CN1291650A - Human hepatome derivative growth factor coding sequence, polypeptide coded with it and its preparing process - Google Patents

Abstract

The present invention disclosed a CDNA sequence of III-type human hepatome cell derivative growth factor (HDGF 3), which is the homolog of Mouse HRP-2. The polypeptide coded by said nucleotide sequence, the application of said polynucleotide and polypeptide and the process for preparing said polynucleotide and polypeptide are also disclosed.

Description

A kind of human hepatoma derivative growth factor encoding sequence, its encoded polypeptides and preparation method

The present invention relates to the genetically engineered field, particularly, relate to a kind of human gene nucleotide sequence.More particularly, the present invention relates to the cDNA sequence of a kind of human hepatoma derivative growth factor (HDGF3), this albumen is the homologue of mouse HRP-2.The invention still further relates to by the application of this nucleotide sequence coded polypeptide, these polynucleotide and polypeptide and the preparation method of described polynucleotide and described polypeptide.

Research shows that the adjusting of cell growth is to realize by a series of cascade reactions that cause after the film surface receptor effect special with it of various cytokines.In tumour cell, the cell that causes out of control of some cascade reaction continues propagation.In liver cancer cell, it has been found that some autocrines and the paracrine cell factor (Proc.Natl. Acad.Sci.83:2448-2452,1986; Proc.Natl.Acad.Sci.86:7432-7436,1989; Cell 61:1137-1146,1990).Hepatome derivative growth factor (HDGF) is that the human hepatoma cell strain in serum-free culture is a cytokine that finds among the HuH-7, it has heparin affinity and can stimulate the DNA synthetic (J.Biol.Chem.269 (40): 25143-25149,1994) of Swiss 3T3 cell.

People such as Nakamura in 1989 are first from HuH-7 middle part of cell separating and purifying and identified HDGF (Clin.Chim.Acta.183:273-284,1989), the complete again cDNA sequence (J.Biol.Chem.269 (40): 25143-25149 that has cloned HDGF of this experimental group in 1994,1994), this experimental group in 1997 has found the homologue of HDGF and has found two member HRP-1 in addition of this gene family in mouse, HRP-2, they all have a quite amino terminal sequence (Biochem.Biophys.Res.Commun.238:26-32 of 98 conservative amino acid longs, 1997), these experimental group in 1999 have been cloned into another member HRP-3 (Biochem.Biophys.Res.Commun.266 (1): 81-87,1999) of HDGF gene family again in people and mouse.Before the present invention came forth, still nobody disclosed the people HDGF3 that relates among the application.

The object of the present invention is to provide a kind of new human hepatoma derivative growth factor polynucleotide sequence, the proteic homologous protein of this polynucleotide sequence coding mouse HRP-2, this polynucleotide sequence is named as people HDGF3.

Another object of the present invention provides a kind of albumen of new human hepatoma derivative growth factor, and this albumen is named as people HDGF3.

A further object of the present invention provides a kind of recombinant technology that utilizes and produces the described new proteic method of people HDGF3.

The present invention also provides this people HDGF3 gene order and proteic application.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people HDGF3 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 6-2033 position among described nucleotide sequence and the SEQ ID No.5; Perhaps described nucleotide sequence can be under the rigorous condition of moderate with SEQ ID No.5 in from the nucleotide sequence hybridization of Nucleotide 6-2033 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID No.6.More preferably, this sequence has among the SEQ ID No.5 nucleotide sequence from Nucleotide 6-2033 position.

In another aspect of this invention, provide a kind of isolating HDGF3 protein polypeptide, it comprises: have polypeptide or its active fragments of SEQ ID No.6 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID No.6 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 polypeptide of HDGF3 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of HDGF3 protein-active operationally is connected in expression regulation sequence, form the HDGF3 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 6-2033 position among described nucleotide sequence and the SEQ ID No.5;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of HDGF3;

(c) be fit to express under the condition of HDGF3 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with HDGF3 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 2118 Nucleotide, and its detailed sequence is seen SEQ ID NO.5, and wherein open reading frame is positioned at 6-2033 position Nucleotide.

