Idiopathic steatorrhea,Idiopathic thrombocythemia,Primary thrombocytopenic purpura,Primary thrombocytopenic purpura (ITP),IDPA,Iga nephrosis,IHSS,Ileitis,Ileocolitis,Illinois type amyloidosis,ILS,IM,IMD2,IMD5,Due to lacking the immunodeficiency of thymus gland,Immune hemolytic anemia paroxysmal is cold,Immune deficiency and ataxia telangiectasia,Immune deficiency cell and abnormal immunoglobulin synthesis,Immune deficiency is often variable not to open classification,Immune deficiency and Hyper-IgM,Immune deficiency and Neuroleptic Leukocytopenia,Immune deficiency -2,Immune deficiency -5 (IMD5),Immunoglobulin deficiency,Hedratresia,Hedratresia and brothers and ear are abnormal,Locking nasolacrimal duct and early ageing syndrome,Impotent neutrophil syndrome,It can not dehisce completely and short flexor digitorum muscle of hand,INAD,Birth defects urea synthesizing arginine-type,Birth defects UreaSynthesis Arginino Succinic types,Congenital urea synthesizing carbamyl phosphate type,Birth defects urea synthesizing citrullinemia type,Congenital urea synthesizing glutamic acid synthesis type,INCL,Inclusion body myositis,Treatment of Complete Atrioventricular Defect,Infull testicular feminization,Incontinentia pigmenti,Incontinenti Pigmenti Achromians,The abnormal simultaneously micrognathia-glossoptosis syndrome of forefinger,Amyloidosis,Indiana type (Equations of The Second Kind),Indolent systemic mastocytosis,The children's aphasia day after tomorrow,Children's recessive hereditary polycystic kidney disease,Infantile beriberi,Children's cerebroganglion glycosides fat,Children's cerebral palsy,Children's abderhalden-Kaufmann-Lignac syndrome,Children epilepsy,Children's Fanconi syndrome and abderhalden-Kaufmann-Lignac syndrome,Baby Finland type neuronal waxy lipophilic disease,Children's Gaucher disease,Pediatric hypoglycemia,Children hypophasphatasia,Children's lobe of the lung pulmonary emphysema,Children's myoclonic encephalopathic,Children's myoclonic encephalopathic and myoclonia,Infantilism Myofibromatosis,Infant's necrotizing encephalopathy,Infantile neuronal ceroid lipofuscinosis,Children's neural axis is malnutritive,Infant's morbidity Schindler diseases,Children's phytanic acid storage disease,Children's Refsum disease (IRD),Children sipoidosis GM-2gangliosideosis (s types),Baby sleeping apnea,Infantile spasms,The myeloid muscular dystrophy of children (all types),Children's spinal muscular atrophy ALS,Children's Duchenne-Arandisease type I types,Infantilism neuronal waxy lipophilic disease,Catarrhal jaundice,IBD,Inflammatory breast cancer,Inflammatory linear sebaceous nevus syndrome,Iniencephaly,The acanthosis nigricans of insulin resistance,Insulin lipodystrophy,Insulin-dependent diabetes mellitus,Purpose myoclonia,Intermediate cystinosis,Intermediate maple syrup urine disease,Episodic ataxia and pyruvic dehydrogenase deficiency,Intermittent maple syrup urine disease,Internal hydrocephalus,Interstitial cystitis,Interstitial deletion includes 4q,Intestinal lipodystrophy,Intestines lipophagia granuloma,Intestinal lymphatic dilatation,Intestinal polyp I,Intestinal polyp II,Intestinal polyp III,Intestinal polyposis-cutaneous pigmentation syndrome,Intestinal pseudo-obstruction and ballet's disease,Intracranial neoplasm,Intracranial tumors,Intracranial vascular malformation,Intrauterine nanism,Intrauterine adhesion,Stand upside down and smile and invisible nervous bladder,Iowa types amyloidosis (IV types),IP,IPA,Iridocorneal endothelial syndrome,Iris corneal endothelium (ICE) syndrome Cogan-Resse types,Iridogoniodysgenesis and body exception,Atrophy of iris and corneal edema and glaucoma,Cogan-Reese syndrome,Iron overload anaemia,Iron overload disease,Irritable bowel syndrome,Irritable colon syndrome,Isaacs syndromes,Isaacs-Merten syndromes,Ischemic myocardium disease,Isolate agyria sequence,The amyloidosis of isoleucine 33,Isovaleric acid CoA dehydrogenase deficiency,Isovaleric acidemia,Isovaleric acidemia,Isovaleryl coenzyme A carboxylation enzyme defect,ITO black (color) plain very few disease,ITO,ITP,IVA,Ivemark syndromes,Iwanoff tumours,Jackknife twitches,Jackson-Weiss craniosynostosises,Jackson-Weiss syndromes,Jacksonian epilepsy,Jacobson syndrome,Jadassohn-Lewandowsky syndrome,Jaffe-Lichenstein diseases,Jakob diseases,Jakob-Creutzfeldt disease,Janeway I,Janeway dysgammaglobulinemias,Jansen metaphyseal dysostosises,Jansen type metaphysial chondrodysplasias,Jarcho-Levin syndrome,Jaw winking (reflection),JBS,JDMS,Jegher syndromes,Imperforate jejunum,Jejunitis,Jejunoileitis,Jervell and Lange-Nielsen syndrome,Jeune syndrome,JMS,Job's syndrome,Job-Buckley syndromes,Johanson-Blizzard syndrome,John Dalton,About [Han Xun]-this [Di Wen] Er Shi diseases,Jonston alopecias,Joseph diseases,Joseph disease I types,Joseph disease II types,Joseph disease type IIIs,Joubert syndromes,Joubert-Bolthauser syndromes,JRA,Juberg Hayward syndromes,Juberg-Marsidi syndromes,Juberg-Marsidi baryencephalia syndromes,France ramaninjana,Maine France ramaninjana,Juvenile arthritis,Childhood recessive hereditary polycystic kidney disease,Juvenile cystinosis,Childhood (children) dermatomyositis (JDMS),Childhood diabetes,Childhood Gaucher disease,Juvenile gout choreoathetosis and baryencephalia syndrome,Childhood vitamin b12 intestinal malabsorption,Childhood vitamin b12 intestinal malabsorption,Juvenile macular degeneration,Pernicious anemia,juvenile,Juvenile retinoschisis,Juvenile rheumatoid arthritis,Adolescent spinal muscular atrophy includes,Adolescent spinal muscular atrophy includes ALS,Adolescent spinal muscular atrophy type III,Jaxta-articular adiposis,Hyperplasia by glomerulus,Kabuki's cosmetic syndrome,Kahler's disease,Kallmann syndromes,Kanner syndromes,Kanzaki diseases,Card ripple disease (non-Kaposi sarcoma),κ light chains are lacked,Karsch-Neugebauer syndromes,Kartagener syndromes-chronic sinobronchial diseases and dextrocardia,Kartagener triad,Hemangioma-thrombocytopenia syndrome,Kast's syndrome,Kawasaki disease,Kawasaki syndrome,KBG syndromes,KD,Kearns-Sayre diseases,Kearns-Sayre syndromes,Kennedy diseases,Kennedy syndromes,Kennedy type spinal and bulbar muscular atrophies,Kennedy-Stefanis diseases,Kenny diseases,Kenny syndromes,Kenny types tubular bone (pulp cavity) is narrow (disease),Kenny-Caffe syndromes,Kera.Palmoplant.Con.Pes Planus Ony.Periodon.Arach.,Keratitis ichthyosis and deafness syndrome,Keratoconus,Keratoconus posticus circumscriptus,Cuticula is separated,Keratolysis exfoliativa congenita,Winter exfoliating erythema,Keratomalacia,Darier's disease,Keratosis follicularis spinulosa decalvans,Keratosis follicularis spinulosa decalvans ichthyosis,Angling acanthosis nigricans,Keratosis palmoplantaris and periodontosis and onychogryp(h)osis,Keratosis palmoplantaris congenital flat foot's onychogryp(h)osis periodontosis arachnodactylies,CDK palmoplantaris,Flat foot,Hook first,Periodontosis,Arachnodactyly,Acroosteolysis,Keratosis rubra figurata,Seborrheic keratosis,Keto-acid decarboxylase enzyme defect,Ketone acid is urinated,Ketosis glycinemia,KFS,KID syndromes,Kidney development is not complete,The dirty retinal aplasia Jouber t syndromes of cystic kidney,Killian syndromes,Killian/Teschler-Nicola syndromes,Kiloh-Nevin syndrome is I,Hair nodules,Dancing eyes syndrome,Kleeblattschadel deformities,Kleine-Levin syndrome,Kleine-Levin hibernation syndromes,Klinefelter,Cervical vertebrae synostosis,Cervical vertebrae synostosis I types,Cervical vertebrae synostosis II types,Cervical vertebrae synostosis type III,Klippel Trenaunay syndromes,Io-osteohypertrophy's syndrome,G-Buji 's syndrome,KMS,Kniest depauperations,Kniest syndrome,Ke Nisike diseases,Koebberling-Dunnigan syndromes,Kohlmeier-Degos disease,Kok diseases,Korsakoff mental diseases,Korsakoff syndromes,Krabbe diseases include,Krabbe leukodystrophies,Kramer syndromes,KSS,KTS,KTW syndromes,Kufs disease,Ku-Wei disease,Kugelberg-Welander syndrome,Kussmaul-Landry paralysis,KWS,L-3- hydroxy-acyls-coenzyme a dehydrogenases (LCHAD) defect,Laband syndromes,Labhart-Willi syndromes,Labyrinthine syndrome,Hydrolabyrinth,Tear stains-ear-tooth-refers to (toe) syndrome,Lactase is not tolerated alone,Alactasia,Lactation atrophy of uterus,Lactic acidosis leber hereditary optic neuropathy,Lactic acid and Pyruvic acid acidaemia and the sensitive sense of carbohydrate,Lactic acid and Pyruvic acid acidaemia and sporadic incoordination and weakness,Lactic acid and Pyruvic acid salt,Hyperlactacidemia,Adult is intolerant to lactose,Lactose intolerance,Children are intolerant to lactose,LADD syndromes,LADD,Lafora disease includes,Lzfora corpusculums disease,Fibrinase,fibrin stabilizing factor-Factor XⅢ deficiency disease,LAM,Lambert type ichthyosis,Lambert-Eaton syndrome,Lambert-Eton myasthenic syndrome,Stratiform recessiveness ichthyosis,Lamellar ichthyosis,Lancereaux-Mathieu-Weil spirochetosises,Landau-kleffner syndromes,Landouzy Dejerine muscular dystrophies,Landry ascending paralysises,Langer-Salidino types achondroplasia (Equations of The Second Kind),Langer Giedion syndromes,Histiocytosis X,Langerhans cell histiocytosis (LCH),Big atrium and ventricular loss,Laron dwarfism,Laron type pituitary dwarfisms,Draw Ademilson (family name) syndrome,Laryngeal dystonia,Lata (in Malaysia's discovery),Late period children's neural axis is malnutritive,Late period children's neural axis is malnutritive,The tardy type of Cockayne syndrome three (c types),Tardive dystonia,Late immunoglobulin deficiency disease,Tardy Pei-plum Er Shi cerebrosclerosis,Lattice corneal dystrophy,Lattice dystrophy,Launois-Bensaude,Launois-Cleret syndrome,Laurence syndromes,Laurence-Moon syndromes,Laurence-Moon-Bardet-Biedl syndrome,Lawrence-Seip syndrome,LCA,LCAD is lacked,LCAD,LCAD,LCADH lacks,LCH,LCHAD,LCPD,Jeune syndrome,Leband syndromes,Leber amaurosises,Leber congenital amaurosises,Amaurosis,Congenital post cone missing,Leber ' s congenital retinals blanket layer is denatured,The congenital blanket retinal developments of Leber ' s are abnormal,Leber ' s diseases,Leber ' s optic atrophies,Leber ' s optic neuropathies,Left ventricle fibrosis,Ulcus cruris,Legg-Calve- Perthes diseases,Leigh ' s diseases,Leigh ' s syndromes,Leigh ' s syndromes (subacute necrotizing cerebrospinal cord disease),Leigh brain necrosis,Lennox-Gastaut syndromes,Freckle-hyperposia-digestion syndrome (Lentigio-Polypose-Digestive Syndr ome),Lenz DysmorphogeneticSyndr ome,Lenz dysplasia,Lenz nanophthalmus syndromes,Lenz syndromes,Leopard syndrome,Donohue's syndrome,Pia-arachnoid hemangioma,Leptospiral jaundice,Leri-Weill Disease,Leri-Weill dyschondrosteosis,Leri-WeilSyndrome,Lermoyez syndrome,Leroy Disease,Lesch Nyhan Syndrome,Lethal baby's caradrin myopathy,Lethal neonate's nanism,Lethal osteochondrodysplasia,Letterer-Siwe disease (letterer-Siwe disease),Leukocyte disorder albinism,Leucocyte embedding is with blood platelet disorders,Leukodystrophy,Leukodystrophy is with Rosenthal fiber,Leukoencephalitis (leukoencephalitis) PeriaxialisConcentric (centrality),Levine-Critchley syndrome,Fructosuria,Levy-Hollister syndromes,Limbus girdle muscular dystrophy,Lactoglobulin,Lerbe hereditary neuropathy,Left internal carotid,Lichen ruber acuminatus,Lichen acuminatus,Lichen amyloidosis,Lichen planus,Lichen psoriasis,Lignac-Debre-Fanconi Syndrome,Lignac-Fanconi syndrome,Ligneous conjunctivitis,Limb girdle type muscular dystrophy,LimbMalformations-Dento-Digital Syndrome (Deformities of Limbs ...),Limit dextrinosis,Linear mole hypermelanosis,Naevus linearis Sebacous syndromes,Linear scleroderma,Linear naevus sebaceus sequence,Linear sebaceous nevus syndrome,Bifid tongue,Fissured tongue,Fissured tongue,Lingual surface dyskinesia,Lip Pseudocleft-hemangiomatous Branchial CystSyndrome (- hemangioma branchial cleft cyst syndrome is split in lip vacation),Lipid granuloma disease,Lipid histocytosis,Angle glucoside epoxy-type,Lipid storage disease,Lipid storage myopathy is not enough (Lipid-Storage myopathy Associated with SCAD Deficiency) with SCAD,Baby's ganglioside lipidosis,Lipoatrophic diabetes (Lipoatrophic DiabetesMellitus),Fat nutritional disorders,Lipid corneal dystrophy,Lipid hyperplasia pseudo (Lipoid Hyperplasia-Male Pseudohermaphroditism),Congenital lipomatosis of pancreas,Lipomucopolysaccharidosis,Lipomyelomeningocele,The Deletional schilder's disease,Familial of lipoprotein lipase,Atomicvapor laser isotope separation,LIS1,Agyria 1,Agyria I types,Agyria abnormity is with corpus callosum cerebellar hypoplasia or other exceptions,Infantile spasm Diplegia,Liver phoshorylase,Liver,Kidney,Spleen,Lactose malabsorption syndrome,Circumscribed atrophy of brain,Intelligence circumscribed atrophy of brain,Lobar holoprosencephaly,Baby's lobe of the lung anxiety pulmonary emphysema,Lobstein disease (I types),Lobster claw deformity,Local epidermolysis bullosa,Local fat dystrophia,Local cingulum membri superioris neuritis,Lv Fule bacterium disease,Lv Fule bacterium cardiac muscle internal membrane of heart fibrosis is with eosinophilia,Lv Fule bacterium mural endocarditises,Lip river Kern's syndrome,Senior Lip river Kern syndrome,Long-chain 3-hydroxyacyl-CoA dehydrogenase,Long-chain fatty acyl-CoA dehydrogenase defect,Long-chain fatty acyl-CoA dehydrogenase,Long acyl-CoA dehydrogenase deficiencies,Without deafness QT syndromes,Hypoglycemia,Low-density beta lipoprotein is lacked,Low level hedratresia,Low potassium syndrome,Oculo cerebro renal syndrome Lowe ' s Syndrome,Lowe-BickelSyndrome,Lowe-Terry-MacLachlan Syndrome,Low back pain,Milk of sulfur,Leukotrienes d,Lubs syndromes,Luft disease,Waist spinal canal stenosis,Lumbar spinal stenosis,Waist sacrum spinal canal stenosis,Lundborg-Unverricht Disease,Lundborg-UnverrichtDisease Included,Lupus,Lupus,Lupus erythematosus,Luschka-MagendieForamina Atresia,Lyell's syndrome,Lyell syndrome,Lymphadenoid goitre,The protein losing enteropathy of lymphangiectasis,Lymphangioleiomyoma,Lymphangioleiomyoma,Lymphangioma,Lymphatic malformation,Lynch Syndromes,Lynch SyndromeI,Lynch Syndrome II,Schindler type lysosomes alpha-N-Acetylgalactosaminidase lacks (Lysosomal Alpha-N-Acetylgalactosaminidase DeficiencySchindler Type),Lysosomal Glycoaminoacid StorageDisease-Angiokeratoma Corporis Diffusum,Lysosomal glycosidase is lacked,Single joint arthritis,Machado Disease,Ma-about sick,Macrencephaly,Megacephaly,Megacephaly hemihypertrophy,Megacephaly is with lipomatosis and Hemangiomata,Megacephaly is with pseudopapilloedema and Multiple (multiple) Hemangiomata,Macroglobulinemia,Macroglossia (disease),Macroglossia-omphlexoche-visceral obesity syndrome,Macrostomia (meloschisis) Ablepheron Syndrome,Bernard-Soulier Type familials Macrothrombocytopenia (Macrothrombocytopenia Familial Bernard-Soulier Type),Macula retinae sexual involution,Macular amyloidosis,Macular degeneration,Macular disciform degeneration,Macular disciform degeneration,Macular disciform degeneration,Mottled corneal nutrition obstacle,Maximum allowable amount,Madelung’s Disease,Maffucci syndrome (multiple chondroma is with internal organ cvernous hemangioma),Grand mal,Malabsorption,Malabsorption-ectodermal dysplasia-wing of nose hypoplasia,Maladie de Roger,Maladie de Tics,Malaria,Male four limbs and deformity of kidney,Male gonad maldevelopment disease,Pernicious acanthosis,Malignant acanthosis nigricans,Malignant astrocytoma,Malignant atrophic papulosis,Malignant malaria,Malignant hyperphenylalaninemia,Malignant fever,Pernicious hyperpyrexia,Malignant mela noma,Central nervous system malignant tumour,Mallory-Weiss tear,Mallory-Weiss tear,Ma-Wei syndrome (orifice of the stomach mucous membrane lacerated wound syndrome),Mammary Paget’s Disease,Mandibular ameloblastoma,Faciomandibular dysostosis,Mannosidosis,Surface-point-finger pattern corneal dystrophy (Map-Dot-Fingerprint Type Corneal Dystrophy),Maple syrup urine disease,Marble bone,Paroxysmal nocturnal hemoglobinuria (marchiafava-Micheli syndrome),Ma Kasigeen jaw winking syndromes,Ma Kasigeen (family name) phenomenon,Ma Kasigeen ptosis is with gunn syndrome,Ridge grace syndrome is (during unilateral ptosis,Ipsilateral upper eyelid is moved with mandibular symphysis),Ma Kasigeen (gunn syndrome) syndrome,Ma Kasigeen sagging (with gunn syndrome),Marden-Walker syndrome (recessive hereditary multiple arthrogryposis deformity),Ma-fertile Er Shi CTDs,Malaysia side nutrition exhaustion,Malaysia side-Achard syndromes,Malaysia side syndrome,Malaysia side syndrome I types,Malaysia side variant,Malaysia side type hyperkinesia syndrome,Marginal corneal dystrophy,Mary's incoordination,Mary's disease,Marie-Strumpell disease,MarieStrumpell Disease,Marie-Strumpell spondylitis (ankylosing spondylitis),Marinesco-Sjogren syndrome (ataxia hereditaria,Cataract,Dwarf and amentia syndrome),Ma-Si-lattice syndrome,Marker X syndrome,Ma Shaoer syndromes,Maroteaux LamySyndrome,Maroteaux Type Acromesomelic Dysplasia,Ma Shaoer is ectodermal dysplasia with vision auditorily handicapped,Ma Shaoer-Smith's syndrome,Ma Shaoer syndromes,Ma Shaoer types are deaf-myopia-flood-saddle nose (Marshall TypeDeafness-Myopia-Cataract-Saddle Nose),Ma-Austria's syndrome (false accessory thyroid glands function is low to subtract disease),Ma-shellfish syndrome,Martorell syndromes,MASA Syndrome,Large area myoclonia,Mast cell leukemia,Mastocytosis,Mastocytosis is with haematological disorders,Maumenee Corneal Dystrophy (corneal dystrophy),Maxilla ameloblastoma,Maxillary bone dysbolism,Maxillonasal dysplasia,Maxillonasal dysplasia adhesion type,Maxillary sinus-eyelid synkinesia,Abnormal (the may-Hegglin anomalies of Mei-sea Er Shi (blood platelet),Heredity nucleic acid is abnormal),Medium-chain acyl-coenzyme A dehydrogenase deficiency,McArdle disease (glycogen storage disease IV types),McCune-Albright,(kidney) medullary cystic disease,McKusick type metaphysial chondrodysplasias,Mitral valve closure speed,Mixed connective tissue disease,U.S. syndrome,Meckel-Gruber syndrome (internal organ tumour-head depauperation syndrome),Median cleft face syndrome,Thalassemia,Medium chain acyl coa dehydrogenase,Medium chain acyl coa dehydrogenase is lacked,Medium chain acyl coa dehydrogenase is lacked,(kidney) medullary cystic disease,Medullary sponge kidney,Maximum expiratory flow volume,Mega-esophagus,Macrencephaly,Macrencephaly is with hyaloid inclusion body,Megalencephaly with Hyaline Panneuropathy,Megaloblastic anemia,Juvenile erythrocyte anemia during pregnancy,Macrocornea-mental retardation's syndrome,Basal cell naevus syndrome,MeigeShi lymphedemas,Meige syndromes (Meige ' s Syndrome),Macrotooth (macroteeth) leukodystrophy,Intestinal mucosa melanoplakia polyposis,Intestinal mucosa melanoplakia polyposis,MELAS Syndrome,MELAS,Melkersson Syndrome,BOR syndrome,Osteodysplasty of Melnick and Needles,Melnick-Needles syndrome,Mucous membrane fat nutritional disorders,Da Costa's syndrome (cardiac neurosis),Meniere disease (auditory vertigo),Meniere disease,Meninx capillary blood tuberculation,Menkes disease,Menkes disease I,Aphasia body disorders and hallux valgus type baryencephalia,Mental retardation-deafness-skeletal abnormality-coarse face and thick lip (Mental Retardation-Deafness-Skeletal Abnormalities-CoarseFace with Full Lips),5th thumb and toe hypoplasia type mental retardation,Osteochondrodysplasia type mental retardation,The short and small type mental retardation of growthing lag-deafness-genitals (Mental Retradation-X-linked with Growth Delay-Deafness-Microgeni-talism),Menzel type olvopontocerebellar atrophies,Mermaid syndrome,Myoclonus epilepsy and ragged red fibrers syndrome,Myoclonus epilepsy and ragged red fibrers syndrome,Merten-Singleton Syndrome,Limb injury syndrome,Glomerular mesangium immunoglobulin-a type nephrosis,Mesenteric fat dystrophia,Mesiodens-Cataract Syndrome,Mesodermal Dysmorphodystrophy,Mesomelic Dwarfism-MadelungDeformity,Metabolic acidosis,Metachromatic leukodystrophy,Metatarsus varus,Metatropic dwarfism syndrome,Metatropic dysplasia,Metatropic dysplasia I,Metatropic dysplasia II,Methylmalonic acidemia,Methylmalonyl CoA carboxyls mutase lacks disease,Meulengracht’s Disease,Faciomandibular dysostosis I,Macroglobulinemia,Malignant histiocytosis,Microangiopathic hemolytic anemia,Micrencephaly,Primary nanocephalic dwarf I,Microcephaly's deformity,Microcephaly-ceasma hernia-nephropathy Galloway types (Microcephaly-Hiatal Hernia-Nephrosis Galloway Type),Microcephaly-ceasma hernia-nephropathy syndrome,Folliculus cornea (epithelium) is malnutritive,Microcytosis,Microlissencephaly,Nanophthalmus,Anophthalmia or nanophthalmus are with relevant abnormalities,Gyrus is small with muscular dystrophy,Knee cap micrognathia syndrome without microtia,Microvillus embedding disease,Mesangial immune deposits,Midsystolic click late systolic murmur syndrome,Miescher’s Type I Syndrome,Mikulicz syndrome,Mikulicz-RadeckiSyndrome,Mikulicz-Sjogren Syndrome,Slight autosomal recessive inheritance,Light moderate maple syrup urine disease,Mild maple syrup urine disease,Miller syndrome,Miller-Dieker syndrome,The syndrome of meter -not,Meige disease,Minkowski-Chauffard syndrome (familial spherocytosis,Congenital hemolytic anemia,Acholuri familial jaundice),Lapse,Von Willebrand's disease (constitutional blood platelet disorders disease),Mirror image dextrocardia,Mitochondria beta oxidation is disorderly,Mitochondria and cytoplasmic,Mitochondrial cytopathies,Mitochondrial cytopathies,Kearn-Sayre Type,Mitochondrial encephalopathy,Mitochondrial encephalomyopathy lactic acidosis and the breaking-out of apoplexy sample,Mitochondrial myopathy,Mitochondrial encephalomyopathy lactic acidosis and the breaking-out of apoplexy sample,Mitochondria phosphoenolpyruvate carboxykinasedeficiency,Mitral valve prolapse,Mixed apnea,Mixed connective tissue disease,Mixed hepatic porphyria,Nonfluent aphasia,Mixed sleep apnea,Tonic-clonic Combination torticollis,MJD,Mounier-Kuhn syndrome (congenital,Eso-ethmoiditis combines presence with bronchiectasis),Malignant lymphoma I (ML I),Malignant lymphoma II,Malignant lymphoma III,Malignant lymphoma IV,Mediterranean lymphoma disorder I (ML Disorder TypeI),ML Disorder TypeII,ML Disorder Type III,ML Disorder TypeIV,Membranous lupus nephritis,Moderate nerve growth retardation syndrome,Motor neuron disease,MNGIE,MNS,MobitzI,MobitzII,Mo Biesi (family name) syndrome (congenital facial diplegia),Moebius syndrome (pain motion can not),Moersch-Woltmann syndromes,Mohr's syndrome (OFD syndrome i I types,Bifid tongue,Conduction deafness,Thumb portion is overlapping),Monomodal typhloexias (Monomodal Visual Amnesia),Mononeuritis multiplex,Mononeuritis,Single nerve endings lesion,Monosomy 3p2 (Monosomy 3p2),Monosomy 9p Partial,Monosomy 11q Partial,Monosomy 13q Partial,Monosomy 18qSyndrome,Monosomy X,Sub-thread fibrous dysplasia,Morgagni-Turner-Albright Syndrome,Scleroderma circumscriptum,Eccentro-osteochondrodysplasia,Morquio syndrome,Morquio syndrome A,Morquio syndrome B,Morquio syndrome syndrome,Syringomyelia,The body 9p of piebald four (MosaicTetrasomy 9p),Motor neuron disease,Motor neuron syndrome,Motor neuron disease,Motor neuron disease,Motor neuron disease,Dynamical system disease (is assembled and slow),Moyamoya disease,Moyamoya disease (due to brain bottom abnormal vascular net rupture caused by block and small bleeding caused by cerebrum ischemia cause progressive nervous function defect,Moyamoya disease),Mucopolysaccharide (storing up) disease,Mucopolysaccharide (storing up) disease I,Mucopolysaccharide (storing up) disease IH,Mucopolysaccharide (storing up) disease 1H/S Hurler/Scheie Syndrome,MPS I S Scheie Syndrome,MPSII,MPS II A,MPS II B,MPS II-AR,Autosomal recessive inheritance Hunter syndrome,MPS II-XR,The serious autosomal recessive inheritances of MPS II-XR,Mucopolysaccharide (storing up) disease V,Mucopolysaccharide (storing up) disease VI,Mucopolysaccharide (storing up) disease VI Severe Intermediate MildMaroteaux-Lamy (severe ... in light),Mucopolysaccharide (storing up) disease VII,Mucopolysaccharide (storing up) disease VII sly syndrome (mucopolysaccharidosis VII types：The disease caused by beta-glucuronidase lacks),Mucopolysaccharide (storing up) disease VIII,Mucopolysaccharide (storing up) is disorderly,MPS DisorderI,MPS Disorder II,MPS Disorder III,MPS Disorder VI,MPS DisorderType VII,(classical symptom is the facial paralysis of recurrent exerbation to the Cotard of Mai-sieve two,Non-inflammation edema of the face and folding property harelip),Malignant stricture,Multi-system atrophy,Multiple sulfuric acid lps deficiency,Symptoms of excess fat,Muscular subaortic stenosis,Maple syrup urine disease,Maple syrup urine disease Ib types,Maple syrup urine disease II types,Viscous liposteatosis III,Mucolipidosis IV,Mucopolysaccharidosis,Mucopolysaccharidosis I-H,Mucopolysaccharidosis I-S,Mucopolysaccharidosis II,Mucopolysaccharidosis III,Mucopolysaccharidosis IV,Mucopolysaccharidosis VI,Mucopolysaccharidosis VII,Mucopolysaccharidosis I types,Mucopolysaccharidosis II types,Mucopolysaccharidosis type III,Mucopolysaccharidosis IV types,Stickiness,Mucosulfatidosis,Mucous colitis,(ductus pancreaticus) mucoviscidosis,Mulibrey Dwarfism,Mulibrey Nanism Syndrome,Gyneduct hypoplasia-renal aplasia-neck chest body segment hypoplasia,Gyneduct-kidney-neck chest-upper limbs volume defect,Gyneduct and renal aplasia are with upper limbs and rib deformity,Gyneduct-kidney-neck chest segment monster,Binswanger type multi infarct dementias,MulticentricCastleman’s Disease,Many focus eosinocyte granulation knurls,Multiple acyl-CoA dehydrogenase missing/glutaric aciduria II types,Multiple Angiomas (complicated hemangioma) andEndochondromas,Multiple carboxylase deficiency,Multiple chondrophyte,Multiple cancellous exostoses,Multiple enchondromatosis,Multiple endocrine deficiency syndrome,Multiple epiphyseal dysplasia,Multiple exostosis,Multiple exostoses syndrome,Multiple familial polyposis,Leopard's syndrome,Huppert's disease,Shoulder belt multiple neuritis,Osteochondromatosis,Multiple peripheral neuritis,Multiple sulfatase deficiency,Symptoms of excess fat,Multi-system atrophy,Multisynostotic osteodysgenesis,Multisynostotic osteodysgenesis is with long osteomiosis,Mulvihill-Smith syndromes,MURGS association (Miller tubes,Kidney,Cervical vertebra defect),Murk Jansen type metaphyseal chondrodysplasias,Muscle carnitine deficiency,Muscle axis of centres disease,Muscle phosphofructokinase deficiency,Muscle central core disease disease,Muscular dystrophy,Muscular dystrophy x linked recessives (heredity),Congenital muscular dystrophy involves with central nervous system,Mental retardation causes the congenital muscular dystrophy of progressive,Facioscapulohumeral muscular dystrophy,Muscular rheumatism,Myotonia-progressive property breaking-out,Musculoskeletal pain,Four limbs shortage disease,Mutism,,Microvascular pressure,Microvascular pressure,MWS,Myasthenia gravis,Goldflam disease,Lambert-Eaton myasthenic syndromes,Myelinoclasis diffusivity is hardened,Myelomatosis,Myhre syndromes,Myoclonia astatic type lapse,Myoclonia dystonia,Baby's myoclonia encephalopathic,Myoclonic epilepsy,Hartung types myoclonic epilepsy (Myoclonic Epilepsy Hartung Type),Lafora's disease is with ragged red fibers,Myoclonic epilepsy and ragged red fibers disease,The progressive myoclonus epilepsy of familial,Familial myoclonus epilepsy,Myoclonic seizure,Myoclonia,Lafora's disease,Myoencephalopathy Ragged-Red Fiber Disease (... ragged red fibers disease),Myofibromatosis,Congenital Myofibromatosis,Myogenicity face-omoplate-fibula syndrome,Myoneurogastointestinal Disorder andEncephalopathy (encephalopathic),Myopathy AMC,Muscle [poison] base deficit disease,Muscle central area fibrosis,Myopathy Congenital (congenital myopathy ...) Nonprogressive,myopathy Congenital Nonprogressive withCentral Axis,Myopathy is lacked with carnitine palmityl transferase,Myopathy-Marinesco-Sjogren syndromes,Myopathy-metabolism DL-carnitine chloride palmitoyl transferase missing,Myopathy mitochondria-encephalopathic-lactic acidosis-apoplexy,With the myopathy of sarcoplasmic bodies and sarcoplasmic bodies,Myophosphorylase is lacked,Myositis ossificans,This too sick (atrophica of nanotesla (family name),Myotonia atrophica),Myotonia congenita,Myotonia congenita intermittens,Myotonia atrophica,Myotonia myopathy dwarf chondrodystrophy and face are abnormal,Myotubular myopathy,X myotubular myopathy,Myproic Acid,Myriachit (Siberia saying),Myxedema,N- acetylgalactosamine -1- phosphotransferases lack,N- acetylglutamates synthesize enzyme defect,Reduced nicotinamide adenine dinucleotide defect,Naegeli EctodermalDysplasias (ectodermal dysplasia),Nager acrofacial dysostosis syndrome,Nager acrofacial dysostosis syndrome,Nager acrofacial dysostosis syndrome,N- acetyl-β-d- UNAGs lack,(refer to,Toe) Onychodystrophy-deafness syndrome,(refer to,Toe) first hypoplasia and metodontiasis,Nail patella syndrome,Nance-Horan syndrome,Bird-headed dwarf of Seckel,Microcephaly's (deformity),Ommatidium,Hypnolepsy,Hypnolepsy syndrome,Non-aqueous reverse-phase chromatography,Nose-volume-(Nasal-fronto-faciodysplasia),Wing of nose hypoplasia hypothyroidism achylia,Atelognathia on nose,Nose fat nutritional disorders (Nasu Lipodystrophy),NBIA1,Neurotic depression,Nerve defect index,No protein diet,Cause encephalomyelopathy (the Necrotizing Encephalomyelopathy of Leigh ' s of necrosis,),Cause the respiratory apparatus granulomatosis of necrosis,Neill-Dingwall Syndrome,(after bilateral adrenal glands excision hypophysis tumor and mucocutaneous pigmentation occur for Nelson syndrome),Nemaline myopathy,Neonate's adrenoleukodystrophy,Neonate's adrenoleukodystrophy (NALD),Neonate's adrenoleukodystrophy (ALD),Neonate's autosomal recessive inheritance POLYCYSTIC KIDNEY DISEASE,Neonate dwarf,Neonatal hepatitis,Hypoglycemia of newborn,Neonate's lactose intolerance,Exudative enteropathy causes neonate's lymphedema,Neonatal necrotizing enterocolitis,Neonate's Progeroid syndromes,Wiedemann-RautenstrauchShi neonate's pesudohydrocephalus Progeroid syndromes,Neoformation archnoiditis,Nephroblastoma,Kidney primary diabetes insipidus,Familial teenager's nephrophthisis,Nephropathic cystinosis,Kidney-pseudohermaphroditism-nephroblastoma,Nephrosis-microcephaly's deformity syndrome,Nephrosis-neopiran dysmigration syndromes (Nephrosis-Neuronal Dysmigration Syndrome),Nephropathy-diabetes-dwarf-rickets-hypophosphoremia syndrome,Netherton Disease,Netherton syndrome (with the ichthyosis of tubercle hair),Netherton syndrome ichthyosis,Nettleship FallsSyndrome (x linked recessives),Neu-Laxova syndromes,Neuhauser syndromes,NTD,Brachial neuritis,Mucopolysaccharide-n- acetyl nerve ammoniacal liquor solution enzyme defect,Nerve melanosis,Auditory nerve nerve-cell tumor,Nerve-cell tumor,Neuroacanthocytosis,Schindler type neuroaxonal dystrophies,Brain iron aggregation I types neurodegeneration (NBIAI),Auditory nerve neurofibroma,Neural congenital multiple arthrogryposis,Neuromyelitis optica,Neuromyotonia,Local nerve myotonia,Neuromyotonia,Diffusion,Family,Neuromyotonia,Local nerve myotonia,Distribute,Schindler type neopiran aixs cylinder dystrophias,Adult neopiran ceroid lipofuscinosis,Juvenile neuronal waxy lipophilic disease,I type neopiran ceroid lipofuscinosis,Neuronophage acute glucose cerebrosidase deficiency disease,Nerve amyloidosis,Nerve athlete's foot,Neural incoordination and retinitis pigmentosa,Brachialpelxus DPN syndromes,Heredity sensorium DPN I types,Transmissibility sensorium DPN II types,Neuropsychiatry porpharia,Neutral lipid storage disease,Nev II,Nevoid basal cell carcinoma syn drome,Mole,Mole is porous,Acne-like nevus,Naevus anaemicus,Jadassohn sebum type moles,Nezelof syndromes,Nezelof type thymic aplasias,Nezelof type severe combined immunodeficients,Multiple neurofibromatosis,Multiple neurofibromatosis 1,Multiple neurofibromatosis 2,Multiple neurofibromatosis -1,Multiple neurofibromatosis -2,Standard serum,Niemann Pick diseases,Niemann Pick disease A types (acute neuronophage type),Niemann Pick disease Type Bs,Niemann Pick diseases c-type (chronic neuronophage type),Niemann Pick disease D types (variation of Nova spills),Niemann Pick disease E types,Niemann Pick disease F types (sea blue tissue cells disease),Yctalopia (disease),Nigrospinodentatal degenerates (Nigrospinodentatal Degeneration),NIIkawakuroki syndromes (NII kawakuroki Syndrome),Neonatal lupus syndrome,Nodular melanoma,Noack syndrome I types,Hereditary primary nocturnal myoclonia,Tubercle corneal degeneration,Non- epidermolysis hyperkeratosis congenitalis sample erythroderma,Non- epidermolysis hyperkeratosis congenitalis sample erythroderma,Obstructive hydrocephalus,Non-deletion type alpha Thalassemia/mental retardation,Syndrome,Non- ketone hyperglycinemia I types (NKHI),Nonketotic hyperglycinemia,Nonlipid reticuloendotheliosis,Non- neuronophage chronic adult Gaucher disease,Non- scarring epidermidolysis bulla generation,Non- arteriosclerotic cerebral calcification,Nonarticular rheumatism,Non- brain type,Teenager's Gaucher disease,Non- diabetic diabetes,Non local ischemic-type caradrin myopathy,Slight coronary artery non-ketosis hypoglycemia and meat [poison] base deficit disease caused by lacking,Non- ketosis hypoglycemia caused by fatty acyl-CoA dehydrogenase missing,Non- ketone glycinemia,Nonne ' s syndromes,Nonne-Milroy-Meige syndromes,Non- opalescence opalescent dentine,Non- postpartum galactorrhoea amenorrhoea,Non-secretory myeloma,Aspherical erythrocyte hemolysis type anaemia,Nontropical sprue,Noonan syndrome (noonan syndrome),Norepinephrine,Standard presses hydrocephalus,Norman-Roberts syndromes,Norrbottnian Gaucher diseases,Norrie diseases,Norway's type heredity cholestasia,Niman-Peek's disease (niemann-Pick disease),NPS,Necrotizing sialometaplasia,Normal serum albumin,Nape dystonia dementia,Nutritional neuropathy,Nyhan syndromes,Ocular spine dysplasia spectrum,Obstructive apnea,Obstructive hydrocephalus,Obstructive sleep apnea,Oat cells cancer syndrome,It is engaged Thromboaortopathy,Oat cells cancer syndrome,Hidden encephalic vascular malformation,Occult spinal dysraphism sequence,Ochoa syndromes,Ochronosus,Ochronotic arthritis,Eye-brain-kidney syndrome,OCRL,Otocephaly,Ocular albinism,Eyes bleb,Eyes myasthenia gravis,Ocular spine dysplasia,Eye ear vertebra (depauperation) syndrome,Behet's disease,Eye brain syndrome is with hypopigmentation,Eye brain skin syndrome,Eye brain kidney,Oculocerebralrenal dystrophy,Eye skin albinism,Eye-brain-kidney syndrome,Oculocraniosomatic syndrome (agensis),Eye skin albinism,Eye skin albinism Chediak-Higashi,Eye,Tooth,Refer to (toe) depauperation,Eye tooth digital synthesis disease,Oculo-dento-osseous dysplasia,Eye intestines and stomach muscular dystrophy,Eye intestines and stomach muscular dystrophy,Oculomandibulodyscephaly is with hypotrichosis,Francois' syndrome,Eye movement contracture and muscle atrophy,Oculosympathetic paralysis,ODD syndromes (eye tooth refers to (toe) syndrome),Odontogenic tumor,Tooth trichodes syndrome,Mouth-face-means (toe),Mouth-face-means (toe) syndrome,Ohio type amyloidosis disease (VII types),Otitis interna,Congenital Inner Ear is scorching,Tardy otitis interna,Old-field syndrome,Oligohydramnios sequence,Oligophrenia,Oligophrenic is malnutritive,Olvopontocerebellar atrophy,Olvopontocerebellar atrophy is with dull-witted and extrapyramidal sign,Olvopontocerebellar atrophy is with retinal degeneration,Olvopontocerebellar atrophy I,Olvopontocerebellar atrophy II,Olvopontocerebellar atrophy III,Olvopontocerebellar atrophy IV,Olvopontocerebellar atrophy V,Osteochondromatosis,Ollier osteochondromatosis,Omphlexoche-visceral obesity-macroglossia (disease) syndrome,Ondine is cursed,Onion-bulbar neurons lesion,Longstamen Onion Bulb DPN,Onycho-osteodysplasia,Onychotrichodysplasia is with neutrophilic granulocytopenia,Olvopontocerebellar atrophy,Olvopontocerebellar atrophy I,Olvopontocerebellar atrophy II,Olvopontocerebellar atrophy III,Olvopontocerebellar atrophy IV,Olvopontocerebellar atrophy V,Ear-palate-refers to (toe) syndrome,Ear-palate-refers to (toe) syndrome i type,Ear-palate-refers to (toe) syndrome i I types,Ear-palate-refers to (toe) I syndromes,Ear-palate-refers to (toe) II syndromes,Ophthalmoplegia disease,Ophthalmoplegia-enteral Pseudo-Obstruction,Ophthalmoplegia,Retina and cadmium myopathy pigment degeneration,Ophthalmoplegia plus syndrome,Ophthalmoplegia syndrome,Opitz bundle-branch block syndromes,Opitz BBB/G compound syndromes,Opitz BBBG syndromes,Opitz-Frias syndromes,OpitzG syndromes (hypertelorism-hypospadia is integrated),OpitzG/BBB syndromes,Opitz g syndromes (hypertelorism-hypospadias syndrome),Opitz-Kaveggia syndromes,Opitz Oculogenitolaryngeal syndromes,Opitz trigonocephalias (deformity) syndrome,(eye distance is wide for opitz syndrome,Hypospadia syndrome),Opsoclonus,Opsoclonus-myoclonia,Opthalmoneuromyelitis,Eyesight atrophy DPN and deafness,Neuromyelitis optica,Optic nerve neuromyelities,Opticomyelitis,Chiasmal arachnoiditis,Mouth facial cleft seam,Mouthful face is done exercises obstacle,Mouth face dystonia,Oral-facial-digital syndrome,Oral-facial-digital syndrome I types,Oral-facial-digital syndrome I,Oral-facial-digital syndrome II,Oral-facial-digital syndrome III,Oral-facial-digital syndrome IV,Socket of the eye (interior) tumour is with brain and Lesional Skin deformity,Ornithine carbamyltransferase,Ornithine transcarbamylase defect,Orocraniodigital syndromes,Oral-facial-digital syndrome,Oromandibular dystonias,Orthostatic hypotension,Hereditary hemorrhagic telangiectasia (hereditary hemorrhagic telangiectasia),Osseous-Oculo-Dento depauperations,Osseous-Oculo-Dento depauperations,Osteitis deformity,Chondro osteodystrophy deformity,Osteochondrosarcoma,Melnick and Needles type osteodysplasties,Osteogenesis imperfecta,Osteogenesis imperfecta,Osteogenesis imperfecta congenita,Delayed osteogenesis imperfecta,Osteohypertrophic capillary hemangiomas,The many types of osteopathy Hyperostotica sclerotitis of baby,The many types of osteopathy Hyperostotica sclerotitis of baby,Osteopathyrosis,Osteopetrosis,Adult type autosomal dominant osteopetrosis,The pernicious autosomal dominant osteopetrosis of infantilism,The intermediate slight autosomal recessive inheritance osteopetrosis of typicalness,Osteosclerosis fragilis generalisata,Osteosclerotic myeloma,Ostium primum defect (including endocardial cushions defect),Ostium secundum defect,Ornithine transcarbamoylase deficiency,Ear nose larynx palate finger-like syndrome,Ear-palate-finger syndrome I types,Ear-palate-finger syndrome II types,Ear-palate-finger syndrome I types,Ear-dysplasia dentalis,Otopalatodigital syndrome,Otopalatodigital syndrome II types,Oudtshoorn skins,Ovary dwarf's syndrome type,Ovarian hypoplasia syndrome type,Ovarian wedge resection,Oxalosis,Oxidizing ferment is not enough,Tip [deformity],Tip [deformity]-tip [deformity],P-V,Shaking plasy,Acrodermatitis papulosa infantum,Onychauxis Ichtyosiforme,Congenital finger (toe) onychauxis is with natal teeth,Congenital finger (toe) onychauxis,Congenital finger (toe) onychauxis spreads seborrheic keratosis (through ketone),Jadassohn-Lewandowsky type is congenital to refer to (toe) onychauxis,Paroxysmal atrial fibrillation is with multi-system atrophy,Paget diseases,Bone Paget diseases,Chest Paget diseases,Nipple Paget diseases,Nipple-areola Paget diseases,Pagon syndrome,Painful ophthalmoplegia,PAIS,Palatal myoclonus,Palate-ear-refers to syndrome II types,List syndrome,Pallister-Hall syndromes,Pallister-Killian piebald syndromes,Li Siteshi piebald aneuploidy,Li Siteshi piebald syndromes,Tetrasomy 12p Pallister chimera syndromes,Pallister-W syndromes,Hyperkeratosis of palms and soles and bald,Paralysis,Pancreatic fibrosis,Aprotinin hyposecretion and thrombophthisis,The ulcerogenic tumour syndrome of aprotinin,Panmyelophthisis,Panmyelopathy,The adjoint neurodegeneration of pantothenate kinase (PKAN),Papillon-Lefevre syndrome (hyperkeratosis of palms and soles-periodontitis complex),Papillotonic Psuedotabes,Paralysis period is tetanic,Atrophic beriberi,Paralytic brachial neuritis,The Paramedian recessed-popliteal pterygium syndrome of lower lip lip,Side center diencephalon syndrome,Paramyloidosis,Complicated myoclonia,Myotonia congenita,Von Eulenburg myotonia congenitas,Parkinson diseases,Paroxysmal anterior chamber's tachycardia,Paroxysmal cold hemoglobinuria,Paroxysmal dystonia,Paroxysmal choreoathetosis,Paroxysmal Kinesigenic dystonias,Paroxysmal nocturnal hemoglobinuria (and having anaemia),Common paroxysmal hemoglobinuria,Hypnolepsy,Parrot syndrome,Parry diseases,Parry-Romberg syndrome,Parsonage-Turner syndromes,Partial androgen insensitivity syndrome,No. 4 the short arm of a chromosome excalations,No. 5 the short arm of a chromosome excalations,No. 9 the short arm of a chromosome excalations,3q replicates syndrome in part,15q replicates syndrome in part,Local facial paralysis is abnormal with uropoiesis,Partial lipodystrophy,The local monosomy of No. 11 chromosome long arms,Local backbone consciousness syndrome,11q partial trisomies,Partington syndromes,Paroxysmal auricular tachycardia,PDA,Pathology myoclonia,Few joint type-starting juvenile arthritis,Paullinia,Pregnancy and childbirth,Atrioventricular beam,PC lacks,PC lacks A clusters,PC lacks B clusters,Myotonia congenita,Eulenburg disease,PCC lacks,The cold property hemoglobinuria of paroxysmal,PCLD,Plasma coagulation time,Diastolic pressure,Patent ductus arteriosus,PDH lacks,Pearson's syndrome pyruvate carboxylase deficiency,Children's obstructive sleep apnea,Cast off a skin syndrome,Pelizaeus Merzbacher disease,Pelizaeus-Merzbacher disease (familial centrolobar sclerosis),Rough skin-cerebellar ataxia-kidney acidaminuria syndrome,Pelvic pain syndrome,Pemphigus vulgaris,Pena ShokeirII pattern synthesis diseases,Pena Shokeir syndrome II types,Penile fibromatosis,Penis fibrosis,Penile induration,Five X syndrome,Cantrell pentalogys,Pentalogy syndrome,Pentasomy X,Phosphoenolpy ruvate carboxy kinase is lacked,Pepper syndromes,Perheentupa syndrome,Periarticular,Muscular rheumatism,Pericardium shrinks not enough with growth,All (enclosing) amyloidosis of collagen,Perinatal period polycystic kidney disease,Perineum anus,Periodicity amyloid syndrome,Periodic sleep morbid hunger,Periodically comprehensive peripheral nerve disorder,Periodicity view membrane vesicle sexual involution,Periodicity dysosteogenesis-nose hypoplasia-mental retardation,Peripheral neuritis,Peripheral neuropathy,Peritonaeum pericardial diaphragmatic hernia,Pernicious anaemia,Peromelia is with micrognathia (disease),Peroneal muscular atrophy,Nervus peronaeus neural paralysis,Peroutka sneezes,Peroxidase acyl coenzyme A is aoxidized,Peroxidase beta oxidation is not normal,Peroxidase bifunctional enzyme,Peroxidase acetyl-CoA acetyltransferase,Peroxidase acetyl-CoA acetyltransferase defect,Truncus arteriosus,persistent,Perthes disease,Lapse,Petit mal variant,Outstanding Gus (family name) syndrome,Peutz-Touraine syndromes,Fibrous cavernositis of penis,Fei Fu,Pfeiffer syndrome I types (acrocephalosyndactylia),Prostaglandin aI,Prostaglandin aII,Prostaglandin aIII,Phosphoglycerate kinase,PH I types,PH I types,Pharyngeal pouch syndrome,Pulmonary heart disease short chain acyl coa dehydrogenase is lacked,Phenylalanine hydroxylase deficiency,Phenylalaninemia,PKU,Folling's disease,Phocomelia,Sea dog limb syndrome,Phosphoenolpyruvate carboxykinase deficiency,Phosphofructokinase deficiency,Phosphoglycerate kinase deficiency,Phosphoglycerokinase,The kinases of phosphorylase six is lacked,Phosphorylase deficiency glycogen storage disease,Hepatic phosphorylase kinase is lacked,Light sneezing reflex,Light sneeze,Phototherapeutic keratectomy,PHS,Physicist's John's dalton,Phytanic acid storage disease,Pi Phenotype ZZ,PI,Asphyxia apoptosis,Pick disease,Pik Wei Kan (family name) syndrome (the low obesity syndrome of aloveolar ventilation),Sieve's Pierre spot (family name) robin,Sieve's Pierre spot (family name) compound,Sieve's Pierre spot (family name) sequence,Sieve's Pierre spot (family name) syndrome,Sieve's Pierre spot (family name) syndrome is with Hyperphalangy and refers to (toe) bending,Pierre-Marie diseases,Globus pallidus black substance rubrum pigmentation is degenerated,Pili Torti and nerve deafness,PiliTorti- sensory nerve hearing impairments,Pituitary dwarfism II types,The postoperative hypophysis of epinephroectomy (swollen) knurl,Pityriasis pilaris,Hair (keratosa) pityriasis rubra,Pancreaticojejunostomy,PKAN,POLYCYSTIC KIDNEY DISEASE,POLYCYSTIC KIDNEY DISEASE 1,POLYCYSTIC KIDNEY DISEASE 2,POLYCYSTIC KIDNEY DISEASE 3,Phenyl ketonuria,Phenyl ketonuria 1,Plagiocephaly,Plasma cell myeloma,Plasma cell myeloma,Plasma cell leukemia,Plasma thromboplastin component deficiency,Plasma transglutaminase is lacked,Plastic induration cavernous body,Plastic induration,Poliomyelitis,Fissured tongue,PLS,Pectoralis major muscle defect,Pneumorenal syndromes,Paroxysmal nocturnal hemoglobinuria,,Peripheral nerve myelin,Purine nucleoside phosphorylase) lack,Polycystic ovarian disease,Paroxypropion,Poikiloderma atrophy and racing current,Poikiloderma congenitale,Poland's anomaly,Polish sequence (side chest muscle is lacked and syndactylism),Polish dactylion,Poland's syndrome (pectoralis major is without development and brachydactylia syndrome),Progressive poliodystrophia cerebri,Panarthritis Enterica,PAN,The juvenile arthritis I types for multi-joint-rise,The juvenile arthritis II types for multi-joint-rise,The juvenile arthritis I types and II types for multi-joint-rise,Polychondritis,POLYCYSTIC KIDNEY DISEASE,Marrow type POLYCYSTIC KIDNEY DISEASE,Polycystic liver disease,Polycystic ovarian disease,Polycystic liver nephrosis,Refer to (toe)-Joubert syndromes more,Polydysplastic epidermolysis bullosa,Many dystrophic oligrophrenia,Growth hormone deficiency,Many (endocrine) gland autoimmune syndromes type IIIs,Many (endocrine) gland autoimmune syndromes II types,Many (endocrine) gland autoimmune syndromes I types,Many (endocrine) gland autoimmune syndromes II types,(hormone after excision of endocrines) deletion syndrome II types,Polyglandular syndrome,Polyadenous body macular degeneration,Polymorphic macular degeneration,Platelet glycoprotein Ib polymorphisies,Polymorphous corneal dystrophy,Rheumatic polymyopathy,Polymyositis and dermatomyositis,Primary agammaglobulinemia,Polyneuritis,DPN-deafness-ophthalmatrophy,Peripheral polyneuropathy,DPN and polyradiculoneuropathy,Multiple bone fibrous dysplasia,Polyostotic,Schilder's disease,Familial,Gardner type polyposises,Polyposis paramnesia intestines,Polyp-osteoma-epidermis capsule syndrome,Polyposis cutaneous pigmentation and nail change,Polyp spot syndrome,Recidivity chronic hyperplastic perihepatitis,Y polysomies,Refer to (toe) (deformity) with unusual shape cranium more,Referring to (toe) (deformity)-dysmorphism cranium Greig types more,Pompe disease,Glycogen storage disease-II types,Popliteal pterygium syndrome,Porcupine people,Hole brain,Hole brain,Pancreatin deaminase,Porpharia,Acute intemittent porphyria,ALA-D porpharias,Porphyria cutanea tarda,Porphyria cutanea tarda hereditaria,Porphyria cutanea tarda symptomatica,Hepatic porphyria gonorrhea Variegate,Sweden's type porphyria,Diversity porphyria,Accute ratio coughs up sclererythrin,Porphyrin,Alopecia areata,Port-wine stain,Amyloidosis,Portuguese type,Polyneuritis after infection,Progressive lafora's disease,Progressive osteodysplasty,The pale degeneration syndrome of progressive,Progressive spinobulbar muscular atrophy,Stein-leventhal syndrome,Progressive systemic sclerosis disease,Progressive tapetochoroidal dystrophy,Proline oxidase deficiency,Propionic acidemia,Propionic acidemia I types (PCCA missings),Propionic acidemia II types (PCCB missings),Propionyl CoA carboxylase is lacked,Red color weakness,Protanopia,Congestive heart failure Secondary cases protein losing enteropathy,Proteus syndromes,Base portion including 4q is lost,Primary retinitis pigmentosa,PRS,Prune belly syndrome,Osteitis deformans,Viscous fat stores up disease type III,False malnutrition,False black (color) acanthosis,Pseudoachondroplasia,Pseudocholinesterase deficiency,Pseudogout,Hemogenia,Pseudo,False hermaphroditism-kidney disorder-Wilm tumours,Erb's atrophy,Pseudohypoparathyroidism,Pseudohypophosphatasia,Pseudopolyembryony,Pseudothalidomide syndrome,,Nevus elasticus,Psoriasis,Psorospermosis follicularis,False gestation,Pellegrini-Si Dide (family name) syndrome,Psychomotor shakes,Psychomotor epilepsy,Psychomotor equivalent epilepsy,PTC deficiency diseases,It is pteryium,Pterygium colli,Large area is pteryium,Pterygolymphangiectasia syndrome,Pulmonary atresia,Pulmonary lymphangiomyomatosis,,Pulmonary stenosis,Lung is narrow-interventricular septal defect,Denticle,Marrow depauperation,Aortic arch syndrome,Simple alymphocytosis,Simple epithelial tissue cells increase disease,Purine nucleoside phosphorylase deficiency,Purine nucleoside phosphorylase deficiency,Purtilo syndromes,Nevus elasticus,Nevus elasticus dominant character,Nevus elasticus recessive character,Pycnodysostosis,Pycnodysostosis,Lapse,Pyroglutamic aciduria (disease),Pyroglutamic aciduria (disease),Pyrrolin carboxylate dehydrogenase is lacked,Pyruvate carboxylase deficiency,Pyruvate carboxylase deficiency A clusters,Pyruvate carboxylase deficiency B clusters,Pyruvic dehydrogenase is lacked,Pyruvate kinase deficiency,q25-qter,q26or q27-qter,q31 or 32-qter,Extend with the QT of extracellular hypocalcipexy,Non- congenital deafness QT extensions,With congenital deafness QT extensions,Cerebral palsy quadriplegia,Cerebral paralysis quadriplegia,Quantum scatters,r4,r6,r14,r18,r21,r22,Rachischisis posterior,Agenesis of radius-megacaryocyte cytopenia,Radial aplasia-thrombocytopenia syn-drome,Radial aplasia-thrombocytopenia syn-drome neural paralysis,Radicular neuropathy consciousness,Radicular neuropathy consciousness is shunk back,Radicular dentin dysplasia,Anxious hair property dystonia Parkinson (family name) syndrome,Rapp-Hodgkin syndromes,Rapp-Hodg kin (hypohidrosis) ectodermal dysplasia's syndrome,Ectodermal dysplasia's syndrome of Rapp-Hodgkin hypohidrosis,Rare ataxia hereditaria changes and deaf with multiple neuritis caused by being hydroxylated enzyme defect by phytanic acid,Rautenstrauch-Wiedemann syndromes,Rautenstrauch-Wiedemann type neonates Ji Fude (family name) syndrome,Raynaud phenomenons,Recombinant dry plasma,Reactive functional hypoglycemia,Congenital stealthy myotonia secondary response hypoglycemia,Neurofibromatosis,Proctoperineoplasty,Recurrent vomiting,Sympathetic reflex neurovascular dystrophia,Reflex sympathetic dystrophy syndrome,It is ametropia,Refractory anemia,Frost paralysis,Refsum disease (heredopathia atactica polyneuritiformis),Refsum disease (heredopathia atactica polyneuritiformis),Regional ileitis,Reid-Barlow syndromes,(sexual gland is insufficient for Reifenstein syndrome,Orchiatrophy Oligospermia,Bifid urethra,Womanlike breast),Reiger exception-growth retardations,Reiger syndromes,Reimann periodic diseases,Reimann syndromes,Reis-Bucklers corneal dystrophies,Reiter syndromes,Recur Guillain-Barre syndromes,Recurrence-remitting type multiple sclerosis,Renal aplasia,Kidney development is bad,Heredity,Bad-Loken-Senior types retinene the hypoplasia of kidney development,Renal glucosuria,Renal glucosuria A types,Renal glucosuria Type B,Renal glucosuria is O-shaped,Renal-oculocerebrodystrophy,Kidney-retinene dysplasia is with (kidney) medullary cystic disease,Intentional myoclonus,Atomic force intentional myoclonus,Miller syndrome,Atomic force intentional myoclonus,Miller syndrome,Postaxial polydactyly,Postencephalitic intentional myoclonus,Corneal dystrophy heredity afterwards,Dejerine-Roussy syndrome,Archnoiditis after myelographin use,Postnatal cerebral palsy,Postoperative cholestasia,Postpartum galactorrhea amenorrhea syndrome,Postpartum hypopituitarism,Postpartum panhypopituitarism syndrome,Postpartum panhypopituitarism,Postpartum pituitary necrosis,Postural hypotension,Potassium-losing nephritis,Lose potassium syndrome,PotterI type infantile polycystic kidney diseases,PotterIII type infantile polycystic kidney diseases,Persistent pulmonary hypertension,PPS,Prader-Willi syndrome,Prader-Labhart-Willi de Toni Fanconi syndromes,77 tyrosine amyloidosis of prealbumin,Pre-excitation syndrome,Pregnenolone is lacked,Premature's atrial contraction,Progeria syndrome,Premature's ventricular contraction,Neonate's ventricular complex,Neonate or childbirth when neuroaxonal dystrophy,Alzheimer's disease,Presenile macular degeneration,Primary adrenal insufficiency,Primary agammaglobulinemia,Primary aldosteronism,Primary alveolar hypoventilation,Primary amyloidosis,Primary anemia,Primary tinea pedis,Primary bile,Primary biliary cirrhosis,Primary brown's syndrome,Primary meat [poison] base deficit disease lacks,Primary maincenter hypoventilation syndrome,Primary ciliary dyskinesia type,Primary cutaneous amyloidosis,Primary dystonia,Initial stage adrenocortical insufficiency,Primary familial maxilla hypoplasia,Primary hemochromatosis,Essential hidrosis (disease),Primary hyperoxaluria [I types],The type of primary hyperoxaluria 1 (PH1),Primary hyperoxaluria I types,Primary hyperoxaluria II types,Primary hyperoxaluria type III,Primary hypogonadism,Primary intestinal lymphangiectasia,Primary lateral sclerosis,Primary extragenetic amyloidosis,Primary inaccessible pulmonary vascular disease,Primary progressive multiple sclerosis,Primary pulmonary hypertension,Primary progressive multiple sclerosis,Primary pulmonary hypertension,Primary Dyslexia,Primary renal glucosuria,Primary sclerosing cholangitis,Hemorrhagic thrombocythemia,Primary tumor of central nervous system,Visual agnosia,Proctocolitis,Proctocolitis,Adult Ji Fude (family name) syndrome,Children Ji Fude (family name) syndrome,Lucky Ford (family name) is short and small,Progeriod short statures are with mole,De Barsy Progeriod syndromes,The autonomous exhaustion of progressive is with multi-system atrophy,Carry out sex bulbus paralysis,Including carrying out sex bulbus paralysis,Progressive cardiomyopathy is denatured lentiginosis,Family's progressive cerebellar ataxia,Progressive poliodystrophia cerebri,Poliodystrophia (brain),Progressive diaphyseal dysplasia,Progressive facial hemiatrophy,Progressive familial myoclonic epilepsy,Progressive hemifacial atrophy,Progressive is insensitive,Progressive infant brain poliodystrophia,Progressive lenticular degeneration,Progressive lipodystrophia,Children's progressive muscular dystrophy,Familial kidney-malnutritive to retina,Kidney-retina syndrome,Rendu-Osler-Weber syndrome,Respiratory acidosis,Respiratory tract dysfunction,Breathe myoclonia restless legs syndrome,RestrictiveCardio myopathy,Retention Hyperlipemia,Rehtore syndromes (unobvious),Reticular dysgenesis,Retinal Aplastic-Cystic Kidneys-JoubertSyndrome,The cone is degenerated,Retinal cone dystrophy,Retinal cone-rod dystrophy,Retinitis pigmentosa,Retinitis pigmentosa and congenital deafness,Retinal neuroblastoma,Retinol defect,Retinoschisis,Teenager's retinoschisis,Retraction syndrome,DPN after eyeball,Dejerine-Roussy syndrome,Rett syndromes,,Reye syndromes,Reye syndromes,RGS,Rh blood factors,Rh diseases,Rh factor incompatibilities,Rh incompatibilities,Rh [blood group] incompatibility,Rheumatic fever,Rheumatic fever,Rheumatic fever,The big cerebral arachnoiditis of nose nasal sinus,Acra chondrodysplasia punctata (RCDP),Acatalasemia,Traditional Refsum is sick (heredopathia atactica polyneuritiformis),Rhythmicity myoclonia,Rib GapDefects with Micrognathia,Ribbing diseases (unobvious),Ribbing diseases (epiphysis end achondroplasia),Richner-Hanhart Syndrome,Rieger syndromes,Rieter syndromes,Right ventricle fibrosis,Riley Day syndrome,Riley-Smith syndrome,Circular chromosome 14,Circular chromosome 18,Ring 4,The chromosome of ring 4,Ring 6,The chromosome of ring 6,Ring 9,The chromosome R9 of ring 9,Ring 14,The chromosome of ring 15 (mosaic pattern),Ring 18,Ring chromosome 18,Ring 21,The chromosome of ring 21,Ring 22,The chromosome of ring 22,Dermatitits exfoliative infantum (exfoliative dermatitis of newborn),Ritter-Lyell syndromes,RLS,RMSS,Roberts SC- manomelia syndromes,Roberts syndrome,Luo Baici tetraphocomelia syndromes,Robertson is ectodermal dysplasia,Robin anomalads,Robin sequences (primary defect-early stage hypoplasia of the mandible),Robin syndromes,Robinow nanisms,Robinow syndromes,Robinow syndrome dominant forms,Robinow syndrome recessive forms,Nemutogen,Roger's disease (congenital ventricular septal defect),Rokitansky diseases,Romano-Ward syndrome,Romberg syndrome (progressive facial hemiatrophy),Without rooted tooth,Rosenberg-Chutorian syndromes,Rosewater syndromes,Rosselli-Gulienatti syndromes,Rothmund-Thomson syndromes (congenital poikiloderma atrophicans vasculare),Roussy-Levy syndrome,RP,X RS,Rs (rubinstein syndrome),RSDS,RSH syndromes,RSS (Russian spring and summer encephalitis),RSTS,RTS (Robinstein (family name) syndrome),Congenital rubella,Rubinstein syndrome,Rubinstein's syndrome,Lu-tower Er Shi thumbs and the broadening syndrome of hallux toe,Rufous albinism,Ruhr syndromes,Russell diencephalon cachexia,Russell syndromes,Russell syndromes (diencephalic syndrome of infancy),Russeil-Silver nanisms,Russell-Silver syndrome,X russell-Silver syndrome,Ruvalcaba-Myhre syndrome (RMSS),Ruvalcaba syndrome,Ruvalcaba type osseous dysplasias are with mental retardation,Sacrum bone deterioration,Congenital sacrum dysosteogenesis,SAE,Saethre-Chotzen syndrome,Sakati,Sakati syndromes,Sakati-Nyhan syndromes,Spasmus nutans,Salivo-sudoriparous syndrome,Salzman nodular corneal dystrophies,Sandhoff disease,Sanfilippo syndromes,Sanfilippo types A,Sanfilippo types B,Santavuori diseases,Santavuori-Haltia diseases,Boeck sarcoidosises,Sarcoidosis,Sathre-chotzen,Saturday night palsy,SBMA (spinobulbar muscular atrophy),SC amputation syndromes,SC syndromes,SCA3,SCAD defects,The SCAD defects of adult's partial seizures,Congenital whole body SCAD defects,SCAD,SCAD defects,Scalded skin syndrome,Congenital scalp defect,Cymbocephaly,Shoulder blade is raised,Scapuloperoneal myopathies,Scapuloperoneal muscular dystrophies,Scapuloperoneal Syndrome disease types,Cicatrization bulla,SCHAD,Schaumann diseases,Her husky syndrome,SchereshevkII-Turner syndromes,Adrenoleukodystrophy (Schilder),Xi Erde encephalitis,Adrenoleukodystrophy,I types adrenoleukodystrophy (baby's breaking-out),The adrenoleukodystrophy of infancy,Adrenoleukodystrophy,II types adrenoleukodystrophy (adult onset),Schinzel syndromes,Schinzel-Giedon syndromes,Schinzel acras explain syndrome,Schinzel-Giedion Middle faces are shunk back syndrome,Fissure,Schizophrenia,Stupid type metaphysial chondrodysplasia,Stupid type metaphyseal dysostosis,Schmid-Fraccaro syndrome,Schmidt syndrome,Schopf-Schultz-Passarge syndromes,Schueller-Christian diseases,Schut-Haymaker types,Schut-Haymaker-Passarge syndromes,1A types and 1B type schwartz-Jampel syndromes,Schwartz-Jampel syndrome,2 type schwartz-Jampel syndromes,SCID,Scleroderma,The gradual family's sclerosis of whole body,Dispersivity family cerebral sclerosis,Sciatic nerve crush,Scott cranium syndromes are with baryencephalia,Ditch tongue,SCS,SD,SDS,SDYS,Season membranous conjunctivitis,Naevus sebaceus syndrome,Naevus sebaceus,Seborrheic keratosis,Seborrheic wart,Seckel syndrome,Plug Kerr-type dwarf levies,Secondary cases CHB,Secondary amyloidosis,Secondary cases blepharospasm,Secondary cases nontropical sprue,Secondary cases brown's syndrome,Secondary cases athlete's foot,Secondary cases whole body amyloid lesion,Secondary cases dystonia,Secondary cases secretory component deficiency,Secondary cases IgA defects,SED is slow,Congenital SED,SEDC,Intermittent wire is without mole,Intermittent dystonia,Intermittent myoclonia,Seip's syndrome,Seitelberger diseases,Epilepsy,IgG subtype-selective defects,Selective mutism,IgG subtype-selective defects,Selective IgM deficiency,Selective mutism,Selective IgA deficiency,Self healing cytosis disease,Semilobar holoprosencephaly,Seminiferous tubule dysgenesis,Senile retinoschisis,Senile wart,Senior-Loken syndromes,I type hereditary sensory neuropathies,II type hereditary sensory neuropathies,I type hereditary sensory neuropathies,Sensory radicular neuropathy,Degeneration sensory radicular neuropathy,The gradual granulomatosis of septicopyemia,Septo-Optic depauperations,Blood plasma circumscribed meningitis,Serum protease inhibitors defect,Blood plasma carnosine enzyme defect,Setleis syndrome,Severe combined immunodeficiency,Severe combined immunodeficiency is with adenosine deaminase deficiency,Severe combined immunodeficiency (SCID),Property conversion,Sexual infantilism,SGB syndromes,Sheehan syndrome,Shields type dentinogenesis imperfectas,Herpes zoster,Varicella virus,Ship beriberi,SHORT syndromes,The disconnected chromosome deficiency syndrome of arm 18,Short chain fatty acyl-CoA dehydrogenase (SCAD) defect,Of short and small stature and face telangiectasis,Property face/skeletal abnormality-retardance-macrotooth (macroteeth) of short and small stature,Of short and small stature-paratonia-Rieger exception-delayed dentitions,Of short and small stature-onychodysplasia,Of short and small stature and face telangiectasia erythema,SHORT syndromes,Shoshin athlete's foots,Shoulder girdle syndrome,Shwachman-Diamond syndrome,Shwachman syndromes,Shwachman-Diamond-Oski syndromes,Shwachmann syndromes,Shy-Drager syndrome,Shy-Magee syndromes,SI defects,Sialidosis type I,II type baby's sialidosises,Sialidosis,Sialolipidosis,Sick sinus syndrome,Sickle-cell anemia,Sickle cell disease,Sickle cell-hemoglobin C disease,Sickle cell-hemoglobin D disease,Microdrepanocytosis,Sickled-shapedcell feature,Sideroblastic anemia,Sideroblastic anemia,Sideroblast stain,SIDS,Siegel-Cattan-Mamou syndromes,The coloured skin disease of Siemens-Bloch types,Siemens syndrome,Silver's syndrome,Sai Erwo-russell's dwarf disease,Silver-Russell syndrome,Simmond diseases,Siemens syndrome,Single epidermolytic bleb,Single malformation syndrome,Simpson-Golabi-Behmel syndromes,Sinding-Larsen-Johansson diseases,Singleton-Merten syndrome,Sinus arrhythmia,Venous sinus,Nodal tachycardia,Sirenomelia sequence,Sirenomelus,Situs inversus viscerum bronchiectasis and nasosinusitis,SJA syndromes,Sjogren Larsson concurrency ichthyosis,Sjogren syndrome,Sj

