CN1257925A - Human galactoside transferase I-type homogenous protein - 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 human galactoside transferase I-type homologous protein.

Description

Human galactose glycosides transferase I type homologous protein

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 a newcomer of naturally occurring galactoside transferase family in the human body, and polypeptide especially of the present invention is accredited as β 1 by deduction, 4-galactoside transferase homologue.The invention still further relates to the effect that suppresses these polypeptide.

Oligonucleotide chain on polysaccharide chain in the organism, glycoprotein and the glycolipid synthetic all finished by various single-minded glycosyltransferases.The glycosyltransferase class is extended familys with numerous members, and shifting different glycosyls has the different transferring enzyme of specificity.Even shift identical glycosyl, owing to the acceptor difference of glycosyl, the glycosidic link difference of formation, also by different enzyme catalysiss.The galactoside transferase class is exactly so: they are glycosyl donor with the UDP-galactoside all, but exist such as α 1 6-galactoside transferase, α 1, tens of kinds of different enzymes of 3-galactoside transferase or the like owing to the synthetic disaccharide bond is different.Among this, β 1, and the 4-galactoside transferase (hereinafter to be referred as β 1, is to be studied one of maximum enzyme 4GT).It generally is responsible for synthetic modal disaccharide bond: N-acetyllactosamine [Gal (β 1-4) GlcNAc].In the mammal galactophore of lactation, it can combine with alpha-lactalbumin, becomes " lactose synthetase ", and specificity ground catalysis galactosyl is transferred on the D-semi-lactosi, thus the lactose that is rich in the synthesise lactic.

In the cell of each tissue of organism, β 1, and 4GT mainly extensively exists with two kinds of forms: 1. be integrated on the Golgi membrane, 2. be integrated on the cytoplasmic membrane.In recent years along with ox, mouse and people's β 1, the clone in succession of 4GT gene, advanced the further investigation of its important biomolecule being learned function, present result of study shows β 1,4GT, especially be integrated in the β 1 on the cytoplasmic membrane, 4GT has confidential relation with all many-sides such as the propagation of the transfer of the migration of the fertilization of sperm, neuronal cell, cancer cells, epidermic cell and autoimmune diseases.

1986, the Shaper of John Hopkins university takes the lead in being cloned into an ox β 1 with the immunoscanning method from λ gt11 expression library, the encoding sequence of 4GT, Northern result shows the long 4.8kb of its mRNA (Shaper NL et al.Proc Natl Acad Sci.USA.1986; 83:1573).The Narimatsu of Japan has then at first successfully measured its cDNA total length (Narimatsu H et al.Proc Natl Acad Sci.USA.1986 the same year; 83:4720).1988, Shaper cloned the β 1 of mouse again, the cDNA total length of 4GT, and find that its 5 ' end has two translation initiation sites (Shaper NL et al.J Biol Chem.1988; 263:10420).1988, Masri etc. used according to β 1, and the dna probe that 4GT C terminal amino acid designs has in proper order obtained human first β 1, the total length of 4GT encoding sequence (MasriKA et al.Biochem Biophys Res Commun.1988; 157:657:663); The full-length cDNA of this gene and genome sequence analysis were in succession finished (Mengle-Gaw L et al.Biochem Biophys ResCommun.1991 with nineteen ninety-five by Mengle-Gaw and Chatteriee etc. in 1991; 15:1269; Chatteriee SK et al.Int J Biochem Cell Biol.1995; 27:329).1997, clone such as Almeida of Denmark also expressed two mankind's β 1, the newcomer of 4GT family ^[7]Yet before the present invention, the human galactose glycosides transferase I type homologous protein that relates among the application was not disclosed.

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding galactoside transferase family, galactoside transferase homologue called after hGTH6 of the present invention (GenBank Accession No.AF069054).

Another object of the present invention provides a kind of new galactoside transferase family member, called after hGTH6.

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

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, polypeptide of the present invention is that the polypeptide of newcomer, especially this aspect of galactoside transferase family is accredited as people β-1 by deduction, the homologue of 4-galactoside transferase.This evaluation is basis and all known galactoside transferase homologue called after hGTH6 of the present invention (GenBankAccession No.AF069054).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 2147 Nucleotide, and its detailed sequence is seen SEQ ID NO:1, and wherein open reading frame is positioned at 146-1177 position Nucleotide.These polynucleotide are so to obtain, with people's white cell λ gt11cDNA library (available from Clontech company) is template, synthetic forward primer 5 '-TCAATGTGGGCTTCAAAGAGGCC-3 ' and reverse primer 5 '-CAAAGGTCATCATCTTCTCCTCC-3 ', carry out PCR, obtain the purpose fragment of 271bp.This fragment of mark then, with this labeled fragment be probe with amplification after the hybridization of people's white cell 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.Identify the full length cDNA sequence that obtains SEQ ID NO:1 after checking order.

