CN1303861A - Novel retinoblastoma protein binding protein, its preparation and application - Google Patents

Abstract

The present invention relates to a novel human gene nucleotide sequence. It is a cDNA sequence coding Creaml (protein containing repetitive eighty-six amino-acid motif). Said gene-coded product is a novel transcriptional control factor, also is a conjugated protein of retinoblastoma protein (Rb protein). Said invention also relates to a polypeptide coded by said nucleotide sequence, the application of these polynucleotide and polypeptide, and the production method of them.

Description

New retinoblastoma protein binding protein and method for making thereof and purposes

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the cDNA sequence of the people Creaml that the present invention relates to encode, this gene encoding production is a kind of new transcriptional regulator, also is the conjugated protein of retinoblastoma protein (Rb albumen).The invention still further relates to by this nucleotide sequence coded polypeptide the application of these polynucleotide and polypeptide, and the production method of described polynucleotide and described polypeptide.

Retinoblastoma gene also is called the Rb gene for short, and it is the tumor suppressor gene (Goodrich and Lee, 1993, Biochim.Biophy.Acta 1155:43-61) of first discovery.Rb genes encoding Rb albumen (retinoblastoma protein).In the cell cycle, Rb albumen is the important composition composition at the cell cycle G1 phase outpost of the tax office.The forfeiture of Rb protein function can cause tumour to take place and developmental defect (Weinberg, Cell 81,323-330 (1995); People such as Lin, Cancer Biol.7,279-89 (1996); Tan and Wang, Trends.Cell Biol.8,116-20 (1998)).

The transforming protein of several DNA tumour viruses comprises the large T antigen of SV40, the E1a of adenovirus and the E7 albumen of 16 type papilloma viruss, can be when virus infection directly and the T antigen binding domain effect of Rb.This step is vital for the subsequent transformation host cell.This effect causes the crucial Rb conjugated protein (as E2F1) of some cell cycle progress to dissociate; thereby walk around the Normocellular mechanism of action (in normal mechanism; when having only Rb by " cell cycle protein dependent kinase " phosphorylation in the G1 later stage, these factors just are released).Therefore, the cell cycle has lost normal regulatory mechanism, and this is the characteristics of tumour cell.The tumour cell of natural generation then adopts and makes the strategy of Rb because of the sudden change inactivation.As a result, the various different sudden changes of finding the Rb function be present in 100% retinocytoma and the some other tumour (as osteosarcoma, lung cancer and bladder cancer) (Goodrich and Lee, 1993, Biochim.Biophy.Acta1155:43-61).

In addition, nearest research finds that also also there is important effect Rb albumen fetal development and tissue differentiation aspect in early days.The Rb deficient mice can be in embryo the 14th and death in 15 days, and neural the generation and people such as (, the same) Lin unusually of bematogenesis arranged.

Rb also needs in the lipogenesis process of mouse.As if different with Rb common restraining effect in the cell cycle, Rb activates C/EBP by direct effect, thus the differentiation (Chen, people such as P.L., Genes.Dev.10,2794-2804 (1996)) of adjusting adipocyte.

In addition, when not having functional Rb albumen, the mouse Skeletal Muscle Cell can not fully break up, and (Cell 72 for Gu, people such as W., 309-324 (1993) because the myocyte of differentiation can enter the cell cycle again; People such as Zacksenhaus, Genes.Dev.10,3051-3064 (1996)).Rb brings into play its effect in the flesh forming process by activating MyoD.In addition, the stoichiometry of Rb and the sub-HBP1 of transcription inhibition factor is compared to myoblastic differentiation also effect (Shih, people such as H.H., Mol.Cell.Biol.18,4732-4743 (1998)).

Research shows that Rb albumen is important modulin in vertebrate cells cycle and the growth.It is by directly and the cell cycle carry out and break up in related protein factor have an effect and bring into play its function.

Transcription factor TF II-I is a kind of multi-functional transcriptional.It is accredited as the binding factor of initial son at first, by participating in transcriptional control (Roy, A.L.Nature365,355-359 (1993)) in conjunction with the initial sub-site in no TATA (TATA-less) promotor.TF II-I has 6 conservative tumor-necrosis factor glycoproteinss, and (Roy, A.L. wait the people, EMBO.J.16:7091-7104 (1997); Grueneberg, people such as D.A., Genes Dev.11:2482-2493 (1997)).Although the physiological action of tumor-necrosis factor glycoproteins it be unclear that, they may participate in DNA in conjunction with (Roy, people such as A.L., the same) to the helix-loop-helix sample in conjunction with the existence in territory hint.Show that TF II-I can be incorporated into different dna sequence dnas.On the other hand, TF II-I also directly acts on other various transcription factors, for example c-myc (Roy A.L., Nature 365,359-361 (1993)), USFl (Roy, A.L., Deng the people, EMBO.J.16:7091-7104 (1997)), SRF and PhoX1 (Grueneberg, people such as D.A., Genes Dev.11:2482-2493 (1997)) and STAT1 and STAT2 (Kim D.W.Mol.Cell.Biol.18,3310-3320 (1998)).In addition, TF II-I (being also referred to as " BAP-135 ") can be incorporated into BrutonShi Tyrosylprotein kinase (btk) [btk is the required a kind of tenuigenin Tyrosylprotein kinase of normal B cell development], and the activation according to btk makes tyrosine by phosphorylation (Yang, W. wait the people, Proc.Natl.Acad.Sci.USA.94:604-609 (1997)).The tyrosine phosphorylation of TF II-I is vital (Novian, people such as C.D., J.Biol.Chem.273,33443-33448 (1998)) for the dependent transcriptional activity of initial son of TF II-I.These characteristic hints, the effect of TF II-I may be to coordinate the basic mechanism of transcribing and one group of upstream regulatory factor that responds some signal pathway.At last, the TF II-I of hemizygosity (hemizygosity) relevant with the Williams syndromes (Perez-Jurado, people such as L.A., Hum.Mol.Genet.7,325-334 (1998)).

The Williams syndromes is a kind of inborn neurologic disorder syndromes.The symptom of Williams syndromes comprises the defective of multiple systems, and for example MR etc. especially shows as cognitive obstacle.About 1: 20 of this disease sickness rate in world population, 000.In order to reduce the sickness rate of this disease, people press for the new effective methods for prenatal diagnosis of exploitation.

Because, Rb albumen and/or " Rb protein-binding protein " unusual relevant with some diseases, therefore, to research and develop Rb proteic conjugated protein for therapeutic purpose, and promptly that interactional albumen takes place is significant with Rb albumen.

The present invention is intended to solve these and other problem of prior art, and the application at aspects such as diagnosis Williams syndromess of a kind of Rb protein-binding protein and this albumen is provided.

An object of the present invention is to provide a kind of new polynucleotide, a kind of Rb protein-binding protein of this polynucleotide encoding, new Rb protein-binding protein gene of the present invention is named as people Creaml gene.

Another object of the present invention provides a kind of new Rb protein-binding protein, and this albumen is named as people Creaml albumen.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people's Creaml polypeptide.

The present invention also provides this people's the Creaml nucleotide sequence and the application of polypeptide, especially in the purposes of the application facet of diagnosis Williams syndromes.

In a first aspect of the present invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people Creaml protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 8-2887 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQID NO.1 in from the nucleotide sequence hybridization of Nucleotide 8-2887 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.2.More preferably, this sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 8-2887 position.

In a second aspect of the present invention, a kind of isolating people Creaml protein polypeptide is provided, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ IDNO.2 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.

In a third aspect of the present invention, a kind of carrier is provided, it contains above-mentioned isolated DNA.

In a fourth aspect of the present invention, provide a kind of described carrier transformed host cells.

In a fifth aspect of the present invention, provide a kind of generation to have the method for the polypeptide of people Creaml protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people Creaml protein-active operationally is connected in expression regulation sequence, form people Creaml protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 8-2887 position among described nucleotide sequence and the SEQ ID NO.1;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people Creaml;

(c) under the condition that is fit to expressing human Creaml protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people Creaml protein-active.

In a sixth aspect of the present invention, simulation, promotion, the active compound of antagonism people Creaml protein polypeptide are provided, and the compound that suppresses the expression of people Creaml protein polypeptide.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of people Creaml protein polypeptide.

In a seventh aspect of the present invention, provide with above-claimed cpd come mediator Creaml albumen in vivo, the method for external activity.

