CN1223677C - Esophagus cancer related gene - Google Patents

Abstract

The invention discloses the isolated DNA, RNA and polypeptide encoded by the new gene DRC1 related to esophagus cancer, as well as a method for producing the polypeptide by using recombinant technology. It also discloses a diagnostic method for detecting diseases related to the abnormality of the gene's nucleic acid and its encoded polypeptide by detecting the mutation, methylation, RNA and polypeptide levels of the gene's nucleic acid sequence, and also discloses the use of the gene's nucleic acid sequence and The polypeptides treat diseases (such as tumors and cancers) associated with abnormalities in this gene.

Description

an esophageal cancer-associated gene

The invention relates to an esophageal cancer-related gene, a polypeptide encoded by the gene, and the use and preparation method of the gene and the polypeptide.

Using molecular biology techniques to understand the pathological root of diseases at the molecular level is a favorable way to discover or provide effective treatments for these diseases. Since there are still a large number of diseases that have not found effective treatments in the prior art, including most cancers, it is urgent to find genes related to the pathology of these diseases, especially those that are negatively related to these diseases and may be used in these diseases. therapeutic gene.

The purpose of the present invention is to provide a new gene negatively related to esophagus cancer and the polypeptide coded by the gene.

The present invention relates to an isolated polynucleotide comprising a member selected from the group consisting of:

(a) a polynucleotide encoding the polypeptide shown in SEQ ID No. 2 or encoding the mature polypeptide encoded by the cDNA contained in the clone of CGMCC deposit number 0402 (cDRC1);

(b) a polynucleotide that hybridizes to and is at least 70% identical to the polynucleotide of (a); and

(c) A polynucleotide fragment of a polynucleotide of (a) or (b).

The present invention further relates to a vector containing the above polynucleotide, a host cell genetically engineered with the vector and a polypeptide production method comprising culturing the host cell to express the polypeptide encoded by the polynucleotide and recovering the target polypeptide from the culture. The present invention also relates to a method for producing a cell capable of expressing a polypeptide, comprising transforming or transfecting the cell with said vector.

Another aspect of the present invention relates to a polypeptide selected from the group consisting of:

(a) a polypeptide having a deduced amino acid sequence of SEQ ID No. 2 or fragments, analogs and derivatives thereof;

(b) The polypeptide encoded by the cDNA contained in the clone with CGMCC deposit number 0402 or its fragments, analogues and derivatives.

The present invention also relates to antibodies against the aforementioned polypeptides.

The present invention also relates to a method for in vitro diagnosis of a disease or disease susceptibility related to the expression of the above-mentioned polypeptide of the present invention, comprising in a sample from a host:

(a) determining mutations, deletions and rearrangements in the nucleic acid sequence of the gene encoding said polypeptide; and/or

(b) determining methylation in the nucleic acid sequence of a gene encoding said polypeptide; and/or

(c) determining abnormalities in the mRNA of the gene encoding said polypeptide.

The invention further relates to an in vitro diagnostic method comprising analyzing a sample from a host for the presence or changes in the amount of a polypeptide of the invention.

These and other aspects of the present invention should be apparent to those skilled in the art from the detailed description herein below.

One aspect of the present invention provides novel polypeptides, which are human esophageal protein polypeptide pDRC1, and its diagnostically or therapeutically useful fragments, analogs and derivatives having biological activity. Another aspect of the present invention provides isolated nucleic acid molecules (DRC1) encoding these polypeptides, including mRNA, DNA, cDNA (cDRC1) and genomic DNA (gDRC1), and diagnostic or therapeutically useful fragments thereof having biological activity, Analogs and Derivatives. Another aspect of the present invention provides a nucleic acid probe, which comprises a nucleic acid molecule of sufficient length and a sequence that specifically hybridizes with the DRC1 gene sequence.

Figure 1 shows the cDNA sequence of DRC1 and the amino acid sequence deduced therefrom. The standard single letter abbreviations for amino acids are used herein. ABIPRISM*377 DNA automatic sequencer (Takara Company) was used for sequencing, and the accuracy of sequencing was expected to be higher than 98%.

Fig. 2 is a graph showing the results of Northern blot analysis.

Fig. 3 is a graph showing the results of RT-PCR analysis.

According to one aspect of the present invention, it provides a kind of isolated nucleic acid (polynucleotide), these nucleotide codes have the polypeptide of deduced amino acid sequence shown in Fig. 1 (SEQ ID No.2) or code 1999 6 The polypeptide encoded by the cloned cDNA deposited with CGMCC deposit number 0402 (cDRC1) on March 23.

