CN1223677C - Gene associated with esophagus cancer - Google Patents

Abstract

The present invention discloses separated DNA and RNA of a new gene DRC1 relevant to esophagus cancer, polypeptide encoded by DNA and RNA, and a method for producing the polypeptide by recombination technology. The present invention also discloses a diagnostic method for detecting the mutation and the methylation of a nucleotide sequence of the gene, the change of RNA and the polypeptide level, and diseases relevant to the abnormality of nucleotide sequence of the gene and the encoding polypeptide, which also discloses an application for treating diseases relevant to the abnormality of the gene (such as tumor and cancer) by utilizing the nucleotide sequence and the polypeptide of the gene.

Description

A kind of esophageal cancer related gene

The present invention relates to the polypeptide of a kind of esophageal cancer related gene, this genes encoding and the Use and preparation method of this gene and polypeptide.

Utilize Protocols in Molecular Biology, the pathology root of being familiar with disease from molecular level is to find or provide the favourable approach of effective methods of treatment of these diseases.Owing to also have a large amount of diseases not find effective methods of treatment in the prior art, comprise most of cancer, therefore press for and find the gene relevant, particularly those and these disease to be negative correlation, may be used for the gene of these treatment of diseases with the pathology of these diseases.

Purpose of the present invention just provides a kind of new and the gene of esophageal carcinoma negative correlation and the polypeptide of this genes encoding.

The present invention relates to a kind of isolating polynucleotide, it comprises the member who is selected from down group:

(a) polynucleotide of the coded mature polypeptide of the contained cDNA of clone of polypeptide or coding CGMCC preserving number 0402 (cDRC1) shown in the coding SEQ ID No.2;

(b) at least 70% conforming polynucleotide can be arranged with the multi-nucleotide hybrid of (a) and with it; With

(c) (a) or (b) polynucleotide passage of polynucleotide.

The invention further relates to the carrier that contains above-mentioned polynucleotide, cultivate this host cell with polypeptide of expressing described polynucleotide encoding and the production process of polypeptide that from culture, reclaims target polypeptides with comprising with the host cell that this carrier is genetically engineered.The invention still further relates to a kind of method that produces the cell of energy express polypeptide, comprise with described carrier transforming or transfectional cell.

The present invention relates to a peptide species that is selected from down group on the other hand:

(a) have polypeptide or its fragment, analogue and the derivative of the putative amino acid sequence of SEQ ID No.2;

(b) the contained cDNA encoded polypeptides of the clone of CGMCC preserving number 0402 or its fragment, analogue and derivative.

The present invention also relates to the antibody of anti-aforementioned polypeptides.

The invention still further relates to the expression diseases associated of a kind of in-vitro diagnosis and aforementioned polypeptides of the present invention or the method for disease susceptibility, be included in the sample from the host:

(a) sudden change, disappearance and the rearrangement in the gene nucleic acid sequence of mensuration coding said polypeptide; And/or

(b) methylating in the gene nucleic acid sequence of mensuration coding said polypeptide; And/or

(c) gene mRNA of mensuration coding said polypeptide is unusual.

The present invention relates to a kind of in-vitro diagnosis method in addition, is included in from analyzing the existence of polypeptide of the present invention or the change of amount in host's the sample.

The present invention above-mentioned with other the aspect for a person skilled in the art, see it is very clearly from this paper the following detailed description.

One aspect of the present invention provides new polypeptide, and they are PDRC2 pDRC1, and the diagnosis of biologically active or treat useful fragment, analogue and derivative.Another aspect of the present invention, the isolated nucleic acid molecule (DRC1) of these polypeptide of encoding is provided, comprise mRNA, DNA, cDNA (cDRC1) and genomic dna (gDRC1), and the diagnosis of biologically active or treat useful fragment, analogue and derivative.The probe that nucleic acid is provided on the one hand more of the present invention, it comprises the nucleic acid molecule of sufficient length and carries out the sequence of specific hybrid with the DRC1 gene order.

Fig. 1 has shown the cDNA sequence of DRC1 and the aminoacid sequence of inferring thus.Here used is the abbreviation of standard amino acid single-letter.(Takara company) checks order with the ABIPRISM*377 automatic dna sequencer, and the accuracy of order-checking is estimated to be higher than 98%.

Fig. 2 is the figure as a result of Northern engram analysis.

Fig. 3 is the figure as a result that RT-PCR analyzes.

According to an aspect of of the present present invention, it provides a kind of isolating nucleic acid (polynucleotide), and these nucleotide codings have the polypeptide of the aminoacid sequence of inferring shown in Fig. 1 (SEQ ID No.2) or the clone cDNA encoded polypeptides of on June 23rd, 1999 with CGMCC preserving number 0402 (cDRC1) preservation of encoding.

The polynucleotide of coding pDRC1 can separate from the cDNA library of many adults and fetus, as the cDNA library of adult's oesophagus.Polynucleotide sequence shown in SEQ ID No.1 and the SEQ ID No.3 has proteinic open reading frame that contains 495 amino-acid residues of coding.This albumen and people Profilaggrin (GenBank No.:A45135) N-end has the homology of top, has 42% amino acid identical in 88 amino acid of successive, and 65% amino acids seemingly.Have 33% amino acid identical in 225 amino acid of successive with people Trichohyalin (GenBank No.:Q07283) N-end, 49% amino acids seemingly.

Polynucleotide of the present invention may exist with the form of RNA or DNA, and wherein DNA comprises cDNA, genomic dna and synthetic DNA.DNA can be two strands or strand, and strand also can be coding strand or non-coding (antisense) chain.The encoding sequence of coded polypeptide can be identical with the encoding sequence shown in Fig. 1 (SEQ ID No.1 or SEQ ID No.3), also can be identical with the clone's of institute preservation encoding sequence, it perhaps can be a different encoding sequence, since the redundancy or the degeneracy of genetic code, it and the identical mature polypeptide of Fig. 1 cDNA coding (SEQ ID No.2) or preservation.

The polypeptide of code pattern 1 (SEQ ID No.2) or can comprise: the encoding sequence that only is mature polypeptide by the polynucleotide of the cDNA encoded polypeptides of preservation; The encoding sequence of mature polypeptide and other arbitrarily encoding sequence and non-coding sequence, for example 5 ' of intron or mature polypeptide encoded sequence end and/or 3 ' end non-coding sequence.

Thus, term " polynucleotide of the peptide species of encoding " comprises and also comprises the polynucleotide that contain extra coding and/or non-coding sequence by the polynucleotide that only contain this polypeptid coding sequence.

The invention further relates to the various variants of polynucleotide described above, their codings contain fragment, analogue and the derivative of the clone cDNA encoded polypeptides of the polypeptide of the aminoacid sequence of inferring shown in Fig. 1 (SEQ ID No.2) or preservation.These polynucleotide variants can be the polynucleotide variants that naturally occurring allele variant or non-natural exist.

So, present invention includes the polynucleotide of phase homopolypeptide shown in the code pattern 1 (SEQ ID No.2) or preservation clone's the coded identical mature polypeptide of cDNA, and the variant of such polynucleotide, fragment, analogue or the derivative of polypeptide shown in these variant code patterns 1 (SEQ ID No.2) or preservation clone's cDNA encoded polypeptide.These nucleotide variants comprise disappearance variant, displacement variant and additional or insertion variant.

Just as mentioned above, these polynucleotide may have the encoding sequence as the naturally occurring allele variant of encoding sequence shown in Fig. 1 (SEQ ID No.1) or preservation sequence.According to known in the art, allelic variation body has been a speciogenesis, and one or more Nucleotide replace, the alternative form of disappearance or the polynucleotide sequence that adds, and it does not change the function of encoded polypeptide basically.

Polynucleotide of the present invention also may contain the encoding sequence that merges mutually with flag sequence in same frame, flag sequence helps the purifying of polypeptide of the present invention.Flag sequence can be six polyhistidine tags of pQE carrier when using host bacterium, be beneficial to merge the purifying of markd mature polypeptide, when perhaps using mammals host (as the fibroblastic COS-7 clone of monkey kidney), flag sequence can be a kind of hemagglutinin (HA) mark.The HA mark is equivalent to by a protein derived epi-position of influenza hemagglutinin (wilson etc.: Cell 37:767,1984).

" gene " speech refers to produce the dna fragmentation that relates in the polypeptide chain, and it comprises before the coding region and the intervening sequence (intron) between zone afterwards (leading and afterbody) and each encode fragment (exon).

The cDNA that the dna fragmentation of full-length gene of the present invention can go to isolate total length as the hybridization probe in cDNA library with isolate other and this cDNA that gene order height is similar or biological function is similar.Such probe preferably has and has 30 bases at least, for example comprises 50 or more base.This probe also can be used for identifying corresponding to the cDNA clone of the transcript of total length and comprise the genomic clone of the complete genome of adjusting and promoter region, exon and intron.An example of screening is to utilize the synthetic oligonucleotide probe of known dna sequence dna to remove the coding region of isolated genes.The oligonucleotide of the mark of sequence and gene complementation of the present invention is used to screen human cDNA, genomic dna or mRNA library, determines the member with probe hybridization.

The invention further relates to the polynucleotide with above-mentioned sequence hybridization, have 70% at least between these two sequences, preferably at least 90%, more preferably at least 95% consistence.The present invention be more particularly directed under stringent condition polynucleotide with above-mentioned multi-nucleotide hybrid.Terminology used here " stringent condition " means between two sequences have 95%, and preferably at least 97% consistence could be hybridized.In an embodiment preferred, kept substantially and (the SEQ ID No.1) of Fig. 1 or the identical biological function or the activity of cDNA encoded polypeptides of preservation with the polypeptide of the polynucleotide encoding of above-mentioned multi-nucleotide hybrid.

Another selection is, contains 20 bases at least with the polynucleotide of multi-nucleotide hybrid of the present invention, preferred at least 30 bases, and more preferably at least 50 bases and have consistence described above can keep or retentive activity not.For example, such polynucleotide can be used as the probe of SEQ ID No.1 polynucleotide; And for example, can be used for the recovery of polynucleotide or as a kind of diagnostic probe or PCR primer.