In a eighth aspect of the present invention, a kind of disease relevant with people Creaml protein polypeptide unconventionality expression or method of disease susceptibility (especially William syndromes) of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide, detect perhaps whether the Creaml gene is in the state of narrowing in the genome.

In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screening and promote the active agonist of people's Creaml protein polypeptide, and perhaps screening suppresses people's active antagonist of Creaml protein polypeptide or is used to the peptide finger print identification.The proteic encoding sequence of people Creaml of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.

In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the people Creaml protein polypeptide of the present invention and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as William syndromes, cancer, cardiovascular disorder.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

In the application, use following abbreviation:

The DNA of BD:Gal 4 is in conjunction with the territory

The proteic conserved regions 1-3 of Ccr1-3:Creaml

The proteic tumor-necrosis factor glycoproteins 1-5 of Cr1-5:Creaml

EST: expressed sequence sign

IIF: direct immunofluorescence

Inr: initial son

MBP: maltose binding protein

NLS: nuclear localization signal

The SIE:c-sis Thr6 PDGF BB is induced element

SRE: serum response element

The conserved regions 1-3 of Tcr1-3 TF II-1

TnIs slow switch fibers specificity troponin I

The tumor-necrosis factor glycoproteins 1-6 of Tr1-6 TF II-I

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 683 Nucleotide, and its detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 8-2887 position Nucleotide.

In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, term " people Creaml albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people Creaml protein-active is as 8-2887 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 8-2887 position Nucleotide of SEQ ID NO.1 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.1 in 8-2887 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQID NO.2 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition, with among the SEQ ID NO.1 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 8-2887 position.Also term also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 8-2887 position, preferably at least 80%, more preferably at least 90%.At least 95% nucleotide sequence best.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.2 sequence of people Creaml identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and add several at 5 ＇ and/or 3 ＇ end and (be generally in 60, preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.

In the present invention, term " people Creaml protein polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of people Creaml protein-active.This term also comprises having and variant form people Creaml identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people Creaml and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people Creaml DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people Creaml polypeptide to obtain.The present invention also provides other polypeptide, as comprises people Creaml polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people Creaml polypeptide.Usually, this fragment have people Creaml peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.As a kind of purposes of the soluble fragments of people Creaml polypeptide, they can be used as immunogenic antigen and come immune animal, to prepare the antibody of anti-Creaml polypeptide.

Invention also provides the analogue of people Creaml albumen or polypeptide.The difference of these analogues and natural human Creaml polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, " people Creaml conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val;Leu;Ile	Val
Arg(R)	Lys;Gln;Asn	Lys
			Asn(N)	Gln;His;Lys;Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro;Ala	Ala
			His(H)	Asn;Gln;Lys;Arg	Arg
Ile(I)	Leu;Val;Met;Ala;Phe	Leu
			Leu(L)	Ile;Val;Met;Ala;Phe	Ile
Lys(K)	Arg;Gln;Asn	Arg
			Met(M)	Leu;Phe;Ile	Leu
Phe(F)	Leu;Val;Ile;Ala;Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr;Phe	Tyr
			Tyr(Y)	Trp;Phe;Thr;Ser	Phe
Val(V)	Ile;Leu;Met;Phe;Ala	Leu

The present invention also comprises people Creaml polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people Creaml in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 of people Creaml polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people Creaml.

The present invention also comprises the method that detects people Creaml nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to the encoding sequence of people Creaml polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence then, can form protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.

On the other hand, the present invention also comprises people Creaml DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people Creaml gene product or fragment.Preferably, refer to that those can combine with people Creaml gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people Creaml, comprise that also those do not influence the antibody of people Creaml protein function.The present invention also comprise those can with modify or without the people Creaml gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) 2 fragments; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people Creaml gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human Creaml or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies andT Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people Creaml function and the antibody that does not influence people Creaml function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people Creaml gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.coli) with the unmodified form bonded antibody of people Creaml gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

People Creaml Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain to contain the DNA of relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.At present, can be fully come the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced then in the various dna moleculars (as carrier) and cell in this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Can be with polypeptide of the present invention and antagonist and suitable pharmaceutical carrier combination back use.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.

The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.

Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.Creaml comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to patient's Creaml will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

The proteic polynucleotide of people Creaml also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the expression of the Creaml of the nothing expression of Creaml or unusual/non-activity.The Creaml that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic Creaml activity.For example, a kind of Creaml of variation can be the Creaml that shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease of Creaml expression or active caused by abnormal.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the Creaml transgenosis to cell.The method that structure carries the recombinant viral vector of Creaml gene is found in existing document (Sambrook, et al.).Recombinant human Creaml protein gene can be packaged in the liposome and then be transferred in the cell in addition.

Suppress the oligonucleotide (comprising sense-rna and DNA) of people Creaml protein mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.

Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.

Polypeptide of the present invention also can be used as the peptide spectrum analysis, for example, the polypeptide available physical, chemistry or enzyme carry out the specificity cutting, and carry out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension.

The present invention also provides the antibody at people Creaml proteantigen determinant.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.

The proteic antibody of anti-people Creaml can be used in the immunohistochemistry technology, detects the people Creaml albumen in the biopsy specimen.

With the also available labelled with radioisotope of the protein bound monoclonal antibody of people Creaml, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.

Antibody among the present invention can be used for treating or prevention and the relevant disease of people Creaml albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people Creaml or activity.

Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people Creaml albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell of people Creaml protein positive.

The production of polyclonal antibody can choose Creaml albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

People Creaml protein monoclonal antibody can with hybridoma technology production (Kohler and Milstein.Nature, 1975,256:495-497).With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison etc., PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.PatNo.4946778) also can be used for producing the proteic single-chain antibody of anti-people Creaml.

Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people Creaml obtains.During screening, must carry out mark to people Creaml protein molecular.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people Creaml protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people Creaml protein level that is detected in the test can be with laying down a definition the importance of people Creaml albumen in various diseases and be used to the disease of diagnosing Creaml to work.

The polynucleotide of Creaml can be used for the diagnosis and the treatment of Creaml relative disease.Aspect diagnosis, the unconventionality expression of the expression that the polynucleotide of Creaml can be used for detecting Creaml Creaml whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the abnormal expression of Creaml as the CreamlDNA sequence.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (Mlcroarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect Creaml with the special primer of Creaml.

The sudden change that detects the Creaml gene also can be used for the disease of diagnosing Creaml relevant.The form of Creaml sudden change comprises that the point mutation compared with normal wild type Creaml dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

The above-mentioned and other purposes of polypeptide of the present invention and polynucleotide comprises: for example, polypeptide of the present invention can be used for the peptide finger print identification; The proteic encoding sequence of people Creaml of the present invention or its fragment can be used as primer and be used for pcr amplification reaction; Perhaps be used for hybridization, perhaps be used to make gene chip or microarray as probe.According to instruction of the present invention, these application are conspicuous for those skilled in the art.

Creaml albumen be of identifying of the inventor new with Rb bonded candidate albumen.It can combine with Rb by the C end under conditions in vitro specifically, is a kind of Rb protein-binding protein therefore.

Structurally, Creaml albumen is 959 amino acid that contain of a wide expression, the nucleoprotein of the about 120Kda of molecular weight, have one to the similar nuclear localization signal of SV40 large T antigen nuclear localization signal.The aminoacid sequence of Creaml contains 5 has the tumor-necrosis factor glycoproteins of certain homology with multi-functional transcription regulaton factor TF II-I.Except these 5 tumor-necrosis factor glycoproteinss, Creaml also has 3 and TF II-I height homologous zone in addition.TF II-I plays an important role in the transcription initiation system of no TATA box, and it is subjected to the regulation and control of some signal transduction pathway.But difference is that Creaml can form homodimer, and can not form heterodimer with TF II-I.

In addition, a new muscle cell enhancer binding protein MusTRD1 height homology of Creaml and recent findings, but MusTRD1 only contains 458 amino acid of the N end of Creaml, and the reason that causes this result is MusTRDl cDNA coding region many one " G ", causes the frame frameshit.Relation between them is hinting that Creaml may participate in the gene expression regulation of muscle cell.

In addition, the Creaml gene is positioned on the karyomit(e) 7q11.23 zone, contains 27 exons, and the gene total length surpasses 150Kb as a result.This gene is positioned at Williams syndromes patient's chromosome deletion district.The disappearance of this gene on a karyomit(e) might be the cause of disease of some illness in the Williams syndromes.Thereby this gene can be used for the antenatal diagnosis of Williams syndromes.