Gren syndromes,SJS (Stevens-Johnson syndrome),Skeleton development is abnormal,Weismann Netter Stuhl types skeleton development is abnormal,Decortication syndrome,Skin early stage of pyogenic infection of skin,Skull is asymmetric and mild mental retardation,Skull is asymmetric and slight and refers to,SLE (systemic lupus erythematosus),Sleep apnea,SLO,Sly syndromes,SMA,The acute SMA of baby,SMA I,SMA III,I types SMA,II types SMA,Type III SMA,SMA3,SMAXl,SMCR,Smith Lemli Opitz syndromes,Smith Magenis syndromes,Smith Magenis chromosome regions,Smith-McCort nanisms,Smith diseases,Smoldering Myeloma,SMS,SNE,Light irradiation causes sneeze,Sodium vedproate,Solitary plasmacytoma of bone,Sorsby diseases,Sotos syndromes,Souques-Charcot syndromes,South African genetic porphyria,Dysphonia spastica,Accessory cramp,Accessory cramp,Spastic cerebral palsy,Spastic colon,Dysphonia spastica,Spastic paraplegia,SPD calcinosises,The specific antibody defect of normal immunoglobulin,Specific reading disability,SPH2,Congenital hemolytic jaundice,Spherocytosis,Spherophakia-brachymorphia syndrome,Sphingomyelin lipidosis,Sphingomyelinase deficiency,Spider finger,This Pierre plum Ilyushin-Vogt disease,Spielmeyer-Vogt-Batten syndromes,Spina bifida,Spina bifida aperta,Spina bifida aperta,Backbone arteriovenous malformation,Familial Occurrence spinal ataxia,Spinal ataxia,Spinal cord is crushed,Spinal cord diffusivity idiopathic hyperostosis,Backbone DISH,Duchenne-Arandisease,Various Duchenne-Arandisease,ALS type Duchenne-Arandiseasies,Duchenne-Arandisease-calf hyperplasia,I type Duchenne-Arandiseasies,Type III Duchenne-Arandisease,3 type Duchenne-Arandiseasies,Duchenne-Arandisease-calf hyperplasia,Spinal cord ossify archnoiditis,Spinal stenosis,Spinocebellar ataxia,I type Spinocerebellar Atrophies,I types Spinocerebellar Atrophy (SCAl),II types Spinocerebellar Atrophy (SCA II),Type III Spinocerebellar Atrophy (SCAIII),Type III Spinocerebellar Atrophy (SCA3),IV types Spinocerebellar Atrophy (SCAIV),V-type Spinocerebellar Atrophy (SCAV),VI types Spinocerebellar Atrophy (SCAVI),VII types Spinocerebellar Atrophy (SCAVII),Leptospiral jaundice,Splenic agenesis syndrome,It is splenoptosia,It is splenoptosia,Split hand-faciomandibular dysostosis,Split hand,Arthritis vertebralis,I type backbone dysplasias of rib,Spondyloepiphyseal dysplasia tarda,Spondylothoracic dysplasia,Spondylotic caudal radiculopathy,Sponge kidney,Spongioblastoma multiforme,Spontaneous hypoglycemia,High scapula,Spring ophthalmia,SRS,ST,Corrupt fish syndrome,Staphylococcus scalded skin syndrome,Stargardt diseases,Startle diseases,Status epilepticus,Steele-Richardson-Olszewski syndrome,Bristle disease,Stein-Levensa Er 's syndrome,Steinert disease,Stengel syndromes,Stengel-Batten-Mayou-Spielmeyer-Vogt-Stock diseases,Cholangitis,stenosing,Waist Taper Pipe is narrow,It is narrow,Steroid sulfatase deficiency,Stevanovic ectodermal dysplasias,Stevens Johnson syndromes,STGD,Stickler syndromes,Unyielding man's syndrome,Unyielding man's syndrome,Still diseases,Stilling-Turk-Duane syndrome,Stillis diseases,The myoclonia of stimuli sensitive,Unyielding man's syndrome,Unyielding man,Streeter Anomaly,Autosomal dominant type striatum substantia nigra degeneration,Striopallidodentate Calcinosis,Interstitial,Descemet membrane,Interstitial corneal dystrophy,Struma lymphomatosa,Sturge's syndrome,Sturge-Weber syndrome,Si Teqi-weber phakomatoss,Subacute spongiform encephalopathy,Subacute necrotizing cerebrospinal cord disease,Subacute spongiform encephalopathy,Subacute necrotizing encephalopathy,Subacute sarcoidosis,It is subacute thermophilic neural,Subaortic stenosis,Binswanger's disease,Subendocardial sclerosis,Succinylcholine sensitivity,Congenital invertase-isomaltase defects,Congenital sucrose-isomaltose malabsorption,Congenital sucrose intolerance,Pelizaeus-Merzbacher disease ADL,Pelizaeus-Merzbacher disease's pelizaeus-Merzbacher disease：Familial centrolobar sclerosis's type,Built-in pelizaeus-Merzbacher disease,SIDS,SudeckShi atrophies,Sugio-Kaj II syndromes,Summerskill syndromes,Acrocephalosyndactylism,SummittShi acrocephalosyndactylisms,Summitt syndromes,Brown's syndrome,Adrenal gland,Supraaortic stenosis,Supraventricular tachycardia,Surdicardiac syndromes,Jervell-Lange Nielsen syndrome,SVT,Sweat gland ulcer,Stink with perspiration syndrome,Sweet's syndrome,Swiss Cheese cartilage syndrome,A Peier (family name) syndrome,I types simultaneously refer to occur together microcephalus and baryencephalia,Syndromatic liver conduit hypoplasia,Syringomyelia,Systemic aleukemic reticuloendotheliosis,SA,Carnitine deficiency,Systemic elastorrhexis,Systemic lupus erythematosus,Systemic mast cell disease,Systemic mastocytosis,Systemic onset juvenile type arthritis,Systemic sclerosis,Systopic spleens,T lymphocyte lacks,Tachyalimentation hypoglycemia,Tachycardia,Acatalasemia,Aortic arch syndrome,Aorto-arteritis,Talipes calcaneus,Equinovarus,Tip-foot,Talipes varus,Talipes valgus,Tandem spinal stenosis,Tangier disease,Tapetoretinal degeneration,Radial aplasia-thrombocytopenia syn-drome,Tardive dystonias,Delayed onset muscular dystrophy,Tardive dyskinesia,Delayed onset mouth movement disorder,Tardive dystonias,Slow ulnar palsy,Target type red cell anaemia,Tarsomegaly,Glycogenosis type VII,Include TAS center lines shortage,TAS center lines lack,Tay Sachs sphingolipidosises,Tay Sachs disease,Tay Ichthyosis syndromes,Tay Sachs sphingolipidosises,Tay Ichthyosis syndromes,I type Taybi syndromes,Taybi syndrome,TCD,TCOF1,Treacher-Collins syndrome,Deformable dystonia,Hair-dental-bone syndrome,I type hair-dental-bone syndromes,II type hair-dental-bone syndromes,Type III hair-dental-bone syndrome,Telangiectasis,Telecanthus and associated malformation,Telecanthus-hypospadia syndrome,Temporal-lobe epilepsy,Temporal arteritis/giant cell arteritis,Temporal arteritis,Toxic epithelial necrosis,Tiltedly adhere on stndon sheath top,Tension force myalgia,Terminal deletion in 4q,Terrian corneal dystrophies,Corneal dystrophy syndrome,Notochord is limited syndrome,Tethered cord malformation sequence,Tethered cord syndrome,Neck notochord is limited syndrome,Tetrahydrobiopterin lacks,Tetrahydrobiopterin lacks,Method pleasure (family name) tetra logy,Tetraphocomelia-thrombocytopenic syndromes,No. 9 the short arm of a chromosome tetrasomies,9p tetrasomies,No. 18 the short arm of a chromosome tetrasomies,Thalamic syndrome,Thalamic pain syndrome,Thalamic hyperesthetic anesthesia,Medium-sized thalassemia,Minor thalassemia,Major thalaseemia,Thiamine deficiency,Thiamine response type maple syrup urine disease,Thin substrate membranous type nephrosis,Acetyl-CoA acetyltransferase deficiency disease,RCDP,Acyl-CoA dihydroxyacetone phosphate acyltransferase,Third and fourth pharyngeal pouch syndrome,Three degree of CHBs,Myotonic cataract,It is malnutritive that chest basin refers to (toe),Dorsal spinal canal,Chest and abdomen syndrome,Chest and abdomen ectopia cordis syndrome,Three M syndromes,The thin bone type nanisms of three M,Glanzmann and Naegeli thromasthenia,Primary thrombocytosis,Thrombocytopenia-absent radius syndrome,Thrombocytopenia hemangioma syndrome,Heredity thrombophilia caused by AT III,Thrombotic thrombocytopenic purpura,Thrombus Ulcerative Colitis,Thymic dysplasia with normal immunoglobulins,Thymic dysgenesis,DiGeorge type thymic dysgenesis,Primary thymic dysgenesis is with agamaglobulinemia,DiGeorge type thymic dysgenesis,Congenital thymic aplasia,Trigeminal neuralgia,Twitch,Tinel ' s syndromes,Tolosa-Hunt syndrome,Tonic spasm torticollis,Stiff pupil' Argyll Robertson pupil syndrome,Tooth and nail syndrome,Toxoplasmosis,Other viruses,Rubella,Cytomegalovirus,Herpes simplex infections,

