CN1257924A - Human Ras correlative GTP bindin kinase - 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 human Ras related GTP bindin kinase.

Description

Human Ras correlative GTP bindin kinase

The present invention relates to a kind of new polynucleotide, by the polypeptide of this polynucleotide encoding, the application of these polynucleotide and polypeptide, and the production of described polynucleotide and described polypeptide.More particularly, polypeptide of the present invention is accredited as human Ras correlative GTP bindin kinase by deduction.

Oncogene ras is a supergene family, and according to the homology of sequence, Ras proto-oncogene family can further be divided into four subgene families such as ras, rho, rab and arf.Rho subgene family is made up of Rho A, Rho B, Rho C, rac, TC10 and CDC42Hs protein such as (being also referred to as G25K).Studies show that Rho albumen has the effect of combination and hydrolysis GTP, it is an important component part of GTP enzyme family in " acceptor-G albumen coupling " signal transduction path, aspect the keeping of the many basic functions of cell, as the cell cycle process, the propagation of cell, differentiation, cytoskeleton forms, the motility of cell, and the transhipment of intracellular protein and secretion etc. have very important effect.Rho albumen is realized bioactive activation and inactivation by the continuous conversion of GTP enzyme catalysis GTP/GDP bonding state.Numerous protein, as Nucleotide exchanger (Nucleotide exchange proteins), GTP enzyme activation albumen (GTPase-activating proteins, GAPs), guanylic acid degradation inhibitor (Guaninenucleotide dissociation inhibitors) can regulate the proteic biological activity of Rho.Yet people understand very few to the proteic downstream effect albumen of Rho, nearest Narumiya, S. by the yeast two-hybrid system analysis, determined a serine/threonine protein kitase (Ser/Thrprotein kinase, PNK), serine/threonine protein kitase associated protein (PNK-related protein, Rhophilin) and the protein (180-kDa coiled coil-containing protein) of the 180-kDa of a band spiral be the proteinic downstream of Rho candidate's effect protein.Their protein sequence analysis shows, these proteinic N-ends all contain common Rho protein binding sequence, thereby these sequences are considered to the conserved sequence of Rho albumen downstream candidate's effect protein, with this sequence is template, we design special primer respectively, successfully clone a nearly full-length cDNA of new gene that contains Rho downstream effect albumen conserved sequence with the PCR method, thereby finished the present invention.

Therefore, an object of the present invention is to provide a kind of new polynucleotide, this polynucleotide encoding human Ras correlative GTP bindin kinase, called after HUMRH (GenBankAccession No.AF049227).

Another object of the present invention provides a kind of new human Ras correlative GTP bindin kinase.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new human Ras correlative GTP bindin kinase.

The invention still further relates to the various application of this peptide species.

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, to be accredited as by deduction be human Ras correlative GTP bindin kinase to polypeptide of the present invention.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, the target cDNA fragment that from people's nephridial tissue cDNA library, amplifies a 392bp size of the present invention, and be probe screening people kidney cDNA library with this fragment, the result has successfully cloned a nearly full-length cDNA of new gene that contains Rho downstream effect albumen conserved sequence, and this gene and mouse Rho downstream effect protein gene Rhotekine have the height homology.It is conveyed into international GenBank database, obtains accession number AF049227.

Conserved sequence according to Rho albumen downstream effect protein gene, design special primer, the target cDNA fragment that the application PCR method is separated to a length from people's kidney cDNA library be 230bp, after order-checking turns out to be expected sequence, move screening with it in the corresponding kidney cDNA library step, the gained positive colony, confirm that through special primer and vector arms primer collocation amplification this is the cDNA clone of a nearly 2.1kb of length, further make up serial deletion clone and carry out sequencing with it, confirm that its full length sequence is 2170bp, include the complete open reading frame (ORF) of 544 amino-acid residues of a codified.