In sum, Creaml albumen is likely a kind of ubiquitous transcription regulaton factor that is under the Rb regulation and control.Because Creaml of the present invention has the natural acid sequence that is derived from the people, therefore, compare with the albumen of the same clan that derives from other species, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour.

In the accompanying drawings, Fig. 1 has shown the aminoacid sequence of Creaml cDNA nucleotide sequence and supposition.

(A) the cDNA clone that Creaml is different, clone S22 has the disappearance of " G " on 1,406 nucleotide position; Clone S21 has lacked 1,977-2,021 nucleotide sequence.Last clone is complete nucleotide sequence, black surround region representation coding region, and indicated the used restriction enzyme site of structure plasmid.

(B) the Creaml aminoacid sequence of cDNA sequence and supposition.The part of line is nuclear localization signal and a string Serine of inferring.The NLS that derives is gone out by frame.

Fig. 2 has shown the comparison of sequence between TF II-I and the Creaml.Wherein, Fig. 2 A has shown two proteinic structural performances, and the frame of same form is represented homologous region.Fig. 2 B has shown that the sequence of TF II-I (Tr1-6) and these 11 tumor-necrosis factor glycoproteinss of Creaml (Cr1-5) compares.In conserved sequence, the frequency of occurrences is high is listed in first row, and other close amino-acid residue is listed in second row (following amino acid is regarded as homology: L, V and I, Y and F, S and T, K and R, E and D).Draw thick line and partly represent the most conservative zone in all tumor-necrosis factor glycoproteinss, length contains 80 amino-acid residues.Fig. 2 C has shown the amino-acid sequence of Tcr13 and Ccr1-3 conservative region.The amino acid of line is the hydrophobic amino acid that participates in " hydrophobic slide fastener " structure that dimer forms.

Fig. 3 A has shown Creaml gene structure figure.The gene structure of Creaml is that doctor R.H.Waterston according to Washington University School of Medicine gene order-checking center is published in DJ0665P05 gene clone (the about 133Kb among the Genbank, the accession number of Genbank is AC004851) and DJ1186P10 (about 149Kb, the accession number of Genbank is AC005231) be arranged in.This gene is distributed in four fragments at present, and the interval of 3 sections unknown lengths is arranged between them.First of each exon all shows according to their arrangement positions on cDNA with last Nucleotide.The length of exon does not draw in proportion.Fig. 3 B has shown the exon among the Creaml-intron border.

Fig. 4 has shown that the Creaml gene is in the critical area of Williams syndromes.Comprise ELN (coding elastin) at the known gene of this critical area, LIMK1 (coding lim kinase 1), RFC2 (coding replication factor C), FZD3 (coding frizled homologue), STX1A (the outstanding fusion rotein 1A of coding), and GTF21 (coding TF II-I).

(A) A figure shows the gene linkage collection of illustrative plates of being delivered by other researchist.Black surround is partly represented the critical area of Williams syndromes, and GTF2IP is the pseudogene copy of GTF2I.

(B) the RH collection of illustrative plates is that human No. 7 karyomit(e) obtains in the zone of D7S2415-D7S669 98 years gene mappings on being published in the NIH website.Distance represents the distance of the short arm of a chromosome telomere, and the site of sequence mark (STS), and the title of corresponding gene is also listed.

Fig. 5 has shown that Creaml can combine with Rb under conditions in vitro.

(A) p56Rb that merges with poly Histidine (His6) that uses in this individual system.Fig. 5 A figure shows be His6p56Rb from bacterial lysate behind the purifying, behind the 10%SDS polyacrylamide gel electrophoresis, the band that the monoclonal antibody by anti-poly Histidine shows through immune marking method.The figure illustrates size and the purity of His6-P56RB.

What (B) Fig. 5 B figure showed is different FLAG fusion roteins, behind the 8%SDS polyacrylamide gel electrophoresis, and the band that shows through immunoblotting with the monoclonal antibody of anti-FLAG.It expresses the corresponding content of different proteins on film.

(C) Fig. 5 C figure expression be with the same film of B figure through with " RB centre-fills " (i.e. 1 μ gHis6P56RB, the formed immunocomplex of goat anti-rabbit igg of the monoclonal antibody of the anti-His6 of 1 μ g and 1 μ l alkaline phosphatase coupling connection) at incubation on ice after 3 hours, the band that shows through immunoblotting.The arrow indication is the position of different FLAG fusion roteins, and other band is bacterioprotein.

What (D) Fig. 5 D figure showed is the structure of two used Creaml mutant of this system.

Fig. 6 has shown the specificity of anti-Creaml polyclonal antibody T8.

(A). swimming lane 2 is that the 12%SDS polyacrylamide gel electrophoresis is after the protein band that Coomassie brilliant blue dyeing shows, swimming lane 1 is the MBP-p05 fusion rotein, swimming lane 2 is that (band about following two treaty 30KDa is P05, and promptly immune rabbit produces the antigen of anti-Creaml polyclonal antibody for MBP-P05 after cutting with ten factor enzymes; Middle one is MBP.)。Swimming lane 3 is MBP-P05 after enzyme is cut and the result of preimmune serum after the immunoblotting colour developing, protein band do not occur, illustrates that preimmune serum can not discern antigen.Swimming lane 4 is MBP-P05 and the T8 results after the immunoblotting colour developing after enzyme is cut, and illustrates that T8 can discern antigen (P05) specifically.

(B) swimming lane 1,3, the 5th, the mutant FLAG-Ap32 of Creaml, swimming lane 2,4, the 6th, the His6-TF II-I of total length, swimming lane 1-2 is and the result of antibody T8 behind immunoblotting, and swimming lane 3-4 is the result of monoclonal antibody behind immunoblotting with anti-poly Histidine, and swimming lane 5-6 is the result of monoclonal antibody behind immunoblotting with anti-FLAG, the figure shows antibody T8 and only discern FLAG-Ap32 specifically, and nonrecognition TF II-I.

Fig. 7. (A) in CV1 (African green monkey kidney cell) cell pyrolysis liquid, identify the protein that Creaml is the 120KDa size.Swimming lane 1,3,5th, MBP-P05 fusion rotein, swimming lane 2,4,6th, CV1 cell pyrolysis liquid.Swimming lane 1,2nd is with the result of antibody T8 through immunoblotting.With the antibody T8 of swimming lane 3,4 reaction in advance with 5 μ gMBP-P05 incubation mistakes, with the antibody T8 of swimming lane 5,6 reactions in advance with 5 μ gMBP incubation mistakes.The albumen of the 120KDa of A figure explanation T8 identification is specific, should be creaml.

(B) swimming lane 1 is the CV1 cell pyrolysis liquid, and swimming lane 2 is the FLAG-Creaml in the bacterial lysate.B figure be by with the protein band of antibody T8 immunoblotting demonstration place.The Creaml size that wild-type in FLAG-Creaml and the cell is described is identical, thereby the cDNA that we are cloned into is a total length.

Fig. 8 has shown the formation of USEB1-albumen composition in the CV1 nuclear extract.The CV1 nuclear extract mixes with the USEB1 probe of the radiolabeled MusTRD1 of containing binding site.Detect the formation of mixture with mobility analysis.Utilize 50,100,300 times unlabelled USEB1, USEB1b (double chain nucleotide of sudden change) is as the rival.The specific combination band is indicated with arrow, and non-specific combination band (NS) is represented with arrow.

Fig. 9 has shown the evaluation of the nuclear localization signal of Creaml.Wherein, the structure of Fig. 9 A, fusion protein F LAG-AP32 and FLAG-AP32N.The plasmid of Fig. 9 B, expression FLAG-AP32 and FLAG-AP32N is transformed in the CV1 cell, through 48 hours, cell is fixed with-20 ℃ ice methyl alcohol, then with the monoclonal antibody (picture 1 of anti-FLAG, 3,5) and the mixture incubation of the polyclonal antibody T8 of anti-Creaml 2 hours, be associated with the sheep anti mouse of fluorescein with lotus root again or two temperature resistances of goat-anti rabbit were educated 1 hour, observe, take pictures with immunofluorescence microscopy.Picture 1 and 2 is the cells of expressing FLAG-AP32, and picture 3,4 also is the cell of expressing FLAG-AP32, but with the mixtures of antibodies of its reaction in advance with mbp-p5 incubation mistake.Picture 5 and 6 is the cells of expressing FLAG-AP32N.Can find out that from B figure the monoclonal antibody of anti-FLAG is consistent with the location of albumen in cell of the polyclonal antibody of anti-Creaml identification.FLAG-AP32 is a nucleoprotein, and the FLAG-AP32N that removes nuclear localization signal is a cytoplasm protein, can illustrate that from this phenomenon the sequence of that section Nucleotide of FLAG-AP32N disappearance contains the nuclear localization signal of Creaml.