Like this, the present invention relates to and the polynucleotide of the SEQ ID No.2 polypeptide of encoding have at least 70% consistence, preferably at least 90%, more preferably at least 95% conforming polynucleotide and fragment thereof, these fragments have 30 bases at least, preferably have 50 bases at least; The polypeptide that also relates to these polynucleotide encodings.

Here it is to be used under the microbial preservation budapest treaty condition of patented procedure in international recognition that mentioned preservation keeps.These preservations only are that those skilled in the art's facility is prepared, and are not to admit according to the 25th needs preservation of patent law of china detailed rules for the implementation.The polynucleotide sequence that is contained in the preserved material and the aminoacid sequence of polynucleotide encoding are incorporated herein by reference, and are used to examine when this paper sequence description is had any dispute.

The invention further relates to the putative amino acid sequence shown in (SEQ ID No.2) that has Fig. 1 or have polypeptide and fragment, analogue and the derivative of the coded aminoacid sequence of the cDNA of preservation.

Shown in Fig. 1 (SEQ ID No.2) or " fragment ", " derivative " and " analogue " of the cDNA encoded polypeptide of preservation refer to keep substantially the biological function or the active polypeptide of this polypeptide.Thus, analogue has comprised proteinogen, and proteinogen is activated after being cut a part, produces an activated mature polypeptide.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides or synthetic polypeptide, preferred recombinant polypeptide.

Fragment, derivative and the analogue of cDNA encoded polypeptide shown in Fig. 1 (SEQ ID No.2) or preservation can be: (i) one or more amino-acid residues are replaced (preferably Bao Shou amino-acid residue) by conservative or nonconservative amino-acid residue, and the amino-acid residue yes or no of replacement is by the coded amino acid of genetic code; Or (ii) one or more amino-acid residues have substituted radical; Or (iii) mature polypeptide and another compound merge, as improving the polypeptide compound (for example polyoxyethylene glycol) of half life; Or (iv) other amino acid merges mutually with mature polypeptide, such as sequence that helps the purifying maturation protein or proteinogen sequence etc.From these disclosures, such fragment, derivative and analogue are believed in the ken that is in those skilled in the art.

Polypeptide of the present invention and polynucleotide preferably provide and are purified to the degree of homogeneous with isolating form.

" isolating " speech refers to material is shifted out among its original environment (for example, if spontaneous its natural surroundings that just refers to).Such as it is exactly not to be separated that spontaneous polynucleotide or polypeptide are present in the Live Animals, but same polynucleotide or polypeptide with some or all in natural system with it the material of coexistence separately be exactly isolating.Such polynucleotide may be the parts of a certain carrier, the part that also possible such polynucleotide or polypeptide are a certain composition.Since carrier or composition are not the compositions of its natural surroundings, they remain isolating.

Polypeptide of the present invention comprises the polypeptide (referring in particular to mature polypeptide) shown in the SEQ ID No.2, also comprise the polypeptide that has 70% similarity (preferably 70% consistence) with SEQ ID No.2 polypeptide at least, the polypeptide that preferably has 90% similarity (preferably 90% consistence) at least, the polypeptide that more preferably has 95% similarity (preferably 95% consistence) at least, the some parts that also comprises these polypeptide, these polypeptide portions contain 30 amino acid, preferred at least 50 amino acid at least usually.

As known in the art, the similarity of two polypeptide determines by comparing the replacement of a polypeptide and another amino acid sequence of polypeptide and conservative amino acid thereof.

The fragment of polypeptide of the present invention or part can be used for through the synthetic full-length polypeptide of being correlated with that produces of peptide.Therefore, these fragments can be used as the intermediate that produces full-length polypeptide.The fragment of polynucleotide of the present invention or part can be used for synthesizing total length polynucleotide of the present invention.

The present invention also relates to comprise the method that the carrier of polynucleotide of the present invention, the genetically engineered host cell that has carrier of the present invention and dependence recombinant technology produce polypeptide of the present invention.

Host cell carries out genetically engineered (transduction, conversion or transfection) with carrier of the present invention, and carrier can be a cloning vector or expression vector.Carrier can be plasmid, virus particle, phage or the like.The host cell of through engineering approaches can cultivated for being suitable for activating on traditional nutritional medium that promotor, screening transformant or amplification DRC1 gene change.The original used expression condition of culture condition such as temperature, pH etc. and selecteed cell is consistent, is clearly to those skilled in the art.

Polynucleotide of the present invention can be used for producing polypeptide through recombinant technology.For example, these polynucleotide may reside in a series of arbitrary carriers that are used for the expression vector of express polypeptide, these carriers comprise chromosomal, achromosomal and the synthetic dna sequence dna, for example, the derivative of SV40, bacterial plasmid, phage DNA, yeast plasmid, be derived from plasmid and phage DNA bonded carrier, viral DNA, baculovirus, vaccinia virus, adenovirus, fowlpox virus and Pseudorabies virus.But, as long as can duplicate in the host and survive, other carrier also can utilize.

Suitable dna sequence dna can be inserted in the carrier by many methods.Usually, dna sequence dna is inserted on the suitable restriction endonuclease sites with method as known in the art.These methods are believed in the ken that is in those skilled in the art.

Dna sequence dna in the expression vector is connected together with a suitable expression control sequenc (promotor) effectively, thereby instructs synthesizing of mRNA.Cited below is other promotor of genetic expression in the PL promotor of the representative of this promotor: LTR or SV40 promotor, colibacillary Lac or trp promotor, lambda particles phage and known control protokaryon or eukaryotic cell or its virus.Expression vector also comprises needed ribosome bind site of translation initiation and transcription terminator.Carrier can also have the proper sequence that strengthens expression.

In addition, expression vector preferably comprises one or more selectable marker genes that phenotypic characteristic can be provided, so that the screening of transformed host cell, available Tetrahydrofolate dehydrogenase of for example eukaryotic cultivation or neomycin resistance, then available tsiklomitsin of intestinal bacteria or amicillin resistance.

Comprise above-mentioned suitable dna sequence dna, suitable promotor or the carrier of control sequence and can be used to transform appropriate host, allow this albumen of host expresses.

What enumerate below is the representative of suitable host: bacterial cell, such as intestinal bacteria, streptomycete, Salmonella typhimurium; The fungal cell is as yeast; Insect cell is as fruit bat S2 and fall army worm Sf9; Zooblast is as CHO, COS (monkey kidney fibroblast) or Bowes melanoma; Adenovirus; Vegetable cell or the like.From these instruction, the selection of suitable host should be in those skilled in the art's ken.

It is worth mentioning that especially the present invention also comprises the recombinant precursor of the one or more sequences that contain top generality description.This construct comprises carrier, as plasmid or virus vector, has wherein inserted a sequence of the present invention forward or backwards.Of this embodiment preferred aspect, this construct also comprises the adjusting sequence, promotor for example, it is effectively connected on this sequence.To those skilled in the art, many carriers and promotor all are known, and can obtain by commercial sources.Enumerate several carriers below.Bacterium: pQE-70, pQE-60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), pTRC99a, pKK223-3, pDR540, pRIT5 (Pharmacia); Eucaryon: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia).But, so long as can duplicate in the host and survive, other plasmid or carrier also can be used.

The carrier that promoter region can utilize CAT (CAT) carrier or other to have selective marker chooses from the gene of wanting.PKK232-8 and pCM7 are two suitable carriers.The special bacterium promotor that claims comprises LacI, LacZ, T3, T7, gpt, λ PR, PL and trp.Eukaryotic promoter comprises CMV immediate early promoter, HSV thymidine kinase, early stage and late period SM40, retroviral LTR and mouse metallothionein(MT) I.The selection of suitable carrier and promotor should be within the common state of the art in this area.

In another embodiment, the present invention relates to comprise the host cell of above-mentioned construct.Host cell can be a higher eucaryotic cells, such as cells of mamma animals, or eukaryotic cell such as low, such as yeast cell, can also be prokaryotic cell prokaryocyte, such as bacterial cell.The method that construct is imported host cell has: transfection, electroporation or the particle gun method of calcium phosphate transfection, the mediation of DEAE-dextran.

Construct in the host cell can produce the gene product of recombination sequence coding by traditional method.Another approach is that available traditional peptide synthesizer synthesizes polypeptide of the present invention.

Under suitable promotor control, can in cells of mamma animals, yeast, bacterium or other cell, express maturation protein.Utilization also can produce this protein with the cell free translation system by DNA construct derived RNA of the present invention.Being suitable for people such as the clone of protokaryon and eucaryon host and expression vector Sambrook is described in " molecular cloning experiment guide " (second edition, cold spring port press, New York, 1989).

In higher eucaryote, transcribing of the DNA of code book invention polypeptide can be enhanced by insert an enhancer sequence in carrier.Enhanser is the cis acting factor of DNA, and about 10-300bp is arranged usually, acts on promotor, improves transcribing of it.For example, the enhanser that is positioned at replication orgin rear side 100-270bp place of SV40, the sub-enhanser of cytomegalovirus early promoter, the polyoma enhanser that is positioned at the replication orgin rear side and adenovirus enhanser.

Usually, recombinant expression vector comprises replication orgin and is used for the selective marker of transformed host cell, for example colibacillary ampicillin resistance gene and cereuisiae fermentum TRiP1 gene also comprise the promotor of coming from efficiently expressing gene, thereby mediate transcribing of downstream configurations sequence.These promotors can get from the operon of coding glycolytic ferment, such as glycerol 3-phosphate acid kinase (PGK), alpha factor, acid phosphatase or heat shock protein(HSP).Allogenic structure sequence is assembled together with suitable orientation and translation initiation and terminator sequence and other preferred sequence.Selectable situation is, the allogenic sequence fusion rotein of can encoding, and this albumen contains a N-terminal confirms peptide, shows the feature of expection, the stabilization of for example expressed recombinant products and purify and simplify.

