CN1246532A - Coding sequence of human heat shock associated protein, its encoded polypeptide and its preparing process - Google Patents

Abstract

The present invention relates to a new nucleotide sequence of human gene, especially the cDNA coding sequence of HSC3 as a new member of heat shock associated protein family. The polypeptide coded by said cDNA sequence, the method for preparing said polynucleotide sequence and said polypeptide, and the application of the polynucleotide and polypeptide are disclosed.

Description

Human heat shock associated protein 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 newcomer's of heat shock associated protein family cDNA encoding sequence.The invention still further relates to the production method of the polypeptide of this cDNA sequence encoding, described polynucleotide sequence and described polypeptide and the application of these polynucleotide and polypeptide.

Heat shock protein(HSP) (hsp) is cell or organism under such as environmental stresss such as heat-shocked, chemical substances, a synthetic proteinoid of being induced or increasing.It should be noted that the type of pressure and the situation of replying that severity is determining hsp, the result causes cell survival or death.Serious pressure after slight pressure can make cell get ready to tackle, and the cell of not getting ready can not be survived usually.According to molecular weight and amino-acid sequence homology degree, hsp can be divided into different families, and hsp70 family is exactly one of them.

The albumen of this family is not only induced under pressure, and some are called as heat shock associated protein (hsc) member and go back constitutive expression, and this shows that they not only work at crucial moment, and also works under normal operation.The nearest proteinic Metabolic activity of this proteinoid wide participation of discovering comprises Protein Folding, strides the transportation of cell inner membrance, proteolysis, albumen assembling, and albumen aggregate and polyprotein structure separate assembling.DnaJ in the intestinal bacteria, DnaK just belong to this proteinoid, and the activity that DnaJ regulates DnaK as regulatory factor mainly is to stimulate the ATP enzyme of DnaK to live, and makes generate energy be used for metabolism activities such as protein folding.

Studies show that in recent years, also existence and DnaJ, DnaK homologous protein family in the eukaryotic cell.(J.Cell.Biol.109 (1989) 2665-1675) has described in the yeast and DnaJ homologous Protein S ec63 people such as Sadler in 1989 first.Protein S ec63 contains one section similar 70 the amino acid whose zone of N end to DnaJ, and this protein participates in albumen is transported to the process of endoplasmic reticulum.After this, from yeast, be isolated and cloned into some DnaJ homologous proteins.People (Biochem.J.284 (1992) such as Cheetham in 1992,469-476) scan human prefrontal cortex and express the cDNA storehouse with the fibrinous polyclonal antiserum of duplex, the DnaJ homology cDNA that at first clones humans, HSJ1, the amino-acid sequence of deriving is very similar to the albumen in other species.Coming years has HDJ1 again, HDJ2, and HSJ1-b etc. are cloned with DnaJ homologous human protein.But before the present invention came forth, still nobody was openly crossed the human HSC newcomer of family who the present invention relates to.

An object of the present invention is to provide a kind of new people's polynucleotide, a newcomer of this polynucleotide encoding heat shock associated protein family, heat shock associated protein homologue called after HSC3 of the present invention.

Another object of the present invention provides a kind of new human heat shock associated protein family member, and it is named as HSC3.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new human heat shock associated protein family member.

The invention still further relates to the application of HSC3 polypeptide and encoding sequence.

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 HSC3 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 108-1037 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 108-1037 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 108-1037 position.

In another aspect of this invention, provide a kind of isolating HSC3 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 polypeptide of HSC3 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of HSC3 protein-active operationally is connected in expression regulation sequence, form the HSC3 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 108-1037 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 proteic reconstitution cell of HSC3;

(c) be fit to express under the condition of HSC3 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with HSC3 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1203 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 108-1037 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 " HSC3 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with HSC3 protein-active is as 108-1037 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 108-1037 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 108-1037 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 108-1037 position.This term also comprise with SEQ ID NO.3 in from Nucleotide 108-1037 position homology at least 70%, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.3 sequence of people HSC3 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 " HSC3 protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of HSC3 protein-active.This term also comprises having and the variant form human heat shock associated protein 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 (being generally in 20, preferably is in 10, more preferably is in 5) 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 HSC3 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 HSC3 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-HSC3 polypeptide to obtain.The present invention also provides other polypeptide, as comprises HSC3 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of HSC3 polypeptide.Usually, this fragment have the HSC3 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 HSC3 albumen or polypeptide.The difference of these analogues and natural HSC3 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 HSC3 polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of HSC3 in the cell.

