CN102516393B - Insulin-simulated peptide fusion protein and mutant and its application - Google Patents

Abstract

The present invention relates to a kind of aminoacid sequence of the fusion protein for treating people I types and type ii diabetes, and production method and application, the present invention relates to a kind of non-insulin diabetic condition therapy for being avoided that insulin resistant.Fusion protein according to the present invention is mutually merged by connection peptides with IgG Fc or IgG Fc mutants by insulin-simulated peptide and is formed, and while insulin-simulated peptide drop glycopenia activity is retained, can significantly improve the Half-life in vivo of insulin-simulated peptide.Wherein, the mutation physical ability of IgG Fc further increases the half-life in fusion protein body.

Description

Insulin-simulated peptide fusion protein and mutant and its application

Invention field

The present invention relates to the prevention and treatment medicine of people's diabetes.Specifically, the present invention provides one kind and is used for detecting and controlling Treat fusion protein of people I types and type ii diabetes and its preparation method and application.The fusion protein contains insulin-simulated peptide And human IgG-Fc (hinge region-CH2-CH3) albumen and/or IgG-Fc mutant (T250Q/M428L (IMP)；QL and T307Q/ N434A；QA), can effectively reduce the blood sugar level of diabeticss, increase the half-life in insulin-simulated peptide body, especially The mutant of IgG-Fc (QL/QA), can further increase the internal metabolism half-life of insulin-simulated peptide, reduce the injection of medicine The frequency.

Background technology

With the improvement of people's living standards, the increase of aged tendency of population and fat incidence rate, the sickness rate of diabetes In ascendant trend year by year.Diabetes are to cause one of Etiological of human death, global about 200,000,000 patients.Diabetes exist The sickness rate of China reaches 2%, and according to statistics, China has 39,800,000 diabetics at present, occupies the whole world second.Expect 2025, diabetes mellitus in China patient was up to 59,000,000 people.China there are about ten thousand new cases more than 100 at present every year.Wherein I types are sugared Urine patient accounts for 10%, and type ii diabetes patient accounts for 90%.Estimate according to expert, the market of country's diabetes medicament holds at present About 7,000,000,000 yuans of amount, shows according to the data analysiss that Ministry of Public Health is announced, the sale of coming years diabetes mellitus in China class medicine Amount will be more than 10,000,000,000 yuans.

Diabetes are broadly divided into type i diabetes and type ii diabetes.Wherein, type i diabetes be a kind of Chang children or The complicated T cell formed during teenager relies on autoimmune disease (Juneia and Palmer, Autoimmunity.1999；29 (1)：65-83).It is to be caused with corresponding insulin deficit and exogenous insulin by the autoimmune destruction of beta Cell of islet Dependence.Mononuclear cell is to pancreatic endocrine cell and the Focal quality and quantity for invading profit and functional beta cells of tissue Substantially reduce be diagnose the disease histopathology the characteristics of, but these damage degree interindividual variations notable.From Body autoimmune destruction causes T cell to invade profit to islets of langerhans (islets of Langerhans), the apoptosis (Lee of β cells as a result occurs Et al., Mol Genet Metab.2004；83(1-2)：82-92；Mandrup-poulsen, Biochem Pharmacol.2003；66(8)：1433-1440；Scsti, Ann Med.2002；34(6)444-450；Mathis et al., Nature.2001；414(6865)792-798).For studying the potential molecular mechanism of diabetes, many animals mould is had been set up Type.For example, the rat of the insulin deficit for (STZ) being induced with streptozotocin (streptozotocin) or mice are simulated in sugar The destruction of the cell-mediated inflammation of visible T- and islet cellss in urine patient's body.Additionally, non-obese diabetes (NOD) is little Mus are the models of another research autoimmune disease, wherein islets of langerhans-antigen-reactive T cells infiltration islets of langerhans and kill islets of langerhans β- Cell, and/or cause inflammatory process to cause death (Aanderson and Bluestone, the Annu of beta Cell of islet Rev Immunol.2005；23：447-485).

Insulin treatment is the Main Means for intervening type i diabetes.By activating Insulin receptor INSR, internal blood glucose water is adjusted Flat.The receptor of insulin is a tetramer, passes through disulfide bond by two α subunits and two β subunits.Two α subunits positions In the outside of cytoplasma membrane, there is the binding site of insulin thereon；Two β subunits are transmembrane proteins, play Role in Plant Signal Transduction.Nothing When insulin is combined, the tyrosine protein kinase of receptor does not have activity.When insulin is combined and change β with the α subunits of receptor After the configuration of subunit, tyrosine protein kinase is just activated, and can be catalyzed two reactions after activation：

1st, the tyrosine residue phosphorylation of β subunits specific site in tetramer complex, this process is made to be referred to as from phosphoric acid Change (autophosphorylation)；

2nd, have the ten of important function by upper for IRS (insulin receptor substrate, IRSs) Several tyrosine residue phosphorylations, the IRSs of phosphorylation can in conjunction with and activate downstream effect thing to adjust blood glucose.

Type ii diabetes be a kind of generally adult age diagnosis polygenes malfunction, its to cause hyperglycemia three With the characteristics of individual main abnormal：1. peripheral insulin resistance, 2. hepatic glycogen excessive generate, 3. pancreas Instreptozotocin Induced obstacle.Pancreas Insulin resistance refers to that peripheral tissues are mainly Skeletal Muscle Cell and the reaction of insulin is reduced, and causes glucose transport to these groups Obstacle (the Kahn and Goldfine, Jdiabetes Complications.1993 for knitting；7(2)：92-105；Weyer et al., Jclin Invest.1999；104(6)：787-794).Insulin resistant equally can occur in liver, cause pancreas in liver Island element can not effectively suppress generation (Kahn and Goldfine, the Jdiabetes of liver glucose Compliations.1993；7(2)：92-105；Lam et al., Am.Jphysiol Endocrinol Metab.2002；283 (4)E682-E691).The result of insulin resistant is increase in demand of the body to insulin.Early stage rank in insulin resistant Section, causes the compensatory mechanism that β cell qualities increase in pancreas by increasing insulin secretion.The blood sugar level of these patients is still (Bonner Weir, Trends Endocrinol Metab.2000 in normal range can be maintained；11(9)：375-378； Bonner-Weir, Endocrinology.2000；141(6)：1926-1929).Many studies have shown that, if pancreas can be maintained The compensation of beta cell, insulin resistant itself be also not enough to excite diabetes outbreak (Weyer et al., Diabetes.1999,48 (11)：2197-2203).But insulin resistant long-time is not corrected and becomes serious, to insulin Excess demand cause beta cell exhaustion, insulin secretion is reduced, and hyperglycemia symptom and obvious diabetes occurs (DeFronzo, Diabetes.1988；37(6)：667-687；Kahn et al., J Nutr.2001；131(2)：354S；Weyer Et al., J Clin Invest.1999；104(6)：787-794).

The conventional therapy of type ii diabetes includes going on a diet and taking exercise and using sulfo group urea (sulphonylureas), first good fortune Bright metformin and insulin carry out Drug intervention.But these treatments generally cannot all prevent blood glucose control in Most patients Dysfunction (Matthews et al., the Diabet Med.1998 of the long-term degradation of system and β cells；15(4)：297-303； Turner et al., JAMA.1999；281(21)：2005-2012) clinically stepped controlled for what type ii diabetes adopted Treatment method is finally also unable to maintain that blood glucose balance, for Most patients, from go on a diet and temper single factor test Drug therapy, from connection Closing treatment most Zhongdao insulin therapy becomes inevitable process (Turner et al., JAMA.1999；281(21)：2005- 2012；Gerich, Eur J Clin Invest.2002；32Suppl 3：46-53).

Insulin receptor INSR be made up of across subunit the cross-film of the extracellular subunit and two 95-kDa of two 135-kDa two The dimer protein that sulfide linkage is connected, containing the intracellular tyrosine kinase domain (Ebina, the Y. that are activated by upstream binding partner Et al., Cell.1985；40,747-758 and Ullrich, A. et al., Nature.1985；313,756-761).When insulin is tied After closing receptor, a series of metabolism and mitosis reaction is activated, these reactions are by including several including IRS1,2 and Shc Tyrosine kinase in individual extracellular protein substrate there is phosphorylation and produce (White, M.F., Diabetologia.1997.40, Suppl.2, S2-S17).IRS1 and IRS2 are main IRSs, cause sugar Metabolism, and have uniqueness and overlap role in different organs.Additionally, the modification of IRS1 and IRS2 is had with diabetes Very direct relation.(Yamauchi, T. et al., Mol.Cell Biol.1996；16,3074-3084 and Withers, D.J. Et al., Nature.1998；391,900-904) .Thirone etc. Mus front myocyte system (L6 cells) be found that IRS1 and The specific function of IRS2, and summarized (Thirone, A.C. et al., Trends Endocrinol.2006；Metab.17, 72-78).

But IRS1 and IRS2 does not also come to a conclusion to the Relative Contribution that insulin signaling and insulin resistant develop.Major part exists The document that this field is delivered shows that IRS1 is main substrate, so as to stimulate the transport of muscle and fatty tissue to glucose, And IRS1 and IRS2 is in insulin signaling and the complementary action of metabolism in liver.Be not in directly to participate in contrast SHC Insulin metabolism signal, but play in insulin-induced mitosiss pivotal role (Thirone, A.C. et al., Endocr.J.2000；47,373-381).IRS and Shc starts mitogenesis activation pathway kinases/extracellular regulated protein and swashs Enzyme (MAPK/ERK) approach, causes genetic transcription and protein translation and cell growth including activating ERK1/2.Other are by IRS eggs The signal path of white matter activation is phosphatidylinositol3 3 kinase (PI3- kinases) approach, including the activation of PKB, causes Glycogen synthesis Protein necessary to the metabolism of enzyme and other enzymes/acute insulin activation (White, M.F.Diabetologia.1997.40, Suppl.2, S2-S17).