The polynucleotide encoding pDRC1 can be isolated from a number of adult and fetal cDNA libraries, such as the adult esophagus cDNA library. The polynucleotide sequences shown in SEQ ID No.1 and SEQ ID No.3 have an open reading frame encoding a protein with 495 amino acid residues. The protein has the highest degree of homology with the N-terminal of human Profilaggrin (GenBank No.: A45135), 42% of the amino acids are identical and 65% of the 88 consecutive amino acids are similar. Among the 225 consecutive amino acids at the N-terminal of human Trichohyalin (GenBank No.: Q07283), 33% of the amino acids are identical and 49% of the amino acids are similar.

The polynucleotides of the invention may be in the form of RNA or DNA, where DNA includes cDNA, genomic DNA and synthetic DNA. DNA can be double-stranded or single-stranded, and the single strand can also be the coding strand or the non-coding (antisense) strand. The coding sequence encoding the polypeptide may be the same as the coding sequence shown in Figure 1 (SEQ ID No.1 or SEQ ID No.3), or may be the same as the coding sequence of the deposited clone, or may be a different coding sequence, Due to the redundancy or degeneracy of the genetic code, it encodes the same mature polypeptide as that of Figure 1 (SEQ ID No. 2) or the deposited cDNA.

The polynucleotide encoding the polypeptide of Figure 1 (SEQ ID No. 2) or the polypeptide encoded by the deposited cDNA may include: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and any additional coding sequences and non-coding sequences , such as introns or 5' and/or 3' non-coding sequences of the mature polypeptide coding sequence.

Thus, the term "polynucleotide encoding a polypeptide" includes polynucleotides containing only coding sequences for the polypeptide, as well as polynucleotides containing additional coding and/or non-coding sequences.

The present invention further relates to variants of the above-described polynucleotides encoding a polypeptide comprising the deduced amino acid sequence shown in Figure 1 (SEQ ID No. 2) or a fragment of a polypeptide encoded by a deposited clone cDNA, similar substances and derivatives. These polynucleotide variants may be naturally occurring allelic variants or non-naturally occurring polynucleotide variants.

Therefore, the present invention includes polynucleotides encoding the same polypeptide as shown in Figure 1 (SEQ ID No. 2) or the same mature polypeptide encoded by the cDNA of the deposited clone, as well as variants of such polynucleotides, which variants Fragments, analogues or derivatives of the polypeptide encoded by the cDNA encoding the polypeptide shown in Figure 1 (SEQ ID No.2) or the deposited clone. These nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As mentioned above, the polynucleotide may have a coding sequence that is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID No. 1) or the deposited sequence. As known in the art, an allelic variant is an alternative form of a polynucleotide sequence that has one or more nucleotide substitutions, deletions, or additions that do not substantially alter the function of the encoded polypeptide .

A polynucleotide of the invention may also contain a coding sequence fused in frame to a marker sequence which facilitates the purification of the polypeptide of the invention. The marker sequence can be the hexahistidine tag of the pQE vector when using a bacterial host to facilitate the purification of the mature polypeptide fused with the marker, or when using a mammalian host (such as the COS-7 cell line of monkey kidney fibroblasts) Alternatively, the tag sequence may be a hemagglutinin (HA) tag. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al: Cell 37:767, 1984).

The term "gene" refers to the segment of DNA involved in producing a polypeptide chain, which includes regions preceding and following the coding region (leader and trailer) and intervening sequences (introns) between each coding segment (exons).

The DNA fragment of the full-length gene of the present invention can be used as a hybridization probe of a cDNA library to isolate the full-length cDNA and other cDNAs that are highly similar to the gene sequence or have similar biological functions. Probes of this type preferably have at least 30 bases, eg, contain 50 or more bases. Such probes can also be used to identify cDNA clones corresponding to full-length transcripts and genomic clones of the complete gene including regulatory and promoter regions, exons and introns. An example of a screen is the use of known DNA sequences to synthesize an oligonucleotide probe to isolate the coding region of a gene. Labeled oligonucleotides with sequences complementary to the genes of the invention are used to screen human cDNA, genomic DNA or mRNA libraries to determine the members that hybridize to the probe.

The present invention further relates to polynucleotides that hybridize to the aforementioned sequences, the two sequences being at least 70%, preferably at least 90%, more preferably at least 95% identical. The present invention particularly relates to polynucleotides which hybridize to the above polynucleotides under stringent conditions. The term "stringent conditions" as used herein means 95%, preferably at least 97% identity between two sequences for hybridization to occur. In a preferred embodiment, the polypeptide encoded by the polynucleotide hybridized with the above-mentioned polynucleotide basically maintains the same biological function or activity as that of the polypeptide encoded by Figure 1 (SEQ ID No. 1) or the deposited cDNA.