The present invention also comprises a kind of probe molecule, and this molecule has 8-100 of HSC3 polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the HSC3 that encodes.

The present invention also comprises the method that detects the HSC3 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 HSC3 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 various carriers.

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 HSC3 DNA or the polypeptide of its fragment coding has specific polyclone and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into HSC3 gene product or fragment.Preferably, refer to that those can combine with HSC3 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 HSC3, comprise that also those do not influence the antibody of HSC3 protein function.The present invention also comprise those can with modify or without the HSC3 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 HSC3 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing HSC3 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 Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the HSC3 function and the antibody that does not influence the HSC3 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of HSC3 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.With the unmodified form bonded antibody of HSC3 gene product, can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli); 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).

In the present invention, the cDNA nucleotide sequence of HSC3 is so to obtain: with people's testis λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is that primer A1:5 '-TACCTATTTGCAGTCACTACCTC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B1:5 '-ATCCTTTTCTGACATACCCATTC-3 ' (SEQ ID NO.2) is a reverse primer, carries out PCR.Electrophoresis detection obtains the purpose fragment of about 1.2kb.After identifying order-checking, obtain the full length cDNA sequence of SEQ ID NO.3.

Find by the homology retrieval, the cDNA sequence of HSC3 of the present invention and the heat shock associated protein gene order height homology of having been delivered and having confirmed as, and HSC3 albumen of the present invention also with known heat shock associated protein (as the heat shock associated protein of mouse) height homology.In addition, HSC3 albumen of the present invention also has the aminoacid sequence of heat shock associated protein family high conservative.Therefore, this shows that the same with heat shock associated protein family members such as known mouse heat shock associated proteins, HSC3 albumen of the present invention also belongs to heat shock associated protein family, and has similar function.

Many chronic inflammatory diseasess are caused by the antigen activates immunity system, and a series of subsequent reaction can be induced expression of heat shock, and these albumen protect cell and tissue to avoid the harmful effect of inflammation conversely.Existing sign shows; some defencive functions of heat shock protein(HSP)s such as hsp70 may depend on the normal cell function of heat shock protein(HSP), promptly accompany sex change or folding incorrect albumen and follow them to pass function of plasma membrane (Experienntia (1994) 50:1031-1038).There is experiment to show that the member of hsp70 family can improve antigen processing and submission, thereby makes immunne response more effective (Int.Immun.6 (1994) 925-930).This all illustrates the provide protection of hsp70 in inflammation, and hsp70 also is essential for impaired proteic renaturation in the eukaryotic cell.The DnaJ major function is the function of assisting hsp70 and homologous protein thereof, for example protein folding, albumen assembling etc.Because with the DnaJ albumen homology, therefore HSC3 of the present invention also has the function that is similar to DnaJ in the protein metabolism process, particularly may play a protective role in inflammatory reaction.

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 HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone of the cDNA of HSC3 and order-checking

1.PCR amplification

With people's testis λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is that primer A1:5 '-TACCTATTTGCAGTCACTACCTC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B1:5 '-ATCCTTTTCTGACATACCCATTC-3 ' (SEQ ID NO.2) is a reverse primer, carries out PCR.The PCR condition of A1/B1 be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 62 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.Electrophoresis detection obtains the purpose fragment of about 1.2kbd.

2.PCR the order-checking of product

With pcr amplification product and the pGEM-T that obtains in the step 1 ^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 1203bp, detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 108-1037 position Nucleotide.

Derive the aminoacid sequence of HSC3 according to the full-length gene group sequence that obtains, totally 309 amino-acid residues, its aminoacid sequence sees SEQ ID NO:4 for details.

Embodiment 2

The HSC3 homology relatively

Full-length cDNA of the present invention carries out Basic BLAST homology relatively in proper order, the database that adopts is the GenBank+EMBL+DDBJ+PDB of nonredundancy, found that (database numbering and title: gb|AF03596|MRJ) the homology degree is the highest, and nucleotide sequence is 90% with corresponding MRJ similarity with the heat shock associated protein of mouse.

With PCGENE software full-length cDNA of the present invention is translated into the protein order in proper order, ORF is positioned at 108-1037 position Nucleotide, 309 the amino acid whose polypeptide of a segment length of encoding.Then the amino-acid sequence that obtains is carried out Basic BLAST homology relatively, the database that adopts is that the GenBank translation CDS+PDB+SwissPort+ of nonredundancy upgrades SP+PIR, found that (database numbering and title: gi|3142372|MRJ) the homology degree is the highest with the heat shock associated protein of mouse, 68 amino-acid residues 98% of aminoterminal are identical, / 4th orders of carboxyl terminal also have 96% similar, and 92% is identical.