The nearly full length cDNA sequence input GenBank of 2170bp is carried out homology relatively, and (GenBank No. is: homology degree MMU54638) reaches 87% for complete sequence and mouse Rhotekine gene as a result.Proteinic homology degree is 80%.Identical Rho is arranged in conjunction with the territory at N-end, thus we to infer it be exactly the homologous gene of mouse Rhotekin the mankind.Protein function to mouse Rhotekin genetic expression studies show that, the albumen of Rhotekin genetic expression is the important reverse feedback regulatory factor on the cell signaling approach, this albumen can combine with the Rho of GTP bonding state, has the Rho of inhibition albumen to GTP, hydrolytic action, thereby suppress the signal transduction path of cell.The sudden change of this gene will cause the change of many cell basic functions.

In one embodiment of the invention, polynucleotide total length of the present invention is 2170 Nucleotide, and its detailed sequence is seen SEQ ID NO:1, and wherein open reading frame is positioned at 151-1785 position Nucleotide.

According to a further aspect in the invention, polynucleotide sequence of the present invention can be used to express or produce the HUMRH 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 HUMRH 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 HUMRH 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.

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 HUMRH and 1. primer amplifications that check order

With people's kidney λ gt10cDNA library (available from Clontech company) is template, with oligonucleotide 5 '-CCTGCTCGCCTGCCTCAGTGGCC-3 ' is forward primer, oligonucleotide 5 '-GTTCTTCAGTGCCATCCTCAAAG-3 ' is a reverse primer, carry out PCR, the PCR condition be 93 ℃ 3 minutes, carried out 35 circulations in 1 minute 30 seconds with 93 ℃ 1 minute, 68 ℃ 1 minute and 72 ℃, last 72 ℃ were extended 5 minutes thereupon.The 506bp 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 kidney λ gt10cDNA library, clone's number reaches 500,000, 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 18 primary dcreening operation positive colonies by above-mentioned screening by hybridization, these 18 positive colonies are carried out multiple sieve, finally obtain 11 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 two positive anti-primers of above-mentioned steps 1 respectively with λ gt10 left and right arms on primer EF and ER (EF:5 '-AGCAGCCAGTCAACACTTACG-3 ': ER:5 '-GAGTTTGCATATCGCCTCCATC-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 5 ' and extend the longest and extend the longest clone to 3 ', the former insertion fragment is about 2.0kb, and the latter's insertion fragment is about 1.0kb.With these two clones EF, ER 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 SequiThenn 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 2170bp, detailed sequence is seen SEQID NO:1, and wherein open reading frame is positioned at 151-1785 position Nucleotide.

Derive the aminoacid sequence of HUMRH according to the cDNA sequence that obtains, totally 544 amino-acid residues, its aminoacid sequence sees SEQ ID NO:2 for details.The expression pattern analysis of embodiment 2 HUMRH

Multiple Northern (the MNT that organizes ^TM) blotting membrane (16 kinds of tissues) is available from Clontech company, the 2.0kb 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 the HUMRH gene is the wide spectrum expressing gene, express at the prostate gland height, expression is also arranged, but expression amount is variant slightly in testis, ovary, spleen, small intestine, colon, peripheral blood leucocyte, the heart, brain, placenta, skeletal muscle and nephridial tissue.The expression of embodiment 3 HUMRH in intestinal bacteria

In this embodiment, will be template with people's kidney λ gt10cDNA, the cDNA sequence of coding HUMRH uses the PCR Oligonucleolide primers corresponding to 5 ' and 3 ' end of this dna sequence dna to increase, and obtains HUMRH cDNA as inserting fragment.

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

5’-CACAGGATCCATGGAGTTCAAACGCGGCC-3’

This primer contains the restriction enzyme site of BamHI restriction enzyme, is 19 Nucleotide of the HUMRH encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-CCAAAAGCTTTCACACTGGTGACTGGAGC-3’

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the HUMRH of HindIII restriction enzyme.

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 BamHI, 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 (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, and the cDNA fragment of sequence verification nek4 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 is inoculated in the large volume substratum culturing cell to 600 optical density(OD) (OD then ₆₀₀) when being 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 HUMRH from solution.With 6M Guanidinium hydrochloride (PH5.0) wash-out HUMRH from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps from nickel-chelate column, isolate purifying protein.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 (PH5.0) 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 61KDa.