Another option is that the polynucleotide hybridized with the polynucleotide of the present invention contains at least 20 bases, preferably at least 30 bases, more preferably at least 50 bases and has the identity described above, may retain or No activity retained. For example, such a polynucleotide can be used as a probe for the polynucleotide of SEQ ID No. 1; for example, it can be used for polynucleotide recovery or as a diagnostic probe or PCR primer.

Thus, the present invention relates to polynucleotides and fragments thereof having at least 70% identity, preferably at least 90%, more preferably at least 95% identity to a polynucleotide encoding a polypeptide of SEQ ID No.2, which fragments have at least 30 bases, preferably at least 50 bases; polypeptides encoded by these polynucleotides are also contemplated.

The deposit referred to here is maintained under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits are only prepared for the convenience of those skilled in the art, and are not an admission that deposits are required under Rule 25 of the Implementing Regulations of the Chinese Patent Law. The polynucleotide sequences contained in the deposited materials and the amino acid sequences encoded by the polynucleotides are incorporated herein by reference for verification in case of any dispute over the sequence descriptions herein.

The present invention further relates to polypeptides having the deduced amino acid sequence shown in Figure 1 (SEQ ID No. 2) or having the amino acid sequence encoded by the deposited cDNA, and fragments, analogs and derivatives thereof.

"Fragment", "derivative" and "analog" of the polypeptide shown in Figure 1 (SEQ ID No. 2) or encoded by the deposited cDNA refers to a polypeptide that can substantially retain the biological function or activity of the polypeptide. Thus, the analog includes the proprotein, which is activated by cleavage of a portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

Fragments, derivatives and analogs of polypeptides encoded by the cDNA shown in Figure 1 (SEQ ID No.2) or deposited may be: (i) one or more amino acid residues are conserved or non-conserved amino acid residues Substituted (preferably conservative amino acid residues), the substituted amino acid residues are or are not amino acids encoded by the genetic code; or (ii) one or more amino acid residues have a substituting group; or (iii) mature The polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol); or (iv) additional amino acids are fused to the mature polypeptide, such as a sequence to aid in the purification of the mature protein or a proprotein sequence, etc. . In view of these disclosures, such fragments, derivatives and analogs are believed to be within the purview of those skilled in the art.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form and purified to a uniform degree.

The term "isolated" refers to the removal of a substance from its original environment (eg, its natural environment if naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide is separated from some or all of the substances with which it coexists in a natural system is isolated. Such polynucleotides may be part of a vector, or such polynucleotides or polypeptides may be part of a composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.

The polypeptides of the present invention include polypeptides shown in SEQ ID No.2 (especially mature polypeptides), and also include polypeptides with at least 70% similarity (preferably 70% identity) with SEQ ID No.2 polypeptides, preferably at least Polypeptides with 90% similarity (preferably 90% identity), more preferably polypeptides with at least 95% similarity (preferably 95% identity), also include some parts of these polypeptides, usually these polypeptide parts contain at least 30% amino acids, preferably at least 50 amino acids.

As is known in the art, the similarity of two polypeptides is determined by comparing the amino acid sequences of one polypeptide with the other and their conservative amino acid substitutions.

Fragments or portions of polypeptides of the invention may be used to generate related full-length polypeptides via peptide synthesis. Thus, these fragments can serve as intermediates in the production of full-length polypeptides. Fragments or portions of polynucleotides of the invention can be used to synthesize full-length polynucleotides of the invention.

The present invention also relates to vectors comprising polynucleotides of the present invention, genetically engineered host cells carrying the vectors of the present invention and methods for producing polypeptides of the present invention by means of recombinant techniques.

The host cell is genetically engineered (transduced, transformed or transfected) with the vector of the present invention, which may be a cloning vector or an expression vector. Vectors can be plasmids, virus particles, phage, and the like. Engineered host cells can be cultured on conventional nutrient media modified for activation of promoters, selection of transformants, or amplification of the DRC1 gene. It will be apparent to those of ordinary skill in the art that the culture conditions, such as temperature, pH, etc., are consistent with the expression conditions originally used by the selected cells.

The polynucleotides of the invention can be used to produce polypeptides via recombinant techniques. For example, the polynucleotide may be present on any of a number of expression vectors for expression of polypeptides, including chromosomal, non-chromosomal and synthetic DNA sequences, e.g., derivatives of SV40, bacterial plasmids, Phage DNA, yeast plasmids, vectors derived from the combination of plasmids and phage DNA, viral DNA, baculovirus, vaccinia virus, adenovirus, fowl pox virus, and pseudorabies virus. However, other vectors may also be used as long as they are capable of replicating and surviving in the host.