Figure 10 has shown that Creaml can form dimer in yeast two-hybrid.

(A) A figure shows is the structure of the mutant of the various TF II-1 used in this system and Creaml.AD represents active region, and it has the effect that reporter gene is transcribed that activates.The DB representative is in conjunction with the zone of DNA, and this zone can make protein bound to DNA.

(B) result shows.The beta galactosidase enzyme activity of listing is the clone of two random chooses, the mean value that records by the ONPG method.His-compensation expression pass through the selective medium results of screening ,+show positive findings ,-negative findings.Scheme visible CreamlN and CreamlN easily forms dimer from B, AP32 and AP2 also can form dimer, but between interaction change a little less than.And can not form heterodimer between Creaml and the TF II-1.

Figure 11 has shown that Creaml can combine with Rb in vivo.The CV1 cell plasmid co-transfection of expressing Rb and FLAG-Creaml.Lysing cell also carries out co-immunoprecipitation (IP) and immunoblotting with specified antibody among the figure after 48 hours.Use the monoclonal antibody of anti-Rb can not only precipitate Rb albumen (swimming lane 1) down, and can also obtain Creaml (swimming lane 3).By contrast, control antibodies (anti-CD4) antibody then can not precipitate Rb (swimming lane 2) and Creaml (swimming lane 4).

Figure 12 A and 12B have shown the structure iron of plasmid pFLAP2 and pFCreaml respectively.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are not used in F explanation the present invention limits the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone and the mensuration of the cDNA sequence of people Creaml

Initial cDNA Segment A p-2 is that the Rb albumen with purifying is (Shan, B.Mol Cell.Biol.12, the 5620-5631 (1992)) that probe separates goes out from HeLa expression cDNA library.This fragment is by radio-labeling, with the longer cDNA clone of further screening.Because the abundance of ZZ cDNA is low in used library, therefore, the plasmid DNA of placenta cdna library (Clontech) and the Marathon cDNA (Clontech) of people's tire spleen are used as template, obtain 5 ＇ encoding sequences with the PCR method.

For the pACT2 library, carry out first round PCR with primer Gal-p (5 ＇-CTATTCGATGATGAAGATACCCCA-3 ＇) and 2p-r3 (5 '-GGCCGACTCAGGCCCAGGTCGCT-3 ＇), use primer Gal-p and 2p-r4 (5 '-CTTCCTGCTTGAGCTCTCGGATG-3 ＇) to carry out second then and take turns.

Marathon cDNA (Clontech) for people's tire spleen, carry out first round PCR with primer 2 p-r5 (5 '-GTACACATCTGAGCCATGTTCCA-3 ＇) and primer AP1 (providing), use primer 2 p-r6 (5 ＇-ACCTCCTTCGGGTGCTDTGCCT-3 ＇) and primer AP2 (providing) to carry out second then and take turns PCR by Clontech by Clontech.

The PCR fragment is gone in the pGEM-T Easy carrier (Promega) by subclone, selects with the Southern blotting.For fear of because of PCR introduces sudden change, at least 3 independent clonings of every kind of product are checked order.Pick out cDNA fragment, construct full-length cDNA (SEQ ID NO:1) with most clone's consensus sequences.

The cDNA (8-2884) that contains whole coding region also increases from the Marathon cDNA (Clontech) of people's tire spleen by PCR.The fragment that amplifies can be directly used in and make up pFCreaml.Used enzyme is a Pfu DNA polymerase (Sangon).Primer is:

Primer 1:5 ＇ CATGGCCTTGCTGGGTAAGCGCTGTGAC 3 ＇

Primer 2: 5 ＇ CTAGTAATTAAGAGGTCCCGGGAGCTGC, 3 ＇

Plasmid construction

(1) is used for expressing the plasmid of the Creaml of FLAG-mark at mammalian cell.

In order to express Creaml albumen total length, that have FLAG epi-position mark, with Nco I-Sca I restriction fragment (containing Nucleotide 6-2893), be connected into pCEP4F carrier (Zhu after filling and leading up end with the Klenow enzyme, X.Mol.Cell.Biol.15,5017-5029 (1995)) Nhe I site, thus construct plasmid pFCreaml (Figure 12 B).

In a similar manner, make up plasmid pFAP32 with the Nco I that contains Nucleotide 762-2893-Sca I restriction fragment.This plasmid expression FLAG-Ap32 (containing amino acid 253-959).

Make up plasmid pFAP32N with the PCR method.This plasmid FLAG-Ap32N (containing amino acid 253-897).

(2) be used for plasmid at the Creaml of expression in escherichia coli FLAG-mark.

For at expression in escherichia coli FLAG-Ap32, will insert from Nco I-Xho I restriction fragment of the 2.1kb of pFAP32 earlier among the pTrcHisA (Invitrogen) with the digestion of same restrictions restriction endonuclease.

In order to express total length FLAG-Creaml albumen, full-length cDNA is connected into pTrcHisA (Invitrogen) by reading frame, obtain plasmid pFLAP2 (Figure 12 A).

With Pst I cutting pFLAP2, the oneself connects then, forms plasmid pFLCreamlp, this plasmid expression FLAG-Creamlp (containing amino acid/11-455).

(3) be used for the plasmid that yeast two-hybrid is analyzed

With Nco I-Sac I restriction fragment (containing Nucleotide 8-2887), insert between the Nco I and Sal I site (filling and leading up) of pAS2-1 (Clontech), to express BD-Creaml (containing amino acid/11-959) with the Klenow enzyme.

Pst I fragment deletion is fallen to express BD-Creamlp (containing amino acid/11-455).

CDNA is cloned the EcoR I fragment of Ap-2 and insert pY1 (Gene 118 for Sadowski, I., 137-141 (1992)), to express BD-Ap2 (containing amino acid 463-959).

(4) be used at other proteic plasmids of expression in escherichia coli

In order to express total length TF II-I (His6-TF II-I) with polyhistidine tag, to be connected into Nhe I and BamH I and cut and fill and lead up among the pTrcHisA (Invitrogen) from 3.4kbNot I (cutting)-BamH I fragment of pI3-CX with mung-bean nuclease.Identify required construction pHis II-I by restrictive diges-tion and Western engram analysis.

In order to express FLAG-E2F1 (containing amino acid/11 23-437), 1.1kb BamH I-EcoR I fragment of pGST-SH5 people such as (, Mol.Cell.Biol.12,5620-5631 (1992)) Shan B. is connected into the pFLAGl (IBI) that digested with Bgl II and Xho I.Before connection, fill and lead up EcoR I and Xho I end with the Klenow enzyme.

In order to express FLAG-mitosin10N, will be from pMBP-10/H (Zhu, X. wait the people, Mol.Cell.Biol.15,5017-5029 (1995)) the EcoR I of 1.3kb-Hind III restriction fragment (filling and leading up with the Klenow enzyme) is cloned between the EcoR I of pFLAG1 and the Sal II site (filling and leading up with the Klenow enzyme), obtains plasmid pF10N.

In order to express His6-p56Rb, with BamH I and EcoR I cutting plasmid pGEX-2T-RBS (Biochim.Biophy.Acta 1155 for Goodrich, people such as D.W., 43-61 (1993)), this plasmid expression has the Rb albumen of the 56kDa form of function.Then the cDNA fragment is as above inserted pTrcHisA (Invitrogen), obtained plasmid pTrcHisA-RBS.

Production of antibodies

With EcoR I-Ava I restriction fragment of the 0.5kb of Ap-2 (part digestion and fill and lead up with the Klenow enzyme), promptly Nucleotide 1394-1925 inserts in the pMAL-c carrier (New England Biolabs) that digests with Pst I and EcoR I.The plasmid pMAL-P05 that forms is used to express MBP fusion rotein MBP-P05.After the sepharose 4B crosslinked with amylose starch (NewEngland Biolabs) carries out affinity purification (method is with reference to the explanation of producer), with factor Xa (New EnglandBiolabs) cleavage of fusion proteins.Behind 12%SDS-PAGE, with immunogen (P05) trace at Western pvdf membrane (Schleicher ﹠amp; Schuell).To contain immunogenic part and downcut, press Knudsen, K.A.Anal Biochem.147, the described method immune rabbit of 285-288 (1985).

