CN1257923A - RaL GTP guanine dissociation stimulator - Google Patents

Abstract

The present invention discloses a new polynucleotide, the polypeptide coded by said polynucleotide and the process for preparing the polypeptide using said polynucleotide. Said polypeptide is a member in the guanine dissociation stimulator family of Ral GTP enzyme (RalGDS).

Description

The guanine dissociation stimulating factor of Ral GTP enzyme

The present invention relates to a kind of new polynucleotide,, and utilize described polynucleotide to produce the method for described polypeptide by the polypeptide of this polynucleotide encoding.More particularly, polypeptide of the present invention is a newcomer of guanine dissociation stimulating factor (RalGDS) family of Ral GTP enzyme.

The growth of Ras gene and cell, differentiation and transitivity have substantial connection, often relevant (the Barbacid of the sudden change of this gene particularly with the generation of cancer, M. (1987) Annu.rev.Biochem.56 .779-827) Ras is by bringing into play its function with the substrate-function that is referred to as effector.Found at present the effector of multiple Ras, comprise (McCormick such as the kinase whose p110 subunit of Raf, PI (3), RalGDS family, Byr2 (S pombe), Adenylyl cyclase (S.cerevisiae) Rin, AF6, canoe, PKC-ζ and MEKK-1, F.and Wittinghofer, A. (1996) Current Opinion in Biotechnology.7,449-456).Ras can be by several different signal pipelines performance functions, and present solving is clear that Raf/MAPK (mitogen-activated protein kinase) approach the most.

But more and more studies show that in recent years, exist the so new approach that does not rely on Raf of Ras-RalGDS-Ral, thereby RalGDS family played an important role in the Ras function.The RalGDS family member great majority that have now found that all are proved to be has GDP (disactivation state) the dissociated function of catalyzed combination on Ral albumen, thereby impels Ral to transform to its active state (combining with GTP).Experiment shows that many RalGDS family members can both act synergistically with Raf, strengthens it in the effect of inducing aspect the canceration, and this synergy is not to realize by the effect that strengthens the Raf approach.The RalGDS family member who has also is found and can combines with the active state of TC21, and they may participate in the effect of TC21 aspect induced tumor.Except that Ras (or TC21), many RalGDS family members can also with the Rap protein binding.Rap also is a member of Ras family, still, distinguishedly is, it can suppress the Ras carcinogenesis, and RalGDS threshold therein must be studied.In addition, in the research to mouse RalGDS, find that also RalGDS has the substrate except that Ral, this explanation RalGDS family also may play a role in other signal pipeline.

1993, Albright, C.F. wait at first in the fibroblast cDNA library of the 3T3/ of mouse Δ XB clone and rat, found the total length (Albright of RalGDSa and RalGDSb gene by the way of seeking coding and CDC25 and ste6 albumen conserved regions homologous gene, C.F.et al. (1993) EMBO is (1) J.12,339-347).Next year, Kikuchi, A. etc. are when searching Ras is conjugated protein, and the utilization yeast two-hybrid system has been found the Ras calmodulin binding domain CaM RID of mouse gene RGL (RalGDS-like), sieves the storehouse and obtains this full length gene as probe.RGL and RalGDS have 70% amino acid identity, and be higher in both homologys of CDC25 homologous region, thus and think RGL and RalGDS constitute a family (Kikuchi, A.et al. (1994) Mol.Cell.Biol.14,7483-7491.).RLF (RalGDS-like factor) then is initial as a new CDC25 family member, in 1996 by Wolthuis, R.M. wait the little musculus cdna of finding, it is (the Wolthuis that obtains in yeast two-hybrid system when seeking Rap 1A conjugated protein, R.M.et al. (1996) Oncogene 13,353-362).This genes encoding 778 amino acid whose albumen, it is identical that this albumen and RalGDS and RGL have 30% amino acid approximately, especially the carboxyl terminal conservative property at RLF is stronger, also is considered to a new member of RalGDS family.When Peterson, S.N. equal to seek Rap1b conjugated protein with yeast two-hybrid system in 1996, found that the part of a people cDNA is cloned.Relatively find by homology, this Partial cDNA and RalGDS and RGL have important homology, and it has comprised the RBD of complete carboxyl terminal and part, and they are its called after RGL2 (peterson, S.N.et al. (1996) J.Biol.Chem.271 (47), 29903-29908).