According to a further aspect in the invention, polynucleotide sequence of the present invention can be used to express or produce the hGTH6 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 hGTH6 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 hGTH6 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 hGTH6 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 hGTH6 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 hGTH6 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-hGTH6.

According to another aspect of the invention, polynucleotide of the present invention and polypeptide can be used for the diagnosis and the treatment of hGTH6 relative disease.Known β 1,4GT participates in the acrosomal reaction in the fertilization process, and the not normal meeting of this enzyme causes Infertility; and β 1; 4GT is a member of galactoside transferase family, and this family comprises other many different members, and different species and individuality also have different members.β 1 of the present invention, 4GT homologue are the newcomers of this family.Therefore the cDNA of the hGTH6 that is separated to of the present invention can be used for detecting and β 1 of the present invention specifically, the Infertility that the inactivation of 4GT homologue causes, and may provide a kind of approach for the treatment of the Infertility of this class in the future.On the other hand, the antibody at polypeptide of the present invention also can be used as contraceptive bian.

In addition; recently studies show that the β 1 on cancer cells surface; the 4GT enzyme is lived in increasing and is made cell be easy to migration; point out the transfer of this enzyme and tumour closely related; therefore the cDNA of the hGTH6 that is separated to of the present invention can be used for detecting and β 1 of the present invention specifically, and 4GT homologue abnormal expression or enzyme are lived and improved the cancer metastasis that causes.On the other hand, polypeptide antibody of the present invention can combine with this polypeptide and make enzyme reduction alive, thus the blocking-up cancer metastasis.

β 1, and the reduction alive of 4GT enzyme may cause human autoimmune diseases such as rheumatic arthritis, and therefore cDNA of the present invention and polypeptide provide an approach for treating this class disease.

The β 1 of epidermal surface, 4GT transmits the cell proliferation signal from a matter, prompting β 1,4GT and epidermic cell differentiation and proliferation are closely related, and therefore cDNA of the present invention and polypeptide are for treating the dermatosis relevant with the epidermic cell differentiation and proliferation and delaying even eliminate cosmetic treatments such as shrunken skin, aging an approach is provided.

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 hGTH6 and 1. primer amplifications that check order

With people's white cell λ gt11cDNA library (available from Clontech company) is template, with oligonucleotide 5 '-TCAATGTGGGCTTCAAAGAGGCC-3 ' is forward primer, oligonucleotide 5 '-CAAAGGTCATCATCTTCTCCTCC-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, 54 ℃ 1 minute and 72 ℃, last 72 ℃ were extended 5 minutes thereupon.The 271bp 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 white cell λ gt11cDNA library, clone's number reaches 500,000, good cDNA library trace (Amersham) to the HybondTM-N+ nylon membrane 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 9 primary dcreening operation positive colonies by above-mentioned screening by hybridization, these 9 positive colonies are carried out multiple sieve, finally obtain 8 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 gt11 left and right arms on primer S1 and S2 (S1:5 '-GTTCAACATCAGCCGCTACA-3 '; S2:5 '-CACCAGACCAACTTTTAATG-3 ') amplification of arranging in pairs or groups, carry out agarose gel electrophoresis then, to judge that each clone moves the length of (extensions) from probe to 5 ' or 3 ' step, therefrom select and extend the longest and to the longest clone of 3 ' extension to 5 '.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 2147bp, detailed sequence is seen SEQ ID NO:1, and wherein open reading frame is positioned at 146-1177 position Nucleotide.

Derive the aminoacid sequence of hGTH6 according to the cDNA sequence that obtains, totally 343 amino-acid residues, its aminoacid sequence sees SEQ ID NO:2 for details.Expression pattern analysis and the chromosomal localization of embodiment 2 hGTH6

Multiple Northern (the MNT that organizes ^TM) blotting membrane (16 kinds of tissues) is available from Clontech company, people/rodents somatic cell hybrid is that Southern blotting membrane (PstI) is available from Oncor company, 271bp cloned sequence in above embodiment 1 step is a probe, 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 hGTH6 gene is the wide spectrum expressing gene, in spleen, thymus gland, small intestine, large intestine, heart, lung, liver, skeletal muscle, kidney, pancreas, 11 kinds of tissues of prostate gland, expression is arranged all, expression amount is variant slightly, is than high expression level in brain, testis, ovary, placenta and peripheral blood leucocyte.Can be on No. 18 karyomit(e) according to the Southern results of hybridization with this assignment of genes gene mapping.