In addition, press Smith, D.E.J.Cell.Biol.99, the described method of 20-28 (1984), the MBP-P05 with being fixed on the Hybond-C film (Amersham) is purified into specific antibody from serum.

External Rb-binding analysis

Press Zhu, X.Mol.Cell.Biol.15, the described method of 5017-5029 (1995) is the host with the e. coli bl21 strain, expresses the fusion rotein of FLAG-mark or His-mark.His6-p56Rb is with the affine resin of TALON metal (Clontech) purifying (method that provides according to producer), and with 100mM EDTA (pH8.0) wash-out.The cell pyrolysis liquid that contains FLAG-Creamlp, FLAG-Ap32, FLAG-mitosin10N and FLAG-E2F1 is carried out 8%SDS-PAGE, and electroblotting is to Optitran BA-S film (Schleicher ﹠amp then; Schuell).Substantially press people such as Shan, B., Mol.Cell.Biol.12, the described method of 5620-5631 (1992) is carried out external Rb binding analysis.Wherein the preparation of Rb sandwich complex be by with 1 microgram His6-p56Rb, 1 microlitre anti--polyhistidyl monoclonal antibody (Sigma) and 1 microlitre coupling have the sheep anti-mouse igg (Promega) of alkaline phosphatase to sneak into 1 milliliter of binding buffer liquid.Develop the color with CDP-Star chemiluminescence substrate (Promega).

Cell cultures and transfection

With normal monkey-kidney cells CV1, gastric carcinoma cells SGC-7901, human lung adenocarcinoma cell SPC-A1 and Proliferation of Human Ovarian Cell H0-8901, maintain in DMEM (Gibico) nutrient solution that is supplemented with 10% calf serum and be under the air that contains 5% carbonic acid gas.Calcium phosphate method with routine carries out transfection.

Coimmunoprecipitation, immunoblotting and indirect immunofluorescence research

CV1 cell with about 1: 1 pFCreaml of 2 * 107 usefulness and pCepRB (providing) cotransfection by Dr.W.-H.Lee, at 1 milliliter of buffer A (20mM Tris-HCl, pH 7.5,100mM KCl, 0.1%Nonidet P40,1mM EDTA, 1mM dithiothreitol (DTT), 10% glycerine, 50mM NaF and proteinase inhibitor) middle cracking.After the centrifugal removal cell debris, with half lysate with 5 micrograms anti--Rb monoclonal antibody 3C8 (being provided by Dr.W.-H.Lee) is 4 ℃ of overnight incubation, and second half lysate is hatched with the resisting of equivalent-CD4 monoclonal antibody (Dako).Will be in advance with 10 microgram rabbits anti--25 microlitre A albumen-Sepharose (Pharmacia) that mouse IgG (Anogen) was hatched add, placed 2 hours.With 1 milliliter of buffer B (20mM Tris-HCl, pH 7.5,150mM KCl, 0.5%Nonidet P40,1mM EDTA, 1mM dithiothreitol (DTT), 10% glycerine, 50mM NaF and proteinase inhibitor) washing 3 times.Sample is carried out 8%SDS-PAGE and immunoblotting.

Immunoblotting and indirect immunofluorescence (II F) microscopic inspection is with suitable one-level antibody, presses Zhu, people such as X., and Mol.Cell.Biol.15, the described mode of 5017-5029 (1995) is carried out.Enzyme link coupled secondary antibody is available from Promega.Coupling has the secondary antibody of FITC available from Sino-America Biotechnology Company, Shanghai, China.Anti--FLAG M2 and anti--polyhistidyl monoclonal antibody are respectively available from Kodak and Sigma company.In competitive trials, one-level antibody and 5 microgram MBP-P05 or MBP were hatched 30 minutes, carry out immunoblotting or II F dyeing then.II F image is recorded on Kodak Gold III (ASA400) film with Olympus BX50 fluorescent microscope.

Yeast conversion and betagalactosidase activity analysis

According to the condition that the Clontech of manufacturer is advised, pass through lithium acetate method transfection yeast Y190 strain (Clontech) with suitable plasmid.Transformant is at the enterprising row filter of synthetic medium that lacks tryptophane.To the clone of random choose, measure betagalactosidase activity with the ONPG method.Data are shown in Figure 10 B.

Interpretation of result

Creaml is a kind of new albumen relevant with TF II-I

The aminoacid sequence of deriving according to the cDNA sequence (SEQ ID NO:2 and Fig. 1) shows that this novel polypeptide has 3 sections conservative tumor-necrosis factor glycoproteinss, and every section tumor-necrosis factor glycoproteins has 86 amino acid.Therefore, the albumen of this total length is named as Creaml (protein containing repetirive eighty-six amino-acid motif).

Because the Northern engram analysis shows, there is the mRNA (data not shown goes out) of an about 3kb who generally expresses, therefore use ³²The Ap-2 of P-mark is that probe is filtered into retinocytoma Y79 cDNA library.Two clone S21 (2.2kb) the longest and S22 (2.2kb) are by further research (Figure 1A).Sequential analysis shows that clone S21 has lacked Nucleotide 1974-2018, does not read frame (data not shown goes out) but do not destroy, and this may be because other montage modes cause.Clone S22 has lacked one " G " at unknown 1406 places, and it is moving that this causes reading to be frameed shift.Further screening to other cDNA libraries does not have to find longer fragment.Therefore, with PCR increased in people's placenta cDNA storehouse (Clontech), one of them primer is corresponding to the sequence on the carrier, and another primer is corresponding to the 5 ＇ end regions of S22.

Yet (B18 0.6kb) still lacks some encoding sequence of about 200bp to the longest PCR product.Therefore, the Marathon cDNA (Clontech) of personnel selection peptide spleen carries out second as template and takes turns pcr amplification, has obtained the PCR fragment (clone A1) of 320bp.Obtain the nucleotide sequence of the 3043bp shown in the SEQ ID NO:1 after the splicing, its open reading frame is positioned at 8-2887,959 the amino acid whose Creaml albumen (Fig. 1) of encoding.Make up cDNA clone then, wherein Nucleotide 1-289 is from clone A1, and Nucleotide 290-762 is from clone B18, and Nucleotide 763-1239 is from clone S22, and Nucleotide 1240-1522 is from clone S2l, and all the other Nucleotide are from Ap-2.All these fragments are all checked order to get rid of possible sequence variation.

Sequence with the frame both sides is a primer, has also amplified same cDNA from tire spleen Marathon cDNA (Clontech) with PCR.Because the popularity that Creaml expresses, the RT-PCR method that this cDNA also can be by routine amplification from the mRNA of people's tissue or cell is come out.

Creaml of the present invention and multi-functional transcriptional regulator TF II-I (being also referred to as BAP-1335 or SPIN) have significant similarity (Fig. 2).At first, it is Cr1-Cr5 that Creaml has one group of 5 conservative tumor-necrosis factor glycoproteins, and this is similar (Fig. 2 A and 2B) to 6 tumor-necrosis factor glycoproteinss (Tr1-Tr6) of TF II-I.In fact, 80 residue zones in each tumor-necrosis factor glycoproteins are (Fig. 2 B, the underscore part) guarded between two kinds of albumen.

Secondly, the N end parts of Creaml (amino acid 30-88, the i.e. Ccr1) Tcr1 (amino acid 22-80) corresponding with TF II-I is height homologous (Fig. 2 A and 2C).Ccr1 and Tcr1 contain at the related conservative hydrophobic slide fastener sample motif (Fig. 2 C) (Roy, people such as A.L., EMBO.J.16,7091-7104 (1997)) of protein-protein effect.

In addition, from the Ccr2 of amino acid 499-510 with from the Ccr3 of amino acid 527-532, also correspond respectively in TF II-I among the Creaml from the Tcr2 of amino acid 801-812 with from the Tcr3 (Fig. 2 A and 2C) of amino acid 224-229.These similaritys show that these two kinds of albumen belong to same new protein families.

Yet Creaml also has its unique feature.Comprising the fragment that 12 Serines of an end are arranged at amino acid 912-923 place, and there is the nuclear localization signal (NLS) of inferring (Figure 1B) in the 898-905 position.This NLS is similar to the NLS of the large T antigen of SV40.