1997, in rabbit, got back one may be RalGDS family member's gene partial sequence (D ' Adamo, D.R.et al. (1997) Oncogene 14,1295-1305).D ' Adamo, D.R. etc. are in DNBA inductive rabbit squamous cell, and utilization nude mice tumorigenesis experiment (nude mouse tumorigenesis assay) is separated to a full-length cDNA---rsc (rabbit aquamous cell carcinoma).Analyze and find that this gene is an oncogene, it is spliced by two different gene breaks: 785bp amino acid sequence coded and people's HHR23A gene has the homogeny greater than 95% before reading in the frame; 1.4kb subsequently then has 60% identically with the RalGDS of mouse, then is 40% on amino acid levels.The latter means that this may be a new gene of RalGDS family, and it is named as rgr (RalGDS related).Rgr also has CDC25 homologous catalysis district, in this zone it with the identical rate of amino acid of RalGDS up to 72%.Rgr albumen has the GDS activity to Ral equally, and Ras or Ran are not then had.Deletion analysis shows that the part with carcinogenic potential exists only in the rgr part of rsc.

Human RalGDS is also by part clone (Spaargaren, M.and Bischoff, L.R. (1994) Proc.Natl.Acad.Sci.USA 91,12609-12613; Hofer, F.et al. (1994) Proc.Natl.Acad.Sci.USA 91,11089-11093).

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding RalGDS family, RalGDS newcomer's called after hRalGDS of the present invention (GenBank Accession No.AF027169).

Another object of the present invention provides a kind of new RalGDS protein family member, called after hRalGDS.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new RalGDS family member.

The present invention relates to a kind of new polynucleotide,, and utilize described polynucleotide to produce the method for described polypeptide by the polypeptide of this polynucleotide encoding.More particularly, polypeptide of the present invention is a newcomer of RalGDS family.RalGDS homologue called after hRalGDS of the present invention (GenBank Accession No.AF027169).The polynucleotide of the present invention of encoding can be rna form and dna form, and DNA comprises cDNA, genomic dna and synthetic DNA, and DNA can be two strands or strand, if strand can be coding strand or noncoding strand.

In the present invention, term " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.As the polynucleotide under the native state in the active somatic cell and polypeptide be do not have isolating, but same polynucleotide or polypeptide as from native state with in other materials of depositing separately, then be isolating.

In one embodiment of the invention, polynucleotide total length of the present invention is 3585 Nucleotide, and its detailed sequence is seen SEQ ID NO:1, and wherein open reading frame is positioned at 160-2718 position Nucleotide.These polynucleotide are so to obtain, with people's placenta λ gt11cDNA library (available from Clontech company) is template, synthetic forward primer 5 '-CAGCTTCGTCCCGGAGTCTCCTG-3 ' and reverse primer 5 '-GATGCCCTTGGCAATCTTGAGTC-3 ', carry out PCR, obtain the purpose fragment of 568bp.This fragment of mark then, with this labeled fragment be probe with amplification after the hybridization of people's placenta λ gt11cDNA library, the picking positive colony after same probe and screening process are carried out multiple sieve to the positive colony that obtains, finally obtains some positive monoclonals.Obtain partial sequence after identifying order-checking.With this sequence is reference, has designed reverse primer 5 '-TGGAGGCTGAGCTGATGCCGGAG-3 ' and 5 '-TCGCATGAAGCTGTTTGGACTCAAG-3 ' again, with they respectively with carrier primer S1, the S2 amplification of arranging in pairs or groups, and amplified production checked order.With the full length cDNA sequence that obtains behind the measured sequence assembly as SEQ ID NO:1.

According to a further aspect in the invention, polynucleotide sequence of the present invention can be used to express or produce the hRalGDS albumen or the polypeptide of reorganization.In general following steps are arranged: transform proper host cell with the dna fragmentation (or varient) of coding of the present invention hRalGDS or the recombinant expression vector that contains this dna fragmentation; The host cell of in suitable medium, cultivating; Separation, protein purification from substratum or cell.

