CN1250098A - Human P47 coding series and the polypeptide therewith and its preparation - Google Patents

Abstract

The present invention provides the cDNA sequence human P47, and the protein encoded by the sequence is the homologue of P47. The present invention also provides the polypeptide encoded by the nucleotide sequence and the application and production method of the described polynucleotides and polypeptides.

Description

People P47 encoding sequence, its encoded polypeptides and preparation method

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 present invention relates to the cDNA sequence of people P47, this albumen is the homologue of P47.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.

P47 albumen is newfound in recent years first cofactor that is closely related with the protein mediated film fusion of P97.P47 albumen is combined into stable, mixture closely with trimerical form and P97 six aggressiveness, the dictyokinesis bead merges the process that forms the golgi body pond after the division of cytoplasm of fellowship zooblast, thereby golgi body rebuild have vital role (Nature 388:75-78,1997).

People such as Kondo in 1997 separate in the cytosol of rat hepatocytes and have obtained P47 albumen and its cDNA sequence.The homologue SHP1 gene of P47 in yeast saccharomyces cerevisiae obtained by people such as Zhang clone in nineteen ninety-five, prove that after deliberation this gene has the active function of the phosphoprotein phosphatase I of adjusting, the metabolism of wide participation glycoprotein, processes (Mol.Cell.Biol.15:2037-2050,1995) such as reduction division differentiation and the growth of mitotic cell cycle.Before the present invention came forth, still nobody disclosed the mankind's that relate among the application P47 gene and protein sequence.

An object of the present invention is to provide a kind of new polynucleotide sequence, people's homologous protein of this polynucleotide sequence coding P47, gene of the present invention is named as people P47.

Another object of the present invention provides a kind of new albumen, and this albumen is named as human P 47 protein.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new human P 47 protein.

The present invention also provides the application of this people P47 gene order and polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the active polypeptide of human P 47 protein, shows at least 70% homology from the nucleotides sequence of Nucleotide 14-1132 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 14-1132 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 14-1132 position.

In another aspect of this invention, provide a kind of isolating human P 47 protein polypeptide, it comprises: have polypeptide or its active fragments of SEQID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.

In another aspect of this invention, provide a kind of described carrier transformed host cells.

In another aspect of this invention, the method that provides a kind of generation to have the active polypeptide of human P 47 protein, this method comprises:

(a) nucleotide sequence that coding is had an active polypeptide of human P 47 protein operationally is connected in expression regulation sequence, form the human P 47 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 14-1132 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the reconstitution cell of human P 47 protein;

(c) under the condition that is fit to expressing human P47 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate and have the active polypeptide of human P 47 protein.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1412 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 14-1132 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, refer to encode has the nucleotide sequence of the active polypeptide of human P 47 protein to term " human P 47 protein (or polypeptide) encoding sequence ", as 14-1132 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 14-1132 position Nucleotide of SEQ ID NO.3 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.3 in 14-1132 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably, under the height stringent condition with SEQ ID NO.3 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 14-1132 position.In addition, this term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 14-1132 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.3 with people P47 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 several (are generally in 60 to hold interpolation 5 ' and/or 3 ', 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 " human P 47 protein polypeptide " refers to have the active SEQ ID of human P 47 protein NO.4 polypeptide of sequence.This term also comprises having and variant form people P47 identical function, SEQ ID NO.4 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 the active fragments and the reactive derivative of human P 47 protein.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of people P47 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people P47 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people P47 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people P47 polypeptide.Usually, this fragment have people P47 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.

Invention also provides the analogue of human P 47 protein or polypeptide.The difference of these analogues and natural human P47 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.

The present invention also comprises the antisense sequences of people P47 polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of people P47 in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of people P47 nucleotide 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 P47.

The present invention also comprises the method that detects people P47 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 P47 polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 20-50 Nucleotide.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.

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 P47 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 P47 gene product or fragment.Preferably, refer to that those can combine with people P47 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 molecule of human P 47 protein, comprise that also those do not influence the antibody of human P 47 protein function.The present invention also comprise those can with modify or without the people P47 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) ₂Fragment; 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 P47 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human P47 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 and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people P47 function and the antibody that does not influence people P47 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people P47 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 P47 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 P47 nucleotide sequence 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 relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of press the currently known methods preparation as template, increase and must be about 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 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, especially fragment length more in short-term.Certainly, by first synthetic a plurality of small segments, and then connect and obtain the very long fragment of sequence.