TORCH syndromes,Deformable dystonia,Torticollis,Ophygeneralized lipodystrophy,Completeness pulmonary vein is connected extremely,Touraine ' s aphthosises,Tourette syndrome,Tourette obstacles,Townes-Brocks syndromes,Townes syndrome,Poisoning paralytic anaemia,Toxic epidermal necrolysis,Toxopachyosteose Diaphysaire Tibio-Peroniere,Toxopachyosteose,Toxoplasmosis and viral rubella,Cytomegalovirus,Herpes simplex,With or without atretolemia tracheoesophageal fistula,Tracheoesophageal fistula,Transient neonatal myasthenia gravis,Transiens atrioventricular septal defect,Transposition of conducting arteries,Guarded through transtelephonic,The thyroxine-binding globulin of methionine -30 amyloidosis (I types),The multiple bone connection syndromes of Trapezoidocephaly-,Te Leixie Collins (family name) syndrome,Treacher Collins-Franceschetti syndrome,Trevor diseases,Three atrial hearts,Hair-dental-bone syndrome,(hair) hair gray malnutrition,Hair-nose-refers to (toe) syndrome,Tricuspid atresia,Three functional protein deficiency diseases,Trigeminal neuralgia,Triglycerides stores obstacle,Long-chain fat acid oxidase is damaged,Trigonitis,Trigonocephalia,Trigonocephalia syndrome,Trigonocephalia C syndromes,Trimethylamino Aciduria,Three finger joint thumb hypoplasia distal end onychodystrophies,Triphalangeal thumb syndrome,Behcet triplet syndromes,Triple X syndrome,Triplo-Ⅳ syndrome,Triploidy syndrome,Triploidy,Triples syndrome,Trismus pseudocamptodactyly syndrome,Trisomy,Trisomy G syndromes,Trisomy X,6q partial trisomies,Part 6q trisomy syndromes,The trisomy of mosaic type 9,9P patau syndromes,Part 11q trisomys,The trisomy of mosaic type 14,The trisomy syndrome of mosaic type 14,21 (number chromosome) Trisomy syndromes,The trisomy of mosaic type 22,The trisomy syndrome of mosaic type 22,TRPS,TRPS1,TRPS2,TRPS3,True hermaphroditism,Truncus artiriosus,Tryptophan malabsorption,Tryptophan oxygenase syndrome,TS,TTP,TTTS,Nodositas (brain) is hardened,Tubular ectasia,Turcot syndrome,Turner syndrome,Heredity bone-refer to (toe) onychodysplasia syndrome,With the Turner syndrome of normal chromosomal (caryogram),Turner-Varny syndromes,Oxycephaly,Monodidymus transfusion syndrome,Twin-to-twin transfusion syndrome,A types,Type B,AB types,It is O-shaped,Type i diabetes,The incomplete male of I type familials,Incomplete male pseudohermaphroditism,I type Gaucher disease (familial splenic anemias,Gaucher disease),I type histocytosises,II types (PCCB deficiency diseases),II type tyrosinemias,Intermittent ischemic cardiac arrest,Congenital end multi-joint contracture,II I type Gaucher disease (familial splenic anemias,Gaucher disease),Type III tyrosinemia,Type III hereditary opalescent dentin,Typicalness retinoschisis,Tyrosinase feminine gender albinism (I types),Tyrosinase positive albinism (II types),I type tyrosinemia acutes,I type tyrosinemia chronic types,Tyrosinosis,Urea cycle enzyme disease,Ulcerative colitis,Non specific chronic ulcerative colitis,Ulna breast syndrome,Pallister ulna breast syndromes,Ulnar nerve paralysis,Diving Medical Association,Open rate of recovering,UnconjugatedBenign Bilirubinemiav,Parathyroid activity is low,Unilateral ichthyosiform erythroderma is accompanied side cacomelia,Unilateral osteochondromatosis,Unilateral chest muscle defect and and refer to,Unilateral half depauperation,Unilateral macrencephaly,Unilateral partial lipodystrophy,Unilateral agenesis of kidney,Colon is irregular,Lafora's disease,Unverricht-Lundborg disease (myoclonia),Unverricht-Lundborg-LafDisease,Progressive lafora's disease,Upper limbs-angiocarpy syndrome (Holt-Oram),Upper motor neuron disease,Upper respiratory tract apnea,Urea cycle disorder,Arginase type urea cycle disorder,Argininosuccinase type urea cycle disorder,Carbamyl phosphate synthetase type urea cycle disorder,Citrullinemia type urea cycle disorder,Typicalness N- acrylic glutamate synthetase type urea cycle disorders,Ornithine transcarbamylase type urea cycle disorder,Urethral syndrome,Urethra-eye-Articular Syndrome,I type severe uridine diphosphate glucose thuja acids (base) shift enzyme deficiency disease,Urethra defect,Urofacial syndromes,Type III uroporphyrinogen cosynthetase,Urticaria pigmentosa,Usher syndrome (usher syndrome),I type usher syndromes,II type usher syndromes,Type III usher syndrome,IV type usher syndromes,Metrosynizesis,Uoporphyrinogen I synzyme,Uveitis,Uveomeningitis syndrome,The creutzfeldt-Jacob disease of variation,VACTEL associations,VACTERL associations,VACTERL syndromes,Valgus calcaneus,Valine aminotransferase deficiency,Valinemia,Valproic acid,Valproate is contacted,Valproic acid is contacted,Valproic acid,Van Buren ' s diseases,Vander Hoeve-Habertsma-Waardenburg-Gauldi syndromes,Irregular morbidity immunoglobulin deficiency dysgammaglobulinemia,The Creutzfeldt-Jakob disease of variation,Varicella embryopathy,Diversity porphyria,Vascular birthmark,Binswanger ' s types vascular is dull-witted,Vascular angioma cavernosum,Vascular hemophilia,Vascular malformation,Cerebrovascular malformation,Vasculitis,Vasomotor ataxia,Vasopressin-resistant diabetes insipidus,Pitressin sensitiveness diabetes insipidus,Method spy's association,Vcf syndromes,vcf,Velocardiofacial syndromes,The arthritis of venereal disease,Venous malformation,Ventricular fibrillation,Interventricular septal defect,Congenital ventricular defect,Interventricular septal defect,Ventricular Tachycardia,Veinlet deformity,VEOHD,Vermis of cerebellum hypoplasia,Cerebellar hypoplasia,Spring angle (film) conjunctivitis,Verruca,The radius of the tracheoesophageal oesophagus of the anus of vertebra,Vertebral ankylosis hypertrophy,The choreoid early onset thereofs of Huntington ' s,Pole long-chain acyl-CoA dehydrogenase deficiency disease,Vestibular schwannomas,Vestibular schwannomas multiple neurofibromatosis,Vestibulocerebellum,Virchow ' s tips,Internal organ xanthogranulomatosis,Internal organ xanthogranulomatosis,Internal organ myopathy-outside ophthalmoplegia,Visceral obesity-umbilical cord protrusion-beckwith-Wiedemann syndrome,Typhloexia,Vitamine A deficiency,Thiamine deficiency,Macular dystrophy,Leucoderma,Head leucoderma,Degeneratio,hyaloideoretinalis,Spring angle (film) conjunctivitis,Vogt-Koyanagi-Harada syndrome,VLCAD,Vogt syndrome (striatal syndrome),Vogt Cephalosyndactyly,Vogt Koyanagi Harada syndromes,(Feng) spondylitis ankylopoietica,Von Eulenburg myotonia congenitas,Von Frey ' s syndromes,Glycogen storage disease,(Feng) angiomatosis retinae et cerebelli syndrome,Von Mikulicz syndromes,Feng's multiple neurofibroma,Von Willebrandt diseases,Vasopressins,Vrolik disease (I types),Ventricular septal defect,Plain edition keratosis,Plain edition ichthyosis,W syndromes,Wa Erdunbao syndromes,Waardenburg-Klein syndrome,Wa Erdunbao syndromes (I types),Wa Erdunbao syndromes (II types),IIA type Wa Erdunbao syndromes,IIB type Wa Erdunbao syndromes,Type III Wa Erdunbao syndromes,IV type Wa Erdunbao syndromes,Waelsch ' s syndromes,WAGR complexs,WAGR syndromes,Waldenstroem ' s macroglobulinemias,Waldenstrom’s Purpura,Waldens trom ' s syndromes,Waldmann diseases,Walker-Warburg syndromes,Splenectopia,Warburg syndromes,Warm antibody hemolytic anemia,Warm type cross reacting antibody disease,Wartenberg syndrome,Bacterial endocarditis,Brain moisture,Bacterial endocarditis,Watson-Alagille syndromes,Fertile-ergeth syndrome,The disease of wax,Viola-Bertier 's syndrome,Weaver syndrome,Weaver-Smith syndromes,Weber-Cockayne diseases,Wegener ' s granulomatosis,Leptospirosis (leptospiral jaundice),Weil syndromes,Weill-Marchesani,Weill-Marchesani syndrome,Weill-Reyes syndromes,Weismann-Netter-Stuhl syndromes,Weissenbacher-Zweymuller syndromes,Vail syndrome,Wenckebach,Werdnig-Hoffman diseases,Werdnig-Hoffman paralyses,Werlhof ' s diseases,Adult progeria,Wernicke ' s (C) I syndromes,Wernicke ' s aphasias,Wernicke-Kor sakoff syndromes,West's syndrome,Beriberi humida,WHCR,Whipple ' s diseases,Intestines lipogranuloma disease,Whistling face syndrome,Whistling face syndrome,White-Darier diseases,Whitnall-Norman syndrome,Verticillate mole sample melanin is excessively sick,WHS,Wieacker syndromes,Wieacker syndromes,Wieacker-Wolff syndromes,Wiedmann-Beckwith syndromes,Wiedemann-Rautenstrauch syndromes,Wildervanck syndrome,Willebrand-Juergens diseases,Hypotonia-hypomentia-hypogonadism-obesity syndrome,Williams syndrome,Williams-Beuren syndromes,The nephroblastoma,Nephroblastoma irideremia-gonadoblastoma-MR syndrome,Nephroblastoma irideremia-gonadoblastoma-MR,Nephroblastoma irideremia-gonadoblastoma-MR syndrome,The nephroblastoma-pseudohermaphroditism-nephrosis,The nephroblastoma and pseudohermaphroditism,The nephroblastoma-pseudohermaphroditism-glomerulopathy,Wilson ' s diseases,Wilson ' s syndromes,Winchester-Grossman syndromes,Victoria-Austrian syndrome,Wiskott-Aldrich type immune deficiencies,Witkop ectodermal dysplasia,Witkop tooth-nail syndrome,Wittmaack-Ekbom syndromes,WM syndromes,Wechsler Memory Scale,WNS,Wohlfart diseases,Wohlfart-Kugelberg-Welander disease (heredity peri position nerve originality muscular atrophy),Wolf syndrome,Wolf-Hirschhorn chromosomal regions (WHCR),Wolf-Hirschhorn syndromes,Wolff-Parkinson-White syndrome,Wolfram syndrome,Primary familial xanthomatosis (lysosomal storage disease caused by acid lipase deficiency),Woody Guthrie ' s diseases,W-P-Ws,Writer ' s spasm,WS,WSS,WWS,Retinal arterio-venous aneurism,Addison ' the s diseases of X-linkage,The adrenoleukodystrophy of X-linkage,Adult's spinobulbar muscular atrophy of X-linkage,The adult spinal muscular atrophy of X-linkage,X linked agammaglobulinemias are with growth hormone deficiency,X linked agammaglobulinemias,The chain syndromes of lymphadenia type X,Cardiomyopathy and neutrophilic granulocytopenia chain X,Central caryogram myopathy chain X,The chain copper deficiency diseases of X-,Copper uptakie chain X is bad,Dominant Kang-Xu Er 's syndrome chain X,Dominant heredity callosal agenesis chain X,Dystonia-parkinson's syndrome chain X,Ichthyosis chain X,X-linked agammaglobulinemia,Neonate's Nectrotizing encephalopathics chain X,Juvenile retinoschisis chain X,Agyria disease chain X,Lymphoproliferative syndrome chain X,Chain X intelligence development is low-the thumb syndrome held,Intelligence development chain X is low with hypotony,Chain X intelligence development is low and huge testis,Progressive variable immunodeficiency chain X,Recessive Kang-Xu Er 's syndrome chain X,Recessive severe combined immunodeficiency syndrome chain X,Retinoschisis chain X,Spine epiphyseal dysplasia chain X,(lithoxiduria lacks xanthine oxidase deficiency disease,Heredity),The xanthogranulomatosis of whole body,Xanthoma tuberosum,Xeroderma pitmentosum,Dominant xeroderma pitmentosum,AIXPA type xeroderma pitmentosums,Type BII XPB xeroderma pitmentosums,Type EVXPE xeroderma pitmentosums,Type C IIIXPC xeroderma pitmentosums,Type D IV XPD xeroderma pitmentosums,Type F VI XPF xeroderma pitmentosums,Type G V II XPG xeroderma pitmentosums,Xeroderma pitmentosum mutation Type XP-V,Xeroderma-clubfoot enamel defect,Xerodermic idiocy,Xerophthalmia,Keratitis sicca,The chain lymphadenias of X,XO syndrome syndrome,Xeroderma pitmentosum,XX male syndrome,Sex is reversed,XXXXX syndromes,XXY syndromes,XYY syndromes,XYY chromosome models,Yellow mutant albinism,Huang refers to (toe) first syndrome,YKL,Young women with arteritis,Yunis-Varon syndromes,YY syndromes,Z-E syndromes,Z and protease inhibitors deficiency disease,Ze Weige syndromes,Zellweger zellweger syndromes,ZE syndrome,Ziehen-Oppenheim disease (deformable dystonia),Zimmermann-Laband syndrome,Congenital zinc deficiency,Zinsser-Cole-Engman syndrome,ZLS,Zollinger-Ellison Syndrome.