Suitable DNA sequences can be inserted into vectors in a number of ways. Typically, the DNA sequence is inserted into appropriate restriction enzyme sites using methods known in the art. These methods are believed to be within the purview of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence (promoter) to direct the synthesis of mRNA. Mentioned below are representatives of such promoters: LTR or SV40 promoter, Lac or trp promoter of Escherichia coli, PL promoter of bacteriophage lambda and others known to control gene expression in prokaryotic or eukaryotic cells or their viruses Promoter. Expression vectors also contain ribosome binding sites and transcription terminators required for translation initiation. The vector may also have suitable sequences to enhance expression.

In addition, the expression vector preferably contains one or more selectable marker genes that can provide phenotypic characteristics, so as to facilitate the selection of transformed host cells, for example, dihydrofolate reductase or neomycin resistance can be used for the cultivation of eukaryotic cells, and E. Available tetracycline or ampicillin resistance.

A vector containing the above-mentioned suitable DNA sequence, suitable promoter or control sequence can be used to transform a suitable host, allowing the host to express the protein.

Listed below are representatives of suitable hosts: bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS (monkey renal fibroblast cell line) or Bowes melanoma; adenovirus; plant cells, etc. From these teachings, the selection of a suitable host should be within the knowledge of those skilled in the art.

It is particularly worth mentioning that the present invention also includes recombinant constructs comprising one or more of the sequences generally described above. Such constructs include vectors, such as plasmid or viral vectors, into which a sequence of the invention has been inserted in forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises a regulatory sequence, such as a promoter, operably linked to this sequence. Many vectors and promoters are known to those skilled in the art and are commercially available. Several carriers are listed below. Bacterial: 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); eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, other plasmids or vectors can also be used as long as they are capable of replicating and surviving in the host.

Promoter regions can be selected from desired genes using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. pKK232-8 and pCM7 are two suitable vectors. Particular bacterial promoters include Lacl, LacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase, early and late SM40, retroviral LTRs, and mouse metallothionein I. Selection of suitable vectors and promoters is within the ordinary level of skill in the art.

In another embodiment, the present invention relates to a host cell comprising the above construct. The host cell may be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or a prokaryotic cell, such as a bacterial cell. Methods for introducing the construct into host cells include: calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation or gene gun methods.

The construct in the host cell enables the production of the gene product encoded by the recombinant sequence by conventional methods. Alternatively, the polypeptides of the invention can be synthesized using conventional peptide synthesizers.

The mature protein can be expressed in mammalian cells, yeast, bacteria or other cells under the control of an appropriate promoter. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the invention. Cloning and expression vectors suitable for prokaryotic and eukaryotic hosts are described in "A Laboratory Guide to Molecular Cloning" (Second Edition, Cold Spring Harbor Press, New York, 1989) by Sambrook et al.

In higher eukaryotes, transcription of the DNA encoding the polypeptide of the present invention can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase its transcription. For example, the SV40 enhancer located 100-270 bp behind the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer located behind the replication origin, and the adenovirus enhancer.

Typically, recombinant expression vectors contain an origin of replication and selectable markers for transforming host cells, such as the ampicillin resistance gene of E. coli and the TRiP1 gene of S. transcription of the sequence. These promoters can be derived from operons encoding glycolytic enzymes, such as 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase or heat shock proteins. The heterologous structural sequences are assembled in proper orientation with translation initiation and termination sequences and other preferred sequences. Alternatively, the heterologous sequence may encode a fusion protein containing an N-terminal confirmatory peptide that exhibits desired characteristics, such as stabilization of expressed recombinant products and ease of purification.

Effective expression vectors suitable for bacteria are constructed by inserting the structural DNA sequence encoding the target protein into an operable reading frame with a functional promoter along with appropriate translation initiation and termination signals. The vector should contain one or more phenotypic selection markers and an origin of replication. The origin of replication ensures that the vector can be maintained in the host, and more ideally, the vector can be amplified. Prokaryotic hosts suitable for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium and various bacteria of the genera Pseudomonas, Streptomyces and Staphylococcus, and other hosts can also be selected.

As a representative but non-limiting example, effective expression vectors suitable for use in bacteria include a selectable marker and bacterial origin of replication derived from the genetic elements of the well-known cloning vector pBR322 (ATCC37017) obtained from commercial sources. plasmid. These commercial vectors include, for example, pKK223-3 (Pharmacia) and pGEM1 (Promega). The "backbone" portion of pBR322 is combined with an appropriate promoter and structural sequence to be expressed.

After transformation of a suitable host strain and growth of the host strain to the proper cell density, the selected promoter is induced by an appropriate method (eg temperature shift or chemical induction) and the cells are cultured for an additional period of time.

Cells are usually harvested by centrifugation, disrupted by physical or chemical methods, and the crude extract is retained for further purification. Microbial cells used to express proteins can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell lysing agents, all of which are well known to those skilled in the art.