By display technique of bacteriophage have been obtained for being capable of bound insulin receptor synthetic peptide (aminoacid quantity 20～ 40) (Pillutla, R.C. et al., J.Biol.Chem.2002；277,22590-22594).These polypeptides can be attached to Three sites on Insulin receptor INSR (site 1,2 and 3).Wherein 1 and 2 two site is combined with the insulin on Insulin receptor INSR Site overlaps, and the Insulin receptor INSR Binding peptide in site 3 is combined near site 1, forms nearest homodimer. Many of which can activate Insulin receptor INSR, with higher potency and specificity, may pass through a kind of similar insulin and its The mechanism of receptor binding reduces blood sugar level (Schaffer, L. et al., Proc.Natl.Acad.Sci.U.S.A.2003；100, 4435-4439).

These insulin-simulated polypeptides can activate Insulin receptor INSR as insulin, reduce the blood sugar level of Mus.He Structure is simpler, volume is less, can effectively prevent the generation of insulin resistant.They be insulin replacement therapy very Good drug candidate, or there is potential purposes with insulin curing diabetic with combined, it is possible to as novel peptide type medicine Design template.In correlational study work, some researcheres find the insulin-simulated of 31 aminoacid under study for action Peptide (IMP), can be highly affine with Insulin receptor INSR, and relative to insulin, with weaker some gene expressions of promotion Act on mitosis promoting.Further study show that, on the one hand, IMP has the bound insulin receptor similar to insulin Affinity, but different from the action pathway of insulin, and this shows that they can be coexisted on Insulin receptor INSR.It moreover has been found that this Presence significant difference between the gene expression pattern of two part inductions, IMP promote fissional function to be significantly lower than pancreas Island element.This shows that IMP has and insulin identical blood sugar decreasing effect, is avoided that insulin resistant, thin with less promotion Born of the same parents' Mitosis, more safe.

Generally polypeptide drug serum half-life in vivo is all shorter.It is thus desirable to be frequently administered, high-frequency Drug injection, has had a strong impact on the quality of life of patient, while bringing higher treatment cost.Therefore, how peptide is not being affected Increase the half-life in vivo while class biological activity, be the focus of numerous scientist's researchs.Such as WO 02/46227 or WO GLP-1 described in 05/000892 forms fusion protein with Human Albumin or human IgG-Fc, can significantly strengthen which in vivo Half-life.The prolonged half-life in vivo of fusion protein, discharges because of its molecular change ambassador kidney and reduces formation.But melt The in vitro study of hop protein shows relatively low effect (Kim et al., Diabetes.2003；52(3)：751-759).This causes People make great efforts the medicine longer to develop stable effect in vivo and Half-life in vivo.Therefore, for diabetes need to develop For I types and the effectively treatment strategy of the potential molecular mechanism rather than the mechanism consequence of type ii diabetes.Preferably invention is both The injection frequency of medicine can be reduced again for Insulin receptor INSR effectively treatment diabetes, reduce medicine use cost, it is to avoid pancreas Insulin resistance.

IgG-Fc is the most widely used fusion egg for extending peptides, cytokine and soluble recepter class Half-life in vivo One of white, there are many obvious advantages compared with other protides.On the one hand it can extend internal serum half-life, in addition Dimer can be formed, the probability with targeted integration is improved, on the other hand for the research of IgG-Fc is more deep, is therefore used Get up more safe.In IgG, positioned at site (Fig. 1) mediation on Fc between CH2 and CH3 domains with neonatal receptor FcRn's Interact, wherein combination can make the endocytosis Fc fusion protein from interior body be recycled in blood flow (Raghavan et Al., 1996, Annu Rev Cell Dev Biol 12：181-220；Ghetie et al, 2000, Annu Rev Immunol 18：739-766).With reference to reducing kidney filtration after macromole, the internal serum for producing 1～3 week partly declines said process Phase.The combination of Fc and FcRn also plays a crucial role in monoclonal antibody transhipment.Site on Fc with reference to FcRn is also antibacterial egg The site that white A and G is combined.With the combining closely protein purification of generally can serve as of these albumen, using protein A or Protein G parent Mode with chromatography antibody purification Fc.Additionally, FcRn receptors also are responsible for for Fc fusion protein in connection being transferred to new life In the enteric epithelium chamber of the intestinal of youngster and adult, increase antibody Fc cell and tissue penetration power (Ghetie and Ward, Annu.Rev.Immunol., 2000,18：739-766；Yoshida et al., Immunity, 2004,20 (6)：769- 783).

During the mutation research of people Fc γ, to being studied with reference to some important residues of FcRn, as a result table The mutation of some aminoacid bright can significantly increase serum half-life.Hinton etc. is in people Fc γ 1 seriatim by three residues It has been mutated into other 19 kinds of common aminoacid.They have found that the double mutant of some point mutation increased FcRn with reference to affine Double mutation of power, wherein T250Q/M428L sites can significantly improve the affinity effect of Fc and FcRn, and then significantly improve in body The interior half-life.In addition, the T307Q/N434A sites by mutation IgG-Fc such as Henry B.Lowman, equally can be significantly Strengthen internal serum half-life.The drug disposition metabolism that these experiments all pass through monkey is confirmed.In addition, based on increase The mutation of FcRn affinitys can also increase the logical of the transcytosis (Transcytosis) and tissue of fusion protein or antibody Permeability, increase in-vivo tissue systems in drug degree of exposure, can play to greatest extent drug action (Hinton et al., 2004, J.Biol.Chem.279 (8)：6213-6216；Henry B.Lowman et al., Cancer Research.2010, 70(8)：3269-3277；Y Zheng et al, Clinical Pharmacology＆Therapeutics.2010,89 (2), 283-290).

Content of the invention

The present invention relates to fusion protein and the production method of prevention and treatment I types and type ii diabetes.The combination of the present invention Thing includes new fusion protein and its mutant.The fusion protein contains the Insulin with the fusion of IgG light chain constants (Fc) area Mimetic peptides (IMP) molecules or its analog or fragment.The IgG for people source, can be selected from IgG1, IgG2, IgG3 or IgG4.The fusion protein of the present invention is referred to herein as IMP/IgG-Fc or IMP/IgG-Fc (T250Q/M428L； ), or IMP/IgG-Fc or IMP/IgG-Fc (T307Q/N434A QL；QA).

In other aspects of the present invention, the mutant of the fusion protein is IMP/IgG-Fc (T250Q/M428L), contains T250Q/M428L one or two is mutated；Can also be IMP/IgG-Fc (T307Q/N434A；QA), comprising one or two Mutation, or the combination of above-mentioned mutation.

Treatment of the fusion protein and its mutant of the present invention to I types and type ii diabetes in experimenter is effective.Equally Ground, the invention provides the method for the I types and type ii diabetes for treating experimenter, wherein if necessary to use to experimenter Described fusion protein and its mutant.

Present invention also offers using mammal expression and antibacterial culturing system, preparing the fusion protein IMP/ of the present invention IgG-Fc and the new method of IMP/IgG-Fc (T250Q/M428L) and/or IMP/IgG-Fc (T307Q/N434A).At both In system, cultured cells clones to prepare including IMP/IgG-Fc and IMP/IgG-Fc (T250Q/M428L) and/or IMP/IgG- The mutant form of the fusion protein of Fc (T307Q/N434A) and effectively prolongation Half-life in vivo, such as but not limited to IMP/ IgG-Fc (QL) and IMP/IgG-Fc (QA).Strengthen the combination to FcRn receptors and extend internal plasma residence time.

Bivalence IMP/IgG-Fc of the present invention or IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion protein have solely Special the characteristics of：

1) the circulating half-life in vivo T1/2 of IMP increased, and especially IgG-Fc mutants (QL) or (QA) can more enter one Step increases Half-life in vivo；

2) compare with insulin, with lower stimulation cell division capacity, safer, insulin resistant can be avoided；

3) fusion protein formed and disome structure is more stable, while with IgG-Fc mutants (QL) or (QA) fusion increase Dosing thing transcytosis (Transcytosis) and the permeability of tissue, increase the exposure that medicine is organized in vivo；

4) produced by biological engineering method, be conducive to obtaining the higher albumen of biological activity.For prepared on a large scale Easy one-step method purification.The fusion protein of the present invention can be supplied to experimenter on demand in a variety of forms.

An aspect of of the present present invention provides a kind of IMP fusion protein for controlling blood glucose balance for experimenter.

An aspect of of the present present invention provides a kind of IMP fusion protein for controlling experimenter's blood sugar concentration.

An aspect of of the present present invention provides a kind of IMP fusion protein, and which activates islets of langerhans by way of being different from insulin Plain receptor, promotes metabolism of blood glucose.

According to an aspect of the present invention, fusion protein of the invention avoids the insulin resistant of experimenter, strengthens Portugal Grape sugared tolerance simultaneously reduces fasting blood glucose level.

An aspect of of the present present invention provide a kind of fusion protein, the fusion protein contain IMP polypeptides or the like or its Mutant or its fragment or IgG polypeptides.

An aspect of of the present present invention provides a kind of heterologous fusion protein, and the fusion protein contains and IgG peptide fusions IMP polypeptides or its variant, wherein described IgG.

According to an aspect of the present invention, the IgG is people source.

According to an aspect of the present invention, the IgG Fc are human IgG1, IgG2 or IgG4.

According to an aspect of the present invention, the IgG is human IgG2.

According to an aspect of the present invention, the IgG-Fc is the heterozygote of humanized IgG 1-Fc, IgG2-Fc or IgG4-Fc.