In the present invention, the cDNA nucleotide sequence of people P47 is so to obtain, with people's testis λ gt11cDNA library (available from Clontech company) is template, carry out PCR with synthetic forward primer A1:5 '-GCACGGGGCGAGAATGGCGGC-3 ' (SEQ ID NO.1) and reverse primer A2:5 '-AGGAGGGAGGCCAGGCAGCTG-3 ' (SEQ ID NO.2), obtain the purpose fragment of 1162bp.Obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.

During the zooblast division, membrane structure organoids such as golgi body all can split into a large amount of vesicles, short tube, and at telophase, these vesicles, short tube can form golgi body by two kinds of film amalgamation mode reorganization.This dual mode is respectively by two similar ATP enzyme mediations.A kind of mode is fusion (Cell 82:869-872,905-914,1995 by the protein mediated homotype vesica subsequently of P97; Nature 388:20-21,1997).The details of this approach is still among research.Separating the P47 albumen that obtains from rat hepatocytes is that first is proved the protein factor directly related with the P97 approach.Human P 47 protein of the present invention is and P of Rats 47 albumen height homologous to be P of Rats 47 proteic homologous proteins therefore, and to have similar function.P97 albumen is the unique binding substances of P47 albumen in cytosol, and P47 albumen also is the mainly conjugated protein of P97 albumen composition, therefore P47 albumen is essential for the protein mediated golgi body pond process of reconstruction of P97, and external source P47 can substitute endogenous P47.The P47 albumen of rat rat each the tissue in expression is all arranged, thus deducibility it participated in basic cell function (Nature 388:75-78,1997).P47 may be by mediation P97 albumen and the proteic combination of syntaxin5 (Cell 92:603-610,1998), thereby realize merging the adjusting of approach, these prompting people are in the tumour generating process, fast growth and asecretory tumour cell may be fused to the master with homotype, thereby quick founder cell device (Cell 92:603-610,1998).In addition, P47, P97 albumen homologue in yeast are proved and have participated in endoplasmic reticulum fusion and reconstruction and nuclear membrane fusion, thereby may be closely related with division of cytoplasm (J, Cell.Bio.114 (3): 443-453,1991; Cell 82:885-893,1995).

In the accompanying drawings,

Fig. 1 is the homology comparison diagram of the nucleotide sequence of people P47 of the present invention and P of Rats 47.Wherein, identical Nucleotide marks with " | ".

Fig. 2 is the homology comparison diagram of human P 47 protein of the present invention and P of Rats 47 proteic aminoacid sequences.Wherein, identical amino acid marks with " | " between two sequences.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit 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:ColdSpring Harbor Laboratory 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 P47

1. primer amplification

With people's testis λ gt11cDNA library (available from Clontech company) is template, with oligonucleotide A1:5 '-GCACGGGGCGAGAATGGCGGC-3 ' (SEQ ID NO.1) is forward primer, oligonucleotide A2:5 '-AGGAGGGAGGCCAGGCAGCTG-3 ' (SEQ ID NO.2) is a reverse primer, carries 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 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR fragment that electrophoresis detection obtains is the purpose fragment of 1162bp.

2.PCR the order-checking of product

With pcr amplification product A1/A2 and pGEM-T ^TMCarrier (Promega) connects, transformed into escherichia coli JM103, 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 1162bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 14-1132 position Nucleotide.

Derive the aminoacid sequence of people P47 according to the full length cDNA sequence that obtains, totally 372 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.

Embodiment 2

Homology relatively

The full length cDNA sequence of personnel selection P47 and proteins encoded thereof carry out nucleic acid and albumen homology retrieval with BLAST in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database.Found that they and P of Rats 47 genes and proteins encoded thereof have high homology, relatively find with PCGENE software, identity on nucleic acid and protein level has reached 90% and 95% respectively, therefore, people P47 gene of the present invention and albumen are the homologue of P of Rats 47 in the people, and have and P of Rats 47 genes or the same or similar function of albumen.