Various mammalian cell culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems are the COS-7 cell line of monkey kidney fibroblasts described by Gluzman (Cell 23:175, 1981), and other cell lines expressing compatible vectors, such as C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors include an origin of replication, appropriate promoters, enhancers and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking non-transcriptional sequence. DNA sequences and polyadenylation sites derived from the SV40 splice sequence can be used to provide the required non-transcribed genetic elements.

Peptides can be recovered and purified from recombinant cell cultures by ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, and affinity chromatography , hydroxyapatite chromatography and lectin chromatography. Formation of the full conformation of the mature protein requires a protein refolding step. High performance liquid chromatography (HPLC) was applied in the final purification step.

The polypeptide of the present invention may be a purified natural product, or a chemically synthesized product, or may be produced from a prokaryotic or eukaryotic host (eg, cultured bacteria, yeast, higher plant, insect or mammalian cells) through recombinant techniques. Depending on the host used in the recombinant production method, the polypeptides of the present invention may or may not be glycosylated. The polypeptide may also have an initial methionine residue.

The polynucleotides and polypeptides of the present invention can be used as research reagents and materials to discover methods of treatment and diagnosis of human diseases.

This polynucleotide and its encoded polypeptide are also used in related scientific research in vitro, DNA synthesis and preparation of DNA vectors, and are also used in the design of methods for treating and diagnosing human diseases.

Fragments of the full-length DRC1 gene can be used as hybridization probes for cDNA libraries, thereby isolating the full-length gene and other genes with high sequence similarity or similar biological activity. Probes of this type generally have at least 20 bases. However, these probes are preferably at least 30 bases and generally no more than 50 bases, although they may have more bases. The probe can also be used to identify cDNA clones corresponding to full-length transcripts or genomic clones with complete genes, including regulatory and promoter regions, exons and introns. One method of screening is to use the known DNA sequence to synthesize an oligonucleotide probe and use it to isolate the coding region of the gene. Labeled oligonucleotides with sequences complementary to the genes of the invention can be used to screen human cDNA libraries, genomic libraries, mRNA libraries to determine which members of the library hybridize to the probe.

The present invention also relates to using the DRC1 gene product as a component of a diagnostic method for detecting diseases related to DRC1 nucleic acid sequence mutation or methylation or susceptibility to diseases. These diseases are related to insufficient expression of the mRNA and encoded protein of the DRC1 gene of the present invention, such as tumors and cancers.

Individuals with mutations in the DRC1 gene can be detected at the DNA level using various techniques. Nucleic acids for diagnosis can be obtained from patient cells, such as blood, urine, saliva, biopsy and autopsy material. Genomic DNA can be directly used for detection, or it can be amplified enzymatically by PCR (Saiki et al.: Nature 324: 163-166, 1986) before detection and analysis. RNA or cDNA can also be used for the same purpose. For example, PCR primers complementary to nucleic acid encoding the DRC1 gene can be used to identify and analyze mutations in the DRC1 gene. For example, deletion and insertion mutations can be detected from changes in the size of the amplified product compared with the normal genotype. Point mutations can be detected by hybridizing amplified DNA to radiolabeled DRC1 RNA or using radiolabeled DRC1 antisense DNA sequences. Digestion with RNase A or from the difference in melting temperature can distinguish perfectly paired sequences from mismatched duplexes.

Genetic testing based on DNA sequence differences can be performed by detecting changes in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Deletions and insertions of small sequences can be visualized by high-resolution gel electrophoresis. For example, DNA fragments of different sizes can be distinguished in denaturing polyacrylamide gel electrophoresis (PAGE). DNA sequences containing point mutations can be distinguished from normal DNA sequences due to different conformations (different swimming speeds) in polyacrylamide gels without denaturing agents after denaturation.

Nuclease protection assays can reveal sequence changes at specific positions, such as RNase and S1 enzyme protection or chemical fragmentation (eg, Cotton et al: PNAS 85:4397-4401, 1985).

The present invention also relates to diagnostic methods for detecting DNA methylation of the DRC1 gene in various tissues. Methylation at the DNA level can directly lead to changes in gene mRNA transcription, and can also change the normal transcription of genes by causing genome instability and causing gene mutations. DNA methylation can be detected using the methylation-specific PCR method described by Herman (PNAS 93:9821-9826, 1996)

In summary, specific DNA sequences can be detected by hybridization, RNase protection, chemical fragmentation, direct DNA sequencing, or using restriction enzymes (eg, restriction fragment length polymorphisms, RFLP) and Southern blotting of genomic DNA .

In addition to the more traditional methods of gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic method for detecting abnormal expression of DRC1 gene mRNA in various tissues. Changes in mRNA levels can be detected by methods such as reverse transcription PCR (RT-PCR), Northern Blot, Dot Blot or RNA in situ hybridization. These methods should be clear to those skilled in the art from the disclosure herein.