According to an aspect of the present invention, the IMP polypeptides are selected from insulin-simulated peptide (SLEEEWAQIECEVYGRGCPSE SFYDWFERQL) and its fragment and the group of derivant composition.

According to an aspect of the present invention, the IMP is SLEEEWAQIECEVYGRGCPSESFYDWFERQL.

According to an aspect of the present invention, the derivant contains about 70% to about 95% with IMP (Sequence ID NO.1) the polypeptide of sequence identical sequence.

According to an aspect of the present invention, the IgG fragments contain at least 5 aminoacid up to 250 aminoacid.

According to an aspect of the present invention, the IgG contains Fc parts or the fragment or derivant of Fc parts of IgG.

One aspect of the present invention provides a kind of aminoacid sequence of coding heterologous fusion protein.

One aspect of the present invention is there is provided a kind of aminoacid sequence containing the fusion protein through codon optimised vectors.

One aspect of the present invention provides a kind of inclined through codon through the aminoacid sequence containing the heterologous fusion protein The host cell of the good carrier transfection for optimizing.

An aspect of of the present present invention provide a kind of Pharmaceutical composition, the compositionss contain above-mentioned heterologous fusion protein or The carrier that aminoacid sequence of the person comprising the heterologous fusion protein loaded with pharmaceutical acceptable carrier optimizes through codon preference.

According to an aspect of the present invention, the Pharmaceutical composition is used for treating I types and type ii diabetes.

According to an aspect of the present invention, the Pharmaceutical composition can by selected from local application, oral, aerosol mode, The mode of the group of peritoneal injection, intravenous injection or intramuscular injection composition is applied.

According to an aspect of the present invention, the Pharmaceutical composition is used for the treatment of the I types and type ii diabetes of subject, The compositionss contain comprising the heterologous fusion egg with the IMP polypeptides or its derivant or active fragment of IgG peptide fusions In vain.

One aspect of the present invention provides a kind of method of the I types and/or type ii diabetes for treating experimenter, methods described bag Include the fusion protein or the compositionss or the carrier for applying therapeutically effective amount.

One aspect of the present invention provides the heterologous fusion protein to be used for treating or preventing I types and/or type ii diabetes Medicine in purposes.

According to an aspect of the present invention, the group that the IgG is constituted selected from human IgG1, IgG2, IgG3 and IgG4.

Other features and superiority of the present invention can be embodied by detailed description below.It should be understood, however, that only Detailed description and specific embodiment are given in an illustrative manner to show embodiment of the present invention, this is because for this area From described detailed description, multiple changes and improvement are apparent to those of skill in the art within the spirit and scope of the present invention 's.

Description of the drawings

Refer to the attached drawing will be further understood that the present invention from the following detailed description, wherein：

Fig. 1：Build the expression plasmid of coding IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA).1A shows Show and use round pcr, respectively the sequence of IMP and IgG-Fc is expanded from respective template, next with above-mentioned PCR primer For template, IMP/IgG-Fc sequences are amplified with primer 1 and 4.Then, by the cDNA connections of coding IMP/IgG-Fc fusion protein To between BmHI the and EcoRV sites of expression vector pcDNA3.1.Using nucleotide site directed mutation method, coding is produced IMP/IgG-Fc (QL) or the cDNA of IMP/IgG-Fc (QA) fusion protein, using method same as described above by IMP/IgG-Fc Or IMP/IgG-Fc (QA) is cloned into pcDNA3.1 expression vectors (QL).Show in 1B by active IMP molecules and comprising human IgG 4 The diagram of the IMP/IgG-Fc fusion protein of the IgG-Fc compositions of CH (hinge, CH2 and CH3 parts).These albumen Secrete in the form of homodimer when matter is expressed, form space structure as depicted.

Fig. 2：It is shown that IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion protein in CHO-S Expression and detection in cell.With coding IgG-Fc, IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) albumen Plasmid vector fusion transfected CHO-S cells, Aspirate supernatant after transfection 48h, with the solidifying stock purified fusion egg of A albumen agarose In vain.Reduced the SDS-PAGE analyses of (reduced) and non-reduced (Non-reduced) respectively, used Coomassie brilliant blue (Comasie Blue) is dyeed.Then, SDS-PAGE glue is transferred in cellulose acetate membrane, carries out Western blot point Analysis.Above-mentioned film is detected with anti-mouse antibody (1: 5000), and is developed with ECL.As a result IMP/IgG-Fc, IMP/IgG-Fc are shown (QL) or IMP/IgG-Fc (QA) be about 70KDa homodimer, consistent with theoretical molecular.Wherein, swimming lane 1,2,3 is distinguished It is the SDS-PAGE of IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) reducing condition.Swimming lane 5,6,7 is IMP/ The SDS-PAGE of the non-reduced state of IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion protein.

Fig. 3：It is shown that the RT-PCR detections that IMP fusion protein is expressed in CHO-S cells.Growth conditions are preferable CHO-S cells are implanted into 150mm²Cultivate in culture dish, the expression plasmid DNA built in secondary daily 80 μ g is transfected.Use after dress dye G418 antibiotic is screened, and after transfection 48h separates total serum IgE using total RNA extraction reagent box.And with being obtained by reverse transcription The cDNA for obtaining carries out RT-PCR analyses for template.Fig. 3 is shown that with Goldenview colour developings on 1% agarose gel RT-PCR products.Wherein, swimming lane 1～6 is followed successively by control, DL2000Maker, blank, IMP-Fc, IMP-Fc (QA), IgG-Fc.

Fig. 4：It is shown that IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion protein to II The effect of the db/db model mouses of patients with type Ⅰ DM.The db/db mices of 4 weeks and/or 6 week old by intramuscular injection IMP/IgG-Fc or IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion protein.Collect the serum of 2,12,16 weeks after before injection and injection.Living Property IMP levels (data do not show) is detected by IMP Elisa test kits.The empty stomach of the two groups of mices of detection in 2 days after injecting first (n=5～6, p ＜ are 0.001) for blood sugar level.

Fig. 5：It is shown that to express IMP/IgG-Fc in vivo, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion protein To the effect in the type i diabetes model of the insulin deficiency that streptozotocin is induced.To CD1 model mouses, every Mus intramuscular injection IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) fusion protein 1nM.2 days before injection fusion protein, tested Mus By reinforcing injection STZ (55mg/kg, peritoneal injection), and received continuous 5 days to inject in the same day daily.Injection STZ a couple of days Afterwards, the tested Mus blood glucose of the control of injection increases significantly (>=17mM), but IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG- The tested Mus that Fc (QA) is processed are protected and are shown the occurrence frequency of low obvious diabetes.

Specific embodiment

The invention provides the fusion protein of prevention and treatment diabetes and method.The fusion protein passes through Insulin receptor INSR Help adjusts blood sugar level.When the fusion protein to individual administration effective dose, the fusion protein of the present invention is by activating islets of langerhans Plain receptor and associated signal paths, treat and prevent the generation of diabetes.

In one embodiment, the compositions and methods of the invention extend the circulating half-life of IMP, enhance its work( Effect.This is realized by the fusion protein (IMP/IgG-Fc) provided containing active IMP and IgG CH.Described IMP peptides are that natural itself there is embodiment.IgG is people source.Humanized IgG can be selected from IgG1, IgG2 and IgG₄.IMP polypeptides It can be the fragment of sequence itself or derivant.IMP polypeptides can be SLEEEWAQIECEVYGRGCPSESFYDWFE RQL ammonia Base acid sequence.

In another embodiment, fusion protein of the invention contains IMP polypeptides, IMP fragments or derivant or which spreads out Biological fragment or IgG-Fc (QL) and IgG-Fc (QA) mutant fragment.

Present invention also offers the carrier of the fusion protein that secretes of the coding present invention, the fusion protein is included but not It is confined to：Active IMP and human IgG1/IgG2/IgG4-Fc aminoacid sequences, for the IMP polypeptides that mammal expresses bivalence； And the IMP-IgG Fc (QL) and IgG-Fc (QA) aminoacid sequence of activity.Those skilled in the art can be as herein Any required IMP sequences are easily prepared in carrier described in embodiment or in the same way.Body outer cell line grinds Study carefully and treated with reference to intramuscular injection fusion protein in I types and type ii diabetes model mouse body, it was demonstrated that the mankind IMP fusions of restructuring Protein I MP/IgG-Fc, IMP/IgG-Fc (QL) and the biological characteristicses and effectiveness of IMP-IgG-Fc (QA).This treatment Method is proved in serious I types and type ii diabetes model effectively.It is experimentally confirmed in the mankind and larger animal effectively, And pass through intramuscular injection fusion protein prove in rodent less efficient.In a word, the present invention be with protein therapeutic and Gene therapy prevents and treats I types in mammalss and type ii diabetes provide a kind of new method.

The fusion protein of the present invention can be IMP fragments, and which has at least 60% or more identical sequence with polypeptide.Described Sequence can have at least 70%, 80%, 90%, 95% or more identical sequence with the IMP polypeptides of form known；Known to these Form includes analog, its derivant and its fragment.The sequence of such as these forms is in United States Patent (USP) 20070775642 Open.Present invention additionally comprises combinations of the above thing to be used for preventing and treating the purposes in the medicine of diabetes, such as I types and II Patients with type Ⅰ DM.Present invention additionally comprises for a kind of Pharmaceutical composition of above-mentioned all purposes, such as preventative compositionss.

The structure of fusion protein is to merge a kind of new molecule of IMP and IgG-Fc molecular compositions, and the molecule can promote IMP's Effect is simultaneously combined with the advantage of IgG-Fc, that is, increase the Half-life in vivo of IMP and reduce and promote fissional risk, it is to avoid pancreas Insulin resistance.The invention provides combining the fusion protein of IgG-Fc mutants, the fusion protein, such as fusion protein are tied Close IMP and IgG-Fc molecules and form recruit, the recruit has functions that IMP is similar to and possesses the advantage of IgG-Fc molecules. Equally, the recruit that IMP is formed with IgG Fc (QL) or IgG-Fc (QA), its have described promotion IMP effects and IgG-Fc (QL) or IgG-Fc (QA) molecule advantage, strengthen the affinity of Fc and FcRn, further increase the internal half-life.