The effective expression carrier that bacterium is suitable for makes up like this: the structural DNA sequence of the target protein of will encoding is inserted in the steerable reading frame that has function in company with suitable translation initiation and termination signal.Carrier should comprise one or more Phenotypic Selection marks and replication orgin, and replication orgin has guaranteed that carrier can remain in the host, and better is to make carrier obtain amplification.The prokaryotic hosts that is suitable for transforming has the various bacteriums of intestinal bacteria, subtilis, Salmonella typhimurium and Pseudomonas, streptomyces and Staphylococcus, and other host also can be selected.

But the example of indefiniteness representational as one, the effective expression carrier that bacterium is suitable for comprises the replication orgin of a selective marker and bacterium, and it is derived from that obtain, that have the genetic elements of well-known cloning vector pBR322 (ATCC37017) from commercial channels plasmid.These business-like carriers comprise such as pKK223-3 (Pharmacia) and pGEMl (Promega) etc." skeleton " part of pBR322 is combined with suitable promotor and the structure sequence of being expressed.

Transforming after appropriate host bacterial strain and host strain grow into appropriate cell density, induce selected promotor with appropriate means (for example temperature transition or pharmaceutical chemicals are induced), and cell is cultivated for some time again.

Usually use the centrifugation method harvested cell, the method smudge cells with physics or chemistry keeps crude extract to be further purified.The microorganism cells that is used for expressing protein can comprise freeze-thaw cycle, ultrasonic wave, Mechanical Crushing with the fragmentation of any method easily, perhaps uses cell lytic agent, and these methods all are well-known to those having ordinary skill in the art.

The culture systems of various mammalian cells also can be used for express recombinant protein.The example of mammals expression system have Gluzman (Cell 23:175,1981) the fibroblastic COS-7 clone of the monkey kidney of describing, and other can express the clone of compatible carrier, for example C127,3T3, CHO, HeLa and bhk cell are.The mammals expression vector comprises replication orgin, suitable promotor, enhanser and any essential ribosome bind site, polyadenous glycosidation site, splicing donor and acceptor site, transcription termination sequence and 5 ' flank non-transcribed sequence.The dna sequence dna and the polyadenous glycosidation site that are derived from SV40 splicing sequence can be used for providing needed non-transcribed genetic elements.

Polypeptide can reclaim from the reconstitution cell culture and purifying comes out, and the method for recovery and purifying has ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and phytohemagglutinin chromatography.Form ripe proteic complete conformation and need proteinic folding step again.High performance liquid chromatography (HPLC) is applied to last purification step.

Polypeptide of the present invention can be the natural product of purifying, also can be the product of chemosynthesis, can also produce through recombinant technology from protokaryon or eucaryon host (for example, the bacterium of cultivation, yeast, higher plant, insect or mammalian cell).According to host's difference used in the recombinant method for production, the polypeptide among the present invention may be by glycosylation or not by glycosylation.This polypeptide also may have initial methionine residues.

Polynucleotide among the present invention and polypeptide can be used as research reagent and material, to find human treatment of diseases and diagnostic method.

This polynucleotide and its encoded polypeptides also are used for the research of external related science, DNA is synthetic and the preparation of dna vector, also are used to design the method for treatment and diagnosing human disease.

The fragment of total length DRC1 gene can be as the hybridization probe in cDNA library, thereby isolates the gene of total length and sequence similarity is highly arranged or other gene of similar biologic activity with it.Such probe generally has 20 bases at least.But these probes preferably have 30 bases at least and generally are no more than 50 bases, although they can have more base.This probe also can be used to identify corresponding to the cDNA clone of total length transcript or have complete genome, comprise the genomic clone of adjusting and promoter region, exon and intron.The screening a kind of method be, utilize known dna sequence dna to synthesize an oligonucleotide probe, remove to isolate the coding region of gene with it.The oligonucleotide of the mark of sequence and gene complementation of the present invention can be used for screening human cDNA library, genomic library, mRNA library, to determine which member and probe hybridization in the library.

The invention still further relates to the DRC1 gene product and suddenly change with the nucleotide sequence of DRC1 or the relevant disease or of methylating the component part of the diagnostic method of the susceptibility of disease as detecting.These diseases are not enough relevant with the mRNA and the expression of encoded protein matter of DRC1 gene of the present invention, for example tumour and cancer.

Can on the level of DNA, detect the individuality of DRC1 transgenation with various technology.Diagnostic nucleic acid can obtain from patient's cell, for example blood, urine, saliva, examination of living tissue and necrotomy material.Genomic dna can be directly used in detection, also can carry out the enzyme process amplification with PCR (Saiki et al.:Nature 324:163-166,1986) before check and analysis.RNA or cDNA also can be used for same purpose.For example, can be used to identify and analyze the sudden change of DRC1 gene with the nucleic acid complementary PCR primer of encoding D RC1 gene.For example, from comparing the variation of generation with normal genotype, the size of amplified production can detect disappearance and insert sudden change.But, perhaps use radio-labeling DRC1 antisense dna sequence to hybridize with the DNA and the sudden change of radiolabeled DRC1 RNA hybridization check point of amplification.Digest or can distinguish the duplex of complete paired sequence and mispairing from the difference of melting temperature (Tm) with RNA enzyme A.

By detecting the variation of the electrophoretic mobility of dna fragmentation in the gel that contains or do not contain denaturing agent, can carry out genetics test based on dna sequence dna difference.The disappearance of little sequence and insertion can be found out from high-resolution gel electrophoresis.For example, the dna fragmentation of different sizes can distinguish in the polyacrylamide gel electrophoresis (PAGE) of sex change.The dna sequence dna that contains point mutation can be distinguished and come in the polyacrylamide gel that does not contain denaturing agent after the sex change respectively with normal dna sequence dna because of different (the mobility speed differences) of conformation.

Nuclease protection experiment can disclose the sequence variation of specific position, such as RNA enzyme and S1 enzyme protection method or chemical break method (for example, Cotton etc.: PNAS 85:4397-4401,1985).

The present invention also relates to be used for detect the diagnostic method of the dna methylation of various tissue DRC1 genes.The change that methylates and can directly cause gene mRNA to be transcribed of dna level, thus also can cause the normal transcription that transgenation changes gene by causing genomic instability.The methylation status of PTEN promoter method that dna methylation can adopt Herman (PNAS 93:9821-9826,1996) to describe detects

In a word, specific DNA sequence can detect with following method: the Southern engram technology of hybridization, RNA enzyme protection, chemistry fracture, direct dna sequencing or use restriction enzyme (for example, the polymorphism RFLP of limited fragment length) and genomic dna.

Except more traditional gel electrophoresis and dna sequencing method, the also available original position analyzing and testing of suddenling change is come out.

The present invention also relates to be used for detect the diagnostic method of the mRNA abnormal expression of various tissue DRC1 genes.Methods such as the available reverse transcription PCR of the variation of mRNA level (RT-PCR), Northern Blot, Dot Blot or RNA in situ hybridization detect.These methods see it is very clearly from disclosing of this paper for a person skilled in the art.

The invention still further relates to and be used for detecting the diagnostic method that various tissue DRC1 gene protein levels change, compare, the minimizing detectable disease of DRC1 gene protein or exist (as the tumour) of disease susceptibility with the normal control tissue sample.The method of measuring DRC1 gene protein level in host's sample is well known to those skilled in the art, comprises radioimmunoassay, competitive binding assay method, Western engram analysis, enzyme-linked immunosorbent assay and sandwich assay.Enzyme-linked immunosorbent assay (ELISA) (Chapter 6,1991 for .:Current Protocols in Immunology Vol.1 such as Coligan, No.2) will be prepared the antigenic specific antibody to DRC1 in advance, preferably monoclonal antibody.In addition, also to prepare the report antibody of anti-monoclonal antibody.Be connected with a detectable reagent on the report antibody, such as radioactivity, fluorescence or horseradish peroxidase.From the host obtain sample and on a solid phase carrier incubation, polystyrene reactant plate for example, its albumen in can adsorption sample.Use a kind of nonspecific proteins such as bovine serum albumin (BSA) incubation then, any free protein binding site on the wrapper plate.Second step added monoclonal antibody incubation in plate, at this moment, monoclonal antibody with combine attached to the DRC1 gene protein on the polystyrene reactant plate.All are not in conjunction with last monoclonal anti body and function damping fluid flush away.The report antibody that links to each other with horseradish peroxidase adds in the entering plate at this moment, the result report antibody just be combined in DRC1 antigen on monoclonal antibody combine.Do not washed off in conjunction with last report antibody yet.The substrate that adds peroxidase then, the depth of formed color and typical curve are made comparisons in the regular hour, just can measure the content of DRC1 gene protein in the patient samples of certain volume.

Competition assay also is used in this detection.The antigenic specific antibody of DRC1 is linked on the solid phase carrier, the sample that allows the DRC1 of mark then and obtain from the host is the quantity by solid phase carrier and certification mark thing together, for example with the liquid phase chromatogram of glimmering, the quantity of the DRC1 in the quantity of marker and the sample is relevant.Sandwich assay is similar to enzyme-linked immunosorbent assay.In sandwich assay, DRC1 is by solid carrier, with the antibodies that is adsorbed on the solid phase carrier.Second kind of antibody is attached on the DRC1 again.The third antibody be labeled and second kind of antibody had specificity, when it just is attached on second kind of antibody during by solid phase carrier, detect its quantity then.

The diagnosis of pDRC1 polypeptide and biologically active thereof or treat useful fragment, analogue and derivative and can use in composition with pharmaceutical acceptable carrier, which will be described below.

The diagnosis of pDRC1 polypeptide and biologically active thereof or treat useful fragment, analogue and derivative and can be used in combination with suitable pharmaceutical carrier.The polypeptide and pharmaceutically acceptable carrier or the vehicle that comprise dose therapeutically effective in such composition.Such carrier has once be not restricted to a salt solution, buffer saline, dextrose, water, glycerine, ethanol and combination thereof.Preparation should be fit to administering mode.