In addition, with ordinary method the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order.The expression of embodiment 4 HUMRH in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, will be template with people's kidney λ gt10cDNA, the cDNA sequence of coding HUMRH uses the PCR Oligonucleolide primers corresponding to 5 ' and 3 ' end of this dna sequence dna to increase, and obtains HUMRH cDNA as inserting fragment.

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

5’-CACAAAGCTTATGGAGTTCAAACGCGGCC-3’，

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

3 ' end primer sequence is:

5’-CCAAGGATCCTCACACTGGTGACTGGAGC-3’

This primer contains the part encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and HUMRH.

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 HindIII, BamHI digestion pcDNA3 carrier and insertion fragment, 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, and the cDNA fragment of sequence verification HUMRH has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin test kit (GiBcoLife).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 61KDa.

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

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): human Ras correlative GTP bindin kinase 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: I98600CB (ix) telecommunication information:

(A) phone: 8610-66211834

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

(A) length: 2170bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( iii ) ：cDNA ( xi ) ：SEQ ID NO：1 1 ACTGGCGGAC GAGCGTCGAG AGAAGAAGCC GAGGAGAGGA GGCTAAGCGA GCGCCCGGGG 61 ACACATGAAG CAGGCAAAGT CGCAGGGCCG CCAGCATGTT CTCCCGAAAC CACCGGAGCC 121 GGGTCACCGT GGCCAGGGTT CCGCGTGGAG ATGGAGTTCA AACGCGGCCG CTTCCGACTC 181 AGCCTCTTCA GCGACCTGCC CGAGGACACG GAGTTGCAGA GGAAGCTAGA CCATGAGATC 241 CGGATGAGGG AAGGGCCTTG TAAGCTGCTG GCACCTTGCT CCCAAGCGAG ACAAGCTCTG 301 GAGGCCACCA AGAGCCTGCT AGTGTGCAAC AGCCGCATCC TCAGCTACAT GGGCGAGCTG 361 CAGCGGCGCA AGGAGGCGCA GGTGCTGGGG AAGACAAACC GGCGGCCTTC TGACAGTGGC 421 CCGCCGCCTG AGCGCTCCCC CTGCCGCGGC CGGGTCTGCA TCTCTGACCT CCGGATTCCA 481 CTCATGTGGA AGGACACAGA ATATTTCAAG AACAAAGGTG ACTTGCACCG CTGGGCTGTG 541 TTCCTGCTGC TGCAGCTGGG GGAACACATC CAGGACACAG AGATGATCCT AGTGGACAGG 601 ACCCTCACAG ACATCTCCTT TCAGAGCAAT GTGCTCTTCG CTGAGGCGGG GCCAGACTTT 661 GAACTGCGGT TAGAGCTGTA TGGGGCCTGT GTGGAAGAAG AGGGGGCCCT GACTGGCGGC 721 CCCAAGAGGC TTGCCACCAA ACTCAGCAGC TCCCTGGGCC GCTCCTCAGG GAGGCGTGTC 781 CGGGCATCGC TGGACAGTGC TGGGGGTTCA GGGAGCAGTC CCATCTTGCT CCCCACCCCA 841 GTTGTTGGTG GTCCTCGTTA CCACCTCTTG GCTCACACCA CACTCACCCT GGCAGCAGTG 901 CAAGATGGAT TCCTCACACA TGACCTCACC CTTGCCAGTC ATGAGGAGAA CCCTGCCTGG 961 CTGCCCCTTT ATGGTAGCGT GTGTTGCCGT CTGGCAGCTC AGCCTCTCTG CATGACTCAG1021 CCCACTGCAA GTGGTACCCT CAGGGTGCAG CAAGCTGGGG AGATGCAGAA CTGGGCACAA1081 GTGCATGGAG TTCTGAAAGG CACAAACCTC TTCTGTTACC GGCAACCTGA GGATGCAGAC1141 ACTGGGGAAG AGCCGCTGTT TACTATTGCT GTCAACAAGG AGACTCGAGT CCGGGCAGGG1201 GAGCTGGACC AGGCTCTAGG ACGGCCCTTC ACCCTAAGCA TCAGTAACCA GTATGGGGAT1261 GATGAGGTGA CACACACCCT TCAGACAGAA AGTCGGGAAG CACTGCAGAG CTGGATGGAG1321 GCTCTGTGGC AGCTTTTCTT TGACATGAGC CAATGGAAGC AGTGCTGTGA TGAAATCATG1381 AAAATTGAAA CTCCTGCTCC CCGGAAACCA CCCCAAGCAC TGGCAAAGCA GGGGTCCTTG1441 TACCATGAGA TGGCTATTGA GCCGCTGGAT GACATCGCAG CGGTGACAGA CATCCTGACC1501 CAGCGGGAGG GCGCAAGGCT GGAGACACCC CCACCCTGGC TGGCAATGTT TACAGACCAG1561 CCTGCCCTGC CTAACCCCTG CTCGCCTGCC TCAGTGGCCC CAGCCCCAGA CTGGACCCAC1621 CCCCTGCCCT GGGGGAGACC CCGAACCTTT TCCCTGGATG CTGTCCCCCC AGACCACTCC1681 CCTAGGGCTC GCTCGGTTGC CCCCCTCCCA CCTCAGCGAT CCCCACGGAC CAGAGGCCTC1741 TGCAGCAAAG GCCAACCTCG CACTTGGCTC CAGTCACCAG TGTGAGAGAG AAAGGTGCTG1801 GCATAGGATC TGCCCAGAAG AGAAAATGAC CCATGCGCAG TTGGGCTCTG GATACGGCGC1861 TGTCTATAGC AAGTTTGCCA GTCTGGCCTC CTGTTCCTCT GCTGGACCTG GGGTAGGCTG1921 CAGGGGTGGG CAGAAGCCCC TCTTAAATTG TGGTTGCCAT GGTACCGAGG GACTCATTCC1981 TGGGGCTCGC TGGGACCTCC CTAAACCCTT CCTGGAAGAA AACTGGAACC AACTCTGCCC2041 TACCTCCCTG CACTAACCAG CTTTGAGGAT GGCACTGAAG AACCCTTGGA GCAAACATAC2101 CTCCCTTGTG ACTCCCACAT CAACCATTAA AGTTATTTAA CAGCAGCCTT CACCTTGGCT2161 CCTGAGGAAA ( 2 ) SEQ ID NO：2： ( i ) ：