Studies show that, during the zooblast division, membrane structure organoids such as golgi body all can split into a large amount of vesicles, short tube, and at telophase, when parent cell was divided into two daughter cells, these vesicles, short tube can form golgi body by two kinds of film amalgamation mode reorganization.This dual mode is respectively by two similar ATP enzyme mediations: the one, and the fusion of the early stage special-shaped vesica of N-ethylomaleimide sensitive factor (NSF) mediation, producing has the pond of fenestra, and vesicle and the expansible deck edge that adheres to arranged; Another kind of mode is the fusion by the protein mediated homotype vesica subsequently of P97, produces the pond of no fenestra and deck edge (Cell 82:869-872,905-914,1995 of blunt end; Nature 388:20-21,1997).This dual mode complements each other, and finishes the reconstruction of golgi body after the cell fission jointly.The molecularity mode of NSF approach has been studied comparatively clearly, wherein relates to the NSF attachment protein (SNAP) of solubility, the NSF attachment protein cofactors (Nature 362:318-324,1993) such as (SNARE) of solubility.But the details of P97 approach is still among research.Separating the human P 47 protein that obtains from rat hepatocytes is that first is proved the protein factor directly related with the P97 approach.Human P 47 protein that the present invention relates to and P of Rats 47 albumen height homologies, thereby have similar function.And in human body, having the approach that is similar to P97, human P 47 protein is directly related with this approach.P97 albumen is the unique binding substances of P47 albumen in cytosol, and P47 albumen also is the mainly conjugated protein of P97 albumen composition, thus P47 albumen to rebuild for the protein mediated golgi body pond of P97 be essential, and external source P47 can substitute endogenous P47.The P47 albumen of rat shows by the Northen engram analysis, and it all has expression in each tissue of rat, thus deducibility it participated in basic cell function (Nature 388:75-78,1997).Current research shows that P47 may realize the proteic film function of P97 (Cell 92:603-610,1998) by mediation P97 albumen and the proteic combination of syntaxin5.P47 can compete the acting in conjunction factor syntaxin5 albumen of two kinds of approach with the SNAP molecule in the NSF approach, thereby realizes the adjustings to two kinds of fusion approach.This prompting people in the tumour generating process, fast the growth and asecretory tumour cell may be fused to the master with homotype, thereby to reach founder cell device (Cell92:603-610,1998) apace.Can provide a new way for tumor research and treatment to human P 47 protein of the present invention research.In addition, P47, P97 albumen homologue in yeast are proved and have participated in endoplasmic reticulum fusion and reconstruction and nuclear membrane fusion, thereby (the J.Cell.Bio.114 (3): 443-453,1991 that may be closely related with division of cytoplasm; Cell 82:885-893,1995), along with going deep into of research, P47, the P97 identity function in other zooblast also can constantly be found and confirm.

People P47 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 P47 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of the N of inventor P47 end with the P47 of mouse exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor P47, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

Embodiment 3

The expression of people P47 in intestinal bacteria

In this embodiment, with the cDNA sequence of coding people P47 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people P47 cDNA as inserting fragment.

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

5’-GTAAGGATCCATGGCGGCGGAGCGACAGG-3′(SEQ?ID?NO.5)

This primer contains the restriction enzyme site of BamHI restriction enzyme, is 19 Nucleotide of the people P47 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-GAACGTCGACTTATGTTAACCGCTGCACG-3’(SEQ?ID?NO.6)

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people P47 of SalI 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 and SalI 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 P47 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 P47 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people P47 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps use the dialysis step to remove Guanidinium hydrochloride, perhaps isolate purifying protein from nickel-chelate column, the albumen behind the purifying can be 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 the protein that obtains 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 41KDa.

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 sequence of SEQ ID NO.4 with ordinary method.

Embodiment 4

The expression of people P47 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with the cDNA sequence of coding people P47 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people P47 cDNA as inserting fragment.

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

5’-GTGTGGATCCATGGCGGCGGAGCGACAGG-3′(SEQ?ID?NO.7)

This primer contains the restriction enzyme site of BamHI restriction enzyme, is 19 Nucleotide of the people P47 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-AAATCTGCAGTTATGTTAACCGCTGCACG-3’(SEQ?ID?NO.8)

This primer contains the part encoding sequence of the restriction enzyme site of PstI restriction enzyme, translation termination and people P47.