The invention still further relates to the expression vector that contains polynucleotide sequence of the present invention and with the genetically engineered host cell of described expression vector.A key character of expression vector is to contain suitable promotor and translation controlling elements.Multiple expression vector can obtain from commercial channels.Carrier commonly used includes, but is not limited to plasmid, phage, clay, Yeast expression carrier, virus, Ti-plasmids etc., but the first-selected plasmid of the most frequently used carrier.Host cell commonly used includes, but is not limited to bacterium, yeast, insect cell, zooblast or vegetable cell etc., but the most frequently used host cell is a bacterium, especially intestinal bacteria.Another expression system commonly used is a mammal cell line, as Hela, and COS clone or Chinese hamster ovary celI system etc.

The known method of skilled person makes up the recombinant expression vector that contains the hRalGDS encoding sequence in available this area.These methods comprise the extracorporeal recombinant DNA technology, DNA synthetic technology, (J Sambrook etc. 1989, molecular cloning laboratory manuals) such as the interior recombinant technologys of body.

The invention still further relates to antibody, have several different methods to can be used for producing antibody at the hRalGDS antigenic determinant at polypeptide of the present invention.These antibody include, but is not limited to polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, the fragment that Fab fragment and Fab expression library produce.

Available hRalGDS albumen of the production of polyclonal antibody 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.

The hRalGDS 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.Pat No.4946778) also can be used for producing the single-chain antibody of anti-hRalGDS.

Below will further describe the present invention by embodiment, but embodiment only being an illustrative, is not limitation of the present invention.The clone of the cDNA of embodiment 1 hRalGDS and 1. primer amplifications that check order

With people's placenta λ gt11cDNA library (available from Clontech company) is template, with oligonucleotide 5 '-CAGCTTCGTCCCGGAGTCTCCTG-3 ' is forward primer, oligonucleotide 5 '-GATGCCCTTGGCAATCTTGAGTC-3 ' is a reverse primer, carry out PCR, the PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 68 ℃ 1 minute and 72 ℃, last 72 ℃ were extended 5 minutes thereupon.The 568bp purpose fragment that electrophoresis detection obtains.2. probe and mark thereof

With above-mentioned PCR purpose fragment is probe, usefulness α- ³²P-dATP (DuPont company) carries out random primer labelling to it, condition be 37 ℃ 1 hour, MegaprimeDNA Mk system specification sheets (Amersham) is seen in concrete operations.3.cDNA amplified library and trace mark (screening library)

Increased in people's placenta λ gh11cDNA library, clone's number reaches 1,000,000, good cDNA library trace to the Hybond TM-N+ nylon membrane (Amersham) then will increase, hybridize with blotting membrane after again that mark is the good probe sex change, wash film, put into the sheet folder of band intensifying screen, under-80 ℃, carry out radioautograph, punching after 72 hours with FUJI MEDIAI X-ray film.4. picking clone and primary dcreening operation clone's multiple sieve

Obtain 13 primary dcreening operation positive colonies by above-mentioned screening by hybridization, these 13 positive colonies are carried out multiple sieve, finally obtain 6 positive monoclonals with above-mentioned identical probe hybridization and screening process.5. monoclonal evaluation and order-checking

With the mono-clonal phage that obtains in the above-mentioned steps 4 is template, with new reverse primer 5 '-TGGAGGCTGAGCTGATGCCGGAG-3 ' and the 5 '-TCGCATGAAGCTGTTTGGACTCAAG-3 ' of probe sequence design, respectively with λ gt11 left and right arms on primer S1 and S2 (S1:5 '-GTTCAACATCAGCCGCTACA-3 '; S2:5 '-CACCAGACCAACTGGTAATG-3 ') amplification of arranging in pairs or groups, carry out agarose gel electrophoresis then, to judge that each clone moves the length of (extension) to 5 ' or 3 ' step from probe, therefrom select to two ends and extend the longest clone, electrophoresis identifies that they are respectively to 5 ' and the 3 ' length of extending, the former insertion fragment is about 2.3kb, and the latter's insertion fragment is about 1.0kb.With these two clones S1, S2 amplified production and pGEM-T ^Carrier (Promega) connects, transformed into escherichia coli JM109, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 3585bp, detailed sequence is seen SEQ ID NO:1, and wherein open reading frame is positioned at 160-2718 position Nucleotide.