The present invention also relates to diagnostic methods for detecting changes in the level of DRC1 gene protein in various tissues, compared to normal control tissue samples, the reduction of DRC1 gene protein can detect the presence of disease or susceptibility to disease (such as tumor). Methods for determining the level of DRC1 gene protein in a host sample are well known to those skilled in the art and include radioimmunoassay, competitive binding assay, Western blot analysis, enzyme-linked immunosorbent assay and sandwich assay. Enzyme-linked immunosorbent assay (ELISA) (Coligan et al.: Current Protocols in Immunology Vol.1, No.2, Chapter 6, 1991) requires the preparation of specific antibodies against the DRC1 antigen, preferably monoclonal antibodies. In addition, prepare a reporter antibody against the monoclonal antibody. The reporter antibody is linked to a detectable reagent, such as radioactive, fluorescent, or horseradish peroxidase. Samples are taken from the host and incubated on a solid support, such as a polystyrene reaction plate, which adsorbs the proteins in the sample. Incubation with a non-specific protein such as bovine serum albumin (BSA) then covers any free protein binding sites on the plate. In the second step, the monoclonal antibody is added to the plate for incubation. At this time, the monoclonal antibody binds to the DRC1 gene protein attached to the polystyrene reaction plate. All unbound monoclonal antibodies were washed away with buffer. A reporter antibody linked to horseradish peroxidase is added to the plate at this point, and the reporter antibody binds to the monoclonal antibody bound to the DRC1 antigen. Unbound reporter antibody is also washed away. Then add a peroxidase substrate, compare the depth of the color formed within a certain period of time with the standard curve, and then measure the content of the DRC1 gene protein in a certain volume of patient samples.

Competition assays were also used in this assay. Link the specific antibody of the DRC1 antigen to a solid phase carrier, then let the labeled DRC1 and the sample obtained from the host pass through the solid phase carrier and detect the amount of the marker, for example, by liquid phase scintillation chromatography, the amount of the marker and The amount of DRC1 in the sample correlates. Sandwich assays are similar to ELISA. In the sandwich assay, DRC1 passes through the solid support and binds to the antibody adsorbed on the solid support. The second antibody in turn binds to DRC1. The third antibody is labeled and specific to the second antibody, and when it passes through the solid support, it binds to the second antibody, and its amount is then detected.

The pDRC1 polypeptides and diagnostically or therapeutically useful fragments, analogs and derivatives thereof having biological activity can be used in compositions together with a pharmaceutically acceptable carrier, as described below.

The pDRC1 polypeptide and its biologically active diagnostically or therapeutically useful fragments, analogs and derivatives can be used in combination with appropriate drug carriers. Such compositions include a therapeutically effective amount of the polypeptide and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. In addition, these polypeptides and their biologically active diagnostically or therapeutically useful fragments, analogs and derivatives may also be used in combination with other therapeutic compounds.

These pharmaceutical compositions may be administered in a convenient manner such as epidermal, intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, intranasal or intradermal routes. These pharmaceutical compositions are used in dosages effective for the treatment and/or prophylaxis of the particular indication.

According to the present invention, the pDRC1 polypeptide can be expressed in vivo for use, which is commonly referred to as "gene therapy".

For example, a patient's cells can be engineered ex vivo with a polynucleotide (DNA or RNA) encoding a polypeptide, and the engineered cells can be administered to the patient to be treated. Such methods are well known in the art. For example, cells can be engineered with retroviral particles comprising RNA encoding a polypeptide of the invention.

Likewise, cells can be engineered in vivo to express polypeptides in vivo using methods known in the art. As is known in the art, producer cells for the production of retroviral particles containing RNA encoding a polypeptide of the invention can be injected into a patient to engineer the cells in vivo to express the polypeptide in vivo. These and other modes of administration of polypeptides will be apparent to those skilled in the art from the teachings of the present invention. For example, in addition to retrovirus, there are other expression vectors for engineering cells, such as adenovirus, which can be used to engineer cells in vivo after combining it with a suitable transport vector.

Retroviruses from which the above retroviral plasmid vectors can be derived include—but are not limited to—Moloney murine leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, gibbon leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammalian tumor virus. One embodiment is that the retroviral plasmid vector can be derived from Moloney murine leukemia virus.