In another embodiment, pharmaceutical composition comprising the GM-CSF of separation or its chimeric molecule can be individually or with other biological agents, medicine or treatment method (such as chemotherapeutics such as operation, radiotherapy, bone-marrow transplantation, peripheral stem cell transplanting, classic chemotherapy, including cis-platinum, streptozotocin, adriamycin, fluorouracil, mitomycin C, busulfan, oblimersen, etoposide, vincristine, endoxan, gemcitabine, taxol, cytabarine；Antiviral therapy includes Didanosine, AZT, virazole, Ribavirin, Zidovudine, dideoycytidine；Other treatment methods include antibiotic, cell factor, growth factor, small-molecule drug) combine for a variety of leukaemia such as acute myelogenous leukemia and acute lymphatic leukemia；The disease related for cancer chemotherapies such as leukopenias (Neuroleptic Leukocytopenia), such as acute myelogenous leukemia or other high risk patients receive the neutrophilic granulocytopenia for including producing after febrile neutropenic neutrophilic granulocytopenia or acute myelogenous leukemia and myelodysplastic syndrome strengthening measures occurred after chemotherapy or occur the neutrophilic granulocytopenia that patient's HIV induction of Kaposi sarcomas is produced；For aiding in bone-marrow transplantation or peripheral stem cell to transplant；Auxiliary treatment failure or the bone marrow graft of delay；Autologous bone marrow or the marrow of peripheral stem cell (peripheral blood progenitor cell, PBPC) transplanting (such as non_hodgkin lymphoma patient) is accelerated to recover；For Hodgkin's disease；Improve mobilization and the yield of PBPC for transplanting.

And in another embodiment, the pharmaceutical composition comprising the GM-CSF of separation or its chimeric molecule can be used in combination individually or with other biological agents, medicine or treatment method, chemotherapy dose-effect intensity is improved as adjuvant；Cause chemotherapy priming effect (e.g., changing cell cycle dynamic increase cytotoxicity)；It is used as the Sculpt element agent in chemotherapy；Neutrophilic granulocytopenia is reversed when being used with chemoradiotherapy plus；The side effect of mucosa lesions (catarrh) caused by prevention or treatment chemotherapy；The fungal infection of palliating leukemia patient；Contribute to the maturation of BMDC and antigen presenting cell as immunologic adjuvant；It is used as the adjuvant of the vaccines such as hepatitis B and influenza virus；For premature's septicopyemia bacterium and the auxiliary treatment of fungal infection；Anti-infective auxiliary treatment for immunosuppressive organ transplant patients；Suppress HIV to replicate, be conducive to improving CD4 numbers and reduce AIDS patient infective dose；As tumor vaccine, strengthen the immune response of melanoma tumor antigen epitope polypeptide；Promote wound reparation, such as intractable leg ulcer；Stimulate arteries to generate, be such as used for Peripheral arteral disease；Axon regeneration can be stimulated in nervous system injury；Brain protection after apoplexy；For pulmonary alveolar proteinosis；Graft-versus-host disease is reduced, during such as stem cell transplantation.

In another embodiment, the a variety of diseases for the treatment of can be used in combination individually or with other biological agents, medicine or treatment method in pharmaceutical composition comprising the IL-3 of separation or its chimeric molecule, not only including marrow abnormality diseases such as myelodysplastic syndrome (MDS), heterologous related clones hematopoiesis disorders；It is characterized so that bone marrow morphology and maturity (myelosis can not) be abnormal, and further results in the marrow abnormality disease of peripheral blood cells reduction, anaemia, Neuroleptic Leukocytopenia and decrease of platelet；Diamond-Blackfan anaemias (DBA)；Bone marrow graft to transplanting hyperplasia after such as hematopoietic system cancer high dose chemotherapy such as leucocyte canceration；Non_hodgkin lymphoma；Breast cancer；Gene transfer is treated；Leukopenia related HIV and bone marrow suppression；Thrombopenia；Secondary hematopoietic deficiency；The certain types of anaemia such as alpastic anemia, FSCCL；The parasitic infections such as trichina；Lung cancer；The virus infection such as herpesviral.

In another embodiment, pharmaceutical composition comprising separation IL-4 or its chimeric molecule can combine the treatment and prevention for a variety of diseases individually or with other biological agents, medicine or treatment method, including autoimmune disease, such as psoriasis, by promoting T_HDemyelinating neuropathies inflammation (such as multiple sclerosis), CrohnShi diseases is immunized in 2 cell effects, Grave ' s hyperthyroidisms, autoimmune diabetes；Anaphylactia is treated, such as promotes the tissue survival (such as skin, heart transplant) of allograft as supportive treatment, is reversed or prevention delayed allergy or enteritis；Treat infective inflammation (gastritis as caused by helicobacter pylori)；Glomerulonephritis；Heymann nephritis, membranous nephropathy, periodontosis, rheumatic arthritis；Prevent the communicating pleurisy of leucocyte；Repair the cellular immunity after inflammatory reaction imbalance (such as burn and other damages)；Adjust sleep behavior (such as treatment dyssomnias).

In another embodiment, pharmaceutical composition comprising separation IL-5 its chimeric molecule can combine the treatment for a variety of diseases individually or with other biological agents, medicine or treatment method, the cancer such as parasitic infection, acute myelogenous leukemia (AML) such as including filaria.

Moreover, the Pharmaceutical compositions of the present invention have a higher drug effect, stronger heat endurance, longer serum half-life or with expression in the protein of non-human cell lines or its chimera compared with when with higher blood dissolubility.The present invention also shows lower Ia scavenging action or related side effects.Due to the characteristic of these improvement, the pharmaceutical composition of the present invention can be administered with lower frequency compared with expression is in the protein of non-human cell lines or its chimera.Reduction administration frequency can strengthen the adaptability of patient to be conducive to treatment results.The quality of life of patient equally can also increase.

Therefore, in one embodiment, pharmaceutical composition of the invention can be administered with therapeutic dose with expression in the protein of non-human cell lines or its chimera identical administering mode.Therapeutic dose refers to the amount for producing the necessary pharmaceutical composition of activity in vivo.The accurate amount for giving compound is determined by factors such as the other compositions in the exact type such as treatment symptom, the physical qualification and pharmaceutical composition for the treatment of patient.Pharmaceutical composition containing albumen of the present invention or chimeric molecule isoform can be prepared to treat the patient with said one or multiple symptoms in favor of the different formulas of administration.The mean treatment significant degree of pharmaceutical composition may be different.It is expected that effective dose in 0.1ng/kg body weight between 20 μ g/kg body weight；Or the suggestion according to qualified physicians or prescription.

Invention further provides application of the protein or chimeric molecule and pharmaceutical composition including at least Partial Protein or the separation of its chimeric molecule in different therapy and/or diagnosis.

More specifically, the invention provides the method for treating or preventing subject mammalian diseases, the amount or activity of the albumen in the present invention or chimeric molecule can wherein be increased to alleviate disease, this method includes giving the protein of separation of effective dose, the chimeric molecule comprising the albumen, the chimeric molecule of the extracellular regions segment comprising the protein or protein or the pharmaceutical composition of chimeric molecule comprising the separation to the subject mammal.

The present invention is further illustrated by with non-limiting embodiment below.

Embodiment 1

(a) preparation of pIRESbleo3-Fc constructs

The DNA sequence dna of encoding human IgG1 Fc domains is expanded by PCR (PCR), from EST cDNA storehouses (Clone ID 6277773, Invitrogen), using forward primer (the SEQ ID NO for being mixed with restriction enzyme BamH1 and BstX1 sites respectively：21) with reverse primer (SEQ ID NO：22).The amplicons cloned enters in pI RESbleo3 (Cat.No.6989-1, BD Biosciences) corresponding restriction enzyme site to prepare construct pIRESbleo3-Fc.PIRESbleo3-Fc discharges the Insert Fragment of 780bp desired sizes with BamH1 and BstX1 digestion, as gel electrophoresis is determined.

(b) preparation of the DNA construct of marking protein

The DNA sequence dna of encoding proteins matter is expanded by PCR, from EST cDNA storehouses, uses the forward primer and reverse primer that restriction enzyme site is introduced according to table 8.After amplification, amplicon is through suitable digestion with restriction enzyme and is cloned into expression vector as shown in table 8, to prepare Carrier-protein construct.Suitable restriction enzyme is used for the carrier for digesting the DNA sequence dna containing encoding proteins matter, to discharge the fragment of desired size as shown in table 8.Carrier-protein construct is sequenced to determine the integrality of cloning procedure described herein.

Table 8

Protein-Fc and related cloning information

Protein

CDNA originates

Forward primer

Reverse primer

Restriction endonuclease sites

Carrier

Size (bp)

GM-CSF

PUMCV1-GM-CSF, Aldevron

SEQ ID NO：23

SEQ ID NO：24

EcoRV, BamH1

PIRESbleo 3 (Cat.No.6989-1, BD Biosciences)

447

IL-3

The RNA extracted in the Jurkatcells of stimulation

SEQ ID NO：33

SEQ ID NO：34

BamHI.BamHI

PIRESbleo3 (Cat.No.6989-1, BD Biosciences)

491

IL-4

pUMCV-IL-4Aldevron

SEQ ID NO：49

SEQ ID NO：50

NotI, BamHI

PIRESbleo 3 (Cat.No.6989-1, BD Biosciences)

481

IL-5

IL-5pBR 322, ATCC

SEQ ID NO：59

SEQ ID NO：60

EcoRI, BamHI

PIRESbleo 3 (Cat.No.6989-1, BD Biosciences)

437

(c) preparation of Megaprep carriers-albumen

By the E.coli incubated overnights of carrier-albumen conversion, 750 μ l bacterium solutions are taken to add the sterile LB meat soups containing ampicillin (100 μ g/ml) 750ml.37 DEG C of concussion and cultivates 16 hours.Plasmid is prepared by QiagenEndofree Plasmid Mega Kit (Qiagen Mega Prep Kit #12381).

Or, the nucleotide sequence for the coding protein being cloned into carrier (such as pIRESbleo3) can be expanded with primer, the primer adds the restriction enzyme site for allowing the DNA sequence dna of encoding proteins matter to be cloned into Fc nucleotide sequences upstream in carrier-Fc (such as pIRESbleo3-Fc), so that protein and Fc nucleotide sequences are merged with meeting frame directly or through connexon.

Embodiment 2

(a) people's cell expression of GM-CSF generation and purifying

(i) GM-CSF of the invention generation

At the 0th day, be for inverting embryo HK cells 3 × 10⁷Cell is inoculated with five 500cm²Tissue culturing plate (Corning), the cell such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene) or 293A (Invitrogen).Cell is inoculated into EagleShi culture mediums/HamShi nutritional blends F12 (DMEM/F12) (JRH Biosciences) of every flat board 90mlDulbeccoShi improvement, culture medium with the addition of 10% (v/v) donor calf serum (FCS, JRH Biosciences), 10mM HEPES (Sigma), 4 mM Glus (Ameresco) and 1% (v/v) Pen .- Strep (JRH Biosciences).In addition, no HEPES FCS can be replaced with 10% (v/v) heat-inactivated donor calf serum (DCS, JRH Biosciences).

1st day, transfected with calcium phosphate.Before transfection, the DMEM/F12 for the above-mentioned supplement of addition newly matched somebody with somebody with 120ml changes liquid to each culture plate.Prepare calcium phosphate/DNA sediment：By DNAs of the 1200 μ g pIRESbleo (Invitrogen) containing GM-CSF Gene and 3000 or 3720 μ l 2.5M CaCl₂Add sterile H₂O, makes total amount to 30ml (solution A).10ml pipettes dropwise add 30ml solution As in 2x HEPES buffer salt solutions (HBS) (solution B).In adition process, solution B is gently blown and beaten.Mixture was in 25 DEG C of vortex oscillation cultures 20 minutes.Mixture also can be without incubation under vortex.12ml mixtures are added to each flat board.After 4 hours, remove contained transfection media, and add DMEM/F12 culture mediums according to every piece of plate 100ml, 10% (v/v) heat-inactivated donor calf serum (DCS, JRHBiosciences), 10mM HEPES are supplemented with the culture medium, 4mM Glus, 1% (v/v) Pen .- Strep, and pH7 is arrived with 3.5mM HCl regulations, it is incubated overnight at 37 DEG C.

At the 2nd day, cells and supernatant is discarded.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added into the culture medium of collection.Content in flat board is washed 2 times with 50ml serum-free DMEM/F12 culture mediums, again the fresh serum-free DMEM/F12 culture mediums of 100ml are added to each culture plate, 40mM N- acetyl-D-MANNOSE amine is supplemented with the culture medium, 10mM Glus, 4.1g/L mannoses, 1% (v/v) Pen .- Strep and ITS solution (5mg/L bovine insulins, the human transferrin and 5 μ g/ml selenium of the infiltration of 5mg/L part ions) (Sigma).In addition, supplement N- containing 40mM acetyl-D-MANNOSE amine, 7mM Pidolidones salt, 0.5g/L mannoses and 1% (v/v) mycillin.Culture plate is incubated under the conditions of 37 DEG C.

3rd day, collect serum-free DMEM/F12 culture mediums and the DMEM/F12 culture mediums of the material added in culture medium of the addition just like the 2nd day of the fresh serum-frees of 100ml are added per flat board.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection and mixture is in 4 DEG C of storages.

4th day, collect serum-free DMEM/F12 culture mediums.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection and pooled together with the gleanings of the 3rd day.With reference to gleanings pass through add 1/10th volumes 200mMMES/50mM MgCl₂PH6 is arrived in pH6 regulations, before-protein adsorption filter (Durapore, Millipore) low with 0.45mm removes particulate.Mixture is in -70 DEG C of storages or uses immediately.

(ii) GM-CSF of the invention purifying

The process of dye-ligand chromatogram (DLC) is used as the first step for purifying GM-CSF.In order to be effectively combined and discharge in the micro form of mass purification, immobilized active dyestuff storehouse is used to screen GM-CSF.Then, suitable dye-protein matter is combined examines in small-scale cylindricality formula.

In small scale purification, the sample for the cells and supernatant that 5ml thaws passes through 0.5ml dye ligand posts under conditions of pH6.0.In the step of the optimization, in order to obtain the maximum recovery of classification, optimal dyestuff ball-cell factor and pH combinations are selected, to carry out high-volume DLC.

For extensive DLC, the High of reactive dye numbering 78 (Zymatrix) is selected as the reactive dye to GM-CSF with best combination and elution property.The cells and supernatant of filtering has 3ml or 6ml 50mM MES/5mMMgCl respectively under gravity stream by 4.0ml or 8.0ml₂Cylinder (Alltech, Extract CleanFilter columns) of the pre-balance to pH6 DLC resins.4 DEG C are stored in until ELISA results confirm to purify successfully by the bulk flow of sample.Pillar washes post with buffer A (20mM MES/5mM MgCl2 pH 6), until can't detect protein fractions by colourity Protein assay (Biorad Protein assays).GM-CSF is eluted with three kinds of elution buffers according to order below.

Eluent 1：Buffer solution C (50mM Tris-Cl/10mM EDTA pH8)

Eluent 2：EN1.0(50mM Tris-Cl/10mM EDTA/1.0M NaCl pH8)

Eluent 3：EN2.0(50mM Tris-Cl/10mM EDTA/2.0M NaCl pH8)

Elutriated fraction is detected with silver staining SDS PAGE, (R＆D Systems Duoset) is monitored using 4-20%Tris-Glycine glue (Invitrogen), and with GM-CSF ELISA methods.GM-CSF is detected in Buffer EN1.0 eluent.Analyzed by SDS PAGE, 90% contaminating protein is removed in basic purification step.The DLC fractions for closing GM-CSF are collected, for size exclusion chromatography.