Hsp70 is a class heat shock protein(HSP), and the basic mechanism of its effect is the polypeptide of combination under the mode of dependency ATP, release non-natural structure picture.Discover that hsp70 does not play a role separately, need the other for example participation effect of DnaJ of heat shock protein(HSP), DnaJ regulates the activity of hsp70 as regulatory factor.Intestinal bacteria DnaJ major function feature is directly to combine with hsp70, stimulates ATP enzyme (Trends.Biochem.Sci.19 (1994), the 20-25 alive of hsp70; Trends.Bochem.Sci.17 (1992), 295-299).Some and intestinal bacteria DnaJ homologous gene in yeast, plant, the mankind, have been identified.

Heredity shows eukaryote DnaJ albuminoid relevant (Cell71 (1992), 1143-1155 with intestinal bacteria DnaJ function with biochemical test; J.Biol.Chem.267 (1992), 20927-20931).The amino-acid sequence that compares the DnaJ family member, find that all DnaJ albuminoids contain the J structural domain, this be one section corresponding to 70 amino acid whose one section orders of intestinal bacteria DnaJ aminoterminal, and DnaJ combine and regulate with hsp70 its ATP activity probably by this structural domain play a role (Trends.Biochem.Sci.19 (1994), 176-181).68 amino-acid residues of HSC3 aminoterminal of the present invention and colibacillary J structural domain similarity reach 81%, show that HSC3 also has the J structural domain, belong to the DnaJ albuminoid, are the newcomers of heat shock associated protein family.

In different biologies, it is movable that the DnaJ albuminoid participates in the born of the same parents directly or indirectly.The activity that it regulates hsp70 as regulatory factor, just by J structural domain identification hsp70 bring into play function (Trends.Biochem.Sci.19 (1994), 176-181).HSC3 of the present invention particularly as the DnaJ albuminoid, also has the function of regulating hsp70 as the newcomer of heat shock associated protein family, and is closely related with proteinic Metabolic activity.

Many chronic inflammatory diseasess are caused by the antigen activates immunity system, and what wherein mainly be activated is monocyte, scavenger cell.These phagocytic cells produce a large amount of reactive oxygen species (ROS) and cytokine, thereby regulate the proteic expression of HSP, and these albumen protect cell and tissue to avoid the harmful effect of inflammation conversely.The mechanism of protection comprises dna break, the lipid peroxidation that hinders the ROS initiation.Existing sign shows that some defencive functions of heat shock protein(HSP)s such as hsp70 may depend on the normal cell function of heat shock protein(HSP), promptly accompanies sex change or folding incorrect albumen and follows them to pass cytolemma (Experienntia, 1038).Report is arranged, and some members of hsp70 family participate in the different step (J.Expl.Med. (1989) 170:1799-1803) of antigen presentation and processing.Someone proposes the assembling that HSP not only participates in the main II of histocompatibility complex, also participate in antigen presentation (Critical.Rev.Immum.13 (1993) subsequently, 71-81), existing experiment shows that the member of hsp70 family can improve antigen processing and submission, thereby make immunne response more effective (Int.Jmmun.6 (1994), 925-930).This all illustrates the provide protection of hsp70 in inflammation, and hsp70 also is essential for impaired proteic renaturation in the eukaryotic cell.With homologous DnaJ protein similar (J.Biol.Chem.271 (1996), 11236-11246), HSC3 albumen major function of the present invention may be the function of assisting hsp70 or homologous protein, comprise Protein Folding, stride the transportation of cell inner membrance, proteolysis, albumen assembling, and albumen aggregate and polyprotein structure separate assembling.This prompting the present invention plays a part similar DnaJ in the protein metabolism process, particularly may play a protective role in inflammatory reaction.

Embodiment 3

The expression of HSC3 in intestinal bacteria

In this embodiment, the dna sequence dna of coding HSC3 uses the PCR Oligonucleolide primers corresponding to 5 of this dna sequence dna ' and 3 ' end to increase, and inserts fragment to obtain HSC3.

PCR reaction 5 ' Oligonucleolide primers sequence is:

5′-GGAAGTCGAC?ATGGTGGATT?ACTATGAAG-3′(SEQ?ID?NO.5)

This primer contains the restriction enzyme site of the restricted restriction enzyme of SalI, is 19 Nucleotide of the HSC3 encoding sequence that begun by initiator codon after restriction enzyme site;

3 ' end primer sequence is:

5’-GTGCTGCAGT?TAACAATTCC?TTTTGGTAG-3′(SEQ?ID?NO.6)

This primer contains the encoding sequence of the restriction enzyme site of the restricted restriction enzyme of PstI, translation termination and HSC3.