What is interesting is, the cDNA (O ＇ Mahoney, people such as J.V., Mol.Cell.Biol.18,6641-6652 (1998)) of a kind of new muscle enhanser-conjugated protein (MusTRD1) that encode, basic identical with Creaml cDNA.Yet only long 458 amino acid of MusTRD1 are because it is moving to cause reading to be frameed shift having inserted extra " G " corresponding to the zone, 1307-1309 position of Creaml cDNA.Therefore, MusTRD1 only contains 2 tumor-necrosis factor glycoproteinss of Creaml.According to sequential analysis, do not have a kind of sequence to contain MusTRD1 CDNA peculiar " G " and insert clone S21, S22 and other existing EST.Therefore, this difference of MusTRD1 may be a kind of special shape in muscle, also might be because sudden change or human factor cause.

The gene structure of Creaml and locus

Also determined the complete genome structure (Fig. 2 D) of Creaml.Structure gene Creaml is found and is positioned among genomic clone DJ0665P05 (about 133kb, Genbank accession number AC004851) and the DJ1186P10 (about 149kb, Genbank accession number AC005231).These two clones are almost finished by order-checking and login in Genbank.

Creaml is arranged in 4 independently contigs.27 exons of 150kb scope constitute (Fig. 2 D and table 1) to this gene by being positioned at least.Exon 2 contains first ATG, and encodes slide fastener sample zone C cr1 hydrophobic with exon 3.The encoding sequence of conservative region Ccr2 and Ccr3 lays respectively at exons 13 and 14.In addition, each Creaml tumor-necrosis factor glycoproteins is by 3 adjacent exons codings.For example, Cr1 is encoded by exon 4-6; Cr2 is encoded by exon 8-10; Cr3 is encoded by exons 1 6-18; Cr4 is encoded by extron 20-22; Cr5 is encoded by exon 2 3-25.In 3 exons of every cover, the degree of a middle exon is fixed as 184bp, except exon 24 is 193bp (Fig. 2 D, a table 1).

Creaml cDNA of the present invention is corresponding to people UniGene Hs.21075.Its corresponding gene is located in human chromosome 7q11.23 (gene map that NIH site http://www.ncbi.nlm.nih.gov announced in 1998).Be used to locate the sequence-tagged site (sts-AA019591) of Creaml, in the intron of part between exon 26 and 27 and extend partially in the exon 27.

Importantly, the Creaml locus is between LIM-kinases and xyntaxin 1A.(Cell 86 for Frangi skakis, people such as J.M., 59-69 (1996) because these two kinds of genes often lack in the William syndromes; Osborne, people such as L.R., Am.J.Hum.Genet.61,449-52 (1997)), so this shows that Creaml also often lacks in this disease.Therefore, whether the disappearance by detecting the Creaml gene, and whether the Creaml protein content is unusual, can detect the William syndromes.

Creaml is a kind of 120kDa protein of generally expressing

In order to probe into the biochemical property of Creaml, prepare rabbit polyclonal antibody with the Creaml polypeptide antigen (P05) (containing amino acid 463-640) of bacterial expression.Express about 70kDa maltose binding protein fusions (MBP-P05) (Fig. 3 A, swimming lane 1) earlier.After handling with factor Xa, MBP-P05 is cut into 2 parts: independent MBP (about 40kDa) and P05 (Fig. 3 A, swimming lane 2).P05 performance is about 30 and the doublet form of 31kDa, and this may be because degraded or factor Xa also have the cause of other cleavage site on this albumen.Because cDNA inserts the reading frame that fragment is not destroyed LacZ α gene among the pMAL-c (New EnglandBiolabs), so P05 also contains some β-gal (about 60 residues).As shown in Figure 3, detect less than albumen (swimming lane 3) in the preimmune serum.Immune serum can be discerned the complete MBP-P05 molecule of P05 doublet and trace specifically, although its abundance of nonrecognition MBP very high (swimming lane 4).

Because P05 contains and TF II-I homologous the 3rd tumor-necrosis factor glycoproteins and 2 additional areas (Fig. 1 and 2), so antagonist is tested to determine whether and TF II-I cross reaction.Shown in Fig. 3 B, in total bacterial lysate, no matter still detect with anti--FLAG M2 monoclonal antibody (swimming lane 5) with anti--Creaml polyclonal antibody T8 (swimming lane 1), the fragment that Creaml mutant FLAG-Ap32 can make to be about 90kDa is detected specifically.Yet, the TF II-I (His6-TF II-I) (swimming lane 2 and 6) of the polyhistidine tag of all nonrecognition total lengths of these two kinds of antibody.On the contrary, anti--Histidine monoclonal antibody can detect TF II-I (swimming lane 4) of 120kDa.These results show, anti--Creaml polyclonal antibody T8 is a high special.

Detect endogenous Creaml (Fig. 4) in the cercopithecus aethiops CV1 cell with T8 antibody then.This antibody can be discerned MBP-P05 (Fig. 4 A, swimming lane 1) in the bacterial lysate and the albumen (swimming lane 2) of the about 120kDa in the total cell pyrolysis liquid of CV1 specifically.For the albumen of testing this 120kDa is Creaml, with MBP or the MBP-P05 preincubate of antibody with the pMAL-c vector expression.Antibody after being neutralized by MBP-P05 can not detect MBP-P05 (swimming lane 3), can not detect the albumen (swimming lane 4) of 120kDa.And do not have influence (swimming lane 5 and 6) with MBP preincubate antagonist.Therefore, these results show that the Creaml in the CV1 cell moves with 120kDa albumen form in SDS-PAGE.In addition, same albumen also is detected in various human cell line and tissue, comprising embryonic kidney cell HEK293, cancer of the stomach SGC-7901, lung cancer SPC-A1, ovarian cancer HO-8901, fetus and adult's skeletal muscle, heart and tire brain (data not shown goes out).Usually, the level of this albumen in institute's specimen is all very low.

Whether contain complete reading frame in order to test Creaml cDNA, the mobility of the total length Creaml (FLAG-Creaml) of the FLAG-mark of Creaml in the cell and expression in escherichia coli is compared (Fig. 4).The result shows that both mobilities are close, and FLAG-Creaml (swimming lane 2) migration is than endogenous Creaml in the CV1 cell (swimming lane 1) slow slightly (because having the FLAG mark).This has confirmed that the cDNA shown in the SEQ ID NO:1 has contained complete Creaml encoding sequence.

Creaml in vivo with the external Rb albumen that all is incorporated into

Isolating Creaml is that a kind of candidate's Rb-is conjugated protein, is named as Rbap2 (Rb-associatedprotein 2, Rb associated protein 2) at first.In order to confirm The selection result, test itself and Rb and interact.

For convenience's sake, the Rb (His6-p56Rb) with the polyhistidyl-mark of affinity purification prepares " Rb is sandwich " (Fig. 5 A).The FLAG-mitosin10N (Fig. 5 B, swimming lane 1) that contains mitosin Argine Monohydrochloride 2484-2927 is used as negative control (Fig. 5 C, swimming lane 1).FLAG-E2F1 (containing residue 123-437) (Fig. 5 B, swimming lane 3) is used as positive control (Fig. 5 C, swimming lane 3).Two kinds of overlapping deletion mutation body FLAG-Creamlp and the FLAG-Ap32 (Fig. 5 D) of Creaml have been tested.FLAG-Ap32 can be obviously and Rb probe effect (Fig. 5 C, swimming lane 2).FLAG-Creamlp (Fig. 5 B, swimming lane 4) but is negative (Fig. 5 C, swimming lane 4).Because clone's Ap2 encoded polypeptide comprises amino acid 463-959, so these results show, can partly have an effect with the C-terminal of Creaml specifically at external Rb.

In vivo test.Because the level of endogenous Creaml is low, the therefore coexpression in the CV1 cell with FLAG-Creaml and Rb is to improve this two kinds of proteinic partial concns.Detect the expression of FLAG-Creaml with II F staining.When the lysate of coexpression cell with anti--after the Rb monoclonal antibody is carried out co-precipitation (figure ll, swimming lane 1), available anti--FLAGl monoclonal antibody M2 detects FLAG-Creaml (swimming lane 3) easily in a plurality of tests.In contrast, control antibodies (anti--the CD4 monoclonal antibody) can not make Rb (swimming lane 2) or Creaml (swimming lane 4) precipitation.

This shows that Creaml can be incorporated into Rb in vivo.We prove further that also Creaml also can combine (Figure 11) in vivo with Rb.For increasing intracellular protein concentration, the way that we utilize cotransfection at cell inner expression Rb and Creaml.Anti-Rb antibody can not only precipitate Rb albumen (swimming lane 1), also makes Creaml also thereupon precipitate (swimming lane 3); Anti-CD 4 antibodies does not in contrast then precipitate Rb or Creaml.This explanation Creaml really is Rb intracellular one of conjugated protein.