The vector contains one or more promoters. Suitable promoters that can be used include - but are not limited to - the LTR of retroviruses, the SV40 promoter, the human cytomegalovirus (CMV) promoter (Miller: Biotechniques, Vol.7, No.9, pp980-990, 1989 ) or other promoters (for example, promoters of cells, such as promoters of eukaryotic cells are - but not limited to - histone, polIII, β-actin promoters). Other useful viral promoters are - but are not limited to - adenovirus promoters, thymidine kinase (TK) promoters and B19 parvovirus promoters. Selection of an appropriate promoter will be clear to the skilled artisan from the teachings herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of the corresponding promoter. Suitable promoters include, but are not limited to, promoters from adenoviruses, such as the adenovirus major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter ; inducible promoters, such as MMT promoter, metallothionein promoter; heat shock promoter; albumin promoter; ApoAI promoter; human globin promoter; viral thymidine kinase promoter, such as herpes simplex the glycoside kinase promoter; the LTR of retroviruses (including the modified retroviral LTRs described above); the beta-actin promoter; and the human growth hormone promoter. It may also be the own promoter controlling the gene encoding the polypeptide.

The packaging cell line is transduced with the retroviral plasmid vector to form a producer cell line. Packaging cells for transfection include, but are not limited to, PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAM12 and DNA cell lines, as described by Miller (Human Gene Therapy Vol. 1, pp5-14, 1990), see full text hereof. The packaging cell line can be transduced with the vector by methods known in the art. These methods include, but are not limited to, electroporation, the use of liposomes, and calcium phosphate precipitation. An alternative approach is to encapsulate the retroplasmic vector into liposomes or conjugate lipids and inject them into the host.

Producer cell lines produce infectious retroviral particles comprising nucleic acid sequences encoding these polypeptides. Such retroviral vector particles can be used to transduce eukaryotic cells in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence encoding the polypeptide. Eukaryotic cells that can be used for transduction include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

Polypeptides and fragments, derivatives or analogs thereof, or cells expressing them, can be used as immunogens to generate antibodies thereto. These antibodies can be polyclonal or monoclonal. The invention also encompasses chimeric, single chain and humanized antibodies and Fab fragments, or the products of Fab expression libraries. Various methods known in the art can be used for the production of such antibodies and fragments.

Antibodies raised against polypeptides corresponding to the sequences of the present invention can be obtained by directly injecting the polypeptides into animals or introducing the polypeptides into animals (preferably not humans). The resulting antibody binds to the polypeptide itself. In this way, antibodies that bind the entire native polypeptide can be generated even with sequences encoding only a fragment of the polypeptide. Antibodies can then be used to isolate the polypeptide from the tissue expressing it.

Production of monoclonal antibodies can be done using continuous cell line culture to produce antibodies. For example, using hybridoma technology (Kohle & Milstein: Nature 256:495-497, 1975), Trioma technology, human B cell hybridoma technology (Kozbor et al.: Immunology Today 4: 72, 1983) and EBV-hybridoma technology to produce human Monoclonal antibodies (Cole et al.: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp77-96, 1985).

Single-chain antibody production techniques (US Patent No. 4,946,778) are suitable for production of single-chain antibodies to the immunogenic polypeptide products of the invention. Transgenic animals can also be used to express humanized antibodies to the immunogenic polypeptide products of the invention.

The present invention will be further described in the following examples. However, the present invention is not limited to these examples. All parts and amounts are by weight unless otherwise indicated.

In order to facilitate the understanding of the following embodiments, some frequently occurring methods and/or terms are explained first.

"Plasmids" are designated by a lowercase p preceded and/or followed by uppercase letters and/or numbers. Starting plasmids are either commercially available, publicly available on an unrestricted basis, or constructed from available plasmids according to published procedures. In addition, equivalent plasmids to the described plasmids are known in the art and will be clear to those of ordinary skill.

"Digestion" of DNA refers to the catalytic cleavage of DNA with restriction enzymes that act only at specific sites in the DNA sequence. Various restriction enzymes used herein are commercially available, and their reaction conditions, cofactors and other requirements are applied as known to those of ordinary skill. For analysis, usually 1 μg of plasmid or DNA fragment and about 2 units of enzyme are added to about 20 μl of buffer solution. To isolate DNA fragments for plasmid construction, 5 to 50 μg of DNA is typically digested with 20 to 250 units of enzyme in a larger volume. The manufacturer will indicate the appropriate amount of buffer and substrate for the specific restriction enzyme. Typically the incubation time used is approximately one hour at 37°C, but may vary according to the manufacturer's instructions. The desired fragments are isolated by electrophoresis on a polyacrylamide gel directly after restriction digestion.

8% polyacrylamide gel can be used for separation according to the size of cut fragments (Goedde et al.: Nucleic Acids Research 8: 4057, 1980).

"Oligonucleotide" refers to a single-stranded polydeoxyribonucleotide or two complementary polydeoxyribonucleotide chains that can be chemically synthesized. These synthetic oligonucleotides do not contain a 5' terminal phosphate, so it cannot be ligated to another oligonucleotide without adding a phosphate with ATP by the action of a kinase. Synthetic oligonucleotides will be ligated to fragments that have not been dephosphorylated.