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

With SalI and PstI 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 HSC3 has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution is inoculated in the large volume substratum culturing cell to 600 optical density(OD) (OD then ₆₀₀) be 0.4-0.6, add subsequently 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 HSC3 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out HSC3 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein from nickel-chelate column.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.

Embodiment 4

The expression of HSC3 in eukaryotic cell (Chinese hamster ovary celI strain)

With reference to embodiment 3, the dna sequence dna of coding HSC3 uses the PCR Oligonucleolide primers corresponding to 5 of this dna sequence dna ' and 3 ' end to increase, with synthetic HSC3 gene as inserting fragment.

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

5′-GCAACTGCAG?ATGGTGGATT?ACTATGAAG-3′(SEQ?ID?NO.7)

This primer contains the restriction enzyme site of the restricted restriction enzyme of PstI, and what connect is 19 Nucleotide of the HSC3 encoding sequence that begun by initiator codon;

3 ' end primer sequence is:

5’-TCGGATATCT?TAACAATTCC?TTTTGGTAG-3’(SEQ?ID?NO.8)

This primer contains the encoding sequence of the restriction enzyme site of the restricted restriction enzyme of EcoRV, translation termination and HSC3.

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 (Invitrogen company), this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (f1 ori), a virus replication starting point (SV40 ori), a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and corresponding polyA order.

With PstI and EcoRV digestion pcDNA3 carrier and insertion segment, 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 BamHI enzyme and insert clip size and direction, and the cDNA of sequence verification HSC correctly inserts carrier.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin test kit (GiBcolife).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mM oTrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mM TrisHCl (pH8.0) solution that contains 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 35kD.

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:4 with ordinary method.

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 3 or 4 is 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 proteic ability of external precipitation HSC3 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.

Information (i) sequence signature of sequence table (2) SEQ ID NO:1

(A) length: 23 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:1TACCTATTTG CAGTCACTAC CTC 23 (2) SEQ ID NO:2 is sequence signature (ii)

(A) length: 23 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:2ATCCTTTTCT GACATACCCA TTC 23 (2) SEQ ID NO:3: (i) sequence signature:

(A) length: 1203bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：3 1 TACCTATTTG CAGTCACTAC CTCTATCACC ACCACCAGCA GAGCCTGAGC TGAGGAAACC61 ACGGTTCTCA ATACCCAGCA CACCCACTTC CAACTATCTG TTAAAACATG GTGGATTACT 121 ATGAAGTTCT AGGACTGCAA AGATATGCTT CACCTGAGGA CATTAAAAAA GCTTATCATA 181 AAGTGGCACT TAAATGGCAC CCTGATAAAA ATCCAGAAAA TAAAGAAGAA GCAGAGAGAA 241 AATTCAAAGA AGTAGCTGAG GCATACGAGG TATTATCAAA TGATGAGAAA CGGGACATTT 301 ATGATAAATA TGGCACAGAA GGATTAAACG GAGGTGGAAG TCATTTTGAT GATGAATGTG 361 AGTACGGCTT CACATTCCAT AAGCCAGATG ATGTTTTTAA AGAAATTTTT CATGAAAGGG 421 ATCCATTTTC TTTTCACTTC TTTGAAGACT CGCTTGAGGA CCTGTTAAAT CGTCCAGGAA 481 GCTCCTATGG AAACAGAAAC AGAGATGCAG GATACTTTTT CTCCACTGCC AGTGAATATC 541 CAATTTTTGA GAAATTTTCT TCATATGATA CAGGATATAC ATCACAGGGT TCATTGGGGC 601 ATGAAGGCCT TACTTCTTTC TCTTCCCTGG CTTTTGATAA TAGTGGGATG GACAACTACA 661 TATCTGTTAC AACTTCAGAC AAAATCGTTA ATGGCAGAAA TATTAATACA AAGAAAATTA 721 TTGAAAGTGA TCAAGAAAGA GAAGCTGAAG ATAATGGAGA GTTGACATTT TTTCTTGTAA 781 ATAGTGTGGC CAATGAAGAG GGCTTTGCAA AAGAATGCAG CTGGAGAACA CAGTCATTCA 841 ACAACTATTC ACCAAATTCT CACAGCTCCA AACATGTATC TCAATATACT TTCGTGGACA 901 ATGATGAGGG AGGTATATCT TGGGTTACCA GCAACAGAGA TCCCCCTATT TTCTCAGCAG 961 GAGTCAAAGA GGGTGGTAAG AGGAAAAAAA AGAAGCGTAA AGAGGTGCAA AAGAAGTCTA1021 CCAAAAGGAA TTGTTAAATT GACTCTTCAA ATATATAACA TTTGAACACA ATTGTGTGTG1081 TTTTGGTTAA TCACAAATTT TGTAGATAAC ACTTAATACT ATACTAAGAG CTTTTCAACA1141 CTTTTAGCAG GATTGTGGAC ATTTGGTTAG TAGTTTTTTT GAATGGGTAT GTCAGAAAAG1201 GAT ( 2 ) SEQ ID NO：4：

