CN1128878C - Coding sequence of human methionine sulfoxide reductase, its encoded polypeptide and its preparing process - Google Patents

Abstract

The present invention relates to a novel human methionine sulfoxide reductase msrA and provides the cDNA coding sequence of the novel methionine sulfoxide reductase, the polypeptide of the coding sequence and a method for producing the novel human methionine sulfoxide reductase by using a reorganization technology. The present invention also provides the application of the novel human methionine sulfoxide reductase.

Description

Human methionine sulfoxide reductase 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 human methionine sulfoxide reductase (msrA).The invention still further relates to the production method of described polynucleotide sequence and described polypeptide, and the production method of these polynucleotide and described polypeptide.

Methionine sulfoxide reductase has important effect aspect active keeping the organism internal protein, and it can be repaired methionine residues reduction oxidized in the protein, thereby avoids the protein inactivation that causes owing to methionine(Met) is oxidized.Inactivation that causes owing to the methionine(Met) oxidation in the known protein matter and multiple disease-related, these diseases comprise respiratory distress syndrome (ivrds), pulmonary emphysema, cataract and rheumatic arthritis etc. ( J.Clin.Invest.1983,71,754-761; Proc.Natl.Acad.Sci.U.S.A.1980,79,2041-2045; Biochim.Biophys.Acta.1977,492,43-52; Proc.Natl.Acad.Sci.U.S.A.1980,77,1274-1277; Biochem.Biophys.Res.Commun.1980,96,1449-1454).Rahman MA equals at first colibacillary methionine sulfoxide reductase (msrA) gene to be cloned in 1992, order-checking and express (Ragman MA, etc, J Biol Chem1992 Aug5; 267 (22): 15549-15551), and finished in 1994 this gene physical map location (Rahman MA, etc, J Bacteriol1994 Mar; 176 (5): 1548-15490).Nineteen ninety-five Brot N etc. also has been the clone of this gene, the experiment of high expression level and purification (Brot N, etc, Methods Enzymol1995; 251:462-470).The proteic homologues of intestinal bacteria msrA of from beef liver, having purified such as Moskovitz J in 1996, clone and expressed this proteic gene of coding, and the distribution (Moskovitz J, the etc that organize in the leach liquor the proteic mRNA of coding msrA have been detected mouse with the Northern analytical method Proc Natl Acad Sci USA1996 Mar 5; 93 (5): 2095-2099), in the same year, they have carried out the differentially expressed experiment of this gene in rat tissue with the immunochemistry blotting, and with this assignment of genes gene mapping in No. 14 karyomit(e) of mouse (Moskovitz J, etc, Proc Natl Acad Sci USA1996 Apr 16; 93 (8): 3205-3208).Yet before the present invention, the newcomer of the human msrA family that relates among the application was not disclosed.

An object of the present invention is to provide new polynucleotide, a newcomer of this polynucleotide encoding methionine sulfoxide reductase family, the homologue called after msrA of methionine sulfoxide reductase of the present invention family.

Another object of the present invention provides a kind of newcomer of new methionine sulfoxide reductase family.

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

The invention still further relates to this new methionine sulfoxide reductase gene order and the application of polypeptide.

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 msrA protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 61-768 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 61-768 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 61-768 position.

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

(a) nucleotide sequence that coding is had a polypeptide of msrA protein-active operationally is connected in expression regulation sequence, form the msrA protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 61-768 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 msrA;

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

(d) isolate polypeptide with msrA protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 853 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 61-768 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 " msrA albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with msrA protein-active is as 61-768 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 61-768 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 61-768 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 61-768 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 61-768 position, 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 msrA 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 " msrA protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of msrA protein-active.This term also comprises having and the variant form human methionine sulfoxide reductase 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 proteic active fragments of msrA 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 msrA DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-msrA polypeptide to obtain.The present invention also provides other polypeptide, as comprises msrA polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of msrA polypeptide.Usually, this fragment have the msrA 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 msrA albumen or polypeptide.The difference of these analogues and natural msrA 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 msrA polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of msrA in the cell.

The present invention also comprises a kind of probe molecule, and this molecule has 8-100 of msrA 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 msrA that encodes.

The present invention also comprises the method that detects the msrA 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 msrA 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 msrA DNA or the polypeptide of its fragment coding has specific polyclonal antibody or monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into msrA gene product or fragment.Preferably, refer to that those can combine with msrA 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 msrA, comprise that also those do not influence the antibody of msrA protein function.The present invention also comprise those can with modify or without the msrA 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, United States Patent (USP) NO.P, 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 msrA gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing msrA 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 prepare (see people such as Kohler, Nature256; 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 msrA function and the antibody that does not influence the msrA function.Each antibody-like of the present invention can utilize the fragment or the functional zone of msrA 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 msrA 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).