The NLS of Creaml is similar to the NLS of SV40 large T antigen

As the potential transcriptional regulator, Creaml should be a kind of nucleoprotein.According to amino acid sequence analysis, Creaml contains a NLS similar to the NLS of SV40 large T antigen (Figure 1B).Change the plasmid of expressing FLAG-Ap32 and FLAG-Ap32N over to the CV1 cell respectively.After transfection 48 hours, use the cold methanol fixed cell, then with anti--FLAG antibody M2 ( swimming lane 1,3,5) and rabbit anti--Creaml antibody T8 ( swimming lane 2,4,6) carries out dual II F and dyes.

The result as shown in Figure 7.FLAG-Ap32 shows a situation of typically appraising and deciding (swimming lane 1: with anti--FLAG antibody colour developing; With swimming lane 2: with anti--Creaml antibody colour developing).In order to get rid of in the labeling process cross reaction possible between antibody, with MBP-P05 add in the one-level mixtures of antibodies with limit optionally neutralization anti--Creaml antibody.This processing causes obviously descending (swimming lane 3) with anti--Creaml antibody labeling, but does not influence the mark (swimming lane 4) of anti--FLAG antibody.This shows that these two kinds of antibody are discerned epi-position separately independently.

When expressing, also observed and appraised and decided a phenomenon with total length FLAG-Creaml.In addition, when the NLS that infers was lacked, corresponding variant protein FLAG-Ap32N (Fig. 7 A) just became the albumen (Fig. 7 B, swimming lane 5 and 6) of cytoplasm type, and this shows that Creaml uses the NLS sequence really.

Creaml has intrinsic and activates active

Whether contain activation in conjunction with the territory in order to test Creaml, the different piece of Creaml is combined territory (BD) merge with the DNA of Gal4, detect the transactivation of formed fusion rotein in yeast then.Yeast strains Y190 contains and is in Gal4-and replys beta-galactosidase enzymes reporter gene under the type promotor control.The result shows with betagalactosidase activity mensuration as shown in Figure 8, express effectively transcribing of residue reporter gene of BD-Creaml in Y190.The BD-Creamlp that contains the amino acid/11-455 of Creaml still has activity.On the contrary, the fusion rotein (being BD-Ap2) that contains amino acid 463-959 only shows the transcriptional activity higher slightly than background.Therefore, as if the activation structure territory is positioned at the N end parts of Creaml.

The potential function of Creaml

Because Creaml generally expresses in multiple different clone and tissue, and its structure is very similar to TF II-I, therefore this shows that Creaml is a kind of transcription regulating and controlling article of action range, is likely a kind of factor in conjunction with Inr of new some no TATA promoter transcription of mediation.

On the other hand, the function of Creaml and TF II-I is by different mechanism regulation.TF II-can be incorporated into btk (a kind of B cell-specific cytoplasmic tyrosine kinase of src superfamily) (Yang, newspaper (Proc.Natl.Acad.Sci.USA) 94 of institute of W. American Academy of Sciences, 604-609 (1997)).In case B cell antigen receptor is got involved, phosphorylation will take place in TF II-I on tyrosine residues.In addition, TF II-I depends on ras-signal transduction path (Kim, D.W., Mol.Cell.Biol.18,3310-3320 (1998)) to the hormesis of c-fos promotor.And to activate for the TF II of V β promotor-I dependent transcription be vital (NoVina, C.D.J.Biol.Chem.273,33443-33448 (1998)) in the src self-phosphorylation site of TF II-I.

Yet Creaml obviously lacks the src phosphorylation site, and it does not react (data not shown goes out) with btk yet.Yet it can be by map kinase by potential phosphorylation site regulation and control (Fig. 1).In addition, data show that Creaml can be by being regulated and control with the association of Rb.According to its express spectra widely, Creaml may be a kind of transcriptional regulator widely that relates in cell cycle progression.In addition, because Rb is very crucial for the final differentiation of skeletal muscle, therefore, Creaml may also play a role under Rb control in the muscle differentiation.

The William syndromes is a kind of congenital disorders, and feature is elf's sample face and mental retardation, and often with cardiovascular and muscle skeleton defective.The hemizygosity of karyomit(e) 7q11.23 is this sick pathogenesis (Ewart, people such as A.K., Nat.Genet.5,11-16 (1993)).Common disappearance breaking point is positioned at about lcM place, elastin gene both sides.So far, found that some genes are hemizygosity in the main crowd of William disease.Comprising elastin gene, LIM-kinases, replication factor C, syntaxin 1A, TF II-I, Wscrl and Fkbp6 etc.As if although the LIM-kinases of high density (a kind of mainly expressed proteins kinases in brain) is vital for this disease, the pathology of all the other these syndromess of gene pairs also has effect.

Importantly, the Creaml locus is between LIM-kinases and xyntaxin 1A.Because these two kinds of genes often lack in the William syndromes, so this shows that Creaml also often lacks in this disease.Further studies show that, in William syndromes patient, the Creaml gene that truly has a copy have disappearance.Therefore, whether the disappearance by detecting the Creaml gene, and whether Creaml protein-active or function be unusual, can detect the William syndromes.

Because Creaml expresses (data do not provide) in brain, so its content decline may influence vital other albumen of neurodevelopment.In addition, research shows also that some relevant with the William syndromes of the state that narrows of Creaml is cardiovascular and unusually also has cognation with skeletal muscle.

People Creaml of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor Creaml can also merge with other members of this family or exchange fragment, to produce new albumen.For example the C end of the C of inventor Creaml end with the Creaml of other species exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor Creaml, be used to screen other members of this family, perhaps be used for affinity purification conjugated protein (as other members of this family).

In addition, inventor Creaml nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people Creaml or the overexpression that suppresses people Creaml.People Creaml albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people Creaml disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table (1) general information:

(ⅱ) denomination of invention: new retinoblastoma protein binding protein and method for making thereof and purposes

(ⅲ) sequence number: the information of 2 (2) SEQ ID NO.1:

(ⅰ) sequence signature:

(A) length: 3043bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ⅱ) molecule type: cDNA