(i) sequence signature:

(A) length: 309 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

( xi ) ：SEQ ID NO：4 1 Met Val Asp Tyr Tyr Glu Val Leu Gly Leu Gln Arg Tyr Ala Ser 16 Pro Glu Asp Ile Lys Lys Ala Tyr His Lys Val Ala Leu Lys Trp 31 His Pro Asp Lys Asn Pro Glu Asn Lys Glu Glu Ala Glu Arg Lys 46 Phe Lys Glu Val Ala Glu Ala Tyr Glu Val Leu Ser Asn Asp Glu 61 Lys Arg Asp Ile Tyr Asp Lys Tyr Gly Thr Glu Gly Leu Asn Gly 76 Gly Gly Ser His Phe Asp Asp Glu Cys Glu Tyr Gly Phe Thr Phe 91 His Lys Pro Asp Asp Val Phe Lys Glu Ile Phe His Glu Arg Asp106 Pro Phe Ser Phe His Phe Phe Glu Asp Ser Leu Glu Asp Leu Leu121 Asn Arg Pro Gly Ser Ser Tyr Gly Asn Arg Asn Arg Asp Ala Gly136 Tyr Phe Phe Ser Thr Ala Ser Glu Tyr Pro Ile Phe Glu Lys Phe151 Ser Ser Tyr Asp Thr Gly Tyr Thr Ser Gln Gly Ser Leu Gly His166 Glu Gly Leu Thr Ser Phe Ser Ser Leu Ala Phe Asp Asn Ser Gly181 Met Asp Asn Tyr Ile Ser Val Thr Thr Ser Asp Lys Ile Val Asn196 Gly Arg Asn Ile Asn Thr Lys Lys Ile Ile Glu Ser Asp Gln Glu211 Arg Glu Ala Glu Asp Asn Gly Glu Leu Thr Phe Phe Leu Val Asn226 Ser Val Ala Asn Glu Glu Gly Phe Ala Lys Glu Cys Ser Trp Arg241 Thr Gln Ser Phe Asn Asn Tyr Ser Pro Asn Ser His Ser Ser Lys256 His Val Ser Gln Tyr Thr Phe Val Asp Asn Asp Glu Gly Gly Ile271 Ser Trp Val Thr Ser Asn Arg Asp Pro Pro Ile Phe Ser Ala Gly286 Val Lys Glu Gly Gly Lys Arg Lys Lys Lys Lys Arg Lys Glu Val301 Gln Lys Lys Ser Thr Lys Arg Asn Cys

(2) information of SEQ ID NO:5

(i) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:5GGAAGTCGAC ATGGTGGATT ACTATGAAG 29

(2) information of SEQ ID NO:6

(i) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (iii): oligonucleotide

(xi) sequence description: information (i) sequence signature of SEQ ID NO:6GTGCTGCAGT TAACAATTCC TTTTGGTAG 29 (2) SEQ ID NO:7

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (iv): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:7GCAACTGCAG ATGGTGGATT ACTATGAAG 29 (2) SEQ ID NO:8

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (v) molecule type: oligonucleotide (xi) sequence description: SEQ ID NO:8TCGGATATCT TAACAATTCC TTTTGGTAG 29

Claims (14)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people HSC3 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 108-1037 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 108-1037 position.

2 dna moleculars 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 has the sequence of Nucleotide 108-1037 position among the SEQ ID NO.3.

4. isolating HSC3 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 polypeptide of HSC3 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of HSC3 protein-active operationally is connected in expression regulation sequence, form the HSC3 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 108-1037 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 proteic reconstitution cell of HSC3;

(c) be fit to express under the condition of HSC3 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with HSC3 protein-active.

11. method as claimed in claim 10 is characterized in that, this nucleotides sequence is classified as among the SEQ ID NO.3 from Nucleotide 108-1037 position.

12. energy and the described HSC3 protein polypeptide of claim 4 specificity 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.