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 (f1 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.

Digest the pcDNA3 carrier respectively and insert fragment with BamHI and PstI, 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 cuts evaluation with the ApaI enzyme and insert clip size and direction, and the cDNA fragment of sequence verification people P47 has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to use lipofection, and (GiBco Life) carries out with the Lipofection test kit.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 41KDa.

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 sequence of SEQ ID NO.4 with ordinary method.

Embodiment 5

Preparation antibody

Embodiment 3 and 4 recombinant proteins that obtain are used for immune animal to produce antibody, specific as follows.Recombinant protein is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people P47 gene translation product with it.

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: (i) applicant: Xinhuangpu-Fudan Gene Engineering Co., Ltd., Shanghai is denomination of invention (ii): people P47 encoding sequence, its encoded polypeptides and preparation method be the sequence number (iii): information (i) sequence signature of 8 (2) SEQ ID NO.1

(A) length: 21 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.1GCACGGGGCG AGAATGGCGG C 21 (2) SEQ ID NO.2

(A) length: 21 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO.2AGGAGGGAGG CCAGGCAGCT G 21 (2) SEQ ID NO.3: (i) sequence signature

(A) length: 1162bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO.3 1 GCACGGGGCG AGAATGGCGG CGGAGCGACA GGAGGCGCTG AGGGAGTTCG TGGCGGTGAC 61 GGGCGCCGAG GAGGACCGGG CCCGCTTCTT TCTCGAGTCG GCCGGCTGGG ACTTGCAGAT121 CGCGCTAGAG CTCTTTTATG AGGACGGAGG GGATGAAGAC ATTGTGACCA TTTCGCAGGC181 AACCCCCAGT TCAGTGTCCA GAGGCACAGC CCCCAGTGAT AATAGAGTGA CATCCTTCAG241 AGACCTCATT CATGACCAAG ATGAAGATGA GGAGGAAGAG GAAGGCCAGA GGAGCAGGTT301 TTATGCTGGG GGCTCAGAGA GAAGTGGACA GCAGATTGTT GGCCCTCCCA GGAAGAAAAG361 TCCCAACGAG CTGGTGGATG ATCTCTTTAA AGGTGCCAAA GAGCATGGAG CTGTAGCTGT421 GGAGCGAGTG ACCAAGAGCC CTGGAGAGAC CAGTAAACCG AGACCATTTG CAGGAGGTGG481 CTACCGCCTT GGGGCAGCAC CAGAGGAAGA GTCTGCCTAT GTGGCAGGAG AAAAGAGGCA541 GCATTCCAGC CAAGATGTTC ATGTAGTATT GAAACTCTGG AAGAGTGGAT TCAGCCTGGA601 TAATGGAGAA CTCAGAAGCT ACCAAGACCC ATCCAATGCC CAGTTTCTGG AGTCTATCCG661 CAGAGGGGAG GTGCCAGCAG AGCTTCGGAG GCTAGCTCAC GGTGGACAGG TGAACTTGGA721 TATGGAGGAC CATCGGGACG AGGACTTTGT GAAGCCCAAA GGAGCCTTCA AAGCCTTCAC781 TGGCGAGGGT CAGAAACTGG GCAGCACTGC CCCCCAGGTG TTGAGTACCA GCTCTCCAGC841 CCAACAGGCA GAAAATGAAG CCAAAGCCAG CTCTTCCATC TTAATCGACG AATCAGAGCC901 TACCACAAAC ATCCAAATTC GGCTTGCAGA CGGCGGGAGG CTGGTGCAGA AATTTAACCA961 CAGCCACAGG ATCAGCGACA TCCGACTCTT CATCGTGGAT GCCCGGCCAG CCATGGCTGC1021 CACCAGCTTT ATCCTCATGA CTACTTTCCC GAACAAAGAG CTGGCTGATG AGAGCCAGAC1081 CCTGAAGGAA GCCAACCTGC TCAATGCTGT CATCGTGCAG CGGTTAACAT AACCGCCCAG1141 CCAGCTGCCT GGCCTCCCTC CT ( 2 ) SEQ ID NO.4：