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

In the present invention, term " HDGF3 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with HDGF3 protein-active is as 6-2033 position nucleotide sequence and degenerate sequence thereof among the SEQ ID No.5.This degenerate sequence is meant, is arranged in the encoder block 6-2033 position Nucleotide of SEQ ID No.5 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.5 in 6-2033 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID No.6 of also encoding out.This term also comprises can be under the rigorous condition of moderate, more preferably, under highly rigorous condition with SEQ ID No.5 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 6-2033 position.In addition, this term also comprise with SEQ ID No.5 in from the homology of nucleotide sequence at least 70% of Nucleotide 6-2033 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 the proteic SEQ ID No.5 with people HDGF3 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 for 20% of the total material of sample at least, preferably 50%, more preferably 80%, and 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 " HDGF3 protein polypeptide " refers to have the SEQ ID No.6 polypeptide of sequence of HDGF3 protein-active.This term also comprises having and variant form people HDGF3 identical function, SEQ ID No.6 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, (preferably by carrying out shown in the table 1) can not change proteinic function usually when replacing with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of HDGF3 and reactive derivative.

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 HDGF3 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-HDGF3 polypeptide to obtain.The present invention also provides other polypeptide, as comprises HDGF3 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of HDGF3 polypeptide.Usually, this fragment have the HDGF3 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 HDGF3 albumen or polypeptide.The difference of these analogues and natural HDGF3 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 HDGF3 polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of HDGF3 in the cell.

The present invention also comprises the nucleic acid molecule that can be used as probe and primer, and this molecule has about 8-100 of HDGF3 polypeptid coding sequence, preferably about 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the HDGF3 that encodes.

The present invention also comprises the method that detects the HDGF3 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 HDGF3 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.

The present invention also comprises HDGF3 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 HDGF3 gene product or fragment.Preferably, refer to that those can combine with HDGF3 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of HDGF3, comprise that also those do not influence the antibody of HDGF3 protein function.The present invention also comprise those can with modify or without the HDGF3 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 HDGF3 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing HDGF3 or its has 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 CellHybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the HDGF3 function and the antibody that does not influence the HDGF3 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of HDGF3 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 HDGF3 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).

The homology of the nucleotide sequence of HDGF3 of the present invention and mouse HRP-2 relatively sees Table 2.Wherein, identical Nucleotide marks with " ︱ ".

The homology of HDGF3 of the present invention and the proteic aminoacid sequence of mouse HRP-2 relatively sees Table 3.Wherein, identical amino acid marks with " ︱ ", and similar amino acid marks with ". ", and the proteic nuclear localization signal of mouse HRP-2 is used Mark.

HDGF3 of the present invention and HDGF family member aminoacid sequence N-terminal highly conserved sequence relatively see Table 4.Wherein, identical amino acid marks with " * "; Similar amino acid marks with ". ".

In the present invention, the cDNA nucleotide sequence of people HDGF3 is so to obtain, with people's testis λ gtllcDNA library (available from Clontech company) is template, using oligonucleotide A1:5 '-TCAGCATGCCACACGCCTTCAAG-3 ' (SEQ ID No.1) and B1:5 '-GACTCCGACTCCAAGGCCGATTC-3 ' (SEQ ID No.3) is forward primer, oligonucleotide A2:5 '-CTTCACAGACACATCGGAGTCGG-3 ' (SEQ ID No.2) and B2:5 '-TCTCTGCTCTGCTTTCCTGCGAG-3 ' (SEQ ID No.4) reverse primer, carry out PCR, the PCR condition be 93 ℃ 4 minutes, thereupon with 93 ℃ 1 minute, 68 ℃ of 1 minute and 72 ℃ carried out 35 circulations in 1 minute, and last 72 ℃ were extended 5 minutes.The PCR fragment that electrophoresis detection obtains is about 800 and the purpose fragment of 1400bp.With Restriction Enzyme NocI both are carried out enzyme then and cut, connect, obtain purpose cDNA sequence and assorted band.The methods such as being with available electrophoresis of mixing is removed, thereby obtains the higher purpose cDNA sequence of concentration.

Hepatome derivative growth factor (HDGF) is the heparin-binding protein that is separated to from people's hepatoma cell strain HuH-7, and it has the activity of stimulate cell growth, can promote the growth of inoblast and some liver cancer cells.The acidic amino acid tail of the C of HDGF end and .HMG-1/-2 height homology, and this section sequence known be the histone calmodulin binding domain CaM, so HDGF probably brings into play the activity of its stimulate cell growth as transcription factor.The mitogen activity of HDGF makes it exist great using value in the treatment of acute pernicious hepatitis and liver injury.