Combining DLC fractions carry out exclusion chromatography (SEC) with Superdex 75 preparative grade 16/70 (Pharmacia, Uppsala, Sweden) post.1% ammonium bicarbonate advection liquid was by speed flowing in 1ml/ minutes.In addition, by 1.5ml/ minutes flow velocitys Sephadex 200 preparative grade (Pharmacia, Uppsala, Sweden) post can be crossed with 50mM MES buffer solutions (pH6.5).Total to spend the post time for 120 minutes, there is peak between 40-100 minutes in Superdex posts；Then there is peak between 20-100 minutes in Sephadex posts.Elutriated fraction is detected with silver staining SDS PAGE, using 4-20%Tris-Glycine glue (Invitrogen).GM-CSF detection times from eluent are that the detection time in 55-65 minutes, Sephadex posts is about 52 minutes in Superdex posts.

The GM-CSF purified with Sephadex posts is further purified by anion exchange resin (Uno Q, Bio-Rad Laboratories), and the latter is in advance with 50mM MES (Sigma) balances to pH 6.5.With linear gradient, from 50mM MES pH6.5 to 50mM containing 1MNaCl, the combining GM-CSF of MES pH 6.5 are eluted from post.With the quick desalination posts of HiPrep 26/10 by the fraction of factor-containing the desalination in PBS.

The GM-CSF apparent molecular weights of purifying are 14-32kDa, are 100% with silver staining SDS PAGE detection purity.After the fraction collector containing GM-CSF, concentrate in one with centrifugal filter device (AmiconUltra, Millipore) and be less than on 2ml post.280nm absorption values are detected with 14230 M-1 cm-1 molar absorption coefficient, the GM-CSF concentration of purifying is 417ug/ml.

(b) IL-3 of people's cell expression production and purifying

(i) IL-3 of the invention production

0th day, by the human embryonic kidney cells of transfection, such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene) or 293A (Invitrogen), it is inoculated in by cell number 3 × 107 in 500cm2 tissue culture dishes (Corning).Added per hole cell in 90ml Dulbecco ' s Modified Eagle ' sMedium/Ham ' s Nutrient Mixture F12 (DMEM/F12) (JRHBiosciences), culture medium and be previously added 10% (v/v) heat-inactivated hyclone (FCS, JRH Biosciences), 4mM Pidolidones salt (Amresco) and 1% (v/v) mycillin (benzyl penicillin 5000U/ml, the μ g/ml of streptomycin sulfate 5000) (JRHBiosciences).Also the FCS without HEPES can be replaced with 10% (v/v) heat-inactivated donor calf serum (DCS, JRHBiosciences).

First day, transfected with calcium phosphate.Before transfection, the DMEM/F12 for the above-mentioned supplement of addition newly matched somebody with somebody with 120ml changes liquid to each culture plate.Prepare calcium phosphate/DNA sediment：The DNA of 1200 μ g pIRESbleo3 (Invitrogen) genes of IL-3 containing people and 3000 or 3720 μ l 2.5M CaCl2 are added into sterile H2O, make total amount to 30ml (solution A).30ml solution As are added 2 x HEPES buffer salt solutions (HBS) (solution B) by 10ml pipettes dropwise.During liquid feeding, with solution B featheriness bubble.25 DEG C of whirlpools are incubated mixed liquor 20 minutes, and mixed liquor also can be without incubation under whirlpool.Each hole is added dropwise in 12ml mixed liquors.37 DEG C of culture plate, 5%CO2 are incubated overnight.

Second day, discard culture supernatant.The culture washed with 50ml DMEM/F12 culture mediums in each culture plate, the fresh serum free DMEM/F12 culture mediums of 100ml Pidolidones containing 4mM salt and 1% (v/v) mycillin are added per culture plate.The DMEM/F12 of addition can also fill into 40mM N- acetyl-D-MANNOSE amine, 7mM Pidolidones salt, 0.5g/L mannoses and 1% (v/v) mycillin.By 37 DEG C of culture plate, 5%CO2 is incubated overnight.

3rd day, collect culture supernatant.Each culture plate adds the fresh serum free DMEM/F12 culture mediums 100ml of the salt of Pidolidone containing 4mM and 1% (v/v) mycillin.37 DEG C, 5%CO2 overnight incubations.100mM PMSF (1% (v/v)) and 500mMEDTA (1% (v/v)) are added in the culture supernatant of collection, 4 DEG C of preservations after mixing.

4th day, collect culture supernatant, add 100mM PMSF (1% (v/v)) and 500mMEDTA (1% (v/v)), before particle is removed with 0.45 μm low protein binding filter (Durapore, Millipore), it is mixed with the supernatant of the 3rd day Collection and conservation.The mixed liquor can immediately using or put -70 DEG C of preservations.

(ii) IL-3 of the invention purifying

The cells and supernatant of 1 liter of filtering is concentrated with tangential flow filter (TFF) device (Pelicon XL, Ultracell, Millipore).With 5KDa it is standard molecular weight burble point until sample is concentrated into 40 or 50ml by sample with regenerated cellulose film of the 150ml/ minutes speed by 150cm2.To make concentrating sample filter completely, continue to be concentrated into another 40ml with the extra buffer solutions of equivalent 50mM MES pH 5.6.Completely filtering the step of repeat 2-3 times to being finally concentrated to 80ml.Afterwards with the complete filtered sample of 0.45 μm low protein binding filter (Durapore, Millipore) filtering and concentrating.

With 50mM MES pH5.6 (Sigma) by cation exchange column (Bio-RadLaboratories, Uno S12) pre-equilibration to pH5.6, IL-3 is purified by the way that the cells and supernatant of concentration is crossed into post from TFF.Eluted with linear gradient from the combining IL-3 of 50mM MES pH5.6 of 50mM MES pH 5.6 to NaCl containing 1M from post.The apparent molecular weight and degree of purification of obtained fractions are analyzed with ELISA and 1DSDS PAGE (4-20%gradient Tris-Glycine gels (Invitrogen)), it is quantitative with anti-IL-3 ELISA (R ＆ DSystems).Using 5KDa as standard molecular weight burble point, the fraction containing IL-3 is concentrated about 50 times with centrifugal filter device (AmiconUltra, Millipore).

IL-3 is further purified by size exclusion chromatography with Superdex 75 preparative grade16/70 (Pharmacia, Uppsala, Sweden) post.1% ammonium bicarbonate advection flow velocity is 0.5ml/ minutes.Furthermore, it is possible to be flowed through with 1 x Dulbecco ' s phosphate-buffered salines (DPBS amendments) (JRH Biosciences) (pH7.3) as advection liquid with 1.5ml/ minutes speed.Ammonium bicarbonate buffer solution is total to spend the post time for 240 minutes, and elution peak value is between 70 and 190 minutes.And when correcting buffer solution with DPBS, total to spend the post time for 120 minutes, elution peak value is between 20 and 100 minutes.With anti-IL-3 ELISA (R ＆ D Systems) and silver staining SDS PAGE detection elutriated fractions, using 4-20%Tris-Glycine glue (Invitrogen).During with ammonium bicarbonate buffer solution, IL-3 elution peak values are between 90 and 120 minutes；And when correcting buffer solution with DPBS, peak value is about 53 minutes.

The IL-3 apparent molecular weights of purifying be 16-32 kDa between, with silver staining SDS PAGE detection purity be at least 99%.After the fraction collector containing IL-3, concentrate in one with centrifugal filter device (Amicon Ultra, Millipore) and be less than on 2ml post.280nm absorption values are detected with 12615 M-1cm-1 molar absorption coefficient, the IL-3 concentration of purifying is 931ug/ml.

(c) IL-4 of people's cell expression production and purifying

(i) IL-4 of the invention production

0th day, by the human embryonic kidney cells of transfection, such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene) or 293A (Invitrogen), by cell number 3 × 10⁷It is inoculated in 500cm²In tissue culture dishes (Corning).Added per hole cell in 90ml Dulbecco ' s Modified Eagle ' sMedium/Ham ' s Nutrient Mixture F12 (DMEM/F12) (JRHBiosciences), culture medium and be previously added 10% (v/v) heat-inactivated hyclone (FCS, JRH Biosciences), 10mM HEPES (Sigma), (JRH Biosciences), 4mML- glutamates (Ameresco) and 1% (v/v) mycillin.Also it can replace not conforming to HEPES FCS with 10% (v/v) donor calf serum (DCS, JRH Biosciences)

First day, transfected with calcium phosphate.Before transfection, the DMEM/F12 for the above-mentioned supplement of addition newly matched somebody with somebody with 120ml changes liquid to each culture plate.Prepare calcium phosphate/DNA sediment：By the DNA and 3000 or 3720 μ l 2.5M CaCl of 1200 μ g pIRESbleo (Invitrogen) genes of IL-4 containing people₂Add sterile H₂O, makes total amount to 30ml (solution A).30ml solution As are added 2xHEPES buffer salt solutions (HBS) (solution B) by 10ml pipettes dropwise.During liquid feeding, with solution B featheriness bubble.25 DEG C of whirlpools are incubated mixed liquor 20 minutes, and mixed liquor also can be without incubation under whirlpool.Each hole is added dropwise in 12ml mixed liquors.The culture medium containing transfection liquid is removed after 4 hours, 100ml hot Dead donor cow's serums containing 10% (v/v) (DCS, JRH Biosciences), 10mM HEPES, 4mM Pidolidones salt, 1% (v/v) mycillin and 3.5mM HCl, pH7 DMEM/F12 are added per culture plate.37 DEG C of overnight incubations.

Second day, DMEM/F12 culture medium supernatants are collected, 100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added to the culture medium collected.Culture is washed in culture plate with 50ml serum-frees DMEM/F12 respectively twice, 100ml fresh serum free DMEM/F12 culture mediums are added per culture plate and (40mM N- acetyl-D-MANNOSE amine are filled into DMEM/F12 culture mediums, 10mM Pidolidone salt, 4.1g/L mannose, 1% (v/v) mycillin and ITS solution (5mg/L bovine insulins, the siderophillin of 5mg/L parts iron ion saturation and 5 μ g/ml selenium) (Sigma) is in addition, 40mM N- acetyl-D-MANNOSE amine can be contained in supplement, 7mM Pidolidone salt, 0.5g/L mannoses and 1% (v/v) mycillin.37 DEG C of overnight incubations.

3rd day, DMEM/F12 culture mediums are collected, each culture plate adds the 100ml fresh serum free DMEM/F12 culture mediums containing supplement used in second day.100 mM PMSF (1% (v/v)) and 500 mM EDTA (1% (v/v)) are added in the culture medium of collection, 4 DEG C of preservations after mixing.

4th day, collect the DMEM/F12 culture mediums in culture plate.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the culture medium of collection.Take out 5ml mixed liquors and carry out ELISA detections, remaining mixed liquor is mixed with the serum-free liquid that the 3rd day collects.Before particle is removed with 0.45 μm low protein binding filter (Durapore, Mililpore), the 200mM MES/50mM MgCl2 pH6 of 1/10 volume are added to mixed liquor, it is 6 to make its pH value.The mixed liquor can immediately using or put -70 DEG C of preservations.

(b) IL-4 of the invention purifying

Ligand dye chromatogram (DLC) is purifying IL-4 basic step.With fixed reactive dye storehouse screening IL-4, it can effectively be combined and be discharged in a collection of micro form of purifying with selecting.Appropriate dyestuff protein binding is tested with small-scale bulk form.

During small scale purification, 5ml cells and supernatants are thawed, 0.5ml, pH6 ligand dye post is run through.The Optimization Steps obtain optimum dye pearl-cell factor and the coefficient information of pH, are conducive to maximizing the fraction for recovering extensive DLC.

Because several 17 High (Zymatrix) of extensive DLC reactive dyes are screened as reactive dye, the characteristics of dyestuff has best combination to IL-4 and eluted.Under gravity, the cells and supernatant of filtering flows through 4.0ml or 8.0ml cylinders (Alltech, Extract CleanFilter columns), both cylinders DLC resins containing 3ml or 6ml, and with 50mM MES/5mM MgCl2 with balancing to pH6 respectively.The gross sample of outflow is stored in 4 DEG C, until ELISA results verifications are purified successfully.Post is washed with buffer A (20mM MES/5mM MgCl2 pH6), until can't detect protein fractions by colourity Protein assay (Biorad Protein assays).IL-4 is eluted successively with following elution buffer solution.

Eluent 1：Buffer solution C (50mM Tris-Cl/10mM EDTA pH8)

Eluent 2：EN1.0(50mM Tris-Cl/10mM EDTA/1.0 M NaCl pH8)

Eluent 3：EN2.0(50mM Tris-Cl/10mM EDTA/2.0 M NaCl pH8)

Elutriated fraction is detected with silver staining SDS PAGE, (R＆D Systems Duoset) is monitored using 4-20%Tris-Glycine glue (Invitrogen), and with IL-4 ELISA methods.The IL-4 of elution is can detect in buffer solution 2 and 3.Analyzed by SDS PAGE, 90% contaminating protein is removed in the basic purification step.The DLC fractions containing IL-4 are collected, are concentrated into centrifugal filter device (Amicon Ultra, Millipore) less than 2ml, for size exclusion chromatography.

Combining DLC fractions carry out exclusion chromatography (SEC) with Superdex 75 preparative grade 16/70 (Pharmacia, Uppsala, Sweden) post.1% ammonium bicarbonate advection liquid was flowed through by 1ml/ minutes speed.It is total to spend the post time for 120 minutes, between peak value comes across 40 and 100 minutes.In addition, by 1.5ml/ minutes flow velocitys Sephadex 200 preparative grade (Pharmacia, Uppsala, Sweden) post can be crossed with 50mM MES buffer solutions (pH 5.6).Now, IL-4 elution times are between 20-100 minutes.Elutriated fraction is detected with silver staining SDS PAGE, (R＆D Systems Duoset) is monitored using 4-20%Tris-Glycine glue (Invitrogen), and with IL-4 ELISA methods.It is about that then IL-4 elution times are about 75 minutes between 65-70 minutes and for Sephadex post SEC for Superdex post SEC, IL-4 elution times.

The IL-4 apparent molecular weights of purifying are 14kDa, are at least 95% with silver staining SDS PAGE detections purity.After the fraction collector containing IL-4, concentrate in one with centrifugal filter device (Amicon Ultra, Millipore) and be less than on 2ml post.280nm absorption values are detected with 8855 M-1 cm-1 molar absorption coefficient, the IL-4 concentration of purifying is 507ug/ml.

(d) IL-5 of people's cell expression production and purifying

(i) IL-5 of the invention production

0th day, by the human embryonic kidney cells of transfection, such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene) or 293A (Invitrogen), it is inoculated in by cell number 3 × 107 in 500cm2 tissue culture dishes (Corning).Added per hole cell in 90ml Dulbecco ' s Modified Eagle ' sMedium/Ham ' s Nutrient Mixture F12 (DMEM/F12) (JRHBiosciences), culture medium and be previously added 10% (v/v) heat-inactivated hyclone (FCS, JRH Biosciences), 10mM HEPES (Sigma), 4mM Pidolidones salt (Amresco) and 1% (v/v) mycillin (JRH Biosciences).Also the FCS without HEPES can be replaced with 10% (v/v) heat-inactivated donor calf serum (DCS, JRH Biosciences).

First day, transfected with calcium phosphate.Before transfection, the DMEM/F12 for the above-mentioned supplement of addition newly matched somebody with somebody with 120ml changes liquid to each culture plate.Prepare calcium phosphate/DNA sediment：The DNA of 1200 μ g pIRESbleo (Invitrogen) genes of IL-5 containing people and 3000 or 3720 μ l 2.5M CaCl2 are added into sterile H2O, make total amount to 30ml (solution A).30ml solution As are added 2 x HEPES buffer salt solutions (HBS) (solution B) by 10ml pipettes dropwise.During liquid feeding, with solution B featheriness bubble.25 DEG C of whirlpools are incubated mixed liquor 20 minutes, and mixed liquor also can be without incubation under whirlpool.Each hole is added dropwise in 12ml mixed liquors.The culture medium containing transfection liquid is removed after 4 hours, 100ml hot Dead donor cow's serums containing 10% (v/v) (DCS, JRH Biosciences), 10mM HEPES, 4mM Pidolidones salt, 1% (v/v) mycillin and 3.5mM HCl, pH7 DMEM/F12 are added per culture plate.37 DEG C of overnight incubations.

3rd day, DMEM/F12 culture mediums are collected, each culture plate adds the 100ml fresh serum free DMEM/F12 culture mediums containing supplement used in second day.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the culture medium of collection, 4 DEG C of preservations after mixing.

4th day, collect the DMEM/F12 culture mediums in culture plate.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the culture medium of collection.Take out 5ml mixed liquors and carry out ELISA detections, remaining mixed liquor is mixed with the serum-free liquid that the 3rd day collects.Before particle is removed with the low protein binding filters (Durapore, Millipore) of 0.45mm, the 200mM MES/50mM MgCl2 pH6 of 1/10 volume are added to mixed liquor, it is 6 to make its pH value.The mixed liquor can immediately using or put -70 DEG C of preservations.

(ii) IL-5 of the invention purifying

The cells and supernatant of 1 liter of filtering is concentrated 20 times with tangential flow filter (TFF) device (Pelicon XL, Ultracell, Millipore).With 5KDa it is standard molecular weight burble point until sample is concentrated into 50ml by sample with regenerated cellulose film of the 150ml/ minutes speed by 150cm2.To make concentrating sample filter completely, continue to be concentrated into another 50ml with the extra buffer solutions of equivalent 20mM Tris-ClpH 8.5.Also the 50mM HEPESpH 8 of extra equivalent can use to continue to be concentrated into another 50ml, so that concentrating sample is filtered completely.Completely filtering the step of repeat 2 (as with Tris buffer solutions) or 3 times (such as with HEPES) to being finally concentrated to 50ml.

With 20mM Tris-Cl pH8.5 (Sigma) pre-equilibration Macroprep-Q posts (Bio-RadLaboratories), the cells and supernatant of concentration is crossed from TFF by post by anion-exchange chromatography (AEC) and purifies IL-5.With linear gradient, from 20mM Tris-Cl pH8.5 to NaCl containing 1M, the combining IL-5 of 20mM Tris-Cl pH8.5 are eluted from post.In addition, it is also possible to which the Uno S12 posts (Bio-RadLaboratories) of 50mM HEPES pH8 (Sigma) pre-equilibration carry out anion-exchange chromatography.The apparent molecular weight and degree of purification of obtained fractions are analyzed with ELISA and 1D SDS PAGE (4-20% gradient Tris-Glycine gels (Invitrogen)), it is quantitative with anti-IL-5 ELISA (R ＆ D Systems).

The fraction containing IL-5 is further purified by size exclusion chromatography (SEC) with (Pharmacia, the Uppsala, Sweden) posts of 75 preparative grade of Superdex 16/70.For some experiments, with centrifugal filter device (Amicon Ultra, Millipore) fraction can be concentrated into about 2ml before SEC.It it is 1ml/ minutes for SEC, 1% ammonium bicarbonate advection flow velocity.It is 120 minutes always to spend the post time, and peak value is between 40-100 minutes.In addition, 50mM1 x Dulbecco ' s phosphate-buffered salines (DPBS amendments) (JRH Biosciences) can be as 75 preparative grade of Superdex 16/70 preparative grade (Pharmacia, Uppsala, Sweden) advection liquid, and flow velocity is 1.5ml/ minutes.Now, IL-5 elution times are 20-100 minutes.

Silver staining SDS PAGE (4-20%Tris-Glycine glue (Invitrogen)) and IL-5 ELISA (R D Systems) detection elutriated fractions can be used.During with ammonium bicarbonate buffer solution, IL-5 elution times are 50-65 minutes；When correcting buffer solution with DPBS, IL-5 elution times are 45 minutes.Fraction containing IL-5 can be concentrated into less than 2ml with centrifugal filter device (Amicon Ultra, Millipore), it is also possible to quick desalination post (Pharmacia, the Uppsala, Sweden) desalinations of HiPrep 26/10 to PBS.

The IL-5 apparent molecular weights of change be 16-36kDa between, with silver staining SDS PAGE (with 4-20%Tris-Glycine glue (Invitrogen)) detect purity be at least 99%.280nm absorption values are detected with 8605 M-1cm-1 molar absorption coefficient, the IL-5 concentration of purifying is 206ug/ml.

Embodiment 3

(a) GM-CSF of the invention sign

(i) two-way polyacrylamide gel electrophoresis

The sample collected by embodiment 2 (a) changes buffer solution into (18 MOhm) water of repurity by dialysis or desalting column (Pharmacia HR10/10 Fast Desalting Column), and is dried with SpeedVac inspissators.In addition, the sample that can be collected into newest TCA or acetone precipitation technical finesse.Then, sample is re-dissolved in 240ml MSD buffer solutions (5M urea, 2M thiocarbamides, 65mM DTT, 2% (w/v) CHAPS, 2% (w/v) DMPT 3-10,0.2% (v/v) carrier ampholyte, 40mM Tris, 0.002% (w/v) bromophenol blue, water) and centrifuged 8 minutes under 15000g.

Isoelectric focusing (IEF) is carried out with prefabricated 11cm or prefabricated 17cm pH of latex gel 3-10 retentive force IEF adhesive tape (BioRad).Rehydration at least 6 hours at room temperature in sample of the IEF adhesive tape in sealed tube.IEF adhesive tape is placed in focus cell and covered with liquid paraffin.IEF is to 11cm adhesive tape in 100V 1 hour, 200V 1 hour, 600V 2 hours, 1000V 2 hours, 2000V 2 hours, 3500V 12 hours and 100V are carried out for more than 12 hours, or are carried out 17cm adhesive tape 85kV a few hours with (using identical V ramp-up process).

Ensuing isoelectric focusing, adhesive tape is reduced and is partially alkylated or alkylated, and is being applied to second to before gel.Adhesive tape is cultivated at least 20 minutes in 1 × Tris/HCl pH8.8,6M urea, 2% (w/v) SDS, 2% (v/v) glycerine, 5mM tributylphosphine oxides (TBP), 2.5% (v/v) acrylamide.

11cm adhesive tape, to separation, passes through Criterion pre-filled (11 × 8cm × 1mm is thick) 10-20% Tris glycine gradient gels (BioRad) second.17cm adhesive tape is separated into the Tris glycine gradient gels for the 10-20% that 17 × 17cm, 1.5mm are thick, irrigate certainly.Precision or Kaleidoscope molecular weight markers (BioRad) are also used for gel.Adhesive tape is put into groove, uses 0.5% agarose containing the bromophenol blue as trace dyestuff.

SDS-PAGE is carried out with Criterion or Protean II electrophoresis systems (BioRad) (200V1h (will be disengaged from gel end until buffer solution is proceeded to) is used for 11cm gels and 15mA constant currents per gel 21h for 17cm gels).Buffer solution used is 192mM glycine, and 0.1% (w/v) SDS, 24.8mM Tris alkali is under pH8.3.

After the completion of second fix 30 minutes to gel-in 10% methanol (MeOH) and 7% acetic acid (HAc) overnight.Then, the gel HAc of Sypro Ruby gel stains (BioRad) dyeing at least 3 hours and the MeOH and 7% with 10% decolourizes at least 30 minutes.Optionally, after fixed, gel Deep Purple fluorescent dyeings.Gel is in 300mM Na₂CO₃、35mM NaHCO₃Culture 2 × 30 minutes, then, is cultivated at least 1 hour in the dark in the Deep Purple dyestuffs of 1: 200 dilution.Then, gel is decolourized by cultivating 2 × 15 minutes in 10% MeOH, 7% HAc.During two, gel is imaged with FX laser densitometers (BioRad) and suitable filter.

(ii) 2 fibrillarin electrophoretograms are analyzed with image analysis software

Software I mageJ(http://rsb.info.nih.gov/ij/)Relative intensity for analyzing protein spotses on each gel.Optical densitometric method is carried out to the spot of the selection area of gel and background subtraction is carried out with the appropriate area of the gel of protein spot.

Calculate the percentage specific strength of each protein spotses and the associated value of the spotted intensity of institute is reached 100% by normalization, relative to other spots in gel, the intensity of each protein spot is measured.

The molecular weight of each spot is by the measurement of each distance between spot and gel bottom and with being also used for relatively determining for the distance that the Precision or Kaleidoscope molecular weight markers of gel are shown.4^thPolynomial exponential function is applied to accurately mark to make up the difference for plaque of protein point location respectively.With this method, the molecular weight of each spot can be accurately determined.

The electric charge (pKa value) of isoform is determined with ImageJ by measuring the distance of the difference on the left of spot and each gel.Because the relation between the pI values of adhesive tape and the physical distance of gel is linear, the pI values corresponding to the different pKa values of isoform spot are easily determined.

Major protein on glue positions corresponding GM-CSF isoforms.Low intensity points are probably GM-CSF or low-level pollution, but are due to low-intensity and can not be confirmed by PMF.Detection to glue finds that the GM-CSF of the present invention has 10-30 isoform.Table 9 shows the main feature of these isoforms：PI values (± 1.0), apparent molecular weight (± 20%) and relative intensity (actual value ± 20%, or sum ± 2%, no matter that is bigger).Numerical tabular reaction selected areas containing dispensing in measurement intensity center, therefore when only reacting once specific interpreting blueprints selected areas pI and molecular weight.In view of the changeability in protein site in 2D glue intrinsic volume and position, based on institute's column data in table 9, the scope of molecule pI values is 2-7；The scope of molecule apparent molecular weight is 16-40kDa.

Table 9

The molecular weight and pI values of GM-CSF isoforms

Spot number	Apparent isoelectric point (pI) (± 1.0)	Apparent molecular weight MW (kDa) (± 20%)	Relative intensity (%) (value of standardization) (± the 20% of total amount or ± 2%）
				2	3.68	33.42	9.56
3	3.94	32.80	13.27
				4	4.15	34.17	10.89
5	4.37	34.76	9.35
				6	4.60	35.36	8.88
7	4.86	36.17	9.65
				8	5.16	36.03	8.75
9	3.71	29.57	1.93
				10	4.62	22.49	4.12
11	5.01	23.32	1.98
				12	4.98	21.40	2.18
13	5.23	23.71	1.75

14	5.23	21.96	2.18
				15	5.22	20.35	1.44
16	5.43	22.40	2.64
				17	5.43	20.43	2.00
18	5.63	22.18	1.68
				19	5.63	20.39	1.40
20	4.54	17.15	0.65
				21	4.72	17.78	0.88
22	5.11	15.78	0.87
				23	5.34	14.76	2.55
24	5.65	15.35	1.41

(ii) unidirectional polyacrylamide gel electrophoresis

The sample drying that will be collected from example 2 (a), then, 100 DEG C heating 5minutes molten with 60 μ l 1D sample buffers (10% glycerine, 0.1%SDS, 10mM DTT, 63mM tris-HCl) weight.In order to carry out PNGaseF processing, take the sample of 30 μ L aliquots and add NP40 to final concentration up to 0.5%.Add PNGase F1 μ L and sialidase A (neuramidase), O- dextranases, β (1-4)-galactosidase and each 1 μ L of β-N-acetylglucosaminidase.Processing and untreated sample is cultivated 3 hours at 37 DEG C.Processing and untreated sample electrophoresis in prefabricated Tris gels, such as Tris 4-20% gradient gels (BioRad) or Tris HCl gradient gels (Invitrogen).Accurate molecular weight mark (BioRad catalogues encode 161-0363) is also used for gel.Criterion 4-20% or 18% gel are used for 1D SDS-PAGE, and (BioRad catalogues are encoded：345-0033 or 345-0024).