Derive the aminoacid sequence of hRalGDS according to the cDNA sequence that obtains, totally 852 amino-acid residues, its aminoacid sequence sees SEQ ID NO:2 for details.Expression pattern analysis and the chromosomal localization of embodiment 2 hRalGDS

Multiple Northern (the MTN that organizes ^TM) blotting membrane (16 kinds of tissues) is available from Clontech company, the 568bp cloned sequence in above embodiment 1 step is a probe, and the marking method of probe is with embodiment 1 step 2, and the user manual operation is pressed in Northern hybridization.

The Northern results of hybridization of 16 kinds of tissues shows that (h) RalGDS gene is the wide spectrum expressing gene, thymus gland, prostate gland, placenta, ovary, small intestine, large intestine, peripheral blood leucocyte, placenta, lung, liver, skeletal muscle, kidney and etc. expression is all arranged in 12 kinds of tissues, expression amount is variant slightly, in spleen, brain, heart and pancreas than high expression level.According to international blastn nr database retrieval, with this assignment of genes gene mapping in No. 9 karyomit(e) 9q34.

Embodiment 3 expression of people RalGDS in intestinal bacteria

In this embodiment, with people's placenta λ gt11cDNA library (available from Clontech company) is template, use 5 ' and 3 ' the PCR Oligonucleolide primers of holding to increase the cDNA sequence of coding people RalGDS, obtain people RalGDS cDNA as inserting fragment corresponding to this dna sequence dna.

5 ' the Oligonucleolide primers sequence of using in the PCR reaction is:

5 '-GTTGGTCGAC ATGGTAGATT GCCAGAGCT-3 ', this primer contains the restriction enzyme site of the restricted restriction enzyme of SalI, is 19 Nucleotide of the people RalGDS encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 '-gcttAAGCTT TCAGAAGATG CCCTTGGC-3 ', this primer contain the part encoding sequence of restriction enzyme site, translation termination and the people RalGDS of the restricted restriction enzyme of HindIII.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp ^r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.

With SalI and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kanr).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people RalGDS has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD600) when the 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people RalGDS from solution.With 6M Guanidinium hydrochloride (PH5.0) wash-out people RalGDS from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein from nickel-chelate column.Protein behind the purifying is incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (PH0.5) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 94kDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the protein sequence of inferring with ordinary method.

The expression of embodiment 4 people RalGDS in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with people's placenta λ gt11cDNA library (available from Clontech company) is template, use 5 ' and 3 ' the PCR Oligonucleolide primers of holding to increase the cDNA sequence of coding people RalGDS, obtain people RalGDS cDNA as inserting fragment corresponding to this dna sequence dna.

5 ' the Oligonucleolide primers sequence of using in the PCR reaction is:

5’-GTTGAAGCTT?ATGGTAGATT?GCCAGAGCT-3’

This primer contains the restriction enzyme site of the restricted restriction enzyme of HindIII, is 19 Nucleotide of the people RalGDS encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-GCTTGAATTC?TCAGAAGATG?CCCTTGGC-3’

This primer contains the part encoding sequence of the restriction enzyme site of the restricted restriction enzyme of EcoRI, translation termination and people RalGDS.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (fl ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With EcoRI and HindIII digestion pcDNA3 carrier, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid, the cDNA fragment of sequence verification people RalGDS has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTrisHCl (PH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mMTrisHCl (PH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (PH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (PH7.4) then.Last freeze-drying is preserved.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 94KDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the protein sequence of inferring with ordinary method.

According to above instruction, the present invention can have various improvement and change, and therefore, within the scope of the appended claims, the present invention can implement with concrete described different mode.

Sequence table (1) general information (i) applicant: Fudan University is denomination of invention (ii): the guanine dissociation stimulating factor of Ral GTP enzyme is sequence number (iii): 2 (iv) contact addresses:

(A) addressee: Ntd Patent ﹠ Trademark Agency Ltd

(B) street: No. 27, financial street

(C) city: Beijing

(D) country: China

(E) postcode: 100032 (vi) the application's data:

(A) application number:

(B) applying date:

(C) classification number: (viii) lawyer/proxy's information:

(A) name: woods Xiao Hong

(B) number of registration: 72002027

(C) reel number: I98602CB (ix) telecommunication information:

(A) phone: 8610-66211834

(B) fax: the information of 8610-66211845 (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 3585bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( iii ) ：cDNA ( xi ) ：SEQ ID NO：1 1 CGAGGGCCGC AGAGTCCTGG GCCCGGCGGG GACCGGAGGC CGCGCCATGA GCCCCGCAGC 61 CGGGCGCACC CCTGCGCCGC GCGCGGGCCC AGGCGAGCGG CCTCTGCGAG CCCCGGGTCC121 CGCCCTGGGG CCGGCGATGT GCCACCGAGG CTGAGGATGA TGGTAGATTG CCAGAGCTCC181 ACGCAGGAGA TCGGTGAGGA GCTGATCAAC GGAGTCATCT ACTCCATCTC CCTGCGCAAG241 GTGCAGCTGC ACCACGGAGG CAACAAGGGG CAGCGCTGGC TCGGGTATGA GAATGAGTCG301 GCCCTGAACC TTTATGAGAC TTGCAAGGTG CGGACCGTGA AGGCTGGCAC GCTGGAGAAG361 CTGGTGGAGC ACCTGGTGCC AGCCTTCCAG GGCAGCGACC TCTCCTACGT CACCATCTTC421 CTGTGTACCT ATAGAGCCTT CACCACCACC CAACAGGTCC TGGACCTGCT GTTCAAAAGG481 TACGGTAGAT GTGACGCCCT CACGGCCTCC TCTAGATACG GCTGCATCCT CCCCTATTCC541 GACGAGGATG GTGGACCCCA GGACCAACTT AAAAATGCCA TCTCCTCCAT CCTGGGCACC601 TGGCTGGACC AGTACTCGGA GGATTTCTGT CAACCTCCGG ACTTTCCCTG CCTCAAGCAG661 CTGGTGGCCT ACGTGCAGCT CAACATGCCA GGCTCAGACC TGGAGCGCCG TGCCCACCTT721 CTCCTGGCCC AGCTGGAGCA CTCGGAACCC ATTGAGGCAG AGCCTGAGGC TCTAAAACCA781 ACTCCAGAGC TCGAGCTAGC TCTAACACCA GCTCGAGCAC CCAGCCCAGT GCCGGCTCCA841 GCCCCGGAGC CAGAGCCAGC TCCAACACCA GCTCCAGGTT CAGAGCTAGA AGTAGCTCCA901 GCACCAGCTC CGGAGCTCCA GCAGGCTCCA GAGCCAGCTG TGGGACTAGA ATCGGCTCCA961 GCGCCAGCTC TGGAACTAGA GCCAGCTCCA GAACAGGATC CAGCTCCCTC ACAAACTCTA1021 GAGCTGGAGC CAGCTCCAGC ACCAGTTCCA TCATTACAGC CTTCCTGGCC TTCACCTGTG1081 GTTGCAGAGA ACGGGCTGAG TGAGGAGAAG CCTCACCTCT TGGTGTTCCC TCCAGATCTG1141 GTGGCAGAGC AGTTTACACT GATGGATGCG GAACTGTTCA AGAAGGTGGT GCCCTACCAC1201 TGCCTGGGCT CCATCTGGTC CCAGCGGGAC AAGAAGGGCA AGGAGCACCT GGCGCCCACC1261 ATCCGCGCCA CTGTCACCCA GTTCAACAGT GTGGCCAACT GTGTCATCAC CACCTGCCTC1321 GGGAACCGAA GCACGAAAGC CCCAGACAGG GCCAGGGTGG TGGAGCACTG GATCGAGGTG1381 GCCAGGGAGT GCCGGATCCT CAAGAACTTC TCGTCACTGT ATGCCATCCT CTCTGCCCTG1441 CAGAGCAACT CCATCCACCG TCTGAAGAAG ACGTGGGAAG ACGTTTCCAG GGACAGTTTC1501 CGGATCTTTC AGAAGCTGTC AGAGATCTTC TCAGATGAGA ACAACTACTC ATTGAGCCGG1561 GAGCTGCTCA TCAAGGAGGG CACCTCCAAG TTTGCCACCC TGGAGATGAA CCCCAAGAGA1621 GCCCAGAAAC GGCCGAAGGA GACGGGCATC ATCCAGGGCA CCGTTCCCTA CCTGGGCACG1681 TTCCTCACCG ACCTGGTGAT GCTGGACACT GCCATGAAGG