In the present invention, the cDNA nucleotide sequence of msrA is so to obtain: with human brain λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-CTGCGGCTCCGCTGCCGGTAG-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotide B1:5 '-CCACGATTGTGAGTCACACAAGC-3 ' (SEQ ID NO:2) is a reverse primer, carry out PCR, obtain 853 purpose fragment respectively.Identify the full length cDNA sequence that obtains SEQ ID NO:3 after checking order.

Cross the homology retrieval and find that the msrA gene of above-mentioned sequence and ox all has high homology on nucleic acid and protein level.

As everyone knows, methionine(Met) in the protein peptide chain can be a methionine sulfoxide by the catalysis of non-enzyme factor, these non-enzyme factors include the by product of oxygen metabolism---(Brot such as superoxide, hydroxyl, hypochlorite, N., and H.Weissbach.1988.Methionine sulfoxide:chemistry and biochemistry, p.851-872.In S.Patai and Z.Rappoport (ed.), The chemistry of sulphones and sulphoxides.John Wiley﹠amp; Sons, Inc., New York.).The oxidation of methionine(Met) can cause protein biological activity forfeiture (Brot, N., and Weissbach, H.1991, Biofactors3,91-96; Brot, N., and Weissbach, H.1983, Arch. Biochem.Biophys.223,271-283), thereby cause the generation of multiple disease, so certainly exist the enzyme that can repair these oxidized methionine residues in the organism.The so just class of enzymes of methionine sulfoxide reductase (msrA).The experiment in vivo and vitro result shows that all msrA can be reduced to methionine(Met) with the methionine sulfoxide in free or the peptide chain, so msrA is playing the part of important role in saving the suffered oxidation harm of albumen, can think a kind of proteinic post-translational control means.

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 msrA

1. primer amplification

With human brain λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-CTGCGGCTCCGCTGCCGGTAG-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotide B1:5 '-CCACGATTGTGAGTCACACAAGC-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.Electrophoresis detection obtains length for being the purpose fragment of 853bp.

2.PCR the order-checking of product

With this section pcr amplification product A1/B1 and pGEM-T ^Carrier (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 853bp, detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 61-768 position Nucleotide.

Derive the aminoacid sequence of msrA according to the full length cDNA sequence that obtains, totally 235 amino-acid residues, its aminoacid sequence sees SEQ ID NO:4 for details.

Embodiment 2

Homology relatively

Full length cDNA sequence and proteins encoded thereof with msrA carry out nucleic acid and albumen homology retrieval with BLAST in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database.The msrA gene and the proteins encoded thereof that found that they and ox have high homology, and identity on nucleic acid and protein level and similarity have all surpassed 85%.Simultaneously, the msrA in ox msrA and yeast and the intestinal bacteria has big homology again, so they are considered to constitute a family, according to the intimate principle of structural similitude, can infer the function of msrA from these genes or proteic function.

As everyone knows, the methionine(Met) in the protein peptide chain can be a methionine sulfoxide by the catalysis of non-enzyme factor, and these non-enzyme factors include the by product of oxygen metabolism---superoxide, hydroxyl, hydrochlorate etc.The oxidation of methionine(Met) can cause forfeiture (Brot, N., and Weissbach, the H. of protein biological activity Biofactors, 1991,3,91-96; Brot, N.and Weissbach, H. Arch.Biochem.Biophys.1983,223,271-283), so certainly exist the enzyme that can repair these oxidized methionine residues in the organism.The so just class of enzymes of methionine sulfoxide reductase (msrA).The experiment in vivo and vitro result shows that all msrA can be reduced to methionine(Met) with the methionine sulfoxide in free or the peptide chain, so msrA is playing the part of important role in saving the suffered oxidation harm of albumen, can think a kind of proteinic post-translational control means.

The oxidation of methionine(Met) is relevant with multiple disease.

The oxidation inactivation of known serum α 1 proteasome repressor (alpha 1-proteinase inhibitor, alpha 1-PI) be exactly since the methionine residues of two keys oxidized due to ( J.Biol.Chem.1979,254,4022-4026).Experiment find grownup's respiratory distress syndrome be exactly probably owing to the methionine(Met) of (bronchoalveolar lavage) alpha 1-PI avtive spot in the bronchovesicular mucus oxidized due to, and the oxidation inactivation of serum α 1 proteasome repressor and grownup's respiratory distress syndrome have close ties ( J.Clin.Invest.1983,71,754-761).The oxidation of alpha 1-PI methionine(Met) is also relevant with smoker's pulmonary emphysema.The inactivation of alpha 1-PI is considered to emophysematous morbidity close ties are arranged, discover that the active of alpha 1-PI in smoker's bronchovesicular mucus significantly reduces, and in the alpha of inactivation 1-PI, have oxidized in a large number methionine(Met)s (the alpha 1-PI of 4mol/mol inactivation) ( Proc.Natl.Acad.Sci.U.S.A.1980,79,2041-2045).