SDS-PAGE is carried out about 1 hour by Mini Protean II or Criterion electrophoresis systems (BioRad) under 200V, or untill glue edge is soon run out of in buffer solution forward position.The buffer solution used is 192mM glycine, 0.1% (w/v) SDS, 24.8mM Tris alkali under pH8.3.

Complete glue is fixed 30 minutes in 10%MeOH and 7%HAc.Then dyed at least 3 hours, then decolourized at least 30 minutes with 10%MeOH and 7%HAc with Sypro Ruby glue coloring agents (BioRad).In addition, it is also possible to which Deep Purple (Amersham) carry out glue dyeing by operation instruction.Glue is imaged under appropriate optical filter with FX laser densitometer instrument (BioRad).

After N connections oligosaccharides release (PNGase processing), GM-CSF apparent molecular weight (as shown in SDS-PAGE) is 12-30kDa.After N- connections and O- connections oligosaccharides release (glucosides ferment treatment), GM-CSF apparent molecular weight (as shown in SDS-PAGE) is 11-25kDa.

(iii) N- end sequencings

Prepare the protein band on glue (may be from two-dimentional or one-dimensional glue) by above-mentioned and cut, be placed in 0.5ml test tubes, add 100ml extraction buffers (100mM sodium acetates, 0.1%SDS, 50mMDTT pH5.5).Gel slice was in 37 DEG C of shaken cultivations 16 hours.Supernatant is used for ProSorb films (ABI) according to manufacturer's technical specification and is sequenced according to manufacturer's technical specification with 494 protein sequencers of automation (Applied Biosystems).The sequence of acquisition is used for the homogeneity for determining protein.

(iv) peptide mapping fingerprinting

Protein band cuts (from two-way gel or unidirectional gel) and with 25 μ l lavation buffer solutions (in 50mM NH from the gel of above-mentioned preparation₄HCO₃In have 50% acetonitrile) washing.Gel slice retains at least 1 hour and through traditional vacuum drying in 30 minutes at room temperature.Gel slice and 12 μ l insulin solutions (20 μ g insulin, 1200 μ l NH₄HCO₃) be placed in each sample groove and cultivated 1 hour at 4 DEG C.Remove remaining insulin solutions and add 20 μ l 50mMNH₄HCO₃.Mixture is in 37 DEG C of gently shaken overnight cultures.Concentrate peptide sample and the prefabricated micro-column desalination with C18Zip-Tips (Millipore, Bedford, MA) or containing Poros R2 (PerseptiveBiosystems, Framingham, MA) chromatographic resin.Binding peptide is directly eluted in purpose flat board in 0.8 μ l matrix solutions (alpha-cyano -4- hydroxycinnamic acids (Sigma), 8mg/ml in 70% acetonitrile/1% formic acid).The peptide mapping fingerprinting of trypsase determines (MALDI-TOF MS) by using Perseptive Biosystems Voyager DE-STR matrix-assisted laser desorption/desorption ionization flight time mass spectrum and produced.Spectrogram is obtained by using the reflective-mode of 20kV accelerating potentials.Mass calibration is carried out using insulin from dissolved peak, 2211.11Da and 842.51Da as internal standard.The data produced by peptide mapping fingerprinting (PMF) are used to determine protein identity.Search for (main Homo sapien (mankind) and mammal entry) to carry out in database, such as the SWISS-PROT and TrEMBL, by PeptIdent(www.expasy.ch/tools/peptident.html)Program.Identification parameter includes the peptide quality of 0.1Da allowable errors, and each peptide escapes the maximum segment of trypsase cracking, and methionine sulfoxide and cysteine-acrylamide modification.The percent of total for the amino acid sequence that peptide quality numbering and these peptides based on matching are covered is recognized, is compared in the input of other databases.General, the peptide quality with least 30% total sequential covering is for determining that homogeneity is necessary, but very low and high-quality protein, and the protein fragments obtained by these albumen, it is impossible to always meet these standards, it is therefore desirable to further identification.

It is inc or obtained without that can be analyzed by MALDI-TOF PMF in place of protein identity, the peptide mixer of reservation or from replicating identical spot that gel cuts through Trypsin Induced and analyzed by electrospray ionization tandem MS (ESI-MS/MS).For ESI-MS/MS, peptide uses the acetonitriles of 1-2 μ l 70% on Poros R2 micro-columns, and 1% formic acid is directly eluted to borosilicate electron spray syringe needle (Micromass, Manchester, UK).Series connection MS is carried out with Q-Tof mixed types three-level/orthogonal acceleration TOF mass spectrographs (Micromass).Electron spray syringe needle containing sample be embedded in source and with capillary voltage 900-1200V acquisition stationary flow in.Precursor ion scans are carried out to detect the quality of peptide and charge ratios (m/z) in mixture.The m/z of each other precursor ion is picked out for being broken and being collided with the argon gas of 18-30eV collision energies.Fragment ion (missing of amino acid of the correspondence from precursor peptide) is recorded and handled with MassLynx Version3.4 (Micromass).Amino acid sequence is inferred to by using the mass discrepancy of y- or b- ions ' gradient ' series of MassSeq (Micromass) program and determined by manual translation.Then, peptide sequence is used to search for NCBI and TrEMBL databases, uses BLASTP programs " short nearly exact matches ".The minimum value of two pairing peptides is necessary for providing given firmly believing for homogeneity.The homogeneity of hydrogel spots is proved to be IL-2.

Point on glue is proved to be GM-CSF.

In addition, the change for the 1Da that tryptic peptide habitat is found illustrates that the asparagine residue (N) of 2NX (S/T/C) die body in the theoretical amino acid sequence of human GM-CSF is changed into aspartic acid (D).This, which meets PNGaseF, to be D residues by the inductions of the N on the related N connections oligosaccharides of removal.It is therefore evident that the GM-CSF N- connections glycosylation site of the present invention is N-44 and N-54 (by signal sequence section start open numbering).

(b) present invention in IL-3 sign

(i) two-dimensional polyacrylamide electrophoresis

The sample collected in method processing and analysis embodiment 2 (b) with embodiment 3 (a) (i).Main protein site correspondence IL-3 isoform on glue.Low intensity points may pollute for IL-3 or low-level, but be due to low intensity and can not be confirmed with PMF.By being carried out to glue it has been observed that IL-3 isoform is 5-15.Table 10 shows the main feature of these isoforms：PI values (± 1.0), apparent molecular weight (± 20%), and relative intensity (actual value ± 20%, or sum ± 2%present, no matter that is bigger).Numerical tabular reaction selected areas containing dispensing in measurement intensity center, therefore when only reacting once specific interpreting blueprints selected areas pI and molecular weight.In view of the changeability in protein site in 2D glue intrinsic volume and position, based on institute's column data in table 10, the scope of molecule pI values is 3.5-7.5；The scope of molecule apparent molecular weight is 15-35kDa.

Table 10

The apparent molecular weight of IL-3 isoforms and pI values

Spot number	Isoelectric point (pI) (± 1.0)	Molecular weight (kDa) (± 20%)	Relative intensity (%) (standardized value) (± the 20% of weight or ± 2%)
				2	5.91	21.22	1.14
3	5.39	20.00	1.24
				4	5.87	20.16	2.49
5	5.30	19.06	1.59
				6	5.85	18.99	3.05
7	5.30	17.97	4.81
				8	5.56	17.70	6.51
9	5.87	17.70	5.35
				10	6.12	17.42	3.30

11	5.28	16.64	3.21
				12	5.87	16.71	3.94
13	5.41	15.75	1.62
				14	5.37	14.89	1.43
15	6.32	19.28	0.47
				16	6.52	19.15	2.75
17	6.73	19.63	6.53
				18	6.88	19.42	3.10
19	6.30	17.45	2.64
				20	6.51	17.29	4.09
21	6.73	17.29	5.93
				22	6.86	17.42	2.79
23	6.51	15.88	5.66
				24	6.73	15.97	5.80
25	6.96	16.44	1.87
				26	6.84	15.96	1.89
27	6.51	14.81	2.42
				28	5.93	12.85	1.53
29	6.49	13.66	3.29
				30	6.73	13.52	0.86
31	6.50	12.34	5.67
				32	6.59	12.33	3.07

(ii) one-dimensional polyacrylamide gel electrophoresis

The sample collected in method processing and analysis embodiment 2 (b) with embodiment 3 (a) (ii).After N connections oligosaccharides release (PNGase processing), IL-3 apparent molecular weights (as shown in SDS-PAGE) are 10-25kDa.

(iii) N- connections protein sequence

The IL-3N end sequences of the present invention are detected by embodiment 3 (a) (iii) methods described.

(iv) peptide quality fingerprinting

The IL-3 peptide quality fingerprintings of the method detection present invention in embodiment 3 (a) (iv).

Point on glue is confirmed to be IL-3.

(c) IL-4 of the invention sign

(i) two-dimensional polyacrylamide electrophoresis

The sample collected in method processing and analysis embodiment 2 (c) with embodiment 3 (a) (i).

Main protein site correspondence IL-4 isoform on glue.By being carried out to glue it has been observed that IL-4 isoform is 1-3.Table 11,12 shows the main feature of these isoforms：PI values (± 1.0), apparent molecular weight (± 20%), and relative intensity (actual value ± 20%, or sum ± 2%present, no matter that is bigger).Numerical tabular reaction selected areas containing dispensing in measurement intensity center, therefore when only reacting once specific interpreting blueprints selected areas pI and molecular weight.In view of the changeability in protein site in 2D glue intrinsic volume and position, based on institute's column data in table 11,12, the scope of molecule pI values is 8-11；The scope of molecule apparent molecular weight is 15-20kDa.

Table 11

The apparent molecular weight of IL-4 isoforms and pI values

Spot number	Isoelectric point (pL) (± 1.0	Molecular weight (kDa) (20 ± %)	Relative intensity (%) (standardized value) (± the 20% of total amount) or ± 2%)
				2	9.45	14.87	13.54
3	9.67	15.17	55.38
				4	9.89	15.25	31.09

Table 12

The apparent molecular weight of IL-4 isoforms and pI values

Spot number	Isoelectric point (pL) (± 1.0)	Molecular weight (kDa) (± 20%)	Relative intensity (%) (standardized value) (± the 20% of total amount) or ± 2%)
				2	9.75	17.56	95.65

3	9.69	10.95	2.55
				4	9.81	11.09	1.79

(ii) one-dimensional polyacrylamide electrophoresis

The processing of sample collected by embodiment 2 (c) and analysis examples above 3 (a) (ii) are described.After N connections oligosaccharides release (PNGase processing), IL-4 apparent molecular weights (as shown in SDS-PAGE) are 10-20kDa.After N connections and O connections oligosaccharides release (being handled respectively by PNGase and glucosides enzymatic mixture), IL-4 apparent molecular weights (as shown in SDS-PAGE) are 10-18kDa

(iii) the N- end sequencings of protein

The IL-4N end sequences of the present invention are detected by embodiment 3 (a) (iii) methods described.

(iv) peptide quality fingerprinting

The IL-4 peptide quality fingerprintings of the method detection present invention in embodiment 3 (a) (iv).

Point on glue is confirmed to be IL-4.

The change for the 1Da that tryptic peptide habitat is found illustrates that the asparagine residue (N) of NX (S/T/C) die body in the theoretical amino acid sequences of people IL-4 is changed into aspartic acid (D).It is therefore evident that the IL-4N- connections glycosylation site of the present invention is N-62 (by signal sequence section start open numbering).

(v) detection of disulfide bond

With insulin (enzyme: albumen=1: 50) in 50mM NH₄HCO₃In 37 DEG C processing the present invention IL-4 stay overnight.Water is dissolved in again after one semifused is dehydrated.The solution injection of 0.5 μ g albumen deciles C18 posts (Zorbax C18,150 × 0.5mm, 5u) Agilent XCTUltra LC-MS are filled into, the acetonitrile/water that the post is corrected using 0.1% formic acid is used as mobile phase.Post is washed 45 minutes with l/ minutes gradients of 12 μ with 0%-90% acetonitrile, peptide fragment is eluted.Mass spectrum is set to positive ion mode by Data-dependentAuto MSn.Peptide fragment is connected with each other by disulfide bond, and two H can be cut according to their molecular weight summations, is detected with MS precursor scans.Then its structure is detected with MS/MS.With Trypsin Induced (the μ l 0.1M NH of 1ug in 25₄HCO₃) add dithiothreitol (DTT) (DTT, 0.1M, 3 μ l) lucifuge to act on 1 hour, then equally handled with acetamide (0.5M, 3 μ l), it may further confirm that disulfide bond.As disulfide bond albumen disappear can further clear and definite disulfide bond presence.

In addition, such as above-mentioned method can not detect disulfide bond, albumen of the invention is glycosylated again, then needs to pass through row detection again after enzyme effect deglycosylation with above-described embodiment 3 (a) (ii) methods described.

Because detecting dipeptides 27-36 and 151-153,46-61 and 89-99, and 67-71 mass spectrums corresponding with 113-126, illustrate that the IL-4 of the present invention includes 3 disulfide bond, respectively positioned at Cys-27 and Cys-151, Cys-48 and Cys-89, and Cys-70 and Cys-123.After peptide fragment sample is reduced or by peptide fragment alkanisation, these mass spectrums disappear.

(d) IL-5 of the invention sign

(i) two-dimensional polyacrylamide electrophoresis

The sample collected in method processing and analysis embodiment 2 (d) with embodiment 3 (a) (i).

Main protein site correspondence IL-5 isoform on glue.Low intensity points may pollute for IL-5 or low-level, but be due to low intensity and can not be confirmed with PF.By being carried out to glue it has been observed that IL-5 isoform is 15-25.Table 13,14 shows the main feature of these isoforms：PI values (± 1.0), apparent molecular weight (± 20%), and relative intensity (actual value ± 20%, or sum ± 2%present, no matter that is bigger).Numerical tabular reaction selected areas containing dispensing in measurement intensity center, therefore when only reacting once specific interpreting blueprints selected areas pI and molecular weight.In view of the changeability in protein site in 2D glue intrinsic volume and position, based on institute's column data in table 13,14, the scope of molecule pI values is 4-9；The scope of molecule apparent molecular weight is 15-25kDa.

Table 13

The apparent molecular weight of IL-5 isoforms and pI values

Spot number	Isoelectric point (pI) (± 1.0)	Molecular weight (kDa) (± 20%)	Relative intensity (%) (standardized value) (± the 20% of total amount or ± 2%)
				2	4.788	18.023	1.371
3	5.153	19.566	0.633
				4	5.156	18.186	1.196
5	5.379	19.131	1.730
				6	5.486	18.322	3.831
7	5.704	21.023	0.910
				8	6.051	20.921	1.063
9	6.464	20.510	0.845
				10	5.994	16.171	2.441
11	6.347	16.172	0.947
				12	6.346	15.432	0.613
13	6.491	13.798	0.478
				14	5.720	18.286	16.824
15	6.062	18.155	22.948
				16	6.449	17.884	20.839
17	6.867	17.443	14.944
				18	7.339	17.542	4.812
19	7.749	17.500	3.576

Table 14

The apparent molecular weight of IL-5 isoforms and pI values

Spot number	Isoelectric point (pI) (± 1.0)	Molecular weight (kDa) (± 20%)	Relative intensity (%) (standardized value) (± the 20% of total amount or ± 2%)
				2	3.41	19.78	4.88
3	3.70	20.15	3.37
				4	4.14	20.31	9.39
5	4.77	21.22	12.02
				6	5.22	20.91	22.38
7	5.51	20.24	5.24
				8	5.66	20.25	13.42
9	5.80	20.37	12.16
				10	5.95	20.22	2.08
11	6.13	20.77	5.68
				12	6.26	20.75	4.18
13	6.51	20.58	1.17
				14	5.94	18.50	1.49
15	6.11	18.73	2.55

(ii) one-dimensional polyacrylamide electrophoresis

The sample collected in method processing and analysis embodiment 2 (d) with embodiment 3 (a) (ii).After N connections oligosaccharides release (PNGase processing), there are two bands in IL-5 apparent molecular weight (as shown in SDS-PAGE)：One is located between 11-16kDa, and another is located between 15-25kDa.After N connections and O connections oligosaccharides release (being handled respectively by PNGase and glucosides enzymatic mixture), there are two bands in IL-5 apparent molecular weight (as shown in SDS-PAGE)：One is located between 10-15kDa, and another is located between 15-22kDa.

(iii) N-terminal sequence

The IL-5N end sequences of the present invention are detected by embodiment 3 (a) (iii) methods described

(iv) peptide quality fingerprinting

The IL-5 peptide quality fingerprintings of the method detection present invention in embodiment 3 (a) (iv).

Point on glue is confirmed to be IL-5.

Embodiment 4

(a) GM-CSF of the invention amino acid, monose, oligosaccharides, phosphate, sulfate and isoform are constituted

(i) it is used for the preparation of amino acid, monose, oligosaccharides, phosphate and the sample of sulfate analysis

For monose and the identification of glycosylated and phosphorylation and sulphation posttranslational modification, glycosyl is removed from polypeptide backbone by the method for hydrolysis or enzyme first.Sample buffer component is also removed, and replaces to eliminate the inhibitory action of hydrolysis and enzyme digestion reaction before analysis starts with water.The GM-CSF solution purified in PBS is dialysed 4 days on a large scale by 4 liters of deionization ultrafiltration water (18MOhm), liquid is changed daily twice, with specified molecular cut off (NMWC) 5KDa regenerated cellulose dialysis membrane (Spectrapore).After dialysis, solution is dried with Savant Speed Vac (New York, USA).Then, dried sample, which is resuspended with 2ml deionization ultrafiltration water (18MOhm) and is divided into each several part, is used for various analyses.

(ii) analyzed by the amino acid composition of vapor phase hydrolysis method

Amino acid in sample is analyzed with the pre-column derivatization by 6- aminoquinolines base-n-hydroxysuccinimide base-carbamate (AQC).Separate stable Fluorescent amino acid derivative and purified by anti-phase (C18) HPLC.Operation is carried out according to Waters AccQTag amino acid analysis methods.

Take the sample of 100 μ l GM-CSF products and dried in Speed Vac.Then, dry sample is hydrolyzed 24 hours at 110 DEG C.After hydrolysis, sample is dried again before following derivative.Drying sample is re-dissolved in adding 15 μ L AQC derivative reagents after 10 μ L amino acid internal standards (butyrine, AABA), 35 μ L borate buffer solutions of addition.Reactant mixture is heated 12 minutes in constant-temperature incubation device at 50 DEG C.Derivative amino sample is transferred to the automatic sampler of HPLC system, and HPLC system includes the separation assemblies of Waters Alliance 2695 of series connection, Waters474 fluorescent probes and the Dual λ absorptance detectors of Waters 2487.Control and analysis software are Waters Empower Pro Module (Waters Corporation, Milford.MA, USA).Sample is used chromatographic parameter known in the art (such as suitable eluent and gradient current) by Waters AccQTag posts (15cm × 3.9mm ID).

(iii) neutral and amino monosaccharide composition analysis

Take the sample of 100 two kinds of GM-CSF products of μ l and handled with two kinds of different modes to discharge monose.Every kind of each three parts of processing method.

1. 100 DEG C are heated to 2M trifluoroacetic acid (TFA) to hydrolyze 4 hours to discharge neutral sugar (galactolipin, glucose, fucose and mannose).

2. 100 DEG C are heated to 4M HCl to hydrolyze 4 hours to discharge amino sugar (N- acetyl-amine-galactose, N- acetyl-aminoglucose).

All hydrolysates are lyophilized with Speed Vac systems, are re-dissolved in 200 μ l and contain in the water of 0.8nmol internal standard compounds.Internal standard compound for neutral and amino sugar is 2-DG.Then, sample centrifuges 30 minutes to remove protein fragments under 10000g.Supernatant is transferred to new test tube and by high pH anion exchange chromatographies, using the systems of LC 50 and GP50 pumps and ED50 pulse current detectors (Dionex Ltd) together.Neutral and amino glycan analysis is carried out with Dionex CarboPac PA-20 posts.Eluted more than 20 minutes with 10mM equal strength hydroxide concentrate.The process elutes generation system with Dionex EG50 and completed.

(iv) acid monosaccharide composition analysis

Take the sample of 100 μ l GM-CSF products and handle to discharge sialic acid monosaccharide with the following method.Three parts of processing.

Sample hydrolyzes 40 minutes to discharge N- acetyl and N- glycoloylneuraminic acids with 0.1M TFA at 80 DEG C.Hydrolysate is freezed with Speed Vac, 200 μ l is re-dissolved in and contains in the water of 0.8nmol internal standard compounds.Internal standard compound for analyzing sialic acid is lactobionic acid.Then, sample centrifuges 30 minutes to remove protein fragments under 10,000g.Supernatant is transferred to new test tube and by high pH anion exchange chromatographies, using the systems of Dionex LC 50 and GP50 pumps and ED50 pulse current detectors together.Saliva acid analysis is carried out with DionexCarboPac PA1, is used chromatographic parameter known in the art (such as suitable eluent and gradient current).

(v) analysis of oligosaccharides composition

In order to analyze oligosaccharides composition, two kinds of 300 μ l, three parts of IL-2 product samples are taken, and handle as follows.

The release of N- connection oligosaccharides is completed with BPTI-N4- (N- acetyl-β-GLUCOSAMINE base) asparagine acid amides enzyme (PNGase).1/5^thThe denaturing soln (2%SDS (Sigma)/1 M beta -mercaptoethanols (Sigma)) of volume is added in sample.Sample is heated to 100 DEG C 5 minutes.1/10^thThe 15%Triton-X100 (Sigma) of volume is added in sample.Sample is gently mixed and allows to cool to room temperature.Add the PNGase (Sigma) of 25 units and in 37 DEG C of cultures.

The release of O- connection oligosaccharides is completed by the method for β-elimination.4M Sodium Borohydrides (brand-new) (Sigma) solution of 1/2 volume is added in sample.The 0.4M NaOH (BDH, HPLC grade) of 1/2 volume are added in sample.Sample is cultivated 16 hours at 50 DEG C.Sample cools down in ice and the 0.4M acetic acid (Sigma) of 1/2 volume is added in sample.

Further processing, to remove buffer solution composition, uses CarboPac graphitised carbon SPE posts for N- connections and O- connections sample.Column equilibration and elution requirement are as follows.Post is with 1 column volume 80%v/v acetonitriles (Sigma) pre-equilibration, then with the water balance of two column volumes.Sample fills post and with the H of two column volumes under gravity stream₂O is rinsed.In order to elute neutral oligosaccharides, 2ml 50%v/v acetonitriles are used in post.In order to elute acidic oligosaccharide, 2ml 50%v/v acetonitriles/0.1%v/v formic acid is used in post.The oligosaccharides of any reservation is eluted by adding 2ml 80%v/v acetonitriles/0.1%v/v formic acid.

Dried by the fraction containing neutral or amino N-connection oligosaccharides and neutral or amino O- connection oligosaccharides of SPE post separations with Speed Vac.Sample is re-dissolved in 200 μ l water, and by high pH anion exchange chromatographies, using the systems of Dionex LC 20 and GP50 pumps and ED50 pulse current detectors together.The analysis of neutral and amino-oligosacchride is carried out with CarboPac PA100 posts and chromatographic parameter known in the art (such as suitable eluent and gradient current).

(vi) analysis of sulfate and phosphate composition

Sulfate/Phosphate analysis is basically by by Harrison and Packer (Harrison and Packer Methods Mol Biol 125：The method of 211-216,2000) description is carried out.

Taking the sample of 100 μ l GM-CSF products is used for sulfate/Phosphate analysis, and is hydrolyzed 4 hours at 100 DEG C in 4M HCl.Pass through the drying sample removing HCl in Speed Vac.Then, sample is re-dissolved in 200 μ l H₂O.In the systems of Dionex LC 50 of the 24 μ l samples injection with GP50 pumps and ED50 pulse current detectors.Separated, used chromatographic parameter known in the art (such as suitable eluent and gradient current) by Dionex IonPacAS11 anion-exchange columns.

Hydroxide ion and SO are neutralized with Dionex self-regeneration anion micro-membrane suppressors (SRMS-1)₄And PO₄Ion is detected with conductance monitor.

(vii) the further separation of protein isoforms

The further separation of GM-CSF isoforms is carried out with pellicular anion exchange column.Suitable sample volume, for example, 24 μ l, are separated by ProPac SAX-10 posts (Dionex Ltd), use the Dionex SUMMIT systems with UV-Vis detectors (Dionex Ltd).Separation is carried out with suitable eluent and gradient known in the art.GM-CSF isoforms find to be eluted in the peak of different shaped.

(viii) result

Amino acid is constituted

GM-CSF is hydrolyzed, amino acid group is surveyed with above-mentioned reversed-phased high performace liquid chromatographic.As a result it is expressed as weight number and each amino acid accounts for percentage (table 15) of the sequence

Table 15

Amino acid is constituted
						Amino acid	1st wheel	2nd wheel	3rd wheel	Average value	Standard deviation
D	8.57	8.68	8.71	8.65	0.07
						S	6.72	6.78	6.35	6.61	0.23
E	18.37	18.20	18.19	18.25	0.10
						G	5.01	5.62	5.14	5.26	0.32
H	2.11	1.68	2.21	2.00	0.28
						R	5.23	5.16	5.17	5.19	0.04
T	8.41	8.38	8.31	8.37	0.06
						A	7.92	7.88	7.87	7.89	0.03
P	7.78	7.65	7.86	7.76	0.11
						Y	1.54	1.34	1.47	1.45	0.10
V	1.54	4.14	4.12	3.27	1.49
						M	2.05	2.16	2.20	2.14	0.07
K	5.45	5.32	5.30	5.36	0.08
						I	3.02	3.07	3.12	3.07	0.05
L	10.03	10.23	10.29	10.18	0.13
						F	3.66	3.72	3.70	3.69	0.03

Amount to

97.42

100

99.14

Monose

Each monose hydrolysis on GM-CSF amino acid backbones is fallen, the analysis of component below is carried out with high pH anion-exchange chromatographies (HP AEC).Testing result is standardized (table 16-18) respectively with GalNAc and 3 times of mannose.Table 19 is that three kinds of detection result of specimen are collected.

Table 16

The monose composition of 1st wheel
			Monose	Using GalNAc as standard	Using mannose as standard
Trehalose	0.19	0.11
			GalNAc	1.00	0.59
GlcNAc	10.80	6.39
			Galactolipin	5.36	3.17
Glucose	46.67	27.59
			Mannose	5.07	3.00
NeuNAC	2.64	1.56
			NeuGly	0	0

Table 17

The monose composition of 2nd wheel
			Monose	Using GalNAc as standard	Using mannose as standard
Trehalose	1.02	1.97
			GalNAc	1.00	1.92
GlcNAc	5.02	9.66
			Galactolipin	2.39	4.59
Glucose	21.69	41.74

Mannose	1.56	3.00
			NeuNAC	0.30	0.59
NeuGly	0.00	0.00

Table 18

The monose composition of 3rd wheel
			Monose	Using GalNAc as standard	Using mannose as mark
Trehalose	0.57	0.79
			GalNAc	1.00	1.40
GlcNAc	5.14	7.17
			Galactolipin	1.85	2.58
Glucose	21.26	29.69
			Mannose	2.15	3.00
NeuNAC	0.00	0.00
			NeuGly	0.00	0.00

Table 19

The monose composition of 3rd wheel
					Monose	Using GalNAc as standard		Using mannose as standard
	It is minimum	It is maximum	It is minimum	It is maximum
					Trehalose	0.19	1.02	0.11	1.97
GalNAc	1.00	1.00	0.59	1.92
					GlcNAc	5.02	10.80	6.39	9.66
Galactolipin	1.85	5.36	2.58	4.59
					Glucose	21.26	46.67	27.59	41.74

Mannose	1.56	5.07	3.00	3.00
					NeuNAC	0.00	2.64	0.00	1.56
NeuGly	0.00	0.00	0.00	0.00

In view of the above-mentioned intrinsic changeability of chromatography process, after being standardized with GalNAc, monose and sialic acid component are 1: 0.1-1.5 trehalose, 1: 2-12GlcNAc in GM-CSF of the invention, 1: 1.0-6.0 galactolipin, 1: 1.0-6.0 mannose and 1: 0-3.0 NeuNAc；It is 3: 0.1-2.5 trehalose, 3: 0.5-2.5 GalNAc, 3: 5.0-10.0 GlcNAc, 3: 2.0-5.0 galactolipin and 3: 0-3.0NeuNAc when being standard with 3 times of mannoses.