The scope of the present invention includes producing its chemical equivalent or the aminoacid sequence being chemically similar and the change that makees.With IMP sequences have the IgG-Fc fused polypeptide of identical sequence within the scope of the invention, and tests determined suitable for the present invention Method in.United States Patent (USP) (application number 20070775642 is entirely incorporated by referring to) describes many insulin-simulated peptides. Multiple polypeptides in the present invention can Lock-in variant, it is also possible to through mutation, or can pass through synthesis, such as by as fixed The protein engineering of point mutation, the technology are amino acid substitution technology well known in the art.Such as one hydrophobic residue picture Glycine can be by another hydrophobic residue as alanine is replaced, and alanine residue can be by the higher bright ammonia of hydrophobicity Acid, L-Valine, isoleucine are replaced.Negatively charged aminoacid such as aspartic acid can be replaced by glutamic acid, positively charged ammonia Base acid such as lysine can be replaced by another positively charged amino acids Arginine.

So, present invention resides in variation with a grain of salt or the IgG-Fc fused polypeptide that replaces on aminoacid sequence.There is guarantor One or more aminoacid are inserted in the replacement that stays, and its reservation is replaced aminoacid identical chemical characteristic.The present invention includes guarantor The alternative sequence of type is kept, and the activity of original sequence is not destroyed after conservative is replaced.The expected IgG-Fc fused polypeptide of the present invention Contain 1 or multiple D- aminoacid.There is the acetylizad polypeptide of one or more aminoacid expected N- ends.Those skilled in the art can Polypeptide analog can be built with multiple technologies to understand, the analog has the required corresponding polypeptide compound with the present invention Have in terms of same or similar property, but the sensitivity in dissolubility, stability and/or to hydrolysis and enzymolysis preferably living Property.For example, Morgan and Gainor (Ann.Rep.Med.Chem., 24：243-252,1989) described in.Polypeptide analog Embodiment is described in United States Patent (USP) No.5,643,873.How other descriptions prepare and apply the patent of polypeptide analog to include (such as US5,786,322,5,753,226,5,654,276,5,643,873,5,683,983,5,677,280,5/767,075, 5,668,110,5,763,571,5,672,584, all above-mentioned document heres are entirely incorporated by referring to).Can also be according to which His techniques known in the art prepares the polypeptide analog of the present invention.For example, by being such as hydroxyl with chemically conversion hydrogen group Or another kind of group of amino processes the IMP-Fc fused polypeptide of the present invention to change the factor of side base.Analog is preferably included It is entirely the sequence of the composition of aminoacid or including aminoacid and the hybrid of modified aminoacid or other organic molecules. The present invention equally includes hybrid and IMP-Fc fused polypeptide, and for example, one of nucleotide sequence is combined with another sequence.

The present invention also includes the segment with identical activity of the IMP-Fc fused polypeptide of the present invention, also includes fused polypeptide Can as inspection polypeptide nature and activity research tool segment.This polypeptide is preferably made up of at least 5 aminoacid. In preferred embodiments, they can by 6～10,11～15,16～25,26～50,51～75,76～100 or 101～ 250 aminoacid compositions.Segment can include the sequence of one or more aminoacid deletion, for example, N in compound sequence End amino acid.

The present invention includes that the fusion protein with mutation, these amino acid mutations can occur portion of area inactive in fusion protein Point, these amino acid mutations can also occur in active region part so as to having increased or decreased the activity of fusion protein.At this In bright, as described in United States Patent (USP) WO.05/00092 (here is entirely incorporated by referring to), those skilled in the art can be passed through The IgG-Fc parts of known technology modification fusion protein are changing (increasing or decreasing) immunogenicity (immunogenicity) Or effector functions (effector function).

The fusion protein of the present invention individually effectively can be used, but it is also possible to other such as medicinal load in pharmaceutical composition The composition of body is used together.Can be in conjunction with the fusion protein of the present invention, you can will be used for such as more than a type of soluble form The mankind or the treated object of animal, to prevent or treat diabetes.

The fusion protein of the present invention can pass through number of ways as medicinal application in the mankind or animal, it is not limited to office Portion applies, such as oral, spraying, tracheal strips implantation, peritoneal injection, intravenous injection, intramuscular injection and gene therapy.Application dosage Depend on patient needs, expectation is obtained effect and route of administration.The amount of application of the such as mankind can be 2nmol/kg body weight or It is 0.02～100nmol/kg body weight.When using gene therapy, the DNA injection concentrations of people can be 1 μ g/kg body weight or 0.1～100 μ g/kg body weight.Fusion protein can be imported cell with vivo liposome or trans-viral vector.It is applied to this Multiple transport vehicle of invention are known to those skilled in the art.The compositionss of the present invention can daily, weekly or abide by Doctor's advice applies the time for persistently needing.

Prepared by the available known method for preparing the pharmaceutical acceptable carrier that can be administered to patient of the Pharmaceutical composition of the present invention, from And make the nucleotide or peptide molecule of effective dose and pharmaceutically useful carrier combine to form mixture.These suitable carriers, in example As Remington pharmacopedicss (Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA) in be described.On this basis, the Pharmaceutical composition can include pharmaceutically acceptable with one or more Carrier or diluent combine and be present in the reactive compound that has in appropriate pH value the buffer isotonic with physiological fluid Or material, such as composite nucleic acid molecule, peptide molecule or fusion egg.Both are tied by binding activity molecule and carrier with diluent The method of conjunction is known to those skilled in the art.The compositionss may include special in tissue for transporting reactive compound Determine the targeting preparation (targeting agent) at position.

The compositionss of the present invention can such as use hypoglycemic medicine with the remedy measures of known I types and/or type ii diabetes Or bound insulin is treated jointly.For example, the medicine for the treatment of diabetes can include：Victoza、Actos、Amaryl、 avandia、 DiaBeta、Diabinese、Dymelor、Glucophage、GlucophageXR、Glucotrol、 GlucotrolXL、Glucovance、Glynase、PresTab、Glyset、Micronase、Orinase、Pandin、 Precose, Starlix and Tolinase.For example, suitable insulin includes：Aspart、Insulin、Glargine (Lantus), Lispro (Humalog), NPH and Ultralente etc..

The present invention is applied to any individuality for needing such treatment.These individualities are possible to develop into diabetes or recently The patient for being diagnosed as diabetes or the patient for being diagnosed as diabetes.Present invention I as described herein with prevention and treatment Type is related with type ii diabetes.For example, such individuality can be fat people or have diabetes Genetic history but not yet fall ill People is diagnosed as or has been diagnosed as the people of diabetes recently.World Health Organization (WHO) (WHO) is according to body-mass index (BMI) Definition is fat.BMI is square getting divided by height rice number with Kg body weight.For 18.5～25 Normal-weights.Individual BMI is 25～30 is overweight, equals or exceeds 30 for obesity.It is higher than same age and body weight that these individualities can also be blood glucose The people of normal value (euglycemia can be according to medical reference data conventional determining), although be also unlikely to be diagnosed as glycosuria Disease.But these individualities can also be the people for having diabetes Genetic history also not developing into diabetes.When blood glucose value is higher than general All over the normal range of accreditation when can be diagnosed as diabetes.

Present invention also offers the plasmid of new encoding fusion protein, fusion protein coding is contained and is overlapped by application People IMP and human IgG-Fc (Fig. 1) that PCR (polymerase chain reaction) is obtained.It is constant heavy that legend shows that IgG4 is contained in IgG-Fc regions Chain (including hinge, CH2 and CH3).

Present invention also offers leader can be produced and importing carrier allows fusion protein to be secreted into cell after expressing The method that goes in outer culture fluid environment.As shown, one section of IgK secretion guides peptide sequence merges guiding synthesis with IMP sequences Polypeptide is secreted in culture fluid.In an embodiment of the present invention, one section of rat immune globulin K (MUS IGK) guide's peptide sequence The polypeptide that (MVSTPEFLVFL LFWIPASRG) merges guiding synthesis with IMP and IgG-Fc sequences is secreted in culture fluid.Each In individual embodiment, this strategy ensure that will make the IMP and IgG- of generation in secretion process after secretion leading peptide is cut off The active histidine residues fusion of Fc fusion protein N-terminals.Representational IMP/IgG-Fc is shown in Fig. 2.This method can reach：

1) as Fc fusion protein is with homogeneous dimeric form secretion, IMP-Fc fusion protein long half time also, Its higher part affinity is so that fusion protein has higher potency.

2) as substantial amounts of Insulin receptor INSR is present with dimeric precursor forms in cell surface, thus IMP is enhanced The drug effect of polypeptide.

3) purge process is simplified.Can be completed to purify with a step using Protein A gels, beneficial to the separation of fusion protein Purification.

This patent also discloses the expression of the new support for demonstrating above-mentioned fusion protein using mammalian expression systems.For Assessment vector expression and the ability of secretion IMP/IgG-Fc fusion protein, by plamid vector transfection to CHO-S cells.After transfection 48h, extracts total serum IgE, the expression for assessing fusion protein with RT-PCR from the transfectional cell of expression.Drawn with gene specific Thing (Fig. 3) detects the expression of IMP-Fc fusion protein and IgG-Fc reference proteins on transcriptional level.Rather than transfection sample does not have Detect expression of the above-mentioned albumen on gene transcription level.