The present invention also provides a kind of drug packages or test kit, comprises one or more containers that the composition of one or more pharmaceutical compositions of the present invention is housed.In addition, the diagnosis of these polypeptide and biologically active thereof or treat useful fragment, analogue and derivative and also can be used in combination with other treatment compound.

These pharmaceutical compositions can be with the administration of a kind of mode easily, such as in epidermis, intravenously, intraperitoneal, intramuscular, the tumour, in subcutaneous, the nose or the intradermal approach.These pharmaceutical compositions use with the effective dose that treats and/or prevents concrete indication.

According to the present invention, the pDRC1 polypeptide can use through expression in vivo, is referred to as " gene therapy " usually.

For example, patient's cell can be supplied with patient to be treated with the cell of through engineering approaches again with polynucleotide (DNA or the RNA) through engineering approaches of a coded polypeptide under the situation of exsomatizing.Such method is that everybody is familiar with in this area.For example, the retroviral particle that can week comprise the RNA of code book invention polypeptide carry out cell engineeringization.

Equally, with method known in the art in vivo the through engineering approaches cell with the expression in vivo polypeptide.As known in the art, the production cell that is used to produce the retroviral particle of the RNA that contains code book invention polypeptide can inject in the patient body, makes the cells in vivo through engineering approaches and expresses this polypeptide in vivo.From instruction of the present invention, these and other polypeptide administering mode is very clearly for those skilled in the art.For example except retrovirus, else be used for the expression vector of through engineering approaches cell in addition, adenovirus for example is it and through engineering approaches cell in vivo after a suitable transport agent combines.

The retrovirus that can derive above-mentioned retroviral plasmid vector comprises-but be not to be limited to-Moloney murine leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus and mammal tumor virus.An embodiment is that retroviral plasmid vector can be derived from the Moloney mouse leukaemia virus.

Carrier comprises one or more promotors.Spendable suitable promotor comprises-but be not to be limited to-retroviral LTR, SV40 promotor, human cytomegalic inclusion disease virus (CMV) promotor (Miller:Biotechniques, Vol.7, No.9, pp980-990,1989) or other promotor (for example, the promotor of cell has-but is not restricted to-promotor of histone, polIII, beta-actin such as eukaryotic promotor).Other available viral promotors has-but is not restricted to-adenovirus promoter, thymidine kinase (TK) promotor and B19 parvovirus promotor.From the instruction here, those skilled in the art will be very clear to the selection of suitable promotor.

The nucleotide sequence of code book invention polypeptide is under the control of corresponding promotor.Suitable promotor comprises, but is not restricted to, and the promotor of adenovirus is such as adenovirus major late promoter; Or allogeneic promoter, such as cytomegalovirus (CMV) promotor; Respiratory syncytial virus (RSV) promotor; Inducible promoter is such as MMT promotor, metallothionein promoter; The heat-shocked promotor; Albumin promoter; The ApoAI promotor; People's globin promotor; Viral thymidine kinase promoter is such as the herpes simplex thymidine kinase promoter; Retroviral LTR (the retroviral LTR that comprises modification described above); The beta-actin promotor; And human growth hormone promotor.It also can be self promotor of the gene of this polypeptide of control coding.

Form production clone with retroviral plasmid vector transduction package cell line.The packing cell that is used for transfection comprises, but be not restricted to, PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψ CRE, ψ CRIP, GP+E-86, GP+envAM12 and DNA clone, as Miller (Human Gene Therapy Vol.1 is described, pp5-14,1990), but reference in its entirety.The currently known methods of available this area carrier transduction package cell line.These methods comprise, but are not restricted to electroporation, use liposome and calcium phosphate precipitation.A kind of selectable method be reverse transcription plasmid vector bag is assisted in the liposome or with the lipid coupling, inject the host then.

Production clone produces the infective retroviral particle of tool, comprises the nucleotide sequence of these polypeptide of encoding in this virion.Such retroviral vector particle can be used for external or the interior transduction of body eukaryotic cell.The eukaryotic cell of being transduceed will be expressed the nucleic acid encoding sequence.The eukaryotic cell that can be used for transduceing comprises, but is not restricted to embryonic stem cell, embryo cells, hemopoietic stem cell, liver cell, inoblast, sarcoplast, keratinocyte, endotheliocyte and bronchial epithelial cell.

Polypeptide and fragment thereof, derivative or analogue or the cell of expressing them can be used as immunogen to produce its antibody.These antibody can be polyclone or monoclonal antibody.The present invention also comprised embedding and, strand and product humanized antibody and Fab fragment or Fab expression library.The whole bag of tricks known in the art can be used for such antibody and segmental generation.

The antibody of the corresponding polypeptide of sequence anti-of the present invention that is produced can obtain by directly polypeptide being injected to animal or polypeptide being imported animal (preferably not being the people).Resulting antibody can combine with polypeptide is own.Also can produce antibody in this way, even with the sequence of the polypeptide fragment of only encoding in conjunction with whole natural polypeptides.Then, antibody can be used to polypeptide is separated from the tissue of expressing it.

The preparation monoclonal antibody can be cultivated the technology that produces antibody with continuous cell line.For example, with hybridoma technology (Kohle﹠amp; Milstein:Nature 256:495-497,1975), Trioma technology, human B cell hybridoma technology (.:Immunology Today 4:72 such as Kozbor, 1983) and the EBV-hybridoma technology produce people's monoclonal antibody (Cole etc.: MonoclonalAntibodies and Cancer Therapy, Alan R.Liss, Inc., pp77-96,1985).

Single-chain antibody generating technique (United States Patent (USP) 4,946,778) is applicable to the single-chain antibody that produces immunogenic polypeptide product of the present invention.Transgenic animal also can be used to express the humanized antibody of immunogenic polypeptide product of the present invention.

The present invention will further describe in the following embodiments.But the present invention is not limited to these embodiment.All parts and amount, except as otherwise noted all by weight.

For ease of understanding the following examples, explain method and/or the term that some often occur earlier.

The name of " plasmid " is with the p of a small letter, in front and/or the back connect the letter of capitalization and/or digital.The plasmid that sets out can obtain from commerce, also can be on unrestricted basis open the acquisition, perhaps by the method for having announced from obtainable plasmid construction.In addition, the plasmid of equal value of the plasmid of describing is known in the art, and is very clearly for general technician.

" digestion " of DNA refers to restriction enzyme DNA be carried out the catalytic cutting, and restriction enzyme only acts on the specific site of dna sequence dna.Here used various restriction enzymes can obtain from commerce, and their reaction conditions, cofactor and other requirement are according to the application known to the those skilled in the art.In order to analyze needs, in the buffered soln of about 20 μ l, add the enzyme of 1 μ g plasmid or dna fragmentation and about 2 units usually.For the DNA isolation fragment is used for plasmid construction, usually in a bigger volume with the DNA of enzymic digestion 5 to the 50 μ g of 20 to 250 units.Manufacturer can indicate the suitable damping fluid of special restriction enzyme and the amount of substrate.Usually the used incubation time approximately be 37 ℃ one hour, but can change to some extent according to the indication of producer.Directly on polyacrylamide gel, carry out electrophoresis after the restrictive diges-tion, isolate the fragment of wanting.

Separate available 8% polyacrylamide gel (.:Nucleic Acids Research 8:4057 such as Goedde, 1980) according to the clip size that is cut into.

" oligonucleotide " refers to strand polydeoxyribonucleotide or two complementary polydeoxyribonucleotide chains of energy chemosynthesis.These synthetic oligonucleotide do not contain 5 ' end phosphoric acid, so if do not add a phosphoric acid with ATP under zymogenesis, it just can not be connected with another oligonucleotide.The synthetic oligonucleotide will be not connected by the fragment of dephosphorylation.

" connection " refers to form the process of phosphodiester bond between two double stranded nucleic acid fragments.Unless method for distinguishing is provided, connection can be carried out under known damping fluid and condition, and the dna fragmentation that is used to connect of the about equimolar amount of promptly every 0.5mg adds the T4 dna ligase of 10 units (" ligase enzyme ").

Except as otherwise noted, Graham﹠amp is all used in conversion; Van der Eb:Virology 52:456-457, the method described in 1973 is carried out.

The clone of embodiment 1:cDRC1

Extract the esophageal carcinoma sample of excision respectively and cut the total RNA that rectifies normal tissue (pathological section confirmation cancer-free cell) with Trizol (Gibco) reagent by specification, digest to remove residual genomic dna with the active DNase of no RNase.After the uv-spectrophotometric instrument is quantitative, respectively get the total RNA of 3mg and in the 20ml system, carry out reverse transcription, reaction conditions is: 1 X PCR damping fluid, 2.5mM MgCl2,10mMDTT, 500mM dNTPs, 10mM GT15N (N represents A, G and C), Superscript II reversed transcriptive enzyme 200 units (Gibco), 42 ℃ of insulations are after 60 minutes, and gained cDNA is in-20 ℃ of preservations.

Get 0.5mlcDNA and carry out following difference demonstration PCR (DD-PCR): 1 X PCR damping fluid, 1.5mM MgCl2,200mM dNTPs in the 20ml reaction system, 1mM 10-mer random primer (Operon), 3mMGT15N (N represents A, G and C), Taq enzyme (Gibco) 1 unit.The PCR loop parameter is 95 ℃ of pre-sex change 1 minute, 95 ℃ 15 seconds, 39 ℃ 4 minutes, after 72 ℃ of 4 circulations in 2 minutes, 95 ℃ 15 seconds, 39 ℃ 2 minutes, 72 ℃ carried out 35 circulations again in 1 minute after, 72 ℃ were extended 5 minutes.PCR is reflected in the PE9600PCR system and carries out.