Amino acid composition combines rank rear with the data of monose, phosphate and sulfate in table 20.In view of the above-mentioned intrinsic changeability of chromatography process, the acid monosaccharide components percentages of GM-CSF of the invention is about 6-11%, and N connections oligosaccharides acidity percentage is about 27-41%, and O connections oligosaccharides acidity percentage is about 43-66%.

Table 20

The content of calculating
		% acidity monose	8.4
N connections oligosaccharides neutrality percentage	67.05
		N connections oligosaccharides acidity percentage	32.95
O connections oligosaccharides neutrality percentage	45.81
		O connections oligosaccharides acidity percentage	54.19

(b) IL-3 amino acid, monose, oligosaccharides, phosphoric acid, sulfate and isoform constituent analysis of the invention

(i) it is used for the preparation of amino acid, monose, oligosaccharides, phosphate, sulfate and the sample of isoform analysis

The IL-3 of purifying in PBS solution is handled according to above-described embodiment 4 (a) (i).

(ii) it is made up of gas phase hydrolysis method analysis of amino acid

The sample of IL-3 products is handled according to above-described embodiment 4 (a) (ii).

(iii) analysis that neutral and amino monose is constituted

The sample of IL-3 products is handled according to above-described embodiment 4 (a) (iii).

(iv) analysis of acid monose composition

The sample of IL-3 products is handled according to above-described embodiment 4 (a) (iv).

(v) analysis of oligosaccharides composition

The sample of IL-3 products is handled according to above-described embodiment 4 (a) (v).

(vi) analysis of sulfate and phosphate composition

The sample of IL-3 products is handled according to above-described embodiment 4 (a) (vi).

(vii) the further separation of protein isoforms

Processing such as example 4 above (a) (vii) to IL-3 sample products is described.It was found that IL-3c isoforms are eluted with the pattern at different peaks.

(Viii) result

Monose

Each monose on IL-3 amino acid backbones, sulfate and sulfuric acid salt hydrolysis are fallen, the analysis of component below is carried out with high pH anion-exchange chromatographies (HP AEC).Testing result is standardized (table 21-23) respectively with GalNAc and 3 times of mannose.Table 24 is that three kinds of sample detection results are collected.It should be noted that glucose is common contaminant, itself it is frequently not the composition of N or O connection oligosaccharides.

Table 21

The monosaccharide component of 1st wheel
			Monose	Using GalNAc as standard	Using mannose as standard
Trehalose	5.02	15.75
			GalNAc	1.00	3.14
GlcNAc	3.88	12.18
			Galactolipin	1.82	5.70
Glucose	24.18	75.84
			Mannose	0.96	3.00
NeuNAC	0.08	0.25
			NeuGly	0.00	0.00

Table 22

The monosaccharide component of 2nd wheel
			Monose	Using GalNAc as standard	Using mannose as standard
Trehalose	3.20	11.84
			GalNAc	1.00	3.70
GlcNAc	3.70	13.69
			Galactolipin	0.90	3.32

Glucose	16.07	59.38
			Mannose	0.81	3.00
NeuNAC	0.06	0.21
			NeuGly	0.00	0.00

Table 23

The monosaccharide component of 2nd wheel
			Monose	Using GalNAc as standard	Using mannose as standard
Trehalose	4.24	9.59
			GalNAc	1.00	2.26
GlcNAc	4.07	9.19
			Galactolipin	1.64	3.70
Glucose	20.96	47.40
			Mannose	1.33	3.00
NeuNAC	0.05	0.12
			NeuGly	0.00	0.00

Table 24

Monosaccharide component
					Monose	Using GalNAc as standard		Using mannose as standard
	It is minimum	It is maximum	It is minimum	It is maximum
					Trehalose	3.20	5.02	9.59	15.75
GalNAc	1.00	1.00	2.26	3.70
					GlcNAc	3.70	4.07	9.19	13.69

Galactolipin	0.90	1.82	3.32	5.70
					Glucose	16.07	24.18	47.40	75.84
Mannose	0.81	1.33	3.00	3.00
					NeuNAC	0.05	0.08	0.12	0.25
NeuGly	0.00	0.00	0.00	0.00

In view of the above-mentioned intrinsic changeability of chromatography process, after being standardized with GalNAc, monose and sialic acid component are 1: 2-6 trehalose, 1: 3-5GlcNAc, 1: 0.5-2 galactolipin, 1: 0.5-2 mannose and 1: 0-2 NeuNAc in IL-3 of the invention；It is 3: 5-16 trehalose, 3: 2-4 GalNAc, 3: 9-14 GlcNAc, 3: 3-6 galactolipin and 3: 0.1-2 NeuAc when being standard with 3 times of mannoses.

(c) IL-4 of the invention amino acid, monose, oligosaccharides, phosphate, sulfate and isoform constituent analysis

(i) amino acid, monose, oligosaccharides, phosphate, sulfate and isoform analysis sample are prepared

The IL-4. for the purifying being dissolved in PBS is handled with embodiment 4 (a) (i) methods described

(ii) it is made up of gas phase hydrolysis method analysis of amino acid

The sample of IL-4 products is handled according to above-described embodiment 4 (a) (ii).

(iii) analysis that neutral and amino monose is constituted

The sample of IL-4 products is handled according to above-described embodiment 4 (a) (iii).

(iv) analysis of acid monose composition

The sample of IL-4 products is handled according to above-described embodiment 4 (a) (iv).

(v) analysis of oligosaccharides composition

The sample of IL-4 products is handled according to above-described embodiment 4 (a) (v).

(vi) analysis of sulfate and phosphate composition

The sample of IL-4 products is handled according to above-described embodiment 4 (a) (vi).

(vii) the further separation of protein isoforms

The sample of IL-4 products is handled according to above-described embodiment 4 (a) (vii).

(viii) result

Each monose on IL-4 amino acid backbones, phosphate and sulfuric acid salt hydrolysis are fallen, analyzed with high pH anion-exchange chromatographies (HP AEC), table 25 is listed in percentage and amino acid composition data.Glucose is common contaminant, itself is frequently not the composition of N or O connection oligosaccharides.In view of the above-mentioned intrinsic changeability of chromatography process, the IL-4 of the invention acid percentage of N connections oligosaccharides is about 0-30%.

Table 25

The content of calculating
		N connections oligosaccharides neutrality percentage	90

N connections oligosaccharides acidity percentage

10

(d) IL-5 bases acid, monose, oligosaccharides, phosphate, sulfate and isoform constituent analysis of the invention

The IL-5. for the purifying being dissolved in PBS is handled with embodiment 4 (a) (i) methods described

(ii) it is made up of gas phase hydrolysis method analysis of amino acid

The sample of IL-5 products is handled according to above-described embodiment 4 (a) (ii).

(iii) analysis that neutral and amino monose is constituted

The sample of IL-5 products is handled according to above-described embodiment 4 (a) (iii).

(iv) analysis of acid monose composition

The sample of IL-5 products is handled according to above-described embodiment 4 (a) (iv).

(v) analysis of oligosaccharides composition

The sample of IL-5 products is handled according to above-described embodiment 4 (a) (v).

(vi) analysis of sulfate and phosphate composition

The sample of IL-5 products is handled according to above-described embodiment 4 (a) (vi).

(vii) the further separation of protein isoforms

The sample of IL-5 products is handled according to above-described embodiment 4 (a) (vii).

(viii) result

Amino acid composition

IL-5 is hydrolyzed, and amino acid composition (table 26) is surveyed with above-mentioned reversed-phased high performace liquid chromatographic.As a result it is expressed as weight number and amino acid accounts for the percentage (including standard deviation (SD)) of the sequence.Amion acetic acid is common contaminant in amino acid analysis, can cause the artificial change of amino acid composition.In consideration of it, result is with comparativity, unless it was noted that the level of alanine (A) and aspartic acid (D) is higher than expection with theoretical value.

Table 26

Amino acid composition
						Amino acid	1st wheel	2nd wheel	3rd wheel	Average value	Standard deviation
D	8.33	8.77	8.55	8.55	0.22
						S	5.01	4.59	4.58	4.73	0.25
E	16.99	16.95	16.89	16.94	0.05
						G	7.11	5.96	5.89	6.32	0.68
H	2.09	2.00	2.06	2.05	0.04
						R	5.26	4.95	4.94	5.05	0.18

Amino acid composition
						Amino acid	1st wheel	2nd wheel	3rd wheel	Average value	Standard deviation
T	8.46	8.27	8.29	8.34	0.11
						A	5.17	5.35	5.10	5.21	0.13
P	4.09	4.15	4.13	4.12	0.03
						Y	1.37	1.69	1.67	1.58	0.18
V	5.99	5.97	6.01	5.99	0.02
						M	0.09	0.53	0.54	0.39	0.26
K	8.37	8.37	8.57	8.43	0.12
						I	6.84	7.11	7.20	7.05	0.19
L	11.99	12.42	12.64	12.35	0.33
						F	2.85	2.91	2.94	2.90	0.04
Amount to	100.00	100.00	100.00	100.00

Monose and sulfate

Each monose sulfuric acid salt hydrolysis on IL-5 amino acid backbones are fallen, component analysis is carried out with high pH anion-exchange chromatographies (HP AEC).Result is standardized (table 27-29) respectively with GalNAc and 3 times of mannose.Table 30 is collecting for three kinds of sample results.Glucose is common contaminant, itself is frequently not the composition of N or O connection oligosaccharides.We also noted that having the pollutant of elution in galactolipin peak forward position, the level of such galactolipin is just enhanced manually, and GlcNAc levels also seem higher.

Table 27

The monosaccharide component of 1st wheel
				Monose	Nmol/nmol albumen	Using GalNAc as standard	Using mannose as standard
Trehalose	0.00	0.00	0.00
				GalNAc	2.06	1.00	2.56
GlcNAc	8.49	4.12	10.56

Galactolipin	3.90	1.89	4.85
				Glucose	4.13	2.00	5.14

The monosaccharide component of 1st wheel
				Monose	Nmol/nmol albumen	Using GalNAc as standard	Using mannose as standard
Mannose	2.41	1.17	3.00
				NeuNAC	0.23	0.11	0.29
NeuGly	0.00	0.00	0.00
				SO₄	4.77	9.25	23.72

Table 28

The monosaccharide component of 2nd wheel
				Monose	Nmol/nmo1 albumen	Using GalNAc as standard	Using mannose as standard
Trehalose	0.72	0.3 3	0.87
				GalNAc	2.19	1.00	2.65
GlcNAc	9.13	4.17	11.02
				Galactolipin	3.75	1.71	4.53
Glucose	4.22	1.93	5.09
				Mannose	2.48	1.13	3.00
NeuNAC	0.26	0.12	0.32
				NouGly	0.00	0.00	0.00
SO₄	3.02	5.29	13.99

Table 29

The monosaccharide component of 3rd wheel

Monose	Nmol/nmol albumen	Using CalNAc as standard	Using mannose as standard
				Trehalose	0.72	0.32	0.87
GalNAc	2.28	1.00	2.50
				GlcNAc	9.32	4.09	10.21
Galactolipin	3.77	1.65	4.13
				Glucose	4.84	2.13	5.31
Mannose	2.74	1.20	3.00
				NeuNAC	0.27	0.12	0.30
NeuGly	0.00	0.00	0.00
				SO₄	4.03	7.07	17.65

Table 30

Monosaccharide component
							Monose	Nmol/nmol albumen		Using GalNAc as standard		Using mannose as standard
	It is minimum	It is maximum	It is minimum	It is maximum	It is minimum	It is maximum
							Trehalose	0.00	0.72	0.00	0.33	0.00	0.87
GalNAc	2.06	2.28	1.00	1.00	2.50	2.65
							GlcNAc	8.49	9.32	4.09	4.17	10.21	11.02
Galactolipin	3.75	3.90	1.65	1.89	4.13	4.85
							Glucose	4.13	4.84	1.93	2.13	5.09	5.31
Mannose	2.41	2.74	1.13	1.20	3.00	3.00
							NeuNAC	0.23	0.27	0.11	0.12	0.29	0.32
NeuGly	0.00	0.00	0.00	0.00	0.00	0.00
							SO₄	3.02	4.77	5.29	9.25	13.99	23.72

Amino acid composition data are shown in table 31 by different classes of jointly with monose, phosphate and sulfate data.In view of the above-mentioned intrinsic changeability of chromatography process, after being standardized with GalNAc, monose, sulfate and sialic acid component are 1: 0-0.5 trehalose in IL-5 of the invention, 1: 2-4.5GlcNAc, 1: 1-2 galactolipin, 1: 1-2 mannose and 1: 0.1-1 NeuNAc, 1: 5-10 sulfate；It is 3: 0-1 trehalose, 3: 2-3 GalNAc, 3: 3-12GlcNAc, 3: 2-5 galactolipin and 3: 0.2-1 NeuNAc, 3: 12-24 sulfate when being standard with 3 times of mannoses.

In view of the above-mentioned intrinsic changeability of chromatography process, it is about 2-10% that the IL-5 sialic acids of the present invention, which account for monose percentage, it is about 10-25% that IL-5 sulfate, which accounts for monose percentage, N- connection IL-5 oligosaccharides acidity percentage is about 25-50%, and the IL-5 acid percentage of O- connections oligosaccharides is about 0-40%.

Table 31

The content of calculating
		Sialic acid accounts for monose percentage	2.6
Sulfate accounts for monose percentage	17.7
		N connections oligosaccharides acidity percentage	36.5
O connections oligosaccharides acidity percentage	8.2

Embodiment 5

Saccharic amount dactylogram

The protein of the present invention is separated with the 2D gel electrophoresis technologies of such as embodiment 3 and is adsorbed onto on Kynoar (PVDF) film.Spot is dyed with the protein dye (Colloidal Coomassie Blue, Sypro Ruby or Deep Purple) of a standard volume, and the relative quantity optical density standard measure of isoform.Cut off each spot and handled with a series of deglycosylating enzymes and/or chemical method, depended on the circumstances, to remove the oligosaccharides of presence, the method described according to this specification.Once oligosaccharides is removed, it is separated and analyzed in the chromatography-electrospray-ionization/mass spectrometry system (LC-MS) using graphitized carbon post and organic solution (MeCN) gradient elution system.The peak shape of generation generates " dactylogram " of oligosaccharides present in isoform.Further, mass spectrometer system produces signal to various sugared quality present in sample, and it is by using the pattern match of GlycoSuite databases to recognize their structure.In addition, each mass peak can many times of fragmentations to provide MSⁿSpectrum.These fragments can obtain the prediction of structure, using methods known in the art, for example, passing through the use of GlycosidIQ program bags.

Above-mentioned separation, deglycosylation and analytical procedure is repeated with corresponding albumen of non-human cell's system as expressed by Escherichia coli, yeast or Chinese hamster ovary celI.It was found that corresponding sugar quality fingerprinting is dramatically different.In structure level, such result shows that the protein and corresponding non-human cell's marking protein of the present invention has different mode (pattern) glycan structures.

Embodiment 6

Fluorescence assisted carbohydrate electrophoresis

The oligosaccharides profile that (FACE protocols) obtains target molecule is tested with fluorescence assisted carbohydrate electrophoresis.Oligosaccharides ammonium hydroxide from the aim cell factor is hydrolyzed from amino acid backbone, then with fluorescence 8- amino naphthyls -1,3,6- trisulfonic acids (ANTS) mark.Polyacrylamide gel electrophoresis is used to separate species and the standard for the distinctive oligosaccharides profile of identifying purpose molecule.Further, oligosaccharides is recognized with the substance assistant laser desorpted and ionisation-time of the flight mass spectrum (MALDI-TOF) dependent on fluorescence and to the special matrix of various sugared ionizations.Determine various sugared quality and with GlycoSuite database identification of protein structures.Potential sugar architectural feature is further described by tandem mass spectrum technology, and it make it that obtain oligosaccharides describes in the presence of part the or whole feature with their relative quantity.Further, reuse and recognize the method for isoform to produce the profile of oligosaccharides present in each isoform of separation by 2D gel electrophoresises.

Embodiment 7

QCM and SPR

With the binding characteristic and activity of quartz crystal microbalance (QCM) or surface plasma resonance (SPR) testing goal molecule.In two methods, the chemical action that suitable molecular receptor is described with manufacturer is attached on chip.Molecules of interest is dissolved in suitable biological buffer and is passed to buffer solution and the acceptor interaction on chip.By changing frequency of oscillation (in QCM methods) or measuring the change of the gross protein quality in wafer surface by changing the light scattering characteristic (in SPR methods) of chip.Then, only with biological buffer process chip to observe that molecules of interest is released back into solution.Then, the speed that acceptor reaches saturation and is kept completely separate is used for the binding curve for calculating molecules of interest.

Embodiment 8

The generation of genetically modified host cell system

(a) the genetically modified host cell system of α -2,6- sialyltransferases is contained

The cDNA of coding for alpha -2,6- sialyltransferase (ST of α 2,6) is expanded by PCR by poly (A)-primed cDNA.PCR primer is connected in suitable carrier, such as pIRESpuro4 or pCEP4, to produce the ST plasmids of α 2,6.The cDNA of clone is sequenced and its homogeneity is confirmed by comparing with disclosed α -2,6 ST cDNA sequences.DNA sequencing is carried out with known method.

Mammalian host cell, includes cell strain system (cell line-molecules of interest) α 2 of the identical pedigree of the high-level molecules of interest of expression, 6ST plasmid transfections, and it also carries antibiotic-resistance marker.By the selection that the transfectional cell that cell is stablized is cultivated in the presence of antibiotic；Collect the cell strain system that antibiotic-resistant is shown after transfection and the inspection active for the intracellular ST of α 2,6.In order to separate the ST of express alpha 2,6 individual cells strain, cell gleanings pass through such as Kronman (Gene 121：295-304,1992) description limitation dilution method clone.Single cell strain system is selected at random, and cell expands and detects the ST of α 2,6 activity of strain.

Cell precipitation is washed, is resuspended in dissolving buffer solution, and it is preposition in ice in ultrasonic degradation.Cellular lysate centrifuges and the supernatant of clarification is used for into protein concentration (via known method) and Sialyltransferase Activitychange In The Rat Mammary analysis.Sialyltransferase Activitychange In The Rat Mammary passes through known method, such as Datta et al. (J Biol Chem 270：1497-1500,1995) described by method analysis.

From high express alpha 2, the molecules of interest of expression is purified in 6ST cell lines-molecules of interest cell and external and/or Half-life in vivo biologicall test is carried out (see embodiment 10).Molecules of interest from the high ST cells of express alpha 2,6 shows increased external and/or Half-life in vivo compared with derived from no molecules of interest of identical parental cell line or the molecules of interest derived from other cell lines by any transgeneic procedure.

(b) the genetically modified host cell system containing fucosyltransferase

The cDNA, such as FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11 of fucosyltransferase (FT) are encoded, is expanded by PCR by poly (A)-primed cDNA.PCR primer is connected in suitable carrier, such as pIRESpuro4 or pCEP4, to produce alpha 2,6ST plasmids.The cDNA of clone is sequenced and confirms its homogeneity by the comparison with disclosed FT cDNA sequences.DNA sequencing is carried out with known method.

Human host cell, includes the cell strain system FT plasmid transfections of the identical pedigree of the high-level molecules of interest molecule (cell line-molecules of interest) of expression, it also carries antibiotic-resistance marker.Pass through the selection of the transfectional cell that the culture of cell is stablized in the presence of antibiotic；Collect the strain for the cell that antibiotic-resistant is shown after transfection and for the inspection of intracellular FT activity.In order to separate expression FT individual cells strain, cell gleanings are cloned by limiting dilution method, Kronman as the aforementioned, described by 1992；Individual cells strain is selected at random, and cell expands and detects the FT activity of strain.

Cell precipitation is washed, is resuspended in dissolving buffer solution, and it is preposition in ice in ultrasonic degradation.The supernatant that cellular lysate is centrifuged and clarified is used for protein concentration (through known method) and FT activity analysis.FT activity is analyzed by known method, such as Mas et al. (Glycobiology 8 (6)：605-13,1998) described by.

The molecules of interest of expression is by height expression FT cell lines-molecules of interest cell purification.Lewis x- specific antibodies, such as L5 and sialylated Lewis x- specific antibodies, such as KM93, HECA493,2H5 or CSLEX, presence for detecting Lewis x or sialylated Lewis x structures, according to methods known in the art, for example, such as Lucka et al. (Glycobiology 15 (1)：87,2005) it is described in detail.Or, the presence of Lewis x or sialylated Lewis x structures by using suitable glucosides ferment treatment sample detection and can detect influence of the glycosidase in parameter, such as with quality during MS or with retention time during HPLC.Saccharic amount dactylogram, as described in Example 5, can be used for predicting the presence of Lewis x or sialylated Lewis x structures.

Embodiment 9

Differential gene expression

The difference of gene expression can be analyzed with the aim cell system of molecules of interest.Aim cell grows into suitable density and molecules of interest or buffer control with a range of concentration handle multiple hours, for example, 72 hours.

In the different time, RNA harvests, purifying and reverse transcription are carried out, according to Affymetrix experimental programs.Then, prepare the cRNA (such as biotin labeling) of mark and hybridize with expression matrix, such as U133 GeneChips.It is washed out and signal amplifies, GeneChips GeneChip scanners (Affymetrix) is scanned, and intensity for hybridization and multiple change information GeneChip software (Affymetrix) acquisitions in different time points.

The unique gene expression of molecules of interest induction and cause the mRNA profile different with the cell factor or the profile of receptor-inducible that are produced by separate sources, such as E.coli, yeast or Chinese hamster ovary celI.

Embodiment 10

The measure of the half-life period of purpose of the present invention molecule

The half-life period of molecules of interest, system was measured in vitro.Composition comprising molecules of interest is mixed into human serum/blood plasma and certain temperature culture certain time (such as 37 degree are cultivated 4 hours, 12 hours etc.).The amount of the molecules of interest retained after this treatment is determined by ELISA method known in the art or dot blotting.The biological activity of the molecules of interest of reservation is determined by the progress of the suitable biological detection method selected by those skilled in the relevant art.Selected serum may come from a variety of human blood types (such as A, B, AB, O etc.).

The half-life period of molecules of interest is also measured in vivo system.Composition comprising molecules of interest by radioactive tracer mark (or other method) and through vein, it is subcutaneous, after eye socket, intramuscular or intraperitoneal injection to study in selected species, for example, mouse, rat, pig, primate or people.The time point blood sampling after injection and presence to molecules of interest is analyzed and (marked by ELISA method, dot blotting or by trichloroacetic acid (TCA)-precipitation, such as radiocounting).The comparison of composition containing the molecules of interest for being produced from separate sources, such as E.coli, yeast, or Chinese hamster ovary celI can be carried out as control.

Embodiment 11

The in vivo studies of purpose of the present invention molecule

The individual subjects of in vivo studies described herein are warm-blooded vertebrates, including people.

Clinical test is by strictly controlling to ensure that individual is not interposing in unnecessary risk and they are fully informed their effects under study for action.

It is preferred that, in order to compensate the psychological application for receiving treatment, treatment is carried out with double-blind fashion.Volunteer is randomly assigned into control or molecules of interest treatment group.Further, relevant clinician is also blind for the treatment system being administered to receptor, to prevent there is prejudice in being observed after their treatment.Use this random fashion, each volunteer has the identical chance for being given new treatment or control.

Volunteer receives molecules of interest or control agent for a period of time, simultaneously, starting (baseline before any treatment) with reference to the morbid state or the biological parameter of health status shown, terminating (after final treatment), and be measured in the interval of rule during studying.The measurement includes the level of molecules of interest in the body fluid compared with level before treatment, tissue or organ.Other measurements include, but not limited to morbid state or the index of treated health status, body weight, blood pressure, the serum titer of the pharmacology indicator of disease, such as special disease indicator or toxicity and ADME (absorb, distribution, metabolism and drain) measurement.

Record each patient information include the age (year), sex, the species of the family history (Yes/No) of height (cm), morbid state or disease, motivation grade (few/in/big) and numbering and the disease for showing or sick first therapy.

The volunteer for participating in the experiment is adult, the age at 18 to 65 years old and add experiment masculinity and femininity number approximate equality.Volunteer with some features is evenly distributed for control agent and molecules of interest treatment.General, the volunteer treated with control agent has seldom or not reacted to treatment, but in off-test, the volunteer treated with molecules of interest shows that their morbid state or the index of disease are intended to the positive.

Embodiment 12

(a) comparison of GM-CSF and non-human system's expression of GM-CSF bioactivity of the invention

GM-CSF can induce TF-1 cells propagation.In 48 orifice plates, 30000/m TF-1 cells are handled 9 days with 0-10ng/ml GM-CSF37 DEG C.Use Flow cytometry cell.With document (Dedov et al.Apoptosis 8：399-406,2003) report FACScan flow cytometers (Becton Dickinson Immunocytometry Systems, San Jose, CA) analysis.CellQuest Software analysis results.

With different two kinds of difference GM-CSF molecules (the R ＆ D SystemsCat # 215-GM for being expressed in E.coli expression；WHO#88/646 above-mentioned experiment) is repeated.

Curve is with after absorbance fitting, calculating ED50, and calculate GM-CSF molecular concentration values with 4 yuan of equations.

The GM-CSF of present invention ED50 is about 0.05-0.08ng/ml, and R ＆ D SystemsGM-CSF ED50 is (0.4-0.6ng/ml), WHO GM-CSF (0.4-0.6ng/ml), both of which is expressed in E.coli (table 2) so, and GM-CSF of the invention is strong 5-12 times compared with the proliferation activity of the E.coli human GM-CSFs expressed.

(b) GM-CSF and non-human system's expression of GM-CSF serum stabilities of the invention comparison

It is incubated the GM-CSF (R＆D Systems) 24 hours of GM-CSF and the E.coli expression of the present invention respectively with hyclone.By operation instruction, reduced amount is detected with ELISA (R＆D Systems DuoSet).Use-case 12 (a) method detects GM-CSF bioactivity.It is similar (result is not shown) that the amount for the GM-CSF that two kinds of E.coli are produced reduces level.By contrast, GM-CSF of the invention is incubated 24 hours successors in serum and keeps higher bioactivity (Fig. 3)

(c) propagation and differentiation of granulocyte and macrophage colonies

It is reported that GM-CSF can adjust propagation and the differentiation of granulocyte and macrophage colonies.200 TF-1 cells are inoculated in 35mm culture dishes, cell is handled respectively with the human GM-CSF of GM-CSF and the E.coli expression of the present invention.37 DEG C, 5%CO₂Under the conditions of be incubated 14 days, count clone number.Clone's number is counted under the microscope with 100mm tallies.One clone includes at least 40 cells.

By counting TF-1 cells, 1.5-2 times many compared with the human GM-CSF that E.coli is expressed of the number of clone (Fig. 4) of the GM-CSF treatment groups generation of various concentrations [1.25,2.5,5ng/ml (p the ＜ 0.01)] present invention.