The Chinese hamster ovary cell CHO-S cells that transfection is also analyzed with Western immunoblottings and anti-mouse antibody Lysate and cell culture fluid are determining expression of the fusion protein in translation skill.As shown in Figure 2 A, in culture fluid and carefully Cellular lysate analyte detection Fc fusion protein.RT-PCR reverse transcriptase polymerase chain reactions and Western immunoblottings can be passed through Carry out detection fusion albumen.In conditioned medium and cell lysate, detection fusion albumen shows that fusion protein is being fed simultaneously Synthesize in newborn zooblast and secrete.CHO-S cell lines distinguish transient transfection the IMP/IgG-Fc plasmids of incremental change and IMP- Fc-only plasmids, after transfection, 48h collects cell culture fluid.From 50ml culture fluid (inoculum density about 1.25 × 10⁵Individual cell/ Ml, 2 days static gas wave refrigerators) utilize Protein A gels one step purified fusion protein (Jungbauer et al., J Chromatogr.1989；46：About 300 μ g fusion protein 257-268) are obtained.Sample is again respectively through reduction and non-reducing 2 kinds Gel electrophoresiss are separated and detect the band (Fig. 2) of an about 35KDa or about 70KDa, table through Coomassie brilliant blue dyeing respectively Bright dimer IMP/IgG-Fc fusion protein is present with native state.

In one embodiment, be shown that IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) and In prevention and treatment I types and type ii diabetes effectively, in for embodiment, 1. pre-diabetes db/db Mus are used as II types sugar The model of urine disease；2. the Mus of insulin are lacked as type i diabetes model by the I types that streptozotocin (STZ) is induced.Db/db is little Mus lack functional leptin receptor, therefore form fat, Hyperinsulinism (hyperinsulincmia) and in 4 to 6 week old When there is glucose intolerance, occur obvious diabetes in 8 week old.Therefore these mices are widely used and are considered as The model of type ii diabetes.By low dosage, multiple medication induces the mice for lacking insulin using streptozotocin (STZ).Chain Urea mycin can specifically destroy beta cell, with the cell-mediated infiltrations of T-.Therefore these mices are widely used simultaneously It is considered as the zootype of the individual type i diabetes of simulation.It is currently available and can apply to other mice sugar of the present invention Urine disease model also has：The mouse model of the insulin resistant of high fat diet induction；This mice causes body because of excessively intake fat Interior fatty excess deposition, thus form fat, tool insulin resistance, and the intolerance of glucose.Another kind of animal model is Non-obese diabetes (NOD) mice.These mices are good autoimmune type 2 diabetes mellitus (type i diabetes) model, wherein pancreas Antigen active T- cellular infiltrations islets of langerhans in island simultaneously kills islet cellss, causes the inflammatory process to make islet cellss death (Anderson And Bluestone, 2005).

Existed by hormone therapy approach administration IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) In prevention and treatment I types and type ii diabetes effectively.Any fusion protein such as IMP/IgG-Fc, IMP/ of the coding present invention IgG-Fc (QL) and IMP/IgG-Fc (QA) molecule are effective for treatment.Injected by fusion protein and to 4 week old db/db Above-mentioned fusion protein is applied in tested Mus injection.In all of tested Mus, after first time injects, carry out second injection within 1 week, And monitor the progress of tested Mus diabetes.Heritability lacks the type ii diabetes that the db/db Mus of leptin receptor are Rodents (Leiter, FASEB are J.1989 for model；3(11)：2231-2241).As it appears from the above, used by fusion protein therapy approach simultaneously The Mus of the age-matched of IMP/IgG-Fc process (1 week after injection) in 16 week old show that blood glucose amount is normal, and matched group is little Mus fasting glucose (FBG) level has then been higher by normal range (Fig. 5).These results show with the treatment of IMP/IgG-Fc can in case Only there are diabetes in db/db Mus.Generation in the tested Mus of db/db of expression IMP/IgG-Fc without diabetes.

By intramuscular injection, IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) are posted to respectively In the tested Mus of CD1.While fusion protein is injected, subsequent injections have been carried out to mice, and start continuous 5 days to note daily Penetrate STZ (55mg/kg, i.p.).As a result find, the blood sugar concentration of IgG-Fc control mices significantly rises, after a few days blood sugar concentration The level (17nM) of diabetes is reached, but IMP/IgG-Fc, IMP/IgG-Fc (QL) or IMP/IgG-Fc (QA) mice have been received To the low incidence rate (Fig. 4) for protecting and showing obvious diabetes.

Many advantages are had based on the medicine of IgG-Fc.Because fusion protein can form the dimeric form of 70KDa, and Will not be removed by kidney at short notice, therefore there is substantially longer half-life (Larrick and Fry, Hum Antibodies Hybridomas.1991；2(4)：172-189；Weir et al., Biochem Soc Trans.2002；30 (4)：512-516).Therefore, the big IMP/IgG-Fc homodimer fusion protein more natural IMP half-life increases.Due to one A little enzymes are easier less polypeptide (Hupe-Sodmann et al., the Regul Pept.1995 that degrades；58 (3) .149-156), and divide The larger IMP/IgG-Fc of son amount is not easy to be degraded.And IMP/IgG-Fc dimers can increase the affinity of part, lead to Cross preformed Insulin receptor INSR dimer and polymer (George et al., Nat Rev Drug Discov.2002； (10)：808-820；Dupuis et al., Brain Res Mol Brain Res.1999；67(1)：107-123) strengthen intracellular Signal transduction, the IMP- fusion protein for therefore existing with homodimer have more potentiality, receive in conjunction with more insulins Body.

IMP/IgG-Fc fusion protein has hypoglycemic effect to obtain in the db/db mices entered by intramuscular injection in vivo Proof is arrived.There is this method continuous release fusion protein in the time of continued for several weeks to enter in blood circulation. In intramuscular injection IMP/IgG-Fc albumen db/db mices, the IMP fusions in blood circulation after two weeks, can have been detected Albumen, but IMP fusion protein is but can't detect in the mice as the only injection IgG-Fc albumen of control.

As serious symptom type ii diabetes model, the tested Mus of Db/db are because that it is present in terms of Leptin signaling pathway and lack Fall into (Herberg and Coleman, Metabolism.1977；26(1)：59-99；Chen et al., Cell.1996；84(3)： 491-495).Caused by fusion protein activation Insulin receptor INSR, blood sugar level is realized normal.The normalization for being possible to blood glucose is more Ground is Insulin receptor INSR to be activated by fusion protein, and then increased the metaboilic level of internal blood glucose.Although in addition, we Effect (Turton etc. to reduction body weight that rodent model is arrived in some examples is not seen with natural IMP Ureteral Calculus People, Nature.1996；379(6560)：69-72).We are also not observed IMP/IgG-Fc the situation of anorexia to animal There is (Kim et al., Diabetes.2003；52(3)：Although 751-759) long-time and effective Insulin receptor INSR agonist The process of IMP changes fasting blood glucose level (Wang and Brubaker, the Diabetologia.2002 of obesity mice；45 (9)：1263-1273).But body weight and peripheral insulin sensitivity still do not change (Wang and Brubaker, Diabetologia.2002；45(9)：1263-1273).This discovery further supports only insulin resistant and is not enough to Cause the generation of type ii diabetes, type ii diabetes (Weyer et al., J Clin only can just occur when β cell dysfunctions Invest.1999；104(6)：787-794；Weyer A, Diabetes.1999；48(11)：Anorectic effects 2197-2203) Depend on effect (Schick et al., the Am J Physiol Regul Integr in the multiple regions of Central nervous system brain Comp Physiol.2003；284(6)：R1427-R1435) fusion protein penetrates the ability of blood brain barrier and also needs to further Probe into.

Intramuscular injection fusion protein simultaneously has proved to be the effective and method of safety with reference to internal electrotransfer.This method is It is widely used, and has been used to cytokine, peptide hormone, soluble recepter and many embrane-associated proteins or Cytoplasm egg In vain.Really thus, this method is systematically injecting this kind of protein-based regulatory factor of such as IMP/IgG-Fc fusion protein Aspect is particularly useful.The advantage of IgG integration technologies is can to obtain laboratory scale by simple step program in test chamber The IMP/IgG-Fc fusion protein of amount.IMP bacterial expression testing results show that the expression efficiency of bacterial expression system will be less than and feed The expression efficiency of newborn animal cell expression system, this may be attributed to the mistake that expresses in expression in escherichia coli compared with CHO-S cells The protein of (misfold) is folded by mistake.Although the correct folding of 2 bacterial cells of chi Rosetta gami expression and functionalization egg White is to greatly improve (Pnnz et al., J Biol Chem.1997 compared with other antibacterials；272(25)：15661-15667) this Raising is special close by increase that the strong formation of two sulfur of albumen in bacterial cell plastid providing lacks in E. coli system The tRNAs of numeral realizes (Kurland and Gallant, Curr Opin Biotechnol.1996；7(5)489-493).

In a word, the present invention be IMP (the Insulin receptor INSR agonist obtained by display technique of bacteriophage) with IgG-Fc and Its mutant merges to form IMP/IgG-Fc to increase the half-life, strengthen activity in vivo and reduce immunogenicity IMP/IgG-Fc Fusion protein is in the form of natural bivalence.In one embodiment of the invention, IMP/IgG-Fc fusion protein can be with Directly applied by injection.Shown with Mus diabetes model in vivo test, can be applied by the injection of fusion protein, this side Method can be with the blood sugar level in effective control body, it is to avoid the generation of insulin resistant；Prove in type ii diabetes db/db mouse models Diabetes can be treated.Meanwhile, IMP/IgG-Fc (QL) and IMP/ is further characterized by by the drug metabolism experiment of Rhesus Macacus IgG-Fc (QA) has longer Half-life in vivo than IMP/IgG-Fc.