The DD-PCR product faces sample on the swimming lane with the esophageal carcinoma mutually with cutting to rectify often to organize, and electrophoresis in the denaturing polyacrylamide gel in 6% (PAGE) is with visible band clearly behind the cma staining.The difference band that cutting-out healthy tissues swimming lane is visible, the cancerous tissue swimming lane lacks is as template, carry out the secondary amplification by above-mentioned DD-PCR condition, amplified production directly reclaims with wizard PCR Preps DNA Purification System (Promega), clone in pGEM-T Easy carrier (Promega), check order with the ABIPRISM*377 automatic dna sequencer.Obtain the 3 ' terminal sequence of cDRC1 thus.

According to the sequencing result synthetic primer, angle the 5 ' upstream sequence of getting cDRC1 with Marathon cDNA RACE System (Clontech), splice with above-mentioned 3 ' terminal sequence behind the cloning and sequencing.According to spliced sequence two ends synthetic primer, with PCR method amplification cDRC1 full-length cDNA, further cloning and sequencing is verified again.The result, obtained to contain the plasmid pGEM-TEasy of nucleotide sequence shown in the SEQ.ID.NO.1, the coli strain DH5 α that contains this plasmid on June 23rd, 1999 in (Zhong Guan-cun, BeiJing, China, China Committee for Culture Collection of Microorganisms common micro-organisms center, 100080) preservation, preserving number are CGMCC 0402.

The Northern marking of embodiment 2:DRC1 gene and RT-PCR analyze

CDRC1 3 ' terminal sequence with the clone is a probe, usefulness Primer-a-Gene random primer labelling test kit α- ³²P-dATP/dCTP carries out mark to probe.Often organize RNA to be transferred to nylon membrane and fixing with cutting to rectify the esophageal carcinoma, probe with mark carries out Northern hybridization with it, evaluation clone's cDNA fragment is estimated the RNA size of DRC1 gene really simultaneously from the band of being cut on the PAGE glue (promptly cut and rectify normal tissue expression, the esophageal carcinoma is not expressed).The result of the Northern marking as shown in Figure 2, T1, T2 represent human esophageal carcinoma among the figure, N1, N2 represent cancer beside organism.

According to cDRC1 3 ' terminal sequence synthetic primer, enlarge specimen amount and carry out reverse transcription PCR (RT-PCR, α-Tubulin is as internal reference) analysis, what RT-PCR analyzed the results are shown in Figure 3.Top delegation represents α-Tubulin contrast among the figure, and following delegation represents DRC1.T represents human esophageal carcinoma, and N represents cancer beside organism.Cut at all according to cDRC1 and to rectify that normal tissue sample all has the PCR product and lack the result of PCR product, judge that cDRC1 with the esophageal carcinoma relevant gene takes place at most of esophageal carcinoma samples.

The bacterial expression of embodiment 3:DRC1 and purifying

The cDNA sequence pcr amplification of coding pDRC1,5 ' and 3 ' end of used Oligonucleolide primers and cDRC1 nucleotide sequence is corresponding.Also joining respectively in 5 ' and the 3 ' end sequence in addition with the corresponding Nucleotide of DRC1 gene.The sequence of 5 ' end Oligonucleolide primers is: 5 '-CCCGCATGC CTCAGTTACTGCAAAACA-3 ' (SEQ ID No.4), it comprises a SphI restriction site (italic), then is 18 Nucleotide (line part) of DRC1 encoding sequence, from the 5th Nucleotide of cDRC1 open reading frame sequence.The ATG codon is included in the SphI site.In that codon after ATG, first base is from the SphI site, and two remaining bases are consistent with the 5-6 Nucleotide of cDRC1 open reading frame sequence.3 ' terminal sequence is: 5 '-CCCGGATCC GAAGTCATGGCTTGGTGC TTCT-3 ' (SEQ ID No.5), it comprises a BamHI site (italic), then is 22 Nucleotide (line part) that comprise the distinguished sequence of gene terminator codon.These restriction sites and bacterial expression vector pQE-70 (Qiagen, Inc.Chatsworth, CA) the restriction site unanimity on.PQE-70 coding antibiotics resistance (amp ^r), the promotor operon (P/O) regulated of IPTG of a bacterium replication orgin (ori), a ribosome bind site (RBS), 6-His mark and restriction site.PCR product and pQE-70 digest with SphI and BamHI.The sequence of amplification is linked among the pQE-70, be inserted in the framework of code set ammonia group acidity scale note and RBS sequence.(Qiagen, Inc.), used method has description in " molecular cloning experiment guide " (second edition, press of cold spring harbor laboratory, 1989) of works such as Sambrook with connecting mixture transformed into escherichia coli bacterial strain M15/rep4.M15/rep4 contains the pREP4 plasmid of multiple copied, and this plasmid expression LacI supressor also shows kalamycin resistance (Kan ^r).Transformant is identified containing on the LB flat board of penbritin and kantlex, selects the resistance bacterium colony that can grow.Isolate plasmid DNA and confirm with restriction analysis.Allow and contain being cloned in of the construct wanted and mend overnight growth (O/N) in the LB liquid nutrient medium that Amp (100 μ g/ml) and Kan (25 μ g/ml) are arranged.Again the ratio of O/N culture with 1: 100 to 1: 250 is inoculated in the big nutrient solution.Allow cell grow into optimum density: O.D.600 is between 0.4 to 0.6.Add the IPTG that final concentration is 1mM (sec.-propyl-β-D-sulfo-arsenic mutter galactoside) then.IPTG induces by making LacI supressor inactivation, and cleaning P/O causes genetic expression to be increased.Allow cell grow again 3 to 4 hours.Pass through centrifugal cell harvesting.Cell precipitation is dissolved in the pH5.0 Guanidinium hydrochloride of 6M.After the clarification, under the condition that the protein that allows to comprise 6-Hi s mark is combined closely, from solution, be purified into dissolved DRC1 (Hochuli et al.:J chromatography 411:177-184,1984) with nickel chelate column chromatography.With the 6M Guanidinium hydrochloride DRC1 (purity＞98%) is eluted from post.Removing Guanidinium hydrochloride makes protein renaturation reach (Jaenicke﹠amp with several schemes; Rudolph:Protein Structure-APractical Approach, IRL Press, New York, 1990).At first, remove Guanidinium hydrochloride with the substep dialysis.Perhaps, the protein binding of the purifying that will separate from the nickel chelate column is to another post, and this post has a linear Guanidinium hydrochloride gradient that reduces.Protein just can renaturation when being attached on this post, gets off with the buffer solution elution that contains 250mM imidazoles, 150mM NaCl, 25mM Tris-HCl pH7.5 and 10% glycerine then.At last, with the store buffer liquid that contains 5mM bicarbonate of ammonia soluble proteins is dialysed.

Embodiment 4: the expression of reorganization DRC1 in the COS cell

Expression plasmid DRC1 HA derived from carrier pcDNAI/Amp (Invitrogen, Inc.), it comprises: 1) SV40 replication orgin; 2) ampicillin resistance gene; 3) intestinal bacteria replication orgin; 4) CMV promotor, after connect a multiple clone site zone, SV40 intron and poly-adenosine site.The dna fragmentation of a whole DRC1 and a 3 ' terminal HA mark that merges with frame thereof of encoding is cloned the into multiple clone site zone of carrier, and recombinant protein is expressed under the domination of CMV promotor.The HA mark is an epi-position of the influenza hemagglutination fibroin (.:Cell 37:767 such as wilson, 1984) described of front.The HA mark is fused to the antibody of being convenient on the target protein with identification HA epi-position and detects recombinant protein.

Be the construction strategy of plasmid below:

The cDNA sequence of coding pDRC1 makes up with two primer PCRs: 5 ' end primer is 5 '-CCCAAGCTT CTTCAAAGATGCCTCAGTTACTGCAAAACA-3 ' (SEQ ID No.6), it comprises a HindIII restriction site (italic), then is 30 Nucleotide (line part, the ATG codon is its 9-11bp) of DEC1 sequence.3 ' terminal sequence is 5 '-CCCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTA TGGCTTGGTGCTTCTCAAGTAGG-3 ' (SEQ ID No.7), it comprises an XbaI site (italic), then is last 23 Nucleotide (line part) of translation stop codon (runic), HA mark and DRC1 encoding sequence (not comprising terminator codon).So fusion translation stop codon and XbaI site after the HA of same framework mark, HA mark that the PCR product comprises a HindIII site, DRC1 encoding sequence, is right after.The dna fragmentation of pcr amplification and carrier pcDNAI/Amp are digested with HindIII and XbaI restriction enzyme and couple together.With connect mixture transformed into escherichia coli bacterial strain SURE (Stratagene, Inc.).Conversion product is layered on the acillin flat board, selects the resistance bacterium colony.From transformant, isolate plasmid DNA, and correct fragment whether occurred with the restriction analysis inspection.For express recombinant DRC1, by DEAE-dextran method (Sambrook etc.: molecular cloning experiment guide, second edition, press of cold spring harbor laboratory, 1989) week expressing the carrier rotaring redyeing COS cell.Available radio-labeling of the proteic expression of DRC1 HA and immuno-precipitation detect (Harlow﹠amp; Lane: antibody laboratory manual, press of cold spring harbor laboratory, 1988).Two days usefulness after the transfection ³⁵S-halfcystine labeled cell 8 hours is collected nutrient solution then, and usefulness stain remover lysing cell (the RIPA damping fluid: 150mM NaCl, 1%NP-40,0.1%SDS, 0.5%DOC, 50mM Tirs, pH7.5).With a kind of HA special monoclonal antibody sedimentation cell lysate and nutrient solution, analyze protein precipitation with SDS-PAGE again.

Embodiment 5: with baculovirus expression system clone and expression DRC1

The proteic dna sequence dna pcr amplification of coding total length DRC1, Oligonucleolide primers is consistent with 5 ' and 3 ' terminal sequence of gene: 5 ' end primer sequence is 5 '-CCCGGATCC TTCAAAGATGCCTCAGTTACT GCAAAACA-3 ' (SEQ ID No.8), it comprises a BamHI restriction site (italic), then is 29 Nucleotide (line part, the ATG codon is its 8-10bp) of DRC1 sequence.3 ' terminal sequence is 5 '-CCCGGTACC TCATGGCTTGGTGCTTCTCAAGTAGG-3 ' (SEQ ID No.9), it comprises an Asp781 site (italic), then is 26 Nucleotide (line part) that comprise the distinguished sequence of gene terminator codon.(Qiagen Inc.) separates from 1% sepharose the sequence of amplification with a commercial available test kit.Digest this fragment with restriction endonuclease BamHI and Asp781, and then on 1% sepharose purifying.This fragment is designated as F2.