Known each member's of HDGF family expression pattern is different, but they all have the very enrichment of high level in testis, and their 5 ' non-translational region all has and is higher than 70% GC ratio, thereby in the male sex-cell growth course, critical function is arranged, and and dna methylation, chromatin conformation and translational control are relevant.

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:Cold Spring 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 HDGF3

1. primer amplification

With people's testis λ gtllcDNA library (available from Clontech company) is template, using oligonucleotide A1:5 '-TCAGCATGCCACACGCCTTCAAG-3 ' (SEQ ID No.1) and B1:5 '-GACTCCGACTCCAAGGCCGATTC-3 ' (SEQ ID No.3) is forward primer, oligonucleotide A2:5 '-CTTCACAGACACATCGGAGTCGG-3 ' (SEQ ID No.2) and B2:5 '-TCTCTGCTCTGCTTTCCTGCGAG-3 ' (SEQ ID No.4) reverse primer, carry out PCR, the PCR condition be 93 ℃ 4 minutes, thereupon with 93 ℃ 1 minute, 68 ℃ of 1 minute and 72 ℃ carried out 35 circulations in 1 minute, and last 72 ℃ were extended 5 minutes.The PCR fragment that electrophoresis detection obtains is about 800 and the purpose fragment of 1400bp.With Restriction Enzyme NocI both are carried out enzyme then and cut, connect, obtain purpose cDNA sequence and assorted band.The methods such as being with available electrophoresis of mixing is removed, thereby obtains the higher purpose cDNA sequence of concentration.

2.PCR the order-checking of product

With purpose cDNA sequence and the pGEM-T that as above obtains ^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 (EpicentreTechnologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 2118bp, detailed sequence is seen SEQ ID No.5, and wherein open reading frame is positioned at 6-2033 position Nucleotide.Derive the aminoacid sequence of HDGF3 according to the full length cDNA sequence that obtains, totally 676 amino-acid residues, its aminoacid sequence sees SEQ ID No.6 for details.

Embodiment 2

Homology relatively

The proteins encoded of personnel selection HDGF3 carries out the albumen homology retrieval with BLAST in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database.Found that it and HDGF family member have certain homology on protein level: 82.8% identity is arranged and 6% similarity (seeing Table 2) is arranged with mouse HRP-2 albumen (bdj ︱ BAA22896.1 ︱ (D63850)), (dbj ︱ BAA90478.1 ︱ (ABO29493)) has 52% identity and 11.9% similarity arranged with mouse HRP-3 albumen, (dbj ︱ BAA03903.1 ︱ (D16431)) has 44.6% identity and 11.3% similarity arranged with people HDGF albumen, and (gb ︱ AAF65469.1 ︱ AF251787_1 (AF251787)) has 43% identity and 11% similarity is arranged with mouse HDGF albumen.HDGF family member N-terminal (hathregion, forhomologous to the amino terminus of the HDGF,) have a high conservative, the characteristic sequence that is considered to this family member, and its N-terminal of people HDGF3 of the present invention and other members of HDGF family have higher homology (seeing Table 4).In view of above-mentioned feature of the present invention thinks that it belongs to hepatome derivative growth factor (HDGF) family, has the function similar to this family member.