Oxidized and the cataractous pathogeny of the methionine residues of crystallin is also relevant.Discover the oxidation that is accompanied by methionine(Met) and halfcystine in the cataractous forming process.In serious cataract patient, 60% or the methionine(Met) of more lens symphysis component oxidation has taken place, then do not exist among the normal people in contrast this phenomenon ( Biochim.Biophys.Acta.1977,492,43-52; Proc.Natl.Acad.Sci.U.S.A.1980,77,1274-1277).

In addition, rheumatic arthritis also relevant with the oxidation of methionine residues ( Biochem.Biophys.Res.Commun.1980,96,1449-1454).It should be noted that experiment in vitro confirm E.coli.msrA can make the serum α 1 proteasome repressor of oxidation inactivation recover active ( Proc.Natl.Acad.Sci.U.S.A.1981,78,7483-7486; J.Clin.Invest.1983,71,754-761).Therefore, thereby utilize MsrA can go back active this character of methionine residues recoverin matter oxidized in the crude protein, provide a kind of possible method for treating above-mentioned disease and other associated conditions.

Embodiment 3

The expression of msrA in intestinal bacteria

The PCR Oligonucleolide primers of the eDNA sequence of coding msrA corresponding to 5 of this dna sequence dna ' and 3 ' end, with human brain λ tllcDNA library (available from Clontech company) for template increases, to synthesize the insertion fragment.

5 ' Oligonucleolide primers sequence is:

5′-TTGGGATCCATGCTCTCGGCCACCCGGAG-3′(SEQ?ID?NO.5)

This primer contains the restriction enzyme site of the restricted restriction enzyme of BamH1, and what connect is 20 Nucleotide of the msrA encoding sequence that begun by initiator codon;

3 ' end primer sequence is:

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

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

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

With BamHI and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification msrA 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 msrA from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out msrA from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps from nickel-chelate column, isolate purifying protein.Protein behind the purifying is incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 26kDa.

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 msrA in eukaryotic cell (Chinese hamster ovary celI strain)

The PCR Oligonucleolide primers of the cDNA sequence (GerBank Accession No.AF064257) of coding msrA corresponding to 5 of this dna sequence dna ' and 3 ' end, for template increases, insert fragment with human brain λ gt11cDNA library (available from Clontech company) to synthesize.

5 ' Oligonucleolide primers sequence is:

5′-TTGGGATCCATGCTCTCGGCCACCCGG-3′(SEQ?ID?NO.5)

This primer contains the restriction enzyme site of the restricted restriction enzyme of BamHI, and what connect is 20 Nucleotide of the msrA encoding sequence that begun by initiator codon;

3 ' end primer sequence is:

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

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

The restriction enzyme site of restriction enzyme 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 (flori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With BamHI 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 SacI enzyme and insert clip size and direction, and the cDNA fragment of sequence verification msrA has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mM TrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mM TrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS-PAGE glue with 12% carries out electrophoresis, identifies the molecular weight size of expressing protein, and the molecular weight size that records msrA is about 26kDa.

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 ability of external precipitation msrA 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.

Information (i) sequence signature of sequence table (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:1:CTGCGGCTCC GCTGCCGGTA G21 (2) SEQ ID NO:2

(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:2CCACGATTGT GAGTCACACA AGC (2) SEQ ID NO:3: (i) sequence signature:

(A) length: 853bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：3 1 CTGCGGCTCCGCTGCCGGTAGCGCCGTCCCCCGGGACCACCCTTCGGCTGGCGCCCTCCC 61 ATGCTCTCGGCCACCCGGAGGGCTTGCCAGCTCCTCCTCCTCCACAGCCTCTTTCCCGTC121 CCGAGGATGGGCAACTCGGCCTCGAACATCGTCAGCCCCCAGGAGGCCTTGCCGGGCCGG181 AAGGAACAGACCCCTGTAGCGGCCAAACATCATGTCAATGGCAACAGAACAGTCGAACCT241 TTCCCAGAGGGAACACAGATGGCTGTATTTGGAATGGGATGTTTCTGGGGAGCTGAAAGG301 AAATTCTGGGTCTTGAAAGGAGTGTATTCAACTCAAGTTGGTTTTGCAGGAGGCTATACT361 TCAAATCCTACTTATAAAGAAGTCTGCTCAGAAAAAACTGGCCATGCAGAAGTCGTCCGA421 GTGGTGTACCAGCCAGAACACATGAGTTTTGAGAGAACTGCTCAAGGTCTTCTGGAGAAT481 CACGACCCGACCCAAGGTATGCGCCAAGGGAACGACCATGGCACTCAGTACCGCTCGGCC541 ATCTACCCGACCTCTGACAAGCAAATGCAGGCAGCCCTGAGCTCCAAAGAGAACTACCAA601 AAGGTTCTTTCAGAGCACGGCTTCGGCCCCATCACTACCGACATCCGGGAGGGACAGACT661 TTCTACTATGCGGAAGACTACCACCAGCAGTACCTGAGCAAGAACCCCAATGGCTACTGC721 GGCCTTGGGGGCACCGGCGTGTCCTGCCCAGTGGGTATTAAAAAATAATTGCTCCCCACA781 TGGTGGGCCTTTGAGGTTCCAGTAAAAATGCTTTCAACAAATTGGGCAATGCTTGTGTGA841 TTCACAATCGTGG ( 2 ) SEQ ID NO：4： ( i ) ：

(A) length: 235 amino acid

(B) type: amino acid

( C ) ： ( ii ) ： ( xi ) ：SEQ ID NO：61 Met Leu Ser Ala Thr Arg Arg Ala Cys Gln Leu Leu Leu Leu His16 Ser Leu Phe Pro Val Pro Arg Met Gly Asn Ser Ala Ser Asn Ile31 Val Ser Pro Gln Glu Ala Leu Pro Gly Arg Lys Glu Gln Thr Pro46 Val Ala Ala Lys His His Val Asn Gly Asn Arg Thr Val Glu Pro61 Phe Pro Glu Gly Thr Gln Met Ala Val Phe Gly Met Gly Cys Phe76 Trp Gly Ala Glu Arg Lys Phe Trp Val Leu Lys Gly Val Tyr Ser91 Thr Gln Val Gly Phe Ala Gly Gly Tyr Thr Ser Asn Pro Thr Tyr106 Lys Glu Val Cys Ser Glu Lys Thr Gly His Ala Glu Val Val Arg121 Val Val Tyr Gln Pro Glu His Met Ser Phe Glu Arg Thr Ala Gln136 Gly Leu Leu Glu Asn His Asp Pro Thr Gln Gly Met Arg Gln Gly151 Asn Asp His Gly Thr Gln Tyr Arg Ser Ala Ile Tyr Pro Thr Ser166 Asp Lys Gln Met Gln Ala Ala Leu Ser Ser Lys Glu Asn Tyr Gln181 Lys Val Leu Ser Glu His Gly Phe Gly Pro Ile Thr Thr Asp Ile196 Arg Glu Gly Gln Thr Phe Tyr Tyr Ala Glu Asp Tyr His Gln Gln211 Tyr Leu Ser Lys Asn Pro Asn Gly Tyr Cys Gly Leu Gly Gly Thr226 Gly Val Ser Cys Pro Val Gly Ile Lys Lys ( 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:5:TTGGGATCCA TGCTCTCGGC CACCCGGAG 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:6:AACAAGCTTT TATTTTTTAA TACCCACTG 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: SEQ ID NO:7:AACGGATCCT TATTTTTTAA TACCCACTG 29

Claims (11)

1. an isolated dna molecular is characterized in that, described dna molecule encode one polypeptide, and this polypeptide contains the sequence shown in the SEQ ID NO.4.

2. dna molecular as claimed in claim 1 is characterized in that, described dna molecule encode one polypeptide, the sequence of this polypeptide such as SEQ ID NO.4.

3. dna molecular as claimed in claim 1 is characterized in that, described dna molecular contains among the SEQ IDNO.3 nucleotide sequence from Nucleotide 61-768 position.

4. an isolating msrA protein polypeptide is characterized in that, it contains SEQ ID NO.4 aminoacid sequence.

5. polypeptide as claimed in claim 4 is characterized in that, this amino acid sequence of polypeptide is shown in SEQ ID NO.4.

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. one kind produces the proteic method of msrA, it is characterized in that this method comprises:

(a) the proteic nucleotide sequence of msrA of will encoding operationally is connected in expression regulation sequence, forms the msrA protein expression vector, a described nucleotide sequence coded polypeptide, and this polypeptide contains the sequence shown in the SEQ ID NO.4

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

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

(d) isolate msrA albumen.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 61-768 position among the SEQ ID NO.3.