Carrier pRG1 (modifier of pVL941 carrier) is used to the proteic expression (Sumers﹠amp of DRC1 in the baculovirus expression system; Smith: baculovirus vector and insect cell cultural method handbook, 1987).This expression vector comprises the strong polyhedrin promotor of autographa california nuclear polyhedrosis virus (AcMNPV) and subsequent the recognition site of restriction enzyme BamHI and Asp781.The polyadenylation site of monkey disease poison SV40 is used to efficient polyadenylation.For the ease of selecting recombinant virus, inserted colibacillary beta-galactosidase gene in the same direction of polyhedrin promotor, the polyadenylation signal of polyhedron gene is followed in the back.Polyhedrin sequence both sides are virus sequences, are used for carrying out cell-mediated homologous recombination with the wild-type virus DNA of cotransfection.Many other baculovirus vectors can replace pRG1, for example pAc373, pVL941 and pAcIM1 etc.

With restriction enzyme BamHI and Asp781 digested plasmid, use calf small intestine Phosphoric acid esterase then through the methods known in the art dephosphorylation.(Qiagen Inc.) isolates DNA from 1% sepharose with test kit that commerce obtained.This carrier DNA is designated as V2.

F2 fragment and dephosphorylized plasmid V2 couple together with the T4 dna ligase, and transformed into escherichia coli HB101 cell identifies the bacterium that contains the plasmid (pBac-DRC1) of being with the DRC1 gene with BamHI and Asp781 enzyme then.Sequence by cloned sequence is confirmed with dna sequencing.

The linearizing baculovirus (" BaculoGoldTM baculovirus DNA " that the commerce of the plasmid pBac-DRC1 of 5 μ g and 1 μ g obtains, Pharmingen, San Diego, CA.) transfection together, use liposome transfection method (Felgner et al.:Proc Natl Acad Sci USA84:7413-7417,1987).

The plasmid pBac-DRC1 of the BaculoGoldTM viral DNA of 1 μ g and 5 μ g mixes in the well of aseptic microtiter plate, contain in the hole 50 μ l serum-frees the GraceShi substratum (LifeTechnologies Inc., Gaithersburg, MD).Add 10 μ l liposome transfection agent and 90 μ l GraceShi nutrient solutions again, mixing, incubation is 15 minutes under the room temperature.Then transfection mixture is added drop-wise in the Sf9 insect cell (ATCC CRL1711), these cells are seeded in the tissue culturing plate of a 35mm with the GraceShi nutrient solution of 1ml serum-free.Before and after rock the solution that culture plate is newly added with mixing, 27 ℃ of incubations 5 hours.Remove transfection solution from culture plate after 5 hours, add the GraceShi insect nutrient solution that 1ml has added 10% foetal calf serum.Culture plate is put back in the incubator again, and 27 ℃ continue to cultivate 4 days.

Collect supernatant after 4 days, carry out the plaque test with the described method of Summers and Smith that is similar to.Changing part has been to use and has " Blue Gal " (it makes that can isolate blueness easily has a liking for bacterial plaque for Life Technologies Inc., sepharose Gaithersburg).(can be about the detailed description of having a liking for the bacterial plaque test referring to Life Technologies Inc., the insect cell of Gaithersburg dispensing is cultivated and baculovirus is learned user guided 9-10 page or leaf.)

Behind the serial dilution four days, virus entered in the cell, gets blueness with the choicest of Eppendorf suction pipe and has a liking for bacterial plaque.The resuspended agar that contains recombinant virus in the Eppendorf pipe that contains 200 μ l GraceShi nutrient solutions.Of short duration centrifugal removal agar, the supernatant liquor that contains recombinant baculovirus is used to be seeded in the Sf9 cell in the 35mm plate.Supernatant liquor after 4 days in the results culture dish is stored in 4 ℃.

The Sf9 cell is grown in the GraceShi substratum that is supplemented with 10% hot deactivation FBS.These cells infect with recombinant baculovirus V-DRC1, and infection multiplicity (MOI) is 2.Remove former substratum after 6 hours, with the SF900II substratum that does not contain methionine(Met) and halfcystine replace (Life TechnologiesInc., Gaithersburg).Add the 35S-methionine(Met) of 5 μ Ci and the 35S-halfcystine (Amersham) of 5 μ Ci after 42 hours.With cell incubation 16 hours, centrifugal cell harvesting can be seen the albumen that is labeled with SDS-PAGE and radioautograph again.

Embodiment 6: gene therapy is expressed

Inoblast can obtain from a Skin biopsy object.The gained tissue is placed to morsel in the tissue culture medium (TCM).Little and thick tissue block is placed on the wet surface of a tissue culture flasks, puts ten for about every bottle.With bottle reversing, lid tightly, placement is spent the night in the room temperature.In the room temperature after 24 hours, at the bottom of inverted bottle, tissue block still are fixed on bottle, add the fresh substratum that contains 10%FBS, penicillin and Streptomycin sulphate (for example Ham ' s F12 substratum).Cultivate an about week at 37 ℃ then.At this moment, add fresh culture, after this changed once every several days.After cultivating fortnight again, an individual layer inoblast has appearred, with it with tryptic digestion and scrape in the bigger culturing bottle.

PMV-7 (Kirschmeier et al.:DNA 7:219-25,1988) flank is the long terminal repeat of Moloney mouse sarcoma virus, digests it with EcoRI and HindIII, handles with calf small intestine squama acid enzyme subsequently.Linear carrier separately and with the granulated glass sphere purifying comes out on sepharose.

The cDNA pcr amplification of code book invention polypeptide, primer conform to 3 ' end sequence with 5 ' respectively.5 ' end primer contains an EcoRI site, and 3 ' end primer contains a HindIII site.The EcoRI of the linear skeleton amplification of the Moloney mouse sarcoma virus of equivalent added with HindIII site fragment be in the same place, add the T4 dna ligase again, remain on and be suitable under the condition that two fragments connect.Connect mixture and be used for transform bacteria HB101, then in order to determine that the gene that has correct insertion on the carrier is taped against bacterium on the agar that contains kantlex.

The pA317 of amphophilic or Gp+am12 packing cell in the improved Eagle substratum of the Dulbecco that contains 10% calf serum (CS), penicillin and Streptomycin sulphate (DMEM) tissue culture to the density that is paved with.In substratum, add the carrier contain required gene, with its packing cell of transduceing.Packing cell produces the infectious virion (packing cell is called the production cell at this) with required gene.

Being added fresh substratum in the production cell of transduceing, gather in the crops nutrient solution from being paved with the 10cm plate of producing cell subsequently.The virion that contains the tool resistance infection in the nutrient solution that exhausts is removed dissociated production cell with filtering with microporous membrane, and this nutrient solution is used to infect inoblast again.The inoblast that is paved with from the Asia is removed substratum, is changed to fast from the nutrient solution of producing cell.Replace this substratum with fresh substratum again.If the titre height of virus, all so basically inoblasts do not need to select all with transfected.If titre is very low, need the reverse transcription carrier that has selective marker such as neo or hi s with so.

The inoblast of through engineering approaches can be injected the host separately, perhaps grows into to inject the host when being paved with on the Cytodex3 microcarrier bead.At this moment, inoblast produces protein product.

According to top instruction, may carry out various modifications and variation to the present invention, so within the scope of the appended claims, the present invention can implement to be different from specifically described mode.

Sequence table

SEQ?ID?No.1：

cDRC1?1,891bp，5’-3’