HDGF is the heparin-binding protein that is separated to from people's hepatoma cell strain HuH-7, and it has the activity of stimulate cell growth, can promote the growth (J.Biol.Chem.269 (40): 25143-25149,1994) of inoblast and some liver cancer cells.The fluorescence immunoassay experiment shows that HDGF albumen mainly is present in tenuigenin (J.Biol.Chem.269 (40): 25143-25149,1994), but all contain one section alkalescence zone-potential nuclear localization signal (NLS) in this family member's the aminoacid sequence.Fibroblast growth factor (FGF) must rely on this segment signal zone, makes it to be positioned to examine interior and brings into play the activity of its mitogen.The mitogen activity of HDGF makes it exist great using value (Clin.Chim.Acta.183:273-284,1989) in the treatment of anxious pernicious hepatitis and liver injury.Studies show that, immune serum stimulated after 8-24 hour, HDGF has high expression level in the smooth muscle cell (SMCs) that increases fast, exophytic and endogenous high expression level HDGF can stimulate a large amount of propagation of SMCs and a large amount of of DNA thereof to synthesize, simultaneously, at the aortic SMCs of rat fetus on the 19th (becoming in the mouse does not have), become mouse aorta abdominalis contraction place SMCs all can detect the HDGF of natural type, this hint HDGF can promote its cell growth when SMCs growth and vascular injury, play opsonization (J.Clin.Invest105 (5): 567-575., 2000).In addition, the acidic amino acid tail of the C of HDGF end and the HMG-1/-2 of HMG family height homology, and this section sequence known in HMG-1/-2 be histone calmodulin binding domain CaM (Biochemistry 29:4419-4423,1990).Comprehensive above-mentioned phenomenon thinks that HDGF may bring into play the activity (Biochem.Biophys.Res.Commun.238:26-32,1997) of its stimulate cell growth as transcription factor after internalization.HDGF3 of the present invention also may have similar activity.

Although HDGF is found in liver cancer cell at first, the Northern hybridization analysis shows that it all has expression (J.Biol.Chem.269 (40): 25143-25149,1994) in each tissue such as people's the heart, brain, lung, liver and various JEG-3.HDGF has the very enrichment of high level in testis, and their 5 ' non-translational region all has and is higher than 70% GC than (Biochem.Biophys.Res.Commun.238:26-32,1997), thereby it may have critical function (J.Cell.Biol.115:887-903,1990 in male sex-cell is grown; Cell 62:503-514,1990).In situ hybridization shows, the kidney etap can detect the mRNA of the profuse HDGF of content in nephron formation place, and content seldom thereby thinks that HDGF may play mitogen effect (J.ClinInvest 15:102 (6) in the kidney growth course in adult's kidney; 1208-19,1998).In addition, after mouse bronchial was pricked knot, the HDGF expression amount obviously increased, hint HDGF relevant with the growth and maturity of lung (J.Pediatr Surg35 (1): 113-8,2000).HDGF3 of the present invention also may have similar functions.

In all known HDGF family members, the proteic homology degree of the present invention and mouse HRP-2 is the highest, and except the general character of HDGF family, mouse HRP-2 albumen also has the characteristics of itself.Mouse HRP-2 albumen is rich in Serine (Ser) and proline(Pro) (Pro) (being respectively 8.5% and 12.4%), and this points out this albumen may have the effect of phosphokinase.This proteic potential nuclear localization signal (aminoacid sequence 321-363 position sees attached list 3) is longer, and wherein acid and basic aminoacids content is all very high, and (.Lys+Arg:20.6% Glu+Asp:19.7%), may have the effect of nucleoprotein.Northern is hybridized demonstration, mouse HRP-2 all has expression in tissues such as the heart, liver, lung, spleen, kidney, moderate is expressed in liver, high expression level in testis, its 5 '-non-translational region has 83% GC than (Biochem.Biophys.Res.Commun., 238:26-32,1997), these characteristics are similar with the gene of some specifically expressings in spermary or fetal development, thereby it may have critical function in male sex-cell is grown.People HDGF3 of the present invention and mouse HRP-2 have 78.2% identity on nucleic acid level, 82.8% identity is arranged on amino acid levels and 6% amino acid similarity is arranged, aminoterminal highly conserved sequence unanimity, nuclear localization signal corresponding position amino acid consistence is 90.7% (seeing Table 3), similarity is 7%, Ser and Pro content also higher (being respectively 12.7% and 7.5%), thereby think that the present invention has the function with mouse HRP-2 protein similar.

HDGF3 of the present invention also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, HDGF3 of the present invention can also merge or exchange fragment with other members of this family, to produce new albumen, exchange with people's other HDGF and the N end of mouse HDGF as N end, to produce the albumen that new activity is higher or have new features with HDGF3 of the present invention.

At the antibody of HDGF3 of the present invention, 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 HDGF3 in intestinal bacteria

In this embodiment, use PCR Oligonucleolide primers to increase the cDNA sequence of coding HDGF3 corresponding to 5 ' and 3 ' of this dna sequence dna-end.