(i) sequence signature

(A) length: 372 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.41 Met Ala Ala Glu Arg Gln Glu Ala Leu Arg Glu Phe Val Ala Val 16 Thr Gly Ala Glu Glu Asp Arg Ala Arg Phe Phe Leu Glu Ser Ala 31 Gly Trp Asp Leu Gln Ile Ala Leu Glu Leu Phe Tyr Glu Asp Gly 46 Gly Asp Glu Asp Ile Val Thr Ile Ser Gln Ala Thr Pro Ser Ser 61 Val Ser Arg Gly Thr Ala Pro Ser Asp Asn Arg Val Thr Ser Phe 76 Arg Asp Leu Ile His Asp Gln Asp Glu Asp Glu Glu Glu Glu Glu 91 Gly Gln Arg Ser Arg Phe Tyr Ala Gly Gly Ser Glu Arg Ser Gly106 Gln Gln Ile Val Gly Pro Pro Arg Lys Lys Ser Pro Asn Glu Leu121 Val Asp Asp Leu Phe Lys Gly Ala Lys Glu His Gly Ala Val Ala136 Val Glu Arg Val Thr Lys Ser Pro Gly Glu Thr Ser Lys Pro Arg151 Pro Phe Ala Gly Gly Gly Tyr Arg Leu Gly Ala Ala Pro Glu Glu166 Glu Ser Ala Tyr Val Ala Gly Glu Lys Arg Gln His Ser Ser Gln181 Asp Val His Val Val Leu Lys Leu Trp Lys Ser Gly Phe Ser Leu196 Asp Asn Gly Glu Leu Arg Ser Tyr Gln Asp Pro Ser Asn Ala Gln211 Phe Leu Glu Ser Ile Arg Arg Gly Glu Val Pro Ala Glu Leu Arg226 Arg Leu Ala His Gly Gly Gln Val Asn Leu Asp Met Glu Asp His241 Arg Asp Glu Asp Phe Val Lys Pro Lys Gly Ala Phe Lys Ala Phe256 Thr Gly Glu Gly Gln Lys Leu Gly Ser Thr Ala Pro Gln Val Leu271 Ser Thr Ser Ser Pro Ala Gln Gln Ala Glu Asn Glu Ala Lys Ala286 Ser Ser Ser Ile Leu Ile Asp Glu Ser Glu Pro Thr Thr Asn Ile301 Gln Ile Arg Leu Ala Asp Gly Gly Arg Leu Val Gln Lys Phe Asn316 His Ser His Arg Ile Ser Asp Ile Arg Leu Phe Ile Val Asp Ala331 Arg Pro Ala Met Ala Ala Thr Ser Phe Ile Leu Met Thr Thr Phe346 Pro Asn Lys Glu Leu Ala Asp Glu Ser Gln Thr Leu Lys Glu Ala361 Asn Leu Leu Asn Ala Val Ile Val Gln Arg Leu Thr ( 2 ) SEQ ID NO.5 ( i )

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.5GTAAGGATCC ATGGCGGCGG AGCGACAGG 29 (2) SEQ ID NO.6

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.6GAACGTCGAC TTATGTTAAC CGCTGCACG 29 (2) SEQ ID NO.7

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.7GTGTGGATCC ATGGCGGCGG AGCGACAGG 29 (2) SEQ ID NO.8

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.8AAATCTGCAG TTATGTTAAC CGCTGCACG 29

Claims (14)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the active polypeptide of human P 47 protein,

Show at least 70% homology from the nucleotides sequence of Nucleotide 14-1132 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 14-1132 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.4.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence comprises the sequence of Nucleotide 14-1132 position among the SEQ ID NO.3.

4. isolating human P 47 protein polypeptide is characterized in that it comprises: have polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

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. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.

9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.

10. a generation has the method for the active polypeptide of human P 47 protein, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had an active polypeptide of human P 47 protein operationally is connected in expression regulation sequence, form the human P 47 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 14-1132 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the reconstitution cell of human P 47 protein;

(c) under the condition that is fit to expressing human P47 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate and have the active polypeptide of human P 47 protein.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 14-1132 position among the SEQ ID NO.3.

12. energy and the described human P 47 protein polypeptid specificity of claim 4 bonded antibody.

13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.

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

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