(A) length: 544 amino acid

(B) type: amino acid

( C ) ： ( iii ) ： ( xi ) ：SEQ ID NO：2 1 MEFKRGRFRLSLFSDLPEDTELQRKLDHEIRMREGPCKLLAPCSQARQALEATKSLLVCN 61 SRILSYMGELQRRKEAQVLGKTNRRPSDSGPPPERSPCRGRVCISDLRIPLMWKDTEYFK121 NKGDLHRWAVFLLLQLGEHIQDTEMILVDRTLTDISFQSNVLFAEAGPDFELRLELYGAC181 VEEEGALTGGPKRLATKLSSSLGRSSGRRVRASLDSAGGSGSSPILLPTPVVGGPRYHLL241 AHTTLTLAAVQDGFLTHDLTLASHEENPAWLPLYGSVCCRLAAQPLCMTQPTASGTLRVQ301 QAGEMQNWAQVHGVLKGTNLFCYRQPEDADTGEEPLFTIAVNKETRVRAGELDQALGRPF361 TLSISNQYGDDEVTHTLQTESREALQSWMEALWQLFFDMSQWKQCCDEIMKIETPAPRKP421 PQALAKQGSLYHEMAIEPLDDIAAVTDILTQREGARLETPPPWLAMFTDQPALPNPCSPA481 SVAPAPDWTHPLPWGRPRTFSLDAVPPDHSPRARSVAPLPPQRSPRTRGLCSKGQPRTWL541 QSPV

Claims (16)

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.