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

E1：5’-CGCCGGATCCATGCCACACGCCTTCAAGC-3’(SEQ?ID?No．7)

This primer contains the restriction enzyme site of BamHI restriction enzyme, is 19 Nucleotide of the HDGF3 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

E2：5’-TGCCAAGCTTTCAGCTCTCCTCGTCCAGG-3’(SEQ?ID?No．8)

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the HDGF3 of HindIII restriction enzyme.

The restriction enzyme site of restriction enzyme BamHI, HindIII 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 mixture, connect subsequently.Transform available from Qiagen with connecting mixture again, 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 cuts with the SalI enzyme and to identify and insert clip size and direction, and the cDNA fragment of sequence verification HDGF3 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 HDGF3 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out HDGF3 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 to containing 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 75kDa.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID No.6 with ordinary method.

Embodiment 4

The expression of HDGF3 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 HDGF3, obtain HDGF3 cDNA as inserting fragment corresponding to this dna sequence dna.

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

C1：5’-CGGCAAGCTTATGCCACACGCCTTCAAGC-3’(SEQ?ID?No．9)

This primer contains the restriction enzyme site of HindIII restriction enzyme, is 19 Nucleotide of the HDGF3 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

C2：5’-TGCCGGATCCTCAGCTCTCCTCGTCCAGG-3’(SEQ?ID?No．10)

This primer contains the part encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and HDGF3.

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 and 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 cuts evaluation with the SalI enzyme and insert clip size and direction, and the cDNA fragment of sequence verification HDGF3 has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to use lipofection, and (GiBco Life) carries out with the Lipofectin 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 75kDa.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID No.6 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 molecule 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 HDGF3 gene translation product with it.The code name that the present invention relates to is described as follows:

1.SEQ the information of ID No.1

(ⅰ) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅲ) sequence description: SEQ ID No.1

TCAGCATGCC?ACACGCCTTC?AAG?????????????23

2.SEQ the information of ID No.2

(ⅰ) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅲ) sequence description: SEQ ID No.2

CTTCACAGAC?ACATCGGAGT?CGG???????????????????23

3.SEQ the information of ID No.3

(ⅰ) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅲ) sequence description: SEQ ID No.3

GACTCCGACT?CCAAGGCCGA?TTC????????????????23

4.SEQ the information of ID No.4

(ⅰ) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅲ) sequence description: SEQ ID No.4

TCTCTGCTCT?GCTTTCCTGC?GAG????????????????23

5.SEQ the information of ID No.5:

(ⅰ) sequence signature

(A) length: 2118bp

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: cDNA

( ⅲ ) ：SEQ ID No．51 TCAGCATGCC ACACGCCTTC AAGCCCGGGG ACTTGGTGTT CGCTAAGATG AAGGGCTACC61 CTCACTGGCC TGCCAGGATC GACGACATCG CGGATGGCGC CGTGAAGCCC CCACCCAACA121 AGTACCCCAT CTTTTTCTTT GGCACACACG AAACAGCCTT CCTGGGACCC AAGGACCTGT181 TCCCCTACGA CAAATGTAAA GACAAGTACG GGAAGCCCAA CAAGAGGAAA GGCTTCAATG241 AAGGGCTGTG GGAGATCCAG AACAACCCCC ACGCCAGCTA CAGCGCCCCT CCGCCAGTGA301 GCTCCTCCGA CAGCGAGGCC CCCGAGGCCA ACCCCGCCGA CGGCAGTGAC GCTGACGAGG361 ACGATGAGGA CCGGGGGGTC ATGGCCGTCA CAGCGGTAAC CGCCACAGCT GCCAGCGACA421 GGATGGAGAG CGACTCAGAC TCAGACAAGA GTAGCGACAA CAGTGGCCTG AAGAGGAAGA481 CGCCTGCGCT AAAGATGTCG GTCTCGAAAC GAGCCCGAAA GGCCTCCAGC GACCTGGATC541 AGGCCAGCGT GTCCCCATCC GAAGAGGAGA ACTCGGAAAG CTCATCTGAG TCGGAGAAGA601 CCAGCGACCA GGACTTCACA CCTGAGAAGA AAGCAGCGGT CCGGGCGCCA CGGAGGGGCC661 CTCTGGGGGG ACGGAAAAAA AAGAAGGCGC CATCAGCCTC CGACTCCGAC TCCAAGGCCG721 ATTCGGACGG GGCCAAGCCT GAGCCGGTGG CCATGGCGCG GTCGGCGTCC TCCTCCTCCT781 CTTCCTCCTC CTCCTCCGAC TCCGATGTGT CTGTGAAGAA GCCTCCGAGG GGCAGGAAGC841 CAGCGGAGAA GCCTCTCCCG AAGCCGCGAG GGCGGAAACC GAAGCCTGAA CGGCCTCCGT901 CCAGCTCCAG CAGTGACAGT GACAGCGACG AGGTGGACCG CATCAGTGAG TGGAAGCGGC961 GGGACGAGGC GCGGAGGCGC GAGCTGGAGG CCCGGCGGCG GCGAGAGCAG GAGGAGGAGC1021 TGCGGCGCCT GCGGGAGCAG GAGAAGGAGG AGAAGGAGCG GAGGCGCGAG CGGGCCGACC1081 GCGGGGAGGC TGAGCGGGGC AGCGGCGGCA GCAGCGGGGA CGAGCTCAGG GAGGACGATG1141 AGCCCGTCAA GAAGCGGGGA CGCAAGGGCC GGGGCCGGGG TCCCCCGTCC TCCTCTGACT1201 CCGAGCCCGA GGCCGAGCTG GAGAGAGAGG CCAAGAAATC AGCGAAGAAG CCGCAGTCCT1261 CAAGCACAGA GCCCGCCAGG AAACCTGGCC AGAAGGAGAA GAGAGTGCGG CCCGAGGAGA1321 AGCAACAAGC CAAGCCCGTG AAGGTGGAGC GGACCCGGAA GCGGTCCGAG GGCTTCTCGA1381 TGGACAGGAA GGTAGAGAAG AAGAAAGAGC CCTCCGTGGA GGAGAAGCTG CAGAAGCTGC1441 ACAGTGAGAT CAAGTTTGCC CTAAAGGTCG ACAGCCCGGA CGTGAAGAGG TGCCTGAATG1501 CCCTAGAGGA GCTGGGAACC CTGCAGGTGA CCTCTCAGAT CCTCCAGAAG AACACAGACG1561 TGGTGGCCAC CTTGAAGAAG ATTCGCCGTT ACAAAGCGAA CAAGGACGTA ATGGAGAAGG1621 CAGCAGAAGT CTATACCCGG CTCAAGTCGC GGGTCCTCGG CCCAAAGATC GAGGCGGTGC1681 AGAAAGTGAA CAAGGCTGGG ATGGAGAAGG AGAAGGCCGA GGAGAAGCTG GCCGGGGAGG1741 AGCTGGCCGG GGAGGAGCTG GCCGGGGAGG AGGCCCCCCA GGAGAAGGCG GAGGACAAGC1801 CCAGCACCGA TCTCTCAGCC CCAGTGAATG GCGAGGCCAC ATCACAGAAG GGGGAGAGCG1861 CAGAGGACAA GGAGCACGAG GAGGGTCGGG ACTCGGAGGA GGGGCCAAGG TGTGGCTCCT1921 CTGAAGACCT GCACGACAGC GTACGGGAGG GTCCCGACCT GGACAGGCCT GGGAGCGACC1981 GGCAGGAGCG CGAGAGGGCA CGGGGGGACT CGGAGGCCCT GGACGAGGAG AGCTGAGCCG2041 CGGGCAGCCA GGCCCAGCCC CCGCCCGAGC TCAGGCTGCC CCTCTCCTTC CCCGGCTCGC2101 AGGAGAGCAG AGCAGAGA6．SEQ ID No．6： ( ⅰ )