(d) IL-3 of the invention and non-human system express the comparison of IL-3 bioactivity

It is reported that IL-3 can induce M-NFS-60 cells propagation.In 48 orifice plates, with 37 DEG C of processing 30000M-NFS-60 cells/ml 4 days of IL-3 of the 0-1ng/ml present invention.Use Flow cytometry cell.For flow cytometry, living cells FACScan flow cytometries (Becton Dickinson Immunocytometry Systems, San Jose, CA) disclosed method (Dedov et al.Apoptosis 8：399-406,2003) count.Data analysis is carried out with CellQuest Software.

With different two kinds of difference IL-3 molecules (the R ＆ D SystemsCat # 203-IL for being expressed in E.coli expression；WHO #91/510) repeat above-mentioned experiment.

Curve is with after absorbance fitting, calculating ED50, and calculate IL-3 molecular concentration values with 4 yuan of equations

The IL-3 of present invention ED50 is about 0.12-0.18ng/ml, and R ＆ DSystemsIL-3 ED50 is (0.20-0.30ng/ml), and WHO IL-3 (0.20-0.30ng/ml), both of which is expressed in E.coli.So, IL-3 of the invention is strong 1.1-2.5 times compared with the E.coli people IL-3 expressed proliferation activity by (Fig. 5).

(e) IL-4 of the invention and non-human system express the comparison of IL-4 bioactivity

(i) IL-4 of the present invention IL-4 expressed with non-human system (CHO and E.coli) are detected.In 96 orifice plates, TF-1 cells (100000 cells/ml) are incubated at 37 DEG C with 0-2ng/ml IL-4 7 days.Survivaling cell is detected with the Aqueous One Solution cell proliferation experiments (Promega) of CellTiter 96..In this analysis, the tetrazolium salt compound MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) being present in electronics coupled reagent (phenazine methosulfate) is reduced to formazan by cell biological

Product.FormazanConcentration reads the absorbance measurement of generation solution by using spectrophotometer (E Max precision microplate reader, MolecularDevices) under 490nm.

(R ＆ D Systems Cat #204-IL, are expressed in E.coli to the two kinds of difference IL-4 molecules expressed with different non-human cells；WHO #88/656, are expressed in Chinese hamster ovary (CHO) cell) repeat above-mentioned experiment.

Curve is with after absorbance fitting, calculating ED₅₀, and calculate IL-4 molecular concentration values with 4 yuan of equations.

The IL-4 of present invention ED₅₀About 0.024-0.036ng/ml, and R ＆ DSystems IL-4 ED50 is (0.9-1.3ng/ml, it is expressed in E.coli cells), WHOIL-4 (0.028-0.042ng/ml is expressed in Chinese hamster ovary celI) (Fig. 6 (a)).So, IL-4 of the invention is strong 25-54 times compared with the E.coli people IL-4 expressed proliferation activity, strong 1.75 times compared with the IL-4 that CHO is expressed.

(ii) it is the stability under further examination criteria cell culture condition, after IL-4 of the invention is incubated 4 days with cell culture medium (RPMI 1640,10% hyclone) under 37 C, adds TF-1 cells.The TF-1 cells (200,000 cells/ml) that the culture medium that the IL-4 preincubates of the 0-2ng/ml present invention are crossed is added in 96 orifice plates.After 3 days, survivaling cell is detected with CellTiter 96Aqueous One Solution cell proliferation experiments (Promega).In this experiment, MTS ((3- (4,5-dimethyl thiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) it is used as a kind of tetrazolium composition, can be a kind of first by cell degradation in the presence of electron coupling agent (azophenlyene amethopterin)

Product.First

Concentration 490nm absorbances can be read and be obtained by spectrophotometer (E maximum precise micropore readers, Molecular Devices).

Above-mentioned experiment is repeated with a kind of E.coli (R ＆ D Systems Cat # 204-IL) people IL-4 molecules expressed.

Curve is with after absorbance fitting, calculating ED₅₀, and calculate IL-4 molecular concentration values with 4 yuan of equations.Under cell culture condition, IL-4 of the invention ED₅₀About 0.24-0.36ng/ml, and the IL-4 of E.coli cells expression ED₅₀For 4.7-7.1ng/ml (Fig. 6 (b)).So, IL-4 of the invention is strong 13-30 times compared with people's IL-4 proliferative induction abilities that E.coli is expressed, and shows the stability under higher cell culture condition.

(f) IL-5 of the invention and non-human system express the comparison of IL-5 bioactivity

Human erythroleukemia cell line TF-1 is to cytokine profiles response.It is reported that IL-5 can induce TF-1 cells propagation.In 96 orifice plates, TF-1 cells (10000/hole) are incubated at 37 DEG C with 0-100ng/ml IL-5 68 hours.

Cell number is counted with the Aqueous One Solution cell proliferation experiments (Promega) of CellTiter 96.In this experiment, MTS ((3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) it is used as a kind of tetrazolium composition, can be a kind of first by cell degradation in the presence of electron coupling agent (azophenlyene amethopterin)

Product.First

Curve is with after absorbance fitting, calculating ED50, and calculate IL-5 molecular concentration values with 4 yuan of equations

Use different non-human cells, such as E.coli, yeast, Chinese hamster ovary celI and ED₅₀, the people IL-5 molecules of expression repeat above-mentioned experiment, as a result show notable difference.

Embodiment 13

(a) GM-CSF and non-human system's expression of GM-CSF ion vitro immunization reaction characteristicses of the invention comparison

Use standard protein detection technique, such as Bradford Protein Assavs (Bradford M AnalBiochem 72：248-254,1976) GM-CSF protein concentrations of the invention are estimated.

After being standardized with standard protein detection technique, the GM-CSF of the present invention is diluted and the ELISA kit of purchase, such as R＆D Systems human GM-CSFs DuoSet is used

ELISA kit (Cat# DY215), are detected by operation instruction.The human GM-CSF that the ELISA kit is expressed using E.coli is standard.

Found after the same protein concentration that detection is tested with standard protein is compared, R＆D Systems human GM-CSFs DuoSet

ELISA kit can not correctly estimate the GM-CSF concentration values of the present invention under OD450nm.

In structure level, the result illustrates that the GM-CSF of the present invention is different with non-human system's expression of GM-CSF ion vitro immunization reaction characteristicses.

(b) IL-3 of the invention and non-human system express the comparison of IL-3 ion vitro immunization reaction characteristicses

The IL-3 protein concentrations of the present invention are estimated with Bradford Protein Assavs (supra of Bradford 1976).

After being standardized with Bradford Protein Assavs to the IL-3 of the present invention, by the IL-3 of the present invention with being expressed in insect cell (BD Pharmingen；Cat. #554604) people IL-3 dilutions, and by operational manual R＆D Systems people IL-3 DuoSet

ELISA kit (Cat.#DY203) are detected.The people IL-3 that ELISA kit is expressed using E.coli is standard items.

After being compared by the made standard curves of the people IL-3 with being expressed according to E.coli, R＆DSystems DuoSet

IL-3 ELISA kit are about 466 pg/ml to the IL-3 Concentration Testing results of the present invention, and OD450nm values are 0.3 (Fig. 7)；And concentration is actually added into for 2000pg/ml (Figure 7) in similar OD450nm values.Equally, after being compared by the made standard curves of the people IL-3 with being expressed according to E.coli, R＆D Systems DuoSet

IL-3 ELISAkit are about 280pg/ml to the IL-3 Concentration Testing results of insect cell expression, and OD450nm values are 0.2 (Fig. 7)；And concentration is actually added into for 2000pg/ml (Fig. 7) in similar OD450nm values.

R＆D Systems people IL-3 DuoSet

The IL-3 that ELISA kit is expressed with E.coli is as standard items, and antibody is people's IL-3 antibody that E.col is expressed, and the kit is used for laboratory and the clinical detection to people's IL-3 levels, and the IL-3 of its testing result display present invention concentration should be higher 4 times than expection.

These results illustrate the IL-3 and non-human system's expression IL-3 immune responses feature of the present invention, and there were significant differences

(c) IL-4 of the invention and non-human system express the comparison of IL-4 ion vitro immunization reaction characteristicses

The IL-4 protein concentrations of the present invention are estimated with Bradford Protein Assavs (supra of Bradford 1976)

After being standardized with Bradford Protein Assavs to the IL-4 of the present invention, the IL-4 of the present invention is diluted, and by operational manual R＆D Systems people IL-4 DuoSet

ELISAkit (Cat.# DY204) is detected.The people IL-4 that ELISA kit is expressed using E.coli is standard items.

After being compared by the made standard curves of the people IL-4 with being expressed according to E.coli, R＆DSystems DuoSet

IL-4 ELISA kit are about 780pg/ml to the IL-4 Concentration Testing results of the present invention, and OD450nm values are 0.55 (Fig. 8)；And concentration is actually added into for 1500g/ml (Fig. 8) in similar OD450nm values.

R＆D Systems people IL-4 DuoSetThe IL-4 that ELISA kit is expressed with E.coli is as standard items, and antibody is people's IL-4 antibody that E.coil is expressed, and the kit is used for laboratory and the clinical detection to people's IL-4 levels, and the IL-4 of its testing result display present invention concentration should be higher 2 times than expection.

These results illustrate the IL-4 and non-human system's expression IL-4 immune responses feature of the present invention, and there were significant differences

(d) IL-5 of the invention and non-human system express the comparison of IL-5 ion vitro immunization reaction characteristicses

Standard protein detection technique is used, such as Bradford Protein Assavs (Bradford 1976supra) are estimated the IL-5 protein concentrations of the present invention.

After being standardized with Bradford Protein Assavs to the IL-5 of the present invention, the IL-5 of the present invention is diluted, and by operational manual R＆D Systems people IL-5 DuoSet

ELISAkit (Cat.# DY218) is detected.The people IL-5 that ELISA kit is expressed using E.coli is standard items.

Found after the same protein concentration that detection is tested with standard protein is compared, R＆D Systems people IL-5 DuoSetELISA kit can not correctly estimate the IL-5 concentration values of the present invention under OD450nm.

The IL-5 concentration of the invention and the difference by standard protein determination of experimental method detected with the ELISA kit of purchase, because the people IL-5 albumen of seizure and/or the detection times non-human cell's expression of antibody used in commercial ELISA Kit is improved.

In structure level, the result illustrates that the IL-5 of the present invention is different with non-human system's expression IL-5 ion vitro immunization reaction characteristicses.

Embodiment 14

Purpose of the present invention molecule is further purified and with ESI-MS/MS peptide mapping fingerprinting

In addition to the purification process described in embodiment 2, the purifying of purpose of the present invention molecule is further carried out by RP-HPLC, uses commercially available post.Elution protein is monitored and is collected by 215 or 280nm absorbance, while in order to be corrected due to the delay that the conduit volume between flow cell and collecting terminal is brought.

The gel piece containing protein example from 1D or 2D gels digests in insulin solutions, as described in Example 3.Or, the solution insulin containing protein sample digests in ammonium bicarbonate buffers (10-25mM, pH 7.5-9).Solution incubated overnight at 37 DEG C.Then by add acetic acid until pH in the range of 4-5 with stopped reaction.Peptide sample is concentrated and with C18 Zip-Tips (Millipore, Bedford, MA) or as described in Example 3, the prefabricated microtrabeculae desalination containing Poros R2 chromatographic resins (Perspetive Biosystems, Framingham, MA).

Rinsed with 0.1% formic acid in protein sample (2-5 μ l) injection C18 pre-columns and in the case where 30 μ is l/ minutes with concentration and desalination.After rinsing 3 minutes, pre-column is converted to the analytical column containing C18 RP silica (Atlantis, 75 μ m 100mm, Waters Corporation) in column.Peptide linear solvent gradient is eluted in post, step by step, from H₂O∶CH₃CN(95∶5；+ 0.1% formic acid) arrive H₂O∶CH₃CN (20: 80 ,+0.1% formic acid) was carried out more than 40 minutes with 200nl/ minutes.LC is eluted in Micromass QTOF Ultima mass spectrographs (Micromass, Manchester, UK) to be analyzed through cation nanoflow electron sprays.

Series connection MS is carried out with Q-Tof mixing level Four/orthogonal acceleration TOF mass spectrographs (Micromass).QTOF is operated according to the data of drainage pattern (DDA).TOFMS detection scannings (m/z 400-2000,1.0s) are obtained, while three kinds of most multiple-charged ions (counting ＞ 15) are continuously analyzed in detection scanning through MS/MS.MS/MS spectrums collect 8s (m/z 50-2000).

LC/MS/MS data are searched for Mascot (Matrix Science, London, UK) and ProteinLynx Global Server (" PLGS ") (Micromass).Protein sample is contemplated to molecules of interest.

Embodiment 15

(a) immunogenicity in non-human animal

(i) with the animal immune of target protein

The group of non-human animal respectively, for example, the protein that mouse is expressed with the protein of the 1-100ug present invention and in non-human cell is through subcutaneously, intraperitoneal or intramuscular (IP) are immune.Animal received secondary immunity at immune latter month.Before immune, protein is emulsified in adjuvant, for example, complete Freud ' the s adjuvants for initial immunity and incomplete Freud ' the s adjuvants for secondary immunity.

(ii) to the identification of the antibody of target protein

In order to determine antibody response, the animal from each group takes blood from afterbody and collects serum.Protein specific antibody passes through of the invention protein determinations of the solid phase ELISA with 50ng/ holes.Different immune immunoglobulin isotypes by using mark show anti-igg 1, IgG2, IgG2b, IgG3, IgM, IgA, IgD detection TPPA.Or, Resistant reaction is measured by the protein of the invention being adsorbed onto on the film for dot blotting or Western blot.The measure of the isotype of different immunoglobulins is according to detection described above.It is expected that the protein of the present invention causes the antibody response of the albumen different from being expressed in non-human cell.

(iii) T cell proliferation assay

By immune animal euthanasia and prepare splenocyte.The splenocyte of suitable quantity, for example, 5 × 10⁵Cell, come the animal of the Western Immuno for the present invention that uses by oneself, cultivated together a period of time with the protein of the invention of various concentrations, and identical quantity is cultivated come the splenocyte of animal of Western Immuno expressed in the non-human cell that uses by oneself and the albumen expressed in the non-human cell of various concentrations together.For T cell proliferation assay, spleen cell cultures 96 hours and last 16 hours with 1 μ Ci [³H] thymidine (6-7 μ Ci/umol) processing.Harvesting on filter and incorporation is determined with standard method [³H] thymidine.Compared with the protein expressed in non-human cell, it is contemplated that protein of the invention causes different breeder reactions.

(iv) IFN gamma are analyzed

In order to which IFN gamma are analyzed, the spleen cell cultures supernatant cultivated together with the protein of the protein of the present invention or non-human cell's expression was harvested at 96 hours and IFN gamma products are detected by sandwich ELISA, for example, the anti-IFN gamma Quantikine of R＆D systems

ELISA kit (Cat # DIF50), according to manufacturer's technical specification.It is expected that it is different that the IFN gamma products for the cell that the IFN gamma products of the culture supernatant for the cell cultivated together come the albumen for the present invention that uses by oneself are cultivated together with the protein expressed in come the non-human cell that uses by oneself, which are compared,.

(b) external people's Analysis of Immunogenicity

(i) human T-cell's response analysis

Human dendritic cell and CD4⁺T cell is prepared from people's whole blood, such as Stickler et al.Toxicological Sciences 77：280-289, described in 2004.BMDC and CD4⁺T cell is placed in 96 orifice plates and co-cultured, and includes 2 × 10⁴BMDC and 2 × 10⁵ CD4⁺T cell.The protein and the protein expressed in non-human cell of the present invention is fragments of peptides through enzymic digestion, with the suitable enzyme identified by cleavage site forecasting software, for example, PeptideCutter(http://au.expasy.org/tools/peptidecutter).Obtained fragments of peptides is purified by suitable technology, for example, liquid chromatogram, and add in coculture to final concentration 5ug/ml.Culture culture 5 days, then by 0.5uCi³H thymidines are added in each culture.By cell harvesting into filter and by [³H] thymidine incorporation measure cell propagation.

It is expected that the protein peptide derived from the present invention can cause weaker breeder reaction, relative to the peptide for the protein expressed in non-human cell.

(ii) human antibody response analysis

Blood is taken to people's donor of the protein therapeutic through being expressed with non-human cell and blood plasma is prepared.Protein specific antibody is determined by the solid phase ELISA for the protein expressed in anti-50ng/well protein of the invention and non-human cell.Different Immunoglobulin Isotypes by using show anti-human igg 1, IgG2, IgG3, IgG4, IgM, IgA, IgD mark detection TPPA.

Or, by adsorbed when dot blotting or Western blot film on protein of the invention and the protein determination antibody response expressed in non-human cell.The measure of different Immunoglobulin Isotypes is according to detection described above.

It is expected that the immunoglobulin being present in the serum of the people for the protein for treatment expressed with non-human cell can combine the protein expressed in non-human cell, and combined weak with the protein of the present invention or do not combined.

Embodiment 16

The preparation of protein of the invention from recombination group construct

Pass through GM-CSF, IL-3, IL-4 and IL-5 of the PCR amplification codings present invention genome sequence SEQ ID NO：69th, 70,71,72), and it is cloned into corresponding expression vectors, such as pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA3.1/GS, pCEP4, pIRESpuro3, pIRESpuro4, pcDNA3.1/Hygro (+), pcDNA3.1/Hygro (-), pEF6/V5-His.By the structure of these restructuring, (embodiment 1 (c)) is prepared and transfected to people's cell as stated above.GM-CSF, IL-3, IL-4 and IL-5 product and purification process of the present invention are obtained from recombinant DNA structure referring to embodiment 2.

It will be appreciated by those skilled in the art that invention described here can be modified to the difference being particularly described with those by change XOR.It the present invention should be understood to include all such change XOR modifications.The present invention also includes respectively or fully all steps that are being related in specification or pointing out, feature, and component and compound and any step or feature described in two or more are optionally combined.

Bibliography

Ackland et al.Chroma togr 540：187-198,1991

Aloj et al.J BiolChem 247：1146-1151,1971

Altschul et al.NuclAcidsRes 25：389,1997

Antibodies：ALaboratory Manual, Harlow and Lane (eds.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, (1988)

Aronsson et al.FEBS Lett 411：359-364,1997

Atherton and Shephard Synthetic Vaccines 9：Blackwell Scientific Publications

Ausubel et al.In：Current Protocols in Molecular Biology John Wiley & Sons Inc.1994-1998)

Baneyx Current Opinion in Biotechnology, 10：411-421,1999

Bernstein Methods Mol Biol 23：195-204,2004

BirdScience 242：423,1988

Blenis and Resh Curr Opin Cell Biol 5(6)：984-9,1993

Bonner and Laskey Eur J Biochem 46：83,1974

Bradford M Anal Biochem 72：248-254,1976

Caprioli et al.Biochem Biophys Res Commun 146：291-299,1987

Carr et al.Anal Biochem 175：492-499,1988

Carr et al.Anal Chem 63：2802-2824,1991

Carr et al.J Biol Chem 264(35)：21286-21295,1989

Clackson et al.Nature 352：624-628,1991

Clarke Curr Opin Cell Biol 5：977 983,1993

Datta et al.J Biol Chem 270：1497-1500,1995

Dedov et al.Apoptosis 8：399-406,2003

Edman Mol Biol Biochem Biophys 8：211-55,1970

Erickson et al.Science 249：527-533,1990

Farruggia et al.Int J Biol Macromol 20：43-51,1997

Figeys and Aeber sold, Electrophoresis 19：885-892,1998

Franks et al.Characterization of proteins, Humana Press, Clifton, NJ, 1988

Fritz et al.PNAS 95：12283-12288,1998

Fukuhara et al.J Biol Chem 260：10487-10494,1985

Gelb et al.Curr Opin Chem Biol 2(1)：40-8,1998

Gramer et al.Biotechnology 13 (7)：692-8,1995

Guedez et al.Am J Pathol 162：14 31-1439,2003

Harrison and Packer Methods Mol Biol 125：211-216,2000

Hearn et al.Methods in Enzymol 104：190-212,1984

Herscovics et al.FASEB J 7：540-550,1993

Herzberg et al.Infrared and Raman Spectra of Polya tomic Molecules, Van NostrandReinhold, New York, NY, 1945

Hodgson Bio/Technology 9：19-21,1991

Holzwarth et al.J Am Chem.Soc 178：350,1965

Honroe et al.Biochem J 258：99-204,1989

Huston et al.ProcNatl Acad Sci USA 85：5879,1988

JBiochem 336：647-658,1998

JBiochem 366：619-631,2002

James and Bottomley Arch Biochem Biophy 356：296-300,1998

Jones et al.Nature 321：522-525,1986

Kennet et al.Monoclonal Antibodies, Hybridomas：A New Dimension in BiologicalAnalyses, Plenum Press, New York, 1980

Kivirikko et al.FASEB Journal 3：1609-1617,1989

Kohler et al.Nature 256：495,1975

Kortt et al.Protein Engineering 10：423,1997

Krimm and Bandekar Adv Protein Chem 38：181-364,1986

Kronman Gene 121：295-304,1992

Kurochkin et al.J Mol Biol 248：414-430,1995

Kwon and Yu Biophim Biophys Acta 1335：265-272,1997

Larrick et al.Bio/Technology 7：934,1989

Larsen et al.J Biol Chem 265：7055-7061,1990

Li et al.Biochemistry 34：5762-5772,1995

Liu et al.JImmunol 158：604-613,1994

Lucka et al.Glycobiology 15(1)：87-100,2005

Marks et al.J Mol Biol 222：581-597,1991

Marmur and Doty J Mol Biol 5：109,1962

Martin et al.Nature Medicine 11 (2)：228-232,2005

Mas et al.Glycobiology 8(6)：605-13,1998

McGettrick et al.Methods Mol Biol 244：151-7 2004

Mire-Sluis et al.J Immunol Methods 289(1-2)：1-16,2004

Moore J Biol Chem 278(27)：24243-24246,2003

Morrison et al.Proc Natl Acad Sci USA 81：6851-6855,1984

Nguyen et al.J Chromatogr A.705：21-45,1995

Packer et al.Glycoconj J 5(8)：737-47,1998

Phillies Anal Chem 62：1049A-1057A, 1990

Pikal et al.Pharm Res 8：427-436,1991

Presta, Curr Op Struct Biol 2：593-596,1992

Rando Biochim Biophys Acta 1300(1)：5-16,1996

Reichmann et al.Nature 332：323-329,1988

Sambrook et al.Molecular Cloning-A Laboratory Manual, Cold Spring Harbour, New York, USA, 1990

Schmid et al.Proteinstructure, a practical approach, Creighton Ed., IRI Press, Oxford, England, 1989

Shepherd et al.Arterioscler Thromb Vasc Biol 24：898-904,2004

Stickler et al.Toxicological Sciences 77：280-289,2004

Triguero et al.J of Neurochemistry 54：1882-1888 1990

Ward et al.Nature 334：544,1989

Wells Methods Enzymol 202：2699-2705,1991

Wilkinson Annu Rev Nutr15：161-89,1995

Winter and Harris TIPS 14：139,1993

Yoshioka et al.Pharm Res 10：103-108,1993

Claims

1. the protein of separation, it has measurable physical and chemical parameter feature, wherein described character representation, the pharmacological characteristics for being associated with one or more characteristics or formed one or more characteristics pharmacological characteristics basis, wherein the protein of described separation, which has, includes multiple measurable physical and chemical parameter { [P_x]₁、[P_x]₂、...[P_x]_n, physicochemical characteristicses, wherein P_xRepresent measurable physical and chemical parameter and " n " be >=1 integer, wherein [P_x]₁To [P_x]_nIn each be respectively different measurable physical and chemical parameters, the value of the measurable physicochemical property of any of which or a series of value of more than one measurable physicochemical property represent, is associated with the pharmacological characteristics Ty of characteristic or the pharmacological characteristics { [T of series of features_y]₁、[T_y]₂、....[T_y]_m, or form the pharmacological characteristics Ty of a characteristic basis or the pharmacological characteristics { [T of series of features_y]₁、[T_y]₂、....[T_y]_mBasis, wherein T_yThe pharmacological characteristics and m for representing a characteristic are >=1 integers, wherein [T_y]₁To [T_y]_mIn each be respectively different pharmacological characteristics, wherein the protein of described separation be selected from GM-CSF, IL-3, IL-4 and IL-5.

2. the protein of the separation of claim 1, wherein described protein has measurable physical and chemical parameter shown in one or more table 2.

3. the protein of the separation of claim 1, wherein described protein has the pharmacological characteristics of the characteristic shown in one or more table 3.

4. chimeric molecule, it includes the protein or its segment for the separation for being fused to one or more peptides, polypeptide or the claim 1 on protein portion.

5. the chimeric molecule of claim 4, wherein peptide, polypeptide or protein portion include the constant region (Fc) or framework region of human immunoglobulin(HIg).

6. the chimeric molecule of claim 4, wherein chimeric molecule are selected from GM-CSF-Fc, IL-3-Fc, IL-4-Fc and IL-5-Fc.

7. pharmaceutical composition, the protein or chimeric molecule of the separation containing any one of claim 1 to 6.

8. the method for disease is treated or prevented in mammalian subject, wherein described disease can be improved by increasing the amount or activity of protein, and described method includes the pharmaceutical composition for giving the chimeric molecule or claim 7 of any one of protein, the claim 4 to 6 of the separation according to any one of claims 1 to 3 of the mammalian subject effective dose.

9. nucleotide sequence, selected from SEQ ID No：25th, 27,29,31,35,37,39,41,43,45,47,51,53,55,57,61,63,65 or 67；Or there is the nucleotide sequence of at least about 90% homogeneity with the above-mentioned any sequence enumerated；Or the nucleotide sequence that can hybridize with any above-mentioned sequence or their complementary type under high stringency conditions.

10. the protein or chimeric molecule of separation, by selected from SEQ ID NO：25th, coded by 27,29,31,35,37,39,41,43,45,47,51,53,55,57,61,63,65 or 67 nucleotide sequence, or as coded by there is at least about nucleotide sequence of 90% homogeneity or the nucleotide sequence that can hybridize with any one above-mentioned sequence or they any complementary type under high stringency conditions with the above-mentioned any sequence enumerated.

11. the nucleic acid molecule of separation, encoding proteins matter or chimeric molecule or its funtion part, including with SEQ ID NO：25th, 27,29,31,35,37,39,41,43,45,47,51,53,55,57,61,63,65 or 67 have at least 90% similitude nucleotide sequence, or after optimal comparison and/or can be with SEQ ID NO：25th, 27,29,31,35,37,39,41,43,45,47,51,53,55,57,61,63,65 or 67, or their complementary type one or more nucleotide sequences hybridized under high stringency conditions.

12. the nucleic acid molecule of separation, including coding have substantially such as SEQ ID NO：26th, the protein or the nucleotide sequence of chimeric molecule of 28,30,32,36,38,40,42,44,46,48,52,54,56,58,62,64,66 or 68 one or more shown amino acid sequences, or coding and SEQ ID NO：26th, one or more nucleotide sequences with least about protein of the amino acid sequence of 90% similitude or chimeric molecule after 28,30,32,36,38,40,42,44,46,48,52,54,56,58,62,64,66 or 68 optimal comparisons.

13. kit, for determining human protein or the chimeric molecule level that people's cell present in biological products is expressed, including (a) solid phase support matrix；(b) antibody of the chimeric molecule of any one of any one of one or more anti-claim 1-3 human protein or claim 4-6；(c) lock solution；(d) one or more substrate reservoirs；(e) substrate buffer solution；(f) standard human protein or chimeric molecule sample and (g) operation instructions.

14. the kit of claim 13, wherein standard human protein or chimeric molecule sample be the separation of Claims 2 or 3 protein or claim 4 chimeric molecule product.

15. the kit of claim 13 or 14, mammal is immunized the product that each of which antibody is derived from the chimeric molecule with the protein of the separation comprising Claims 2 or 3 or claim 4.

16. the human protein of the kit of any one of claim 13 to 15, wherein people's cell expression is naturally occurring human GM-CSF, IL-3, IL-4 and IL-5.