Embodiment 3 expression of people hGTH6 in intestinal bacteria

In this embodiment, be masterplate with people's white cell λ gt11cDNA library, use 5 ' and 3 ' the PCR Oligonucleolide primers of holding to increase the cDNA sequence of coding people hGTH6 corresponding to this dna sequence dna, obtain people hGTH6 cDNA as inserting fragment.

5 ' the Oligonucleolide primers sequence of using in the PCR reaction is: 5 '-TCAGGGATCCATGTCTGTGCTCAGGCGGA-3 ', this primer contains the restriction enzyme site of the restricted restriction enzyme of BamHI, is 19 Nucleotide of the people hGTH6 encoding sequence that begun by initiator codon after this restriction enzyme site; 3 ' end primer sequence is: 5 '-AAGGAAGCTTTTAATAGTCTTCGATTGGA-3 ', this primer contain the part encoding sequence of restriction enzyme site, translation termination and the people hGTH6 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 BamHI 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 (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people hGTH6 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) (OD 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 people hGTH6 from solution.With 6M Guanidinium hydrochloride (PH5.0) wash-out people hGTH6 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 40KDa.

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 hGTH6 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment,, use 5 ' and 3 ' the PCR Oligonucleolide primers of holding to increase the cDNA sequence of coding people hGTH6, obtain people hGTH6 cDNA as inserting fragment corresponding to this dna sequence dna with people's white cell λ gt11cDNA library masterplate.

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

5’-TCAGAAGCTT?ATGTCTGTGCTCAGGCGGA--3’

This primer contains the restriction enzyme site of the restricted restriction enzyme of HindIII, is 19 Nucleotide of the people hGTH6 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-AAGGGGATCC?TTAATAGTCTTCGATTGGA-3’

This primer contains the part encoding sequence of the restriction enzyme site of the restricted restriction enzyme of BamHI, translation termination and people hGTH6.

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 BamHI 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 hGTH6 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 40KDa.

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 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): galactoside transferase I type homologous protein is sequence number (iii): 2 (iv) contact addresses:

(A) addressee: Ntd Patent ﹠ Trademark Agency Ltd

(B) street: Building A, No. 27 investment squares, financial street 10 floor

(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: I98599CB (ix) telecommunication information:

(A) phone: 8610-66211834

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

( A ) ：2147bp ( B ) ： ( C ) ： ( D ) ： ( iii ) ：cDNA ( xi ) ：SEQ ID NO：1 1 CTCGGGCCCT CTCCCCAGAG CCTCCCCGCG CGGACCCCGC TGCAGTGTCG GGGTTCTCAG 61 CACATTTATG GAAGTTTAGG GCCTGGACAG TGGCCCCGAG TCCGGCCTGA GAGCGCAGCC 121 TGGGCTGCTG GCAGGGAAGA GGAAGATGTC TGTGCTCAGG CGGATGATGC GGGTTTCCAA 181 TCGCTCTCTC CTCGCCTTCA TCTTCTTTTT CTCCCTCTCT TCGTCCTGTC TGTACTTCAT 241 CTATGTGGCC CCAGGCATCG ATTATCCCGA AGGCAATAAT TCAAGTGATT ATCTTGTTCA 301 AACAACAACG TATCTCCCGG AAAACTTCAC ATACTCACCA TACCTCCCCT GTCCAGAAAA 361 GCTGCCTTAT ATGCGAGGAT TCCTCAATGT CAATGTAAGC GAAGTCAGTT TTGATGAAAT 421 TCATCAACTC TTCTCCAAGG ATTTAGATAT TGAGCCAGGG GGTCATTGGA GGCCAAAAGA 481 CTGTAAACCC AGATGGAAGG TGGCAGTTCT CATTCCTTTC CGTAATCGCC ATGAACATCT 541 TCCAATTTTT TTCTTACATC TGATTCCAAT GCTCCAGAAG CAGCGGCTGG AATTTGCGTT 601 TTATGTCGTT GAACAGACTG GCACACAACC TTTTAACCGT GCGATGCTTT TCAATGTGGG 661 CTTCAAAGAG GCCATGAAAG ACAGTGTCTG GGACTGTGTA ATCTTCCACG ATGTGGATCA 721 TCTACCTGAA AATGACCGGA ACTATTACGG ATGTGGAGAA ATGCCACGTC ATTTTGCTGC 781 AAAGCTGGAT AAATACATGT ATATTCTTCC ATATAAAGAA TTTTTTGGTG GTGTAAGTGG 841 GCTGACAGTG GAACAATTTA GAAAGATCAA TGGTTTTCCT AATGCCTTCT GGGGATGGGG 901 AGGAGAAGAT GATGACCTTT GGAACAGAGT TCACTATGCT GGATATAATG TAACCAGACC 961 AGAGGGAGAC TTAGGAAAAT ACAAGTCAAT TCCTCATCAC CATAGAGGTG AAGTCCAGTT1021 TTTAGGACGG TATAAATTAC TAAGGTATTC CAAGGAGCGT CAGTACATCG ATGGACTGAA1081 CAATTTAATA TATAGGCCAA AAATACTGGT TGATAGGTTG TATACAAACA TATCTGTAAA1141 CCTCATGCCA GAGTTAGCTC CAATCGAAGA CTATTAAAAG AAGTGGCTGT CGTGGCAAGG1201 TAGACCACAA TGCTGGATCA TAAACTTGGA GCGCGCTCTT AGTGGAGGTC AGTGATTGGC1261 TGTGTCACAG TGCCCTTTTC TCTGAGAAGA GCCAGCAGTC CACAGTGTTT ACAGAGTGGG1321 ACTATATACA GTCACCCTTC TCTCACCCCG TCCTCCCTGC TCTGAGACAC ACCCCTGTGG1381 CGAGCACCGC AGAAACGGGA AGCCACTACT TAAGATCGGA AGTTAAGAGA GCTCCCTCCG1441 AAGAGAAAAT TTTTATACTA AAATCTATAA TTTAATTCAA GAGAATGCTT TTATTTCCGT1501 TTAAACATAT TTTGTATATA TGTGATATAA ATTAATGTGT GCAAATTGTT TAAAAATTAG1561 ATGTGTTGCA GTTTTGCATG TAATCGGTTA TACCTTTATT GGACTTTTAT AGACATTTTT1621 TATTTGCATG AAAAAAACTC ACTAAATTTA CATCACTAAA CAAAGGTTAA CCCTTGTGTG1681 AAATGAAGGA ACTGTCAATA ATTGACAGCC AACTAATACA GTAAACTGTT ATACTAGTTT1741 TGAGCTTTAG ACCTCAGCCT TTTGTGTGGA AGAAGTCACA GCTTTCTTAG GCTTTAAAGG1801 AAAAGAAGGA AGGACTTAAA TAGCTTTTCT TCCTACCGGG ATTACCTATG TTTTTCCTTG1861 CTTGCAATCT CATCTGATTT TGCTAGAAAT CACAACCATA TTGTTTATGC ATATTGCATG1921 AGTATTACCA AGAAAAATCT TTAAAAGTTG TGATGTGACA TGATATAAAG GATCTCTTTA1981 TGTTAAATGT CTTTCCATGT ACCTCTGGTG TGTCAGGGAT TTTGTGCCTC AAAAAATGTT2041 TCCAAGGTTG TGTGTTTATA CTGTGTATTT TTTTTAAATT CACGGTGAAC AGCACTTTTA2101 TTATTTCCAG TTCAGAAGAG CCAAAAAAAA AAAAAAAAAA AAAAAAA ( 2 ) SEQ ID NO：2： ( i ) ：

(A) length: 343 amino acid

(B) type: amino acid

(C) topological structure: linear (iii) molecule-type: polypeptide (x) sequence description: SEQ ID NO:2 1 MSVLRRMMRVSNRSLLAFIFFFSLSSSCLYFIYVAPGIDYPEGNNSSDYLVQTTTY LPEN 61 FTYSPYLPCPEKLPYMRGFLNVNVSEVSFDEIHQLFSKDLDIEPGGHWRPKDCKPR WKVA121 VLIPFRNRHEHLPIFFLHLIPMLQKQRLEFAFYVVEQTGTQPFNRAMLFNVGFKEA MKDS181 VWDCVIFHDVDHLPENDRNYYGCGEMPRHFAAKLDKYMYILPYKEFFGGVSGLTVE QFRK241 INGFPNAFWGWGGEDDDLWNRVHYAGYNVTRPEGDLGKYKSIPHHHRGEVQFLGRY KLLR301 YSKERQYIDGLNNLIYRPKILVDRLYTNISVNLMPELAPIEDY

Claims (18)

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.

18, the application of the described polypeptide of claim 15 in the medicine of preparation diagnosis and treatment and the unusual relevant disease of described polypeptide.