(A) length: 676 amino acid

(B) type: amino acid

( D ) ： ( ⅱ ) ： ( ⅲ ) ：SEQ ID No．61 Met Pro His Ala Phe Lys Pro Gly Asp Leu Val Phe Ala Lys Met16 Lys Gly Tyr Pro His Trp Pro Ala Arg Ile Asp Asp Ile Ala Asp31 Gly Ala Val Lys Pro Pro Pro Asn Lys Tyr Pro Ile Phe Phe Phe46 Gly Thr His Glu Thr Ala Phe Leu Gly Pro Lys Asp Leu Phe Pro61 Tyr Asp Lys Cys Lys Asp Lys Tyr Gly Lys Pro Asn Lys Arg Lys76 Gly Phe Asn Glu Gly Leu Trp Glu Ile Gln Ash Asn Pro His Ala91 Ser Tyr Ser Ala Pro Pro Pro Val Ser Ser Ser Asp Ser Glu Ala106 Pro Glu Ala Asn Pro Ala Asp Gly Ser Asp Ala Asp Glu Asp Asp121 Glu Asp Arg Gly Val Met Ala Val Thr Ala Val Thr Ala Thr Ala136 Ala Ser Asp Arg Met Glu Ser Asp Ser Asp Ser Asp Lys Ser Ser151 Asp Asn Ser Gly Leu Lys Arg Lys Thr Pro Ala Leu Lys Met Ser166 Val Ser Lys Arg Ala Arg Lys Ala Ser Ser Asp Leu Asp Gln Ala181 Ser Val Ser Pro Ser Glu Glu Glu Ash Ser Glu Ser Ser Ser Glu196 Ser Glu Lys Thr Ser Asp Gln Asp Phe Thr Pro G1u Lys Lys Ala211 Ala Val Arg Ala Pro Arg Arg Gly Pro Leu Gly Gly Arg Lys Lys226 Lys Lys Ala Pro Ser Ala Ser Asp Ser Asp Ser Lys Ala Asp Ser241 Asp Gly Ala Lys Pro Glu Pro Val Ala Met Ala Arg Ser Ala Ser256 Ser Ser Ser Ser Ser Ser Ser Ser Ser Asp Ser Asp Val Ser Val271 Lys Lys Pro Pro Arg Gly Arg Lys Pro Ala Glu Lys Pro Leu Pro286 Lys Pro Arg Gly Arg Lys Pro Lys Pro Glu Arg Pro Pro Ser Ser301 Ser Ser Ser Asp Ser Asp Ser Asp Glu Val Asp Arg Ile Ser Glu316 Trp Lys Arg Arg Asp Glu Ala Arg Arg Arg Glu Leu Glu Ala Arg331 Arg Arg Arg Glu Gln Glu Glu Glu Leu Arg Arg Leu Arg Glu Gln346 Glu Lys Glu Glu Lys Glu Arg Arg Arg Glu Arg Ala Asp Arg Gly361 Glu Ala Glu Arg Gly Ser Gly Gly Ser Ser Gly Asp Glu Leu Arg376 Glu Asp Asp Glu Pro Val Lys Lys Arg Gly Arg Lys Gly Arg Gly391 Arg Gly Pro Pro Ser Ser Ser Asp Ser Glu Pro Glu Ala Glu Leu406 Glu Arg Glu Ala Lys Lys Ser Ala Lys Lys Pro Gln Ser Ser Ser421 Thr Glu Pro Ala Arg Lys Pro Gly Gln Lys Glu Lys Arg Val Arg436 Pro Glu Glu Lys Gln Gln Ala Lys Pro Val Lys Val Glu Arg Thr451 Arg Lys Arg Ser Glu Gly Phe Ser Met Asp Arg Lys Val Glu Lys466 Lys Lys Glu Pro Ser Val Glu Glu Lys Leu Gln Lys Leu His Ser481 Glu Ile Lys Phe Ala Leu Lys Val Asp Ser Pro Asp Val Lys Arg496 Cys Leu Asn Ala Leu Glu Glu Leu Gly Thr Leu Gln Val Thr Ser511 Gln Ile Leu Gln Lys Asn Thr Asp Val Val Ala Thr Leu Lys Lys526 Ile Arg Arg Tyr Lys Ala Asn Lys Asp Val Met Glu Lys Ala Ala541 Glu Val Tyr Thr Arg Leu Lys Ser Arg Val Leu Gly Pro Lys Ile556 Glu Ala Val Gln Lys Val Asn Lys Ala Gly Met Glu Lys Glu Lys571 Ala Glu Glu Lys Leu Ala Gly Glu Glu Leu Ala Gly Glu Glu Leu586 Ala Gly Glu Glu Ala Pro Gln Glu Lys Ala Glu Asp Lys Pro Ser601 Thr Asp Leu Ser Ala Pro Val Asn Gly Glu Ala Thr Ser Gln Lys616 Gly Glu Ser Ala Glu Asp Lys Glu His Glu Glu Gly Arg Asp Ser631 Glu Glu Gly Pro Arg Cys Gly Ser Ser Glu Asp Leu His Asp Ser646 Val Arg Glu Gly Pro Asp Leu Asp Arg Pro Gly Ser Asp Arg Gln661 Glu Arg Glu Arg Ala Arg Gly Asp Ser Glu Ala Leu Asp Glu Glu676 Ser7．SEQ ID No．7 ( ⅰ )