Content disclosed above has carried out description substantially to the present invention.By reference to specific examples below to the present invention It is more completely understood.These embodiments are used for the purpose of further instruction, rather than in order to limit the scope of the present invention.Change Yan Zhi, some are pro forma to become XORs and replaces considering of being used as expressing one's ideas.Therefore, some special terms that example is quoted below It is intended to express one's ideas, and the unrestricted purpose which is anticipated.

Embodiment

Embodiment 1：The structure of carrier

The carrier that the encoding fusion protein containing IMP and IgG1-Fc is constructed using over-lap PCR (overlap PCR), IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) carrier.Contain IgG4 constant heavies (hinge-CH2-CH3 portions in IgG4-Fc areas Point).Mus IgK is secreted guide's peptide sequence with the fusion of IMP sequences so that the fusion protein of guiding synthesis is secreted in culture fluid.Compile Code IMP/hIgG-Fc fusion protein cDNA be chemosynthesis, and be connected to encoding human IgG4-Fc (hinge, CH2 and On the product of PCR amplifications CH3), it is inserted between EcoRV the and BamHI sites of pcDNA3.1 carriers, building IMP/hIgG- Fc/pcDNA3.1 carriers.The IMP/hIgG-Fc and its mutant fusion protein that can be secreted contain IgG4 constant heavies (hinge- CH2-CH3).The protein excretion that Mus IgK secretions leader peptide sequence is synthesized with the fusion guiding of IMP sequences is in cell culture fluid.Should Method guarantees that in secretion process, after secretion guiding peptide is removed, the aminoterminal of the IMP of fusion protein can be with an activity Histidine residues merge.Fig. 1 shows the diagram of the IMP/IgG-Fc fusion protein of secretion.It is characterized in：

1. IMP half-life shorts are solved the problems, such as, because fusion protein is secretion in the form of homodimer, in body Interior high-titer (Ozmen et al., J has been reached with the long half-life and due to the high affinity of part Immunol.1993,150 (7)：2698-2705；Kurschner et al., J Immunol.1992；149(12)：4096-4100； Kurschner etc., J Biol Chem.1992；267(13)：9354-9360).

2. as most of Insulin receptor INSRs are to be pre-formed dimer (George et al., Nat Rev in cell surface Drug Discov.2002；1(10)：808-820；Dupuis et al., Brain Res Mol Brain Res.1999；67(1)： 107-123) thus the combination of polypeptide and its receptor will more effectively；

3. the purification process in being conducive to producing, can pass through one step purification (Jungbauer etc. of A albumen polydextran gel People, Chromatogr.1989；476：257-268).

Using gene-specific primer and over-lap PCR from IMP (chemosynthesis, Shenggong, Shanghai, China) CDNA and IgG plasmid amplifications total length IMP and IgG-Fc cDNA.For first round over-lap PCR, using 5 '- GATATCATGGTGAGCACCCCCGAGTTCCTGGTGTTCCTGCTGTTCTGGATCCCCGCCAGCCGGGGCAGCCTGGAGG- 3 ' and 5 '-TGCTGACCAAGCTCGCCGTCGACCCGCCGCCGCCGTCGCCG-3 '；5′- AGCGGCAGCTGGGCGGCGGCGGCAGGGCGGCGGCGGCAG-3 ' and 5 '- TGATGTGGGTCTTCTCGGACTCGGACTCGGACCCGTTCCCTAAG-3’.PCR and product used in the second wheel over-lap PCR Thing prepares standby IMP/IgG-Fc cDNA.The product of amplification is subcloned into carrier B am HI and Eco RV sites, using 5 '- ATGGTGAGCACCCCCGAGTTCCTGGTGTTCCTGCTGTTCTGGATCCCCGCCAGCCG GGGCAGCCTGGAGG-3 ' and 5 '-TGATGTGGGTCTTCTCGGACTCGGACTCGGACCCGTTCCCTAAG-3 ' primers.

For building the control vector of coding IMP-IgG-Fc, using 5 '-GGCGGCGGCGGCAGCGGCGGCGGCG GCAG- 3 ' and 5 '-TGATGTGGGTCTTCTCGGACTCGGACTCGGACCCGTTC-3 ', by PCR individually expand IgG cDNA and gram Grand to carrier B am HI and Eco RV sites.PCR amplifications for the cDNA fragments of encoding human IgG4-Fc (hinge-CH2-CH3) Primer be：5 '-GGCGGCGGCGGCAGCGGCGGCGGCGGCAG-3 ' and 5 '- TGATGTGGGTCTTCTCGGACTCGGACTCGGACCCGTTCCCTAAG-3’.Carrier contain CMV directly-in early days (immediate-early) enhancer and promoter, single eukaryote transcriptional units.Purine tail and transcription terminator.For IMP sequence secretories are made, Mus IgK- chain signal peptide sequences are introduced in its 5 ' end by PCR.It is expression IMP/IgG-Fc in antibacterial Fusion protein, fusion cDNA sequence by PCR amplifications simultaneously sub-clone to pET32a carriers (Novagen, EMD Bioscience, San Diego, CA).The construction method of IgG-Fc (QL) and IMP/IgG-Fc (QA) expression plasmid ibid, T250Q/M428L and Two pairs of PCR primers are designed in T307Q/N434A mutational sites in addition, carry out over-lap PCR reaction, finally obtain IMP/IgG-Fc (QL) With IMP/IgG-Fc (QA) expression plasmid (Fig. 1).

Embodiment 2：IMP/IgG-Fc, IMP/IgG-Fc (QL) and the Chinese hamster ovary celI table of IMP/IgG-Fc (QA) fusion protein Reach

In order to evaluate carrier in expression and the ability for secreting IMP/IgG-Fc fusion protein, the fusion protein carrier of structure turns Transient expression in contaminating to CHO-S cells.48h after transfection, extracts total serum IgE from transfectional cell and is imitated with RT-PCR assessment expression Really.Application gene-specific primer, detects expression and IgG-Fc of the IMP/IgG-Fc antigen-4 fusion protein genes on transcriptional level The expression (Fig. 2A) of crt gene.The expression of transcriptional level is not detected by control sample.

The CHO-S cell lysates and cell culture fluid that transfection be have detected with Western Diagnosis of Sghistosomiasis notation and anti-mouse antibody To determine expression of the fusion protein in translation skill.As shown in Figure 2 B, while detecting in culture fluid and cell lysate Arrive Fc fusion protein.Fusion protein is to merge egg under the conditions of the SDS-PAGE of reduction in electrophoretic migration position about in 35kDa The molecular weight of Bai Danti.Fusion protein is detected simultaneously by conditioned medium and cell lysate shows that fusion protein exists Mammal detail synthesizes and secretes.

Expression in mammalian cell, is that the cDNA of IMP/IgG-Fc or IgG-Fc is passed through liposome (Lipofectamine) 2000 (Invitrogen, Carlsbad, CA) are transfected in CHO-S cells.Its process is first by density It is every hole 2.5 × 10⁵Individual CHO-S cell growths are in 6 porocyte culture plates.Add and contain 4 μ gIMP/IgG-Fc cDNA and 10 DMEM (Invitrogen) of the serum-free of μ l transfection liposomees without antibiotics is cultivated.After transfection 6h, full culture is converted to Liquid.After transfection, 48h collects culture fluid and cell respectively.In order to express IMP/IgG-Fc fusion protein on a large scale, will be grown in CHO-S cells cation transfection reagent in 150mm culture dishs, poly- acetimide) (Poly (ethyleneimine) (PEI, 25kDa) with 80 μ g are related.CDNA transfection.Dilution DNA and PEI is distinguished with 150Mm Nacl, cultivate 20 minutes after mixing.Then DNA/PEI complex is added in cell, and 6h is cultivated in culture fluid of the serum-free without antibiotics.With added with 10%FBS and The DMEM of P/S replaces culture fluid.The transfection efficiency of the method may be up to 85%.

For setting up the CHO-S cells for stably expressing IMP/IgG-Fc, will be in 6 porocyte culture plates (2.5 × 10⁵Individual cell/ Hole) the 4 μ g linearizing IMP/IgG-Fc or IgG-Fc transfection of the upper cell for growing.After transfection 24h, cell is containing G418 The DMEM culture fluid division of (500 μ g/ml) and culture, to screen those by recombiant plasmid stable integration in its genome Cell.In incubation, culture fluid was changed per 3 days once until cell clonal formation.Separate monoclonal cell and be extended to steady The supernatant of fixed cell line the tissue culture for growing these cell lines on 24 orifice plates is detected with SDS-PAGE or Elisa Fusion protein.Select to secrete fusion protein in a large number, and stable cell is passed on as later specificity analysises.

Embodiment 3：From purification IMP/IgG-Fc in mammaliancellculture liquid, IMP/IgG-Fc (QL) and IMP/IgG- Fc (QA) fusion protein

For carrying out small-scale purification, the culture fluid that collects from transfectional cell (is usually taken per hole in 6 well culture plates The 70 μ ls full volume (packed of buffer pre-flush with containing 100mM Tris pH value 8.0 and 150mM Nacl 2.5ml) is added volume).In the polydextran gel quick eluting resin (Amersham-Pharmacia, Piscataway, NJ) of A albumen.4 At DEG C after overnight incubation, with Tris wash buffers, then the sample buffer with 30 μ l containing SDS is directly by albumen from resin Eluting.