( ⅹⅰ ) ：SEQ ID NO.1：ggcgaccatg gccttgctgg gtaagcgctg tgacgtcccc accaacggct gcggacccga 60ccgctggaac tccgcgttca cccgcaaaga cgagatcatc accagcctcg tgtctgcctt 120agactccatg tgctcagcgc tgtccaaact gaacgccgag gtggcctgtg tcgccgtgca 180cgatgagagc gcctttgtgg tgggcacaga gaaggggaga atgttcctga atgcccggaa 240ggagctacag tcagacttcc tcaggttctg ccgagggccc ccgtggaagg atccggaggc 300agagcacccc aagaaggtgc agcggggcga gggtggaggc cgtagcctcc ctcggtcctc 360cctggaacat ggctcagatg tgtaccttct gcggaagatg gtagaggagg tgtttgatgt 420tctttatagc gaggccctgg gaagggccag tgtggtgcca ctgccctatg agaggctgct 480cagggagcca gggctgctgg ccgtgcaggg gctgcccgag ggcctggcct tccgaaggcc 540agccgagtat gaccccaagg ccctcatggc catcctggaa cacagccacc gcatccgctt 600caagctcaag aggccacttg aggatggcgg gcgggactcg aaggccctgg tggagctgaa 660tggtgtctcc ctgattccca aggggtcacg ggactgtggc ctgcatggcc aggcccccaa 720ggtgccaccc caggacctgc ccccaaccgc cacctcctcc tccatggcca gcttcctgta 780cagcacggcg ctccccaacc acgccatccg agagctcaag caggaagCAC CTTCCTGCCC 840CCTTGCCCCC AGCGACCTGG GCCTGAGTCG GCCCATGCCA GAGCCCAAGG CCACCGGTGC 900CCAAGACTTC TCCGACTGTT GTGGACAGAA GCCCACTGGG CCTGGTGGGC CTCTCATCCA 960GAACGTCCAT GCCTCCAAGC GCATTCTCTT CTCCATCGTC CATGACAAGT CAGAGAAGTG 1020GGACGCCTTC ATAAAGGAAA CCGAGGACAT CAACACGCTC CGGGAGTGTG TGCAGATCCT 1080GTTTAACAGC AGATATGCGG AAGCCCTGGG CCTGGACCAC ATGGTCCCCG TGCCCTACCA 1140GAAGATTGCC TGTGACCCGG AGGCTGTGGA GATCGTGGGC ATCCCGGACA AGATCCCCTT 1200CAAGCGCCCC TGCACTTATG GAGTCCCCAA GCTGAAGCGG ATCCTGGAGG AGCGCCATAG 1260TATCCACTTC ATCATTAAGA GGATGTTTGA TGAGCGAATT TTCACAGGGA ACAAGTTTAC 1320CAAAGACACC ACGAAGCTGG AGCCAGCCAG CCCGCCAGAG GACACCTCTG CAGAGGTCTC 1380TAGGGCCACC GTCCTTGACC TTGCTGGGAA TGCTCGGTCA GACAAGGGCA GCATGTCTGA 1440AGACTGTGGG CCAGGAACCT CCGGGGAGCT GGGCGGGCTG AGGCCGATCA AAATTGAGCC 1500AGAGGATCTG GACATCATTC AGGTCACCGT CCCAGACCCC TCGCCAACCT CTGAGGAAAT 1560GACAGACTCG ATGCCTGGGC ACCTGCCATC GGAGGATTCT GGTTATGGGA TGGAGATGCT 1620GACAGACAAA GGTCTGAGTG AGGACGCGCG GCCCGAGGAG AGGCCCGTGG AGGACAGCCA 1680CGGTGACGTG ATCCGGCCCC TGCGGAAGCA GGTGGAGCTG CTCTTCAACA CACGATACGC 1740CAAGGCCATT GGCATCTCGG AGCCCGTCAA GGTGCCGTAC TCCAAGTTTC TGATGCACCC 1800GGAGGAGCTG TTTGTGGTGG GACTGCCTGA AGGCATCTCC CTCCGCAGGC CCAACTGCTT 1860CGGGATCGCC AAGCTCCGGA AGATTCTGGA GGCCAGCAAC AGCATCCAGT TTGTCATCAA 1920GAGGCCCGAG CTGCTCACTG AGGGAGTCAA AGAGCCCATC GTGGATAGTC AAGGAACTGC 1980CTCCTCACTT GGCTTCTCTC CCCCTGCCCT GCCCCCAGAG AGGGATTCCG GGGACCCTCT 2040GGTGGACGAG AGCCTGAAGA GACAGGGCTT TCAAGAAAAT TATGACGCGA GGCTCTCACG 2100GATCGACATC GCCAACACAC TAAGGGAGCA GGTCCAGGAC CTTTTCAATA AGAAATACGG 2160GGAAGCCTTG GGCATCAAGT ACCCGGTCCA GGTCCCCTAC AAGCGGATCA AGAGTAACCC 2220CGGCTCCGTG ATCATCGAGG GGCTGCCCCC AGGAATCCCG TTCCGAAAGC CCTGTACCTT 2280CGGCTCCCAG AACCTGGAGA GGATTCTTGC TGTGGCTGAC AAGATCAAGT TCACAGTCAC 2340CAGGCCTTTC CAAGGACTCA TCCCAAAGCC TGATGAAGAT GACGCCAACA GACTCGGGGA 2400GAAGGTGATC CTGCGGGAGC AGGTGAAGGA ACTCTTCAAC GAGAAATACG GTGAGGCCCT 2460GGGCCTGAAC CGGCCGGTGC TGGTCCCTTA TAAACTAATC CGGGACAGCC CAGACGCCGT 2520GGAGGTCACG GGTCTGCCTG ATGACATCCC CTTCCGGAAC CCCAACACGT ACGACATCCA 2580CCGGCTGGAG AAGATCCTGA AGGCCCGAGA GCATGTCCGC ATGGTCATCA TTAACCAGCT 2640CCAACCCTTT GCAGAAATCT GCAATGATGC CAAGGTGCCA GCCAAAGACA GCAGCATTCC 2700CAAGCGCAAG AGAAAGCGGG TCTCGGAAGG AAATTCCGTC TCCTCTTCCT CCTCGTCTTC 2760CTCTTCCTCG TCCTCTAACC CGGATTCAGT GGCATCGGCC AACCAGATCT CACTCGTGCA 2820ATGGCCAATG TACATGGTGG ACTATGCCGG CCTGAACGTG CAGCTCCCGG GACCTCTTAA 2880TTACTAGACC TCAGTACTGA ATCAGGACCT CACTCAGAAA GACTAAAGGA AATGTAATTT 2940ATGTACAAAA TGTATATTCG GATATGTATC GATGCCTTTT AGTTTTTCCA ATGATTTTTA 3000CACTATATTC CTGCCACCAA GGCCTTTTTA AATAAGTAAA AAA 3043 ( 2 ) SEQ ID NO.2： ( ⅰ ) ：

(A) length: 959 amino acid

(B) type: amino acid

( D ) ： ( ⅱ ) ： ( ⅹⅰ ) ：SEQ ID NO.2：MALLGKRCDV PTNGCGPDRW NSAFTRKDEI ITSLVSALDS MCSALSKLNA EVACVAVHDE 60SAFVVGTEKG RMFLNARKEL QSDFLRFCRG PPWKDPEAEH PKKVQRGEGG GRSLPRSSLE 120HGSDVYLLRK MVEEYFDVLY SEALGRASVV PLPYERLLRE PGLLAVQGLP EGLAFRRPAE 180YDPKALMAIL EHSHRIRFKL KRPLEDGGRD SKALVELNGV SLIPKGSRDC GLHGQAPKVP 240PQDLPPTATS SSMASFLYST ALPNHAIREL KQEAPSCPLA PSDLGLSRPM PEPKATGAQD 300FSDCCGQKPT GPGGPLIQNV HASKRILFSI VHDKSEKWDA FIKETEDINT LRECVQILFN 360SRYAEALGLD HMVPVPYQKI ACDPEAVEIV GIPDKIPFKR PCTYGVPKLK RILEERHSIH 420FIIKRMFDER IFTGNKFTKD TTKLEPASPP EDTSAEVSRA TVLDLAGNAR SDKGSMSEDC 480GPGTSGELGG LRPIKIEPED LDIIQVTVPD PSPTSEEMTD SMPGHLPSED SGYGMEMLTD 540KGLSEDARPE ERPVEDSHGD VIRPLRKQVE LLFNTRYAKA IGISEPVKYP YSKFLMHPEE 600LFVVGLPEGI SLRRPNCFGI AKLRKILEAS NSIQFVIKRP ELLTEGVKEP IVDSQGTASS 660LGFSPPALPP ERDSGDPLVD ESLKRQGFQE NYDARLSRID IANTLREQVQ DLFNKKYGEA 720LGIKYPVQVP YKRIKSNPGS VIIEGLPPGI PFRKPCTFGS QNLERILAVA DKIKFTVTRP 780FQGLIPKPDE DDANRLGEKV ILREQVKELF NEKYGEALGL NRPVLVPYKL IRDSPDAVEV 840TGLPDDIPFR NPNTYDIHRL EKILKAREHV RMVIINQLQP FAEICNDAKV PAKDSSIPKR 900KRKRVSEGNS VSSSSSSSSS SSSNPDSVAS ANQISLVQWP MYMVDYAGLN VQLPGPLNY 959

Claims (11)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people Creaml protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 8-2887 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 8-2887 position.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.2.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 8-2887 position, or among the SEQ ID NO.1 from the nucleotide sequence of Nucleotide 1-3043 position.

4. isolating people Creaml protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.2 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4, it is characterized in that, this polypeptide is selected from down group: have SEQ ID NO.2 polypeptide of sequence, polypeptide with amino acid 253-959 among the SEQ ID NO:2, polypeptide with amino acid 253-897 among the SEQ ID NO:2, have the polypeptide of amino acid/11-455 among the SEQ ID NO:2, or have the polypeptide of amino acid 463-959 among the SEQID NO:2.

6. a carrier is characterized in that, it contains the described DNA of claim 1.

7. one kind with the described carrier transformed host cells of claim 6.

8. a generation has the method for the polypeptide of people Creaml protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people Creaml protein-active operationally is connected in expression regulation sequence, form people Creaml protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 8-2887 position among described nucleotide sequence and the SEQ ID NO.1;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people Creaml;

(c) under the condition that is fit to expressing human Creaml protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people Creaml protein-active.

9. energy and the described people Creaml of claim 4 protein polypeptide specificity bonded antibody.

10. a probe molecule is characterized in that, it contains 8-100 continuous nucleotide in the described dna molecular of claim 1.

11, a kind of pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.

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