ORF：53-1,540(1,488bp)495aa

1 CACTTAACAG?CCACTTGTTT?CATCCCACCT?GGGCATTAGG?TTGACTTCAA

51 AGATGCCTCA?GTTACTGCAA?AACATTAATG?GGATCATCGA?GGCCTTCAGG

101 CGCTATGCAA?GGACGGAGGG?CAACTGCACA?GCGCTCACCC?GAGGGGAGCT

151 GAAAAGACTC?TTGGAGCAAG?AGTTTGCCGA?TGTGATTGTG?AAACCCCACG

201 ATCCAGCAAC?TGTGGATGAG?GTCCTGCGTC?TGCTGGATGA?AGACCACACA

251 GGGACTGTGG?AATTCAAGGA?ATTCCTGGTC?TTAGTGTTTA?AAGTTGCCCA

301 GGCCTGTTTC?AAGACACTGA?GCGAGAGTGC?TGAGGGAGCC?TGCGGCTCTC

351 AAGAGTCTGG?AAGCCTCCAC?TCTGGGGCCT?CGCAGGAGCT?GGGCGAAGGA

401 CAGAGAAGTG?GCACTGAAGT?GGGAAGGGCG?GGGAAAGGGC?AGCATTATGA

451 GGGGAGCAGC?CACAGACAGA?GCCAGCAGGG?TTCCAGAGGG?CAGAACAGGC

501 CTGGGGTTCA?GACCCAGGGT?CAGGCCACTG?GCTCTGCGTG?GGTCAGCAGC

551 TATGACAGGC?AAGCTGAGTC?CCAGAGCCAG?GAAAGAATAA?GCCCGCAGAT

601 ACAACTCTCT?GGGCAGACAG?AGCAGACCCA?GAAAGCTGGA?GAAGGCAAGA

651 GGAATCAGAC?AACAGAGATG?AGGCCAGAGA?GACAGCCACA?GACCAGGGAA

701 CAGGACAGAG?CCCACCAGAC?AGGTGAGACT?GTGACTGGAT?CTGGAACTCA

751 GACCCAGGCA?GGTGCCACCC?AGACTGTGGA?GCAGGACAGC?AGCCACCAGA

801 CAGGAAGAAC?CAGCAAGCAG?ACACAGGAGG?CCACCAATGA?CCAGAACAGA

851 GGGACTGAGA?CCCACGGTCA?AGGCAGGAGC?CAGACCAGCC?AGGCTGTGAC

901 AGGAGGACAT?GCTCAGATAC?AGGCAGGGAC?ACACACCCAG?ACACCCACCC

951 AGACCGTGGA?GCAGGACAGC?AGCCACCAGA?CAGGAAGCAC?CAGCACCCAG

1001 ACACAGGAGT?CCACCAATGG?CCAGAACAGA?GGGACTGAGA?TCCACGGTCA

1051 AGGCAGGAGC?CAGACCAGCC?AGGCTGTGAC?AGGAGGACAC?ACTCAGATAC

1101 AGGCAGGGTC?ACACACCGAG?ACTGTGGAGC?AGGACAGAAG?CCAAACTGTA

1151 AGCCACGGAG?GGGCTAGAGA?ACAGGGACAG?ACCCAGACGC?AGCCAGGCAG

1201 TGGTCAAAGA?TGGATGCAAG?TGAGCAACCC?TGAGGCAGGA?GAGACAGTAC

1251 CGGGAGGACA?GGCCCAGACT?GGGGCAAGCA?CTGAGTCAGG?AAGGCAGGAG

1301 TGGAGCAGCA?CTCACCCAAG?GCGCTGTGTG?ACAGAAGGGC?AGGGAGACAG

1351 ACAGCCCACA?GTGGTTGGTG?AGGAATGGGT?TGATGACCAC?TCAAGGGAGA

1401 CAGTGATCCT?CAGGCTGGAC?CAGGGCAACT?TGCATACCAG?TGTTTCCTCA

1451 GCACAGGGCC?AGGATGCAGC?CCAGTCAGAA?GAGAAGCGAG?GCATCACAGC

1501 TAGAGAGCTG?TATTCCTACT?TGAGAAGCAC?CAAGCCATGA?CTTCCCCGAC

1551 TCCAATGTCC?AGTACTGGAA?GAAGACAGCT?GGAGAGAGTT?TGGCTTGTCC

1601 TGCATGGCCA?ATCCAGTGGG?TGCATCCCTG?GACATCAGCT?CTTCATTATG

1651 CAGCTTCCCT?TTTAGGTCTT?TCTCAATGAG?ATAATTTCTG?CAAGGAGCTT

1701 TCTATCCTGA?ACTCTTCTTT?CTTACCTGCT?TTGCGGTGCA?GACCCTCTCA

1751 GGAGCAGGAA?GACTCAGAGC?AAGTCACCCC?TTTGTACTGA?ATTGTCCTCA

1801 TCTTGTGGGG?GGTTTCAGGA?CTATTTTTAT?CTCTGACATC?TCTCTATTGC

1851 CCCATCTACC?CTAATGCATC?AATAAAACCT?TAAGCCGCTG?G

SEQ?ID?No.2：

pDRC1(495aa)

1 MPQLLQNINGIIEAFRRYARTEGNCTALTRGELKRLLEQEFADVIVKPHD

51 PATVDEVLRLLDEDHTGTVEFKEFLVLVFKVAQACFKTLSESAEGACGSQ

101 ESGSLHSGASQELGEGQRSGTEVGRAGKGQHYEGSSHRQSQQGSRGQNRP

151 GVQTQGQATGSAWVSSYDRQAESQSQERISPQIQLSGQTEQTQKAGEGKR

201 NQTTEMRPERQPQTREQDRAHQTGETVTGSGTQTQAGATQTVEQDSSHQT

251 GRTSKQTQEATNDQNRGTETHGQGRSQTSQAVTGGHAQIQAGTHTQTPTQ

301 TVEQDSSHQTGSTSTQTQESTNGQNRGTEIHGQGRSQTSQAVTGGHTQIQ

351 AGSHTETVEQDRSQTVSHGGAREQGQTQTQPGSGQRWMQVSNPEAGETVP

401 GGQAQTGASTESGRQEWSSTHPRRCVTEGQGDRQPTVVGEEWVDDHSRET

451 VILRLDQGNLHTSVSSAQGQDAAQSEEKRGITARELYSYLRSTKP

SEQ?ID?No.3：

gDRC1(5,009bp，5’-3’)