For obtaining substantial amounts of fusion protein, apply A albumen sephadex column carry out the protein purification of middle scale. The column volume of 50ml can be with the transfection IMP/lgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG- of growth in purification 15cm culture dishs The conditioned medium of the CHO-S cells of Fc (QA) fusion vector.The 50ml DMEM culture fluid of 48h after collection cell transfecting, or The cell of stable expressed fusion protein is collected, the full volumes of A albumen polydextran geies 1mL, (Amersham- is added into Pharmacia).Add 1%TritonX-100 overnight incubations at 4 DEG C.Then buffered with the PBS containing 1%TritonX-100 Liquid wash, then wash with 150mM Nacl, finally use 1mM Nacl glycines (pH2.7) by albumen the eluting from resin. The albumen PD-10 gel column desalination (Amersham- of Tris buffer (pH9.0) neutralization and purification used immediately by eluent Pharmacia) and PBS eluting is used.As seen in Fig. (Fig. 2A), most of fusion protein can be gathered by two step elution process by Portugal Elute on sugared gel column.Sample group lease making SDS-PAGE dissolving and through Coomassie brilliant blue dyeing development with assess yield and (result shows per ml culture fluid inoculation 1.25 × 10 purification rate⁵After cell culture 2d, fusion protein yield is about 6 μ g/ml).

Embodiment 4：IMP/IgG-Fc, IMP/IgG-Fc (QL) and the bacterial expression of IMP/IgG-Fc (QA) albumen

Expression IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) fusion eggs in Bacillus coli cells In vain.In order to compensate the codon bias (Codon bias) of e. coli bl21 cell, use and can promote disulfide formation And 2 cells of the Rosetta gami (Merck, Germany) that can additionally carry plasmid to express 7 rare tRNAs use IMP/ IgG-Fc/pET32a, IMP/IgG-Fc (QL) pET32a, IMP/IgG-Fc (QA) pET32a or IgG-Fc/pET32a carriers (Novagen), after transform bacteria, wherein several clones are selected and filter out with optimum expression fusion protein.Protein expression is By monospecific polyclonal culture in 2 × YT culture fluid of the 50ml for having kanamycin in 37 DEG C of incubated overnight.Then that culture fluid is dilute (1: 50) new culture fluid is interpreted into, until cell density reaches OD₆₀₀After reading value 0.6, then the IPTG for adding 1mM in culture fluid (EMD) 3 hours with abduction delivering.Collect antibacterial and fungus ball (Pallet) is preserved to carry out other steps.

The extraction of protein be in brief by centrifugation after antibacterial be resuspended in ice bath containing protease inhibitor chicken Ultrasonic treatment cell is used after the PBS of tail wine (Sigrma, St.Louis, MO).Add 1%TritonX-100 with molten Solution albumen, centrifugation (12000g, 10min) after 30min collect the supernatant containing fusion protein.The fusion protein of expression passes through A Albumen polydextran gel purification simultaneously passes through SDS-PAGE；(data do not show) is verified with Coomassie brilliant blue staining analysiss.From 3L's IMP/IgG-Fc the and Fc-only fusion protein of about 120 μ g can be purified in inoculum.In expression IMP fusion protein It is observed that IMP levels increase in peptide dose dependent in mammalian cell and antibacterial.But it was found that total IMP is in antibacterial Expression is less than the level in mammalian cell.

Embodiment 5：In IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) Prevention db/db mices The generation of (type ii diabetes model) diabetes

The female db/db Mus (Weitong lihua, Beijing, China) of 4 week old.The control Mus of age Background matching CJ57BLKS and CD1 (Weitong lihua, Beijing, China).(12 illumination/12 hour are black there is control illumination for tested Mus Raise secretly) and under constant temperature, there is provided sufficient (suitable Rodents) food and drinking-water.All programs are all according to Chinese real The rules for testing animal welfare committee is carried out and is ratified through laboratory animal Ethics Committee.

Diabetes db/db tested Mus (Prud ' homme and Chang, Gene are processed by foregoing intramuscular injection Ther.1999；6(5)：771-777).In short, to the tested Mus tibiofibula intramuscular injection of anaesthetic treatment containing 1nM's The PBS of IMP/IgG-Fc fusion protein, injection limit needle injection depth for 5mm using No. 27 syringe needles for having plastic sheath.Per It is monitored to its body weight and fasting blood glucose level.Saphena blood under the conditions of taking for 2,3,5,7 days after protein injection on an empty stomach.With The basal plasma glucose measurement amount fasting blood glucose level of Roche companies production, IMP, IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/ The measurement of IgG-Fc (QA) level is as described below：

Expression in IMP/IgG-Fc fusion protein bodies is to make of this research institution and coated IMP Elisa reagents Box detects plasma activities IMP contents to be estimated.As illustrated, after injecting 2 days for the first time, injecting the tested of IMP/IgG-Fc Mus are significantly high compared with the tested Mus serum I MP level of injection IgG-Fc.IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) fusion eggs White IMP levels and IMP/IgG-Fc basically identical (Fig. 5).

In process of the test, there is no significant change (not shown) in the body weight of two groups of tested Mus.After injection In one month, two groups of tested Mus fasting blood glucose levels are also without significant difference (not shown).However, 1 week after injection, injection The tested Mus fasting blood glucose level for crossing IMP/IgG-Fc is substantially less than matched group (P ＜ 0.05) (Fig. 5).This result shows, body The IMP/IgG-Fc of interior injection, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) have the hypoglycemic work(of drop to the tested Mus of db/db Effect, its reason are likely due to have activated Insulin receptor INSR, promote the metabolism of internal saccharide.

Embodiment 6：IMP/IgG-Fc, IMP/IgG-Fc (QL) and the insulin of IMP/IgG-Fc (QA) prevention STZ inductions The generation of diabetes in shortage property mouse type i diabetes model

Recent research result indicate that, insulin-simulated peptide (IMP) can be simulated insulin and be adjusted blood glucose alleviation I type glycosurias Disease (Maja Jensen et al., J Biol Chem.2007,282：35179-35186).In order to determine IMP/IgG-Fc fusion eggs The white effectiveness to pancreaticβ-cell damage model, we have studied its diabetes CD1 mice induced by streptozotocin (STZ) Effect.Respectively fusion protein (1nM/ mices) intramuscular injection of coding IMP/IgG-Fc, IgG-Fc or IgG-Fc is received to CD1 Examination Mus.Simultaneously, injection STZ (55mg/kg, i.p.) result shows that IgG-Fc matched groups blood glucose shows to injection fusion protein within continuous 5 days Writing increases, and reaches the level (blood glucose >=17mM) of diabetes, but inject IMP/IgG-Fc or IMP/IgG-Fc (QL) in several days Or the tested Mus of IMP/IgG-Fc (QA) fusion protein are protected the low incidence rate (Fig. 4) that shows obvious diabetes.

Embodiment 7：RT-PCRization method detection IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) fusion egg White expression

Using One step RT-PCR test kit (Qiagen, Valencia, CA) and gene-specific primer detection IgG fusions Expression of the albumen on transcriptional level.For detecting IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA) fusion egg White gene expression, extracts cell total rna 100ng as template and the specific primer of 0.6 μM of interpolation：

(SEQ IDNo：18)

5′-AGCCTGGAGGAGGAGTGGGCCCAGATCGAGTGCGAGGTGTACGGCCGGGGCTGC-3′

With

(SEQ ID No：19)

5′-TCGAAGATGATGACCAAGATCGCCGTCGAC-3′

It is the expression of detection IgG Fc simultaneously, using specific primer

(SEQ ID No：20)

5 '-GAGAGCAAGTACGGCCCCCCCTGCCCCAGCTGCCCCGCCCCCGAGTTCCTGGGCGG CCCC-3 ' and

(SEQ ID No：21)

5′-TCGACGTCGCACTACGTGCTCCGGGACGTGTTGGTGATGTGGGTCTTCTCGGACTCGGACTCGGAC CCGTTC-3′)

The condition of one-step RT-PCR is 50 DEG C of 30min, 95 DEG C of 5min, 94 DEG C of 30min of 40 circulations, 55 DEG C 30 seconds and 72 DEG C 60 seconds, at 72 DEG C, subsequently carry out the extension of 10min.RT-PCR products are separated by 1% agarose gel electrophoresiies, Goldenview dyeing detections.

Embodiment 8：SDS-PAGE and/or immunoblotting detection IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/ IgG-Fc (QA) fusion protein

Detection in translation skill is carried out to the fusion protein that expresses.Take small-scale extraction is dissolved in SDS sample-loading buffers 30 μ l of fusion protein separate through 10%SDS PAGE gel electrophoresiss after be transferred to cellulose acetate membrane, and with anti-mouse antibody (1: 5000, Amersham-Pharmacia) detect, with the medium-scale extractions of ECL development (Amersham-Pharmacia) 30 μ l Fusion protein is detected with Coomassie brilliant blue dyeing after then separating through 10%SDS PAGE gel electrophoresiss.

Experiment 9：Pharmacokineticss are detected

By Rhesus pharmacokinetics experiment, to medicine kinetics in vivo, especially the half-life is detected.Choose Healthy adult Rhesus Macacus 9, male and female half and half, subcutaneous injection IMP, IMP/IgG-Fc, IMP/IgG-Fc (QL) and IMP/IgG-Fc (QA), afterwards with the concentration of medicine in ELISA method detection blood.Concentration v. time data is calculated through pharmacokinetic parameter, shows medicine Thing is eliminated and meets two compartment models, and blood concentration-time curve and each medicine are shown in Table 1 for parameter.Wherein, IMP/IgG-Fc injections Group, the average internal elimination half-life of 9 Rhesus Macacus is 98 ± 19.2h.Average out to peak concentration is 24.0 ± 4.2 μ g/ml, averagely Supersession rate is 0.190 ± 0.026ml/kg/h, and area under the drug-time curve is 4,915 ± 539 μ g.h/ml, and Half-life in vivo is bright Aobvious 7 ± the 1.8h for being longer than IMP.IMP/IgG-Fc (QL) can further extend the half-life to 153 ± 41.5h simultaneously.