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅲ) sequence description: SEQ ID No.7

Information (ⅰ) sequence signature of CGCCGGATCC ATGCCACACG CCTTCAAGC 298.SEQ ID No.8

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅲ) sequence description: SEQ ID No.8

Information (ⅰ) sequence signature of TGCCAAGCTT TCAGCTCTCC TCGTCCAGG 299.SEQ ID No.9

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅲ) sequence description: information (ⅰ) sequence signature of SEQ ID No.9 CGGCAAGCTT ATGCCACACG CCTTCAAGC 2910.SEQ ID No.10

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅲ) sequence description: SEQ ID No.10

It is as follows that TGCCGGATCC TCAGCTCTCC TCGTCCAGG 29 the present invention relates to form:

The replacement of table 1. conservative property variation polypeptide amino acid

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

Table 2. people HDGF3 and mouse HRP-2 nucleic acid homology are relatively

Annotate: people HDGF3 and mouse HRP-2 nucleic acid identity are 78.2%.

Table 3. people HDGF3 and mouse HRP-2 amino acid identity are relatively

Annotate: people HDGF3 and mouse HRP-2 amino acid identity are 82.8%.

Table 4. human hepatoma-derived growth factor 3 compares human hepatoma-derived growth factor 3 albumen mpha-----fkpgdlvfakmkgyphwpariddiadgavkpppnkypifff 45 mouse HRP-2 albumen mpha-----fkpgdlvfakmkgyphwpariddiadgavkpppnkypifff 45 human hepatoma-derived growth factor albumen msrsnrqkeykcgdlvfakmkgyphwparidempeaavkstankyqvfff 50 mouse HDGF albumen msrsnrqkeykcgdlvfakmkgyphwparidempeaavkstankyqvfff 50 with HDGF family member amino terminal characteristic sequence

* ..* * * * * * * * .***.*******.....*** * .*..*** human hepatoma-derived growth factor 3 albumen gthetaflgpkdlfpydkckdkygkpnkrkgfneglweiqnnphasysap 95 mouse HRP-2 albumen gthetaflgpkdlfpydkckdkygkpnkrkgfneglweiqnnphasysap 95 human hepatoma-derived growth factor albumen gthetaflgpkdlfpyeeskekfgkpnkrkgfseglweiennp-------93 mouse HDGF albumen gthetaflgpkdlfpyeeskekfgkpnkrkgfseglwiennp--------93

* * * * * * * * * * * * * * ..*.*.*********.******.*** annotates: people HDGF3 and HDGF family member aminoacid sequence N-terminal characteristic sequence comparison identity are 62.4%.

Claims (11)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people HDGF3 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 6-2033 position among described nucleotide sequence and the SEQ ID No.5; Perhaps described nucleotide sequence can be under the rigorous condition of moderate with SEQ ID No.5 in from the nucleotide sequence hybridization of Nucleotide 6-2033 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.6.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has the sequence of Nucleotide 6-2033 position among the SEQ ID No.5.

4. isolating HDGF3 protein polypeptide is characterized in that it comprises: have polypeptide or its active fragments of SEQ ID No.6 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.6 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, it is characterized in that it comprises prokaryotic cell prokaryocyte and eukaryotic cell.

8. a generation has the method for the polypeptide of HDGF3 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of HDGF3 protein-active operationally is connected in expression regulation sequence, form the HDGF3 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 6-2033 position among described nucleotide sequence and the SEQ ID No.5;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of HDGF3;

(c) be fit to express under the condition of HDGF3 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with HDGF3 protein-active.

9. energy and HDGF3 protein polypeptide specificity bonded antibody is characterized in that it comprises monoclonal antibody and polyclonal antibody.

10. antibody as claimed in claim 9 is characterized in that, also comprises having immunogenic antibody plasmid heavy chain of antibody, light chain of antibody, genetically engineered Fv molecule and chimeric antibody.

11. a probe molecule is characterized in that, it contains about 8-100 continuous nucleotide in the dna molecular with HDGF3 polypeptid coding sequence.