1 CACTTAACAG?CCACTTGTTT?CATCCCACCT?GGGCATTAGG?TAAGTCCCCT

51 CATAAGAAAC?CTCTTTCTCA?TTCTCAGTGT?CTTGGTGATC?TGAGCTCATA

101 AAACTGGGGC?AGTCAGGTAT?GGACTATGCA?TCCTTCAGAG?CTAGCTGTGA

151 GCACTGGGCA?AACCAACGCT?ACCGTTGGGA?AACATGCTCT?CCTGAAGCAA

201 TCAGGCTTTC?TCCTCCTCCC?TGAGGCTGGC?CTGGGAGCAG?CTCCTCTCAC

251 TGGGAAACTG?TGTGGGCAGC?GGCTATGGGG?CCACCCATGT?GCCTTCCTGG

301 ATCAGCAAAG?GTTTCTTTTT?TCTAAGGCTC?TGGAAGCTTC?TTTGCAGTGC

351 TGAGAGTCTA?TGGGATCAGA?ATCAGTTTAC?TTATGCCAAC?CTAGACAATA

401 AGATCAAACT?GTGTCATGGA?TGAAGGGGTT?TACATGATTC?CCCTCTCCTA

451 CACCAGGGTG?ATATTTAGGC?AAAATATGTG?TAGATTTTTC?TAAGGAATCT

501 AAAATGTAAC?TAAAAGGTCA?TCTTATTATT?TTATTATCTA?AAGGTCAGTG

551 GTTAAAGTCT?GCTACATGGT?TTTAAAAAAA?AGAAAGATAT?TTTTCATCTA

601 TGTTGAGGAA?AACATCCCCA?GTTTTTTACC?TTGATGAAAA?GTTTGCCTGA

651 AATTGTTGGT?TACCAGGTCC?TAGAAAGGGT?TTCTCCTGAA?CAGCCCACCT

701 TTTGCTATGA?CTTACTGAGT?CCTCATGGCC?ACACTAATCT?GCTTTTTCTA

751 GAACTCAAGT?CTCCTTCCTT?CCTTTTTTCT?CTTTCTTCTC?CTACCTATAT

801 CTGCCTCGTC?CCATCCTCTC?TCTGGCTTTC?CAGCTGCTAC?AGGCTCCATC

851 TCCCCTTGCA?TTTGAGACTT?GTCATCTTTG?ATACCATCTC?CTCCTTTGGG

901 TCTCTCCAAG?GCTTCTGCTT?AATGAATCTT?CAAGTCTCTT?TTCCTTTTGC

951 TCATGCAACC?AAACCCAGGC?CTCACCTCAA?CCTACCTCTA?GATTTCTGGC

1001 TAATGAAAAA?GAAAAGCTTT?CCCTTTGATT?AGGAACCAAC?TCATAGGTCA

1051 CCAGAAATCT?GGGCCTGATG?GAGCCACGTG?CCTGTTGGGC?AAGCTGACTT

1101 CTCTGATAAG?TCTCAGGGCT?GTGGACAGAG?GCGACATGCA?GAGAAACTTG

1151 GACCCTCAGA?ACTGGAAGGC?CTCCCACCCA?AAGAAGGTTC?CCCCCTCCTG

1201 AGCATTCCCA?GCAGGTGGTA?GCCCAGTTTC?TTCCCACTTT?CCCAAAAAAA

1251 ACAAGAAGGG?AGGGCTGTGT?CCCTGGAATT?TCTGCTTGGC?TCCCATCCAA

1301 GACAGGGGTT?GACTCACAGA?TGTATTTACT?TCCTTTTGGC?GTTCCCGATG

1351 GGACCCTAAC?ACCCTTGTGA?TAAATAAATA?AATCCTGTCC?ACAGGGTACA

1401 TTCTAGAAAG?CCCATTGCAT?TTGGGTTAAG?GAAAAATGGA?TCCTAGGATT

1451 TCTTGGCTCC?TTAAAAATTG?TGTGGCCTAA?CTTCTCTGAG?CCTGTTTCCT

1501 CACCTATAAA?AAAAAGTGGA?AATAATAAGG?ATTAGAAGAG?ATGATGTACA

1551 GGAAAGTGAT?TATATAGGTG?TACAAAAATT?ATATGTGTGT?ATATGTATAT

1601 AATAGCATAA?TAATTATATT?TAATAAGGAG?CACTTAATAT?AAGCCAGGCA

1651 TCTGCCAAGT?GCTTTTTATG?AATTATGTCT?TTAAATCTTC?ACAATAATCC

1701 CACAGAGTAC?TAATATCACC?TTCAATTACA?ATGAGTAGTA?AACTGAGGCA

1751 TGGAGAGGCA?AAGCAACTTG?TCCCAGCTCA?CATCATACTT?AAGTGGTGGG

1801 GCCAGCATTT?AATCCTGACT?CAGGAACCTG?CCTCCATCCT?GTGTGCTCCT

1851 CCTTCCCATA?CATAAGATGC?ATTATGTAGG?AAAGGACAAA?GGAAGACTAA

1901 AAACAGAGCT?GAACGTGCAA?GGAAAGACCT?AGCAGCAGAC?GTGCTATAAA

1951 GGAAATAGCT?GAGGTTGATT?ATGGAGCCTC?CAAGGGAACT?TTTCATCTTT

2001 TCCAGGTTGA?CTTCAAAGAT?GCCTCAGTTA?CTGCAAAACA?TTAATGGGAT

2051 CATCGAGGCC?TTCAGGCGCT?ATGCAAGGAC?GGAGGGCAAC?TGCACAGCGC

2101 TCACCCGAGG?GGAGCTGAAA?AGACTCTTGG?AGCAAGAGTT?TGCCGATGTG

2151 ATTGTGGTAC?GGTGTGCTGA?GCCGGGTGGA?GAGGGGACAT?AGCAGGAGAG

2201 TGAAACCTGG?TTTGCCTGCA?GAGGCCTTGA?CCTGGGGAAT?TTGAGGAGGC

2251 AGCAGCTAAA?CCCAGGCCTG?CCGGGACAGA?TGGCAGCTGT?GCAGGCAGAA

2301 AAAAAGGTGA?AAGAACCAGA?GATGGTCATG?GGAGTTGGCA?AGTCCTGGCT

2351 CTTTAGATTA?AAACCTTGGT?TTTAATTAAT?TCTAACTTAA?AGACAAGGTA

2401 AAAGGGCTCT?AAAAGGACAA?CTCAGACAGG?AGCAGAGCCT?TGGAATATTT

2451 CAAAATGAAA?ATAATTGCTG?CTTTCTGCCG?CCTCTTAAAT?TTGATACAGT

2501 AAATATTTCC?CACGTCTATC?TGAAATGTAA?TCATCCATTC?ATAGACATTC

2551 ATTAGAAGCA?TAGCTCTGGG?CTTGCACGAA?GCAGTTACTC?AAAAATATTA

2601 GCAGACTGAT?CACATCAGAA?ATGAAATTTT?GAAGAGCAGG?TTGTTAATAG

2651 CTAGGGGAGA?CTTTGGAGCC?TCACCCCACC?CCACCTGGCA?AACCAGAACC

2701 AAGGCCTTGA?TGCACTTTCC?TGTCTTTGGT?TTGCATCTAA?AGCAACCAGG

2751 ATGATGATGG?CCTIAGGGAC?AAGGACATAT?GGGCACAGAA?GGATGCTGCA

2801 TCCACATGCT?CAGGGCAGCG?CTGCAGGGGC?CCACTGCTTC?CCTCCCTCTT

2851 TATCATGGGG?AAACATCTGG?GCCTCAATGA?GGAGCGCACA?GAATTCCCAT

2901 GGGGCTGTGT?TCCCAACCTG?CTGCTCTTTG?TGCTGGGCCT?GCTGAAGAGA

2951 CTAAGGCCTC?AGTGCCAGGG?GCAAGGTGCC?AAGGGCAGCC?CAGACAGTCA

3001 ACTTGAGAGC?CCAAACAGTT?GCATTGTGAA?TTCAATAATT?TATTAACTCT

3051 TCAATAAATC?TTTATCTAAT?TTTCCTGTAG?CCCAGAAATT?GTGCCAAATA

3101 GAGGCTACCA?AACAAAAAAT?GCTCTCTACA?CTTGAGGGAG?AGAGACGGGA

3151 CTAAAAAAAA?ATAAAGGCAA?TTAAGTCTTG?CTGCTGCTCT?AGCCGTATGT

3201 GTGTGTAGTG?TGGGGTCTGA?GGCAGGGGAA?GCTGGCAGCA?GATTGGAAGG

3251 GACCTGCCCA?TGTCCTCCTC?AGGGGAGGGA?TGCTGACTCC?ACCTCATCTT

3301 CTCCTCAGAA?ACCCCACGAT?CCAGCAACTG?TGGATGAGGT?CCTGCGTCTG

3351 CTGGATGAAG?ACCACACAGG?GACTGTGGAA?TTCAAGGAAT?TCCTGGTCTT

3401 AGTGTTTAAA?GTTGCCCAGG?CCTGTTTCAA?GACACTGAGC?GAGAGTGCTG

3451 AGGGAGCCTG?CGGCTCTCAA?GAGTCTGGAA?GCCTCCACTC?TGGGGCCTCG

3501 CAGGAGCTGG?GCGAAGGACA?GAGAAGTGGC?ACTGAAGTGG?GAAGGGCGGG

3551 GAAAGGGCAG?CATTATGAGG?GGAGCAGCCA?CAGACAGAGC?CAGCAGGGTT

3601 CCAGAGGGCA?GAACAGGCCT?GGGGTTCAGA?CCCAGGGTCA?GGCCACTGGC

3651 TCTGCGTGGG?TCAGCAGCTA?TGACAGGCAA?GCTGAGTCCC?AGAGCCAGGA

3701 AAGAATAAGC?CCGCAGATAC?AACTCTCTGG?GCAGACAGAG?CAGACCCAGA

3751 AAGCTGGAGA?AGGCAAGAGG?AATCAGACAA?CAGAGATGAG?GCCAGAGAGA

3801 CAGCCACAGA?CCAGGGAACA?GGACAGAGCC?CACCAGACAG?GTGAGACTGT

3851 GACTGGATCT?GGAACTCAGA?CCCAGGCAGG?TGCCACCCAG?ACTGTGGAGC

3901 AGGACAGCAG?CCACCAGACA?GGAAGAACCA?GCAAGCAGAC?ACAGGAGGCC

3951 ACCAATGACC?AGAACAGAGG?GACTGAGACC?CACGGTCAAG?GCAGGAGCCA

4001 GACCAGCCAG?GCTGTGACAG?GAGGACATGC?TCAGATACAG?GGAGGGACAC

4051 ACACCCAGAC?ACCCACCCAG?ACCGTGGAGC?AGGACAGCAG?CCACCAGACA

4101 GGAAGCACCA?GCACCCAGAC?ACAGGAGTCC?ACCAATGGGC?AGAACAGAGG

4151 GACTGAGATC?CACGGTCAAG?GCAGGAGCCA?GACCAGCCAG?GCTGTGACAG

4201 GAGGACACAC?TCAGATACAG?GCAGGGTCAC?ACACCGAGAC?TGTGGAGCAG

4251 GACAGAAGCC?AAACTGTAAG?CCACGGAGGG?GCTAGAGAAC?AGGGACAGAC

4301 CCAGACGCAG?CCAGGCAGTG?GTCAAAGATG?GATGCAAGTG?AGCAACCCTG

4351 AGGCAGGAGA?GACAGTACCG?GGAGGACAGG?CCCAGACTGG?GGCAAGCACT

4401 GAGTCAGGAA?GGCAGGAGTG?GAGCAGCGCT?CACCCAAGGC?GCTGTGTGAC

4451 AGAAGGGCAG?GGAGACAGAC?AGCCCACAGT?GGTTGGTGAG?GAATGGGTTG

4501 ATGACCACTC?AAGGGAGACA?GTGATCCTCA?GGCTGGACCA?GGGCAACTTG

4551 CATACCAGTG?TTTCCTCAGC?ACAGGGCCAG?GATGCAGCCC?AGTCAGAAGA

4601 GAAGCGAGGC?ATCACAGCTA?GAGAGCTGTA?TTCCTACTTG?AGAAGCACCA

4651 AGCCATGACT?TCCCCGACTC?CAATGTCCAG?TACTGGAAGA?AGACAGCTGG

4701 AGAGAGTTTG?GCTTGTCCTG?CATGGCCAAT?CCAGTGGGTG?CATCCCTGGA

4751 CATCAGCTCT?TCATTATGCA?GCTTCCCTTT?TAGGTCTTTC?TCAATGAGAT

4801 AATTTCTGCA?AGGAGCTTTC?TATCCTGAAC?TCTTCTTTCT?TACCTGCTTT

4851 GCGGTGCAGA?CCCTCTCAGG?AGCAGGAAGA?CTCAGAGCAA?GTCACCCCTT

4901 TGTACTGAAT?TGTCCTCATC?TTGTGGGGGG?TTTCAGGACT?ATTTTTATCT

4951 CTGACATCTC?TCTATTGCCC?CATCTACCCT?AATGCATCAA?TAAAACCTTA

5001 AGCCGCTGG

SEQ?ID?No.4：5’-CCCGCATG CCTCAGTTACTGCAAAACA-3’ 27bp

SEQ?ID?No.5：5’-CCCGGATCC GAAGTCATGGCTTGGTGCTTCT-3’ 31bp

SEQ?ID?No.6：5’-CCCAAGCTT CTTCAAAGATGCCTCAGTTACTGCAAAACA-3’

39bp

SEQ?ID?No.7：5’-CCCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTA

TGGCTTGGTGCTTCTCAAGTAGG-3’ 62bp

SEQ?ID?No.8：5’-CCCGGATCC TTCAAAGATGCCTCAGTTACTGCAAAACA-3’38bp

SEQ?ID?No.9：5’-CCCGGTACC TCATGGCTTGGTGCTTCTCAAGTAGG-3’ 35bp

Claims (13)

1. isolating polynucleotide, its coding contains the polypeptide of from 1 to 495 amino acids among the SEQ ID No.2.

2. the polynucleotide of claim 1, wherein these polynucleotide are DNA.

3. the polynucleotide of claim 2, it is the polynucleotide of polypeptide shown in the coding SEQ ID No.2.

4. the polynucleotide of claim 2, its nucleotide sequence is selected from the nucleotide sequence of SEQ ID No.1 or SEQ ID No.3.

5. isolating polynucleotide, the polynucleotide of the mature polypeptide that the cDNA of the DRC1 that its coding CGMCC preserving number 0402 is contained is coded.

6. the carrier that contains the DNA of claim 2.

7. use the carrier transformed host cells of claim 6.

8. method that produces polypeptide comprises the host cell of cultivating claim 7 expressing the polypeptide of described dna encoding, and reclaim target polypeptides from culture.

9. a method that produces the cell of energy express polypeptide comprises that the carrier with claim 6 transforms or transfectional cell.

10. be selected from down a peptide species of group:

(a) has the polypeptide of the aminoacid sequence of SEQ ID No.2;

(b) mature polypeptide of the cDNA coding of the contained DRC1 of CGMCC preserving number 0402.

11. the antibody of the polypeptide of anti-claim 10.

12. contain the polynucleotide of claim 1 or 5 and the pharmaceutical composition of pharmaceutical carrier.

13. contain the polypeptide of claim 10 and the pharmaceutical composition of pharmaceutical carrier.

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