ACTATCTGTA TGGCAGACTC1741 ATCAACTTTG AGAAGAGGAG GAAGGAGTTC GAGGTGATCG CCCAGATCAA GCTGCTGCAG1801 TCGGCCTGCA ACAACTACAG CATCGCGCCA GATGAGCAAT TTGGGGCCTG GTTCCGGGCC1861 GTGGAGCGGC TCAGCGAGAC TGAGAGCTAC AACCTGTCGT GCGAGCTGGA GCCCCCATCC1921 GAGTCAGCCA GCAACACCCT CAGGACCAAG AAGAACACAG CCATTGTCAA GCGCTGGAGC1981 GACCGCCAGG CCCCCAGCAC TGAGCTCAGT ACCAGTGGCA GCTCCCACTC CAAGTCCTGT2041 GACCAGCTCA GGTGTGGCCC CTACCTCAGC AGCGGGGACA TCGCTGACGC GCTCAGCGTG2101 CACTCGGCCG GCTCCTCTAG CTCCGACGTG GAGGAGATCA ACATCAGCTT CGTCCCGGAG2161 TCTCCTGATG GCCAGGAAAA GAAGTTCTGG GAATCAGCCT CACAGTCATC CCCGGAGACC2221 TCCGGCATCA GCTCAGCCTC CAGCAGCACC TCGTCCTCCT CAGCCTCCAC CACGCCCGTG2281 GCTGCCACAC GCACCCACAA GCGCTCTGTC TCAGGGCTCT GCAACTCCAG CTCCGCGCTG2341 CCGCTCTACA ACCAGCAGGT GGGCGACTGC TGTATCATCC GCGTCAGCCT GGACGTGGAC2401 AATGGCAACA TGTACAAGAG CATCCTGGTG ACCAGCCAAG ATAAGGCTCC GGCTGTAATC2461 CGCAAGGCCA TGGACAAACA CAACCTGGAG GAGGAGGAGC CGGAGGACTA TGAGCTGCTG2521 CAGATTCTCT CAGATGACCG GAAGCTGAAG ATCCCTGAAA ACGCCAACGT CTTCTATGCC2581 ATGAACTCTA CTGCCAACTA TGACTTTGTC CTGAAGAAGC GGACCTTCAC CAAGGGAGTG2641 AAGGTCAAGC ACGGAGCCAG CTCCACCCTC CCTCGCATGA AGCAGAAAGG ACTCAAGATT2701 GCCAAGGGCA TCTTCTGAGG GCATCCTCCC AGGGTCTGGC TGGCTGGTAG CCAAGCACTT2761 ATGGACCAGA GTGGCCCAGG CCAGCTGGGC GCCTTCCTCC CACCTGCCAG CCCAGGGTAC2821 CCCAGACTCC AGTTTCATCC TGAACCTCTC CCGCTGCTGG GATTGACGCC TGCCATTGGT2881 CAGGCTGACC TGGCCTCCCG TGGACCACTC GCTGCCTTAG GTGCCTTCTG CTCTCTGGAA2941 CCAGAGGACT AGCTGACTTT TGCCAAGGAG CAGTGCCAAC GGGCATGGCA TGGTGCCCTG3001 CCTGCCCCCG GGCGCCACCT CTGTACACTT CCCTGACACC TTCCCAGGTG TGGGTCACTG3061 CCACCTGTGC CCATGGGCAC CCCAGAGCAC CCACTGTGAC CACTGCAGTT CTCTCATGCC3121 CACAGGCACT GGCCTGTGAC CTTCGCAGGG GTCCCGGCCC CTCCCACCAC TCTAGCCTTT3181 CTCAGGCTGC ACCAAAGATT CCATCATCAG GGCCAACTGA GAGTGAGGGA GTCTCACCCA3241 CCGCTTACCC CAGCCCTCCC CTGGGAGCAG AGAGAGAAAC CCTCTTCATG GACCAGACTC3301 TGCACCCGGT GAGTGAGGAC AGTCCCAGCT GAGTCCCATC GATGTTGAAT CTCATGCCAC3361 TGCAAGTGCC ATTCACCACT GCGTCCTGGG CTTTACGAGA CCATGCAAGA CGGGGGTTAG3421 TGAGGAAGGA GGATTTGGGG TGGGGGTGGG GTGATTGAAT ATTTGTATAA AAAGCAAAAA3481 GAAAAAAAAA ATGTTTGTTT ACTGATTGGG GAGGGGCAAT ATTTATTTGT TGTAAATAGC3541 AAATGCTAGA CTTGAATATT ATATTAAAAT CCTGTTTCTA CTATA ( 3 ) SEQ ID NO：2： ( i ) ：