Table 1：The pharmacokinetic parameter of IMP/IgG-Fc

Statistical analysis：All data are all represented in the form of mean+SD (mean+SEM).Use SAS softwares (Statistical Analysis Systems, Cary, NC) carries out student t inspections or variance analyses (ANOVA), uses " n- 1 " hypothesis testing (post hoc custom hypotheses test), represents significant difference with p ＜ 0.05.

Sequence table：

IMP, human IgG1-Fc, human IgG2-Fc, human IgG 3-Fc, human IgG 4-Fc and its mutant (QL) and (QA), Mus IgK Homing sequence：

<110>Haitao Zhang

<120>Insulin-simulated peptide fusion protein and mutant and its application

<160>

<210>1

<211>30

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<310>

<311>2011-11-26

<312>

<400>1

Ser Leu Glu Glu Glu Trp Ala Gln Ile Glu Cys Glu Val Tyr Gly

1 5 10 15

Arg Gly Cys Pro Ser Glu Ser Phe Tyr Asp Trp Phe Glu Arg Gln

16 20 25 30

<210>2

<211>5

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>2

Gly Gly Gly Gly Ser (SEQ ID NO：2)

<210>3

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>3

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO：3)

<210>4

<211>15

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>4

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO：4)

<210>5

<211>20

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>5

(GlyGlyGlyGlySer)4 (SEQ ID NO：5)

<210>6

<211>25

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>6

(GlyGlyGlyGlySer)5 (SEQ ID NO：6)

<210>7

<211>30

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>7

(GlyGlyGlyGlySer)6 (SEQ ID NO：7)

<210>8

<211>4

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>8

(GlyGlyGlySer) n wherein n are 0,1,2,3,4,5 or 6. (SEQ ID NO：8)

<210>9

<211>237

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>9

IgG1-Fc(Hinge-CH2-CH3)：(SEQ ID NO：9)

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

16 20 25 30

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

31 35 40 45

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

46 50 55 60

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

61 65 70 75

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

76 80 85 90

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

91 95 100 105

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

106 110 115 120

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

121 125 130 135

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

136 140 145 150

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

151 160 165 170

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

171 175 180 185

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

186 190 195 200

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

201 205 210 215

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

216 220 225 230

Leu Ser Leu Ser Pro Gly Lys

231 235

<210>10

<211>228

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>10

IgG2-Fc(Hinge-CH2-CH3)：(SEQ ID NO：10)

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro

1 5 10 15

Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

16 20 25 30

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

31 35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

46 50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

61 65 70 75

Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val

76 80 85 90

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

91 95 100 105

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

106 110 115 120

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

121 125 130 135

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

136 140 145 150

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

151 155 160 165

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

166 170 175 180

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

181 185 190 195

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

196 200 205 210

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

211 215 220 225

Pro Gly Lys

226

<210>11

<211>249

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>11

IgG3-Fc(Hinge-CH2-CH3)：(SEQ ID NO：11)

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg

1 5 10 15

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

16 20 25 30

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

31 35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

46 50 55 60

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

61 65 70 75

Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

76 80 85 90

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Val

91 95 100 105

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

106 110 115 120

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

121 125 130 135

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val

136 140 145 150

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

151 155 160 165

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

166 170 175 180

Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr

181 185 190 195

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

196 200 205 210

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe

211 215 220 225

Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

226 230 235 240

Lys Ser Leu Ser Leu Ser Pro Gly Lys

241 245

<210>12

<211>229

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>12

IgG4-Fc(Hinge-CH2-CH3)：(SEQ ID NO：12)

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu

1 5 10 15

Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

16 20 25 30

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

31 35 40 45

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

46 50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

61 65 70 75

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Arg Val Leu Thr Val

76 80 85 90

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

91 95 100 105

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

106 110 115 120

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

121 125 130 135

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

136 140 145 150

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

151 155 160 165

Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu

166 170 175 180

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

181 185 190 195

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

196 200 205 210

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

211 215 220 225

Ser Leu Gly Lys

226

<210>13

<211>247

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>13

Mutation IgG1-Fc (Hinge-CH2-CH3)：(SEQ ID NO：13)

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

16 20 25 30

Lys Pro Lys Asp Xaa₁Leu Met Ile Ser Arg Thr Pro Glu Val Thr

31 35 40 45

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

46 50 55 60

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

61 65 70 75

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

76 80 85 90

Leu Xaa₂Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

91 95 100 105

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

106 110 115 120

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

121 125 130 140

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

141 145 150 155

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

156 160 165 170

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

171 175 180 185

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

190 195 200 205

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

210 215 220 225

Ser Val Xaa₃His Glu Ala Leu His Xaa₄His Tyr Thr Gln Lys Ser

226 230 235 240

Leu Ser Leu Ser Pro Gly Lys

241 245

<210>14

<211>227

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>14

Mutation IgG2-Fc (Hinge-CH2-CH3)：(SEQ ID NO：14)

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro

1 5 10 15

Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

16 20 25 30

Xaa₁Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

31 35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

46 50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

61 65 70 75

Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Xaa₂Val Val

76 80 85 90

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

91 95 100 105

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

106 110 115 120

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

121 125 130 135

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

136 140 145 150

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

151 155 160 165

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

166 170 175 180

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

181 185 190 195

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Xaa₃His

196 200 205 210

Glu Ala Leu His Xaa₄His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

211 215 220 225

Pro Gly Lys

226

<210>15

<211>249

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>15

Mutation IgG3-Fc (Hinge-CH2-CH3)：(SEQ ID NO：15)

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg

1 5 10 15

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

16 20 25 30

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

31 35 40 45

Pro Pro Lys Pro Lys Asp Xaa₁Leu Met Ile Ser Arg Thr Pro Glu

46 50 55 60

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

61 65 70 75

Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

76 80 85 90

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Val

91 95 100 105

Ser Val Leu Xaa₂Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

106 110 115 120

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

121 125 130 135

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val

136 140 145 150

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

151 155 160 165

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

166 170 175 180

Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr

181 185 190 195

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

196 200 205 210

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe

211 215 220 225

Ser Cys Ser Val Xaa₃His Glu Ala Leu His Xaa₄Arg Phe Thr Gln

226 230 235 240

Lys Ser Leu Ser Leu Ser Pro Gly Lys

241 245

<210>16

<211>234

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>16

Mutation IgG4-Fc (Hinge-CH2-CH3)：(SEQ ID NO：16)

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu

1 5 10 15

Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

16 20 25 30

Asp Xaa₁Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

31 35 40 45

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

46 50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

61 65 70 75

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Arg Val Leu Xaa₂Val

76 80 85 90

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

91 95 100 105

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

106 110 115 120

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

125 130 135 140

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

141 145 150 155

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

156 160 165 170

Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu

171 175 180 185

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

186 190 195 200

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Xaa₃

201 205 210 215

His Glu Ala Leu His Xaa₄His Tyr Thr Gln Lys Ser Leu Ser Leu

216 220 225 230

Ser Leu Gly Lys

231

<210>16

<211>234

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesis construct

<400>16

Mouse IgK signal leader：(SEQ ID NO：17)

Met Val Ser Thr Pro Glu Phe Leu Val Phe Leu Leu Phe Trp Ile

1 5 10 15

Pro Ala Ser Arg Gly

16 20

Claims (7)

1. a kind of fusion protein, is mutated the bodily form by connection peptides (Linker) with IgG-Fc or IgG-Fc by insulin-simulated peptide Into its formula is IMP (Insulin mimetic peptides)-Linker-IgG-Fc or IgG-Fc-Linker-IMP (Insulin mimetic peptides)；

The sequence of the IMP polypeptides be SLEEEWAQIECEVYGRGCPSE SFYDWFERQL or SLEEEWAQIECEVYGRGCPSE SFYDWFERQA；

IgG Fc (hinge region-CH2-CH3) sequence is human IgG1, the Fc of IgG2, IgG3 or IgG4 and its mutation or disappearance Body, above-mentioned sequence are selected from SEQ ID NO：9；SEQ ID NO：10；SEQ ID NO：11 or SEQ ID NO：12, wherein IgG-Fc Sequence is numbered with reference to EU (Kabat) index, and above-mentioned IgG-Fc mutants are selected from sequence SEQ ID NO：13；SEQ ID NO： 14；SEQ ID NO：15；SEQ ID NO：16, above-mentioned mutational site can be that single-site mutant or multisite mutation are combined, its In：

Xaa₁=Gln or Thr

Xaa₂=Gln or Thr

Xaa₃=Leu, Met or His

Xaa₄=Ala or Asn；

The insulin-simulated peptide is connected with Human Albumin or transferrinss；Or the insulin-simulated peptide and Human Albumin Or transferrin moiety fragment, mutant are connected with deletant.

2. the fusion protein as described in claim l, it is characterised in that the Linker is selected from sequence SEQ ID NO：2；SEQ ID NO：3；SEQ ID NO：4；SEQ ID NO：5；SEQ ID NO：6；Or SEQ ID NO：7, (GGGGS) n, wherein n are l, 2,3,4,5 or 6.

3. the fusion protein as described in claim l, it is characterised in that the Linker can be selected from SEQ ID NO：8, wherein N is l, 2,3,4,5 or 6, or be directly connected to.

4. fusion protein described in any one of claim l～3 is in the prevention and therapy-related medicine for preparing people I types and type ii diabetes Application in thing, or application of its DNA sequence in gene therapy related drugs are prepared.

5. fusion protein as claimed in claim 1, it is characterised in that the fusion protein is affine with FcRn based on increasing Power is further increasing the IMP half-life in vivo.

6. the fusion protein as described in any one of claim l～3, it is characterised in that the fusion protein uses eukaryotic cell Expressed, and purification is carried out using A or G-protein sephadex chromatography post to the albumen for obtaining.

7. the fusion protein as described in any one of claim l～3, it is characterised in that the fusion protein uses prokaryotic cell Expressed.