(A) length: 852 amino acid

(B) type: amino acid

( C ) ： ( iii ) ： ( xi ) ：SEQ ID NO：2 1 MVDCQSSTQEIGEELINGVIYSISLRKVQLHHGGNKGQRWLGYENESALNLYETCKVRTV 61 KAGTLEKLVEHLVPAFQGSDLSYVTIFLCTYRAFTTTQQVLDLLFKRYGRCDALTASSRY121 GCILPYSDEDGGPQDQLKNAISSILGTWLDQYSEDFCQPPDFPCLKQLVAYVQLNMPGSD181 LERRAHLLLAQLEHSEPIEAEPEALKPTPELELALTPAPAPSPVPAPAPEPEPAPTPAPG241 SELEVAPAPAPELQQAPEPAVGLESAPAPALELEPAPEQDPAPSQTLELEPAPAPVPSLQ301 PSWPSPVVAENGLSEEKPHLLVFPPDLVAEQFTLMDAELFKKVVPYHCLGSIWSQRDKKG361 KEHLAPTIRATVTQFNSVANCVITTCLGNRSTKAPDRARVVEHWIEVARECRILKNFSSL421 YAILSALQSNSIHRLKKTWEDVSRDSFRIFQKLSEIFSDENNYSLSRELLIKEGTSKFAT481 LEMNPKRAQKRPKETGIIQGTVPYLGTFLTDLVMLDTAMKDYLYGRLINFEKRRKEFEVI541 AQIKLLQSACNNYSIAPDEQFGAWFRAVERLSETESYNLSCELEPPSESASNTLRTKKNT601 AIVKRWSDRQAPSTELSTSGSSHSKSCDQLRCGPYLSSGDIADALSVHSAGSSSSDVEEI661 NISFVPESPDGQEKKFWESASQSSPETSGISSASSSTSSSSASTTPVAATRTHKRSVSGL721 CNSSSALPLYNQQVGDCCIIRVSLDVDNGNMYKSILVTSQDKAPAVIRKAMDKHNLEEEE781 PEDYELLQILSDDRKLKIPENANVFYAMNSTANYDFVLKKRTFTKGVKVKHGASSTLPRM841 KQKGLKIAKGIF

Claims (17)

1, a kind of isolated polynucleotide, its coding has the polypeptide of deduced amino acid of SEQ ID NO:2 or fragment, analogue or the derivative of described polypeptide, and described fragment, analogue or derivative have the biological function identical with full-length polypeptide.

2, polynucleotide as claimed in claim 1, wherein these polynucleotide are DNA.

3, polynucleotide as claimed in claim 1, wherein these polynucleotide are RNA.

4, polynucleotide as claimed in claim 1, wherein these polynucleotide are genomic dnas.

5, polynucleotide as claimed in claim 1, its coding have the polypeptide of the deduced amino acid of SEQ ID NO:2.

6, polynucleotide as claimed in claim 1, it has the encoding sequence shown in the SEQ ID NO:1.

7, the carrier that contains the described polynucleotide of claim 2.

8, a kind of host cell, it uses carrier conversion, transfection or the transduction of claim 7.

9, host cell as claimed in claim 8, this host cell are prokaryotic cell prokaryocyte.

10, host cell as claimed in claim 9, this host cell are intestinal bacteria.

11, host cell as claimed in claim 8, this host cell are eukaryotic cell.

12, host cell as claimed in claim 11, this host cell are COS-7, CHO, Hela cell.

13, host cell as claimed in claim 11, this host cell are yeast cell.

14, produce the method for polypeptide, be included in the host cell of cultivating claim 8-13 under the appropriate condition, make it to express described polypeptide.

15, a peptide species, described polypeptide are to have the polypeptide of deduced amino acid of SEQ ID NO:2 or fragment, analogue or the derivative of described polypeptide, and described fragment, analogue or derivative have the biological function identical with full-length polypeptide.

16, polypeptide as claimed in claim 15, described polypeptide has the deduced amino acid of SEQ ID NO:2.

17, the antibody of anti-claim 15 or 16 described polypeptide.