"Ligation"refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments. Unless otherwise provided, ligation can be performed under known buffers and conditions, i.e., 10 units of T4 DNA ligase ("ligase") per 0.5 mg of approximately equimolar amounts of DNA fragments for ligation .

Transformations were performed as described in Graham & Van der Eb: Virology 52:456-457, 1973 unless otherwise stated.

Example 1: Cloning of cDRC1

Trizol (Gibco) reagent was used to extract total RNA from surgically resected esophageal cancer specimens and incised normal tissues (no cancer cells confirmed by pathological sections) according to the instructions, and digested with DNase without RNase activity to remove residual genomic DNA. After quantification by UV spectrophotometer, take 3mg of total RNA and carry out reverse transcription in 20ml system. The reaction conditions are: 1X PCR buffer, 2.5mM MgCl2, 10mM DTT, 500mM dNTPs, 10mM GT15N (N stands for A, G and C) , Superscript II reverse transcriptase 200 units (Gibco), after incubation at 42°C for 60 minutes, the resulting cDNA was stored at -20°C.

Take 0.5ml of cDNA in a 20ml reaction system for differential display PCR (DD-PCR) as follows: 1X PCR buffer, 1.5mM MgCl2, 200mM dNTPs, 1mM 10-mer random primer (Operon), 3mMGT15N (N stands for A, G and C ), Taq enzyme (Gibco) 1 unit. PCR cycle parameters are 95°C for 1 minute, 95°C for 15 seconds, 39°C for 4 minutes, 72°C for 2 minutes, 4 cycles at 95°C for 15 seconds, 39°C for 2 minutes, and 72°C for 1 minute for 35 cycles , 72°C for 5 minutes. The PCR reaction was carried out in the PE9600PCR system.

The DD-PCR product was loaded on adjacent lanes of esophageal cancer and incised normal tissue, electrophoresed in 6% denaturing polyacrylamide gel (PAGE), and clear bands could be seen after staining with silver nitrate. Excise the differential band that is visible in the normal tissue lanes and missing in the cancer tissue lanes as a template, and perform secondary amplification according to the above DD-PCR conditions. The amplified product is directly recovered by wizard PCR Preps DNA Purification System (Promega), and cloned in pGEM-T Easy vector (Promega) was sequenced with ABIPRISM*377 DNA automatic sequencer. The 3' end sequence of cDRC1 was thus obtained.

Primers were synthesized according to the sequencing results, and the 5' upstream sequence of cDRC1 was captured by Marathon cDNA RACE System (Clontech), cloned and sequenced, and spliced with the above 3' end sequence. Then, primers were synthesized according to the two ends of the spliced sequence, the full-length cDNA of cDRC1 was amplified by PCR method, and further cloned and sequenced for verification. As a result, the plasmid pGEM-TEasy containing the nucleotide sequence shown in SEQ.ID.NO.1 was obtained, and the Escherichia coli strain DH5α containing the plasmid was registered in China Microorganism Culture Collection Management Committee on June 23, 1999. Center (Zhongguancun, Beijing, China, 100080) preservation, the preservation number is CGMCC 0402.

Example 2: Northern blot and RT-PCR analysis of DRC1 gene

The cloned cDRC1 3' end sequence was used as a probe, and the probe was labeled with Primer-a-Gene Random Primer Labeling Kit α- ³² P-dATP/dCTP. The esophageal cancer and cut-end normal tissue RNA were transferred to nylon membrane and fixed, and the labeled probe was used to carry out Northern hybridization, and the cloned cDNA fragment was identified to be from the cut band on the PAGE gel (that is, the cut-end normal tissue expression, esophageal cancer does not expression) while estimating the RNA size of the DRC1 gene. The results of Northern blot are shown in Figure 2, in which T1 and T2 represent esophageal cancer tissues, and N1 and N2 represent paracancerous tissues.

Primers were synthesized according to the cDRC1 3' end sequence, and the sample volume was enlarged for reverse transcription PCR (RT-PCR, α-Tubulin was used as an internal control) analysis. The results of RT-PCR analysis are shown in Figure 3. The upper row in the figure represents α-Tubulin control, and the lower row represents DRC1. T represents esophageal cancer tissue, N represents paracancerous tissue. According to the results that cDRC1 has PCR products in all cut-end normal tissue samples, but lacks PCR products in most esophageal cancer samples, it is judged that cDRC1 is a gene related to esophageal carcinogenesis.

Example 3: Bacterial expression and purification of DRC1

The cDNA sequence encoding pDRC1 was amplified by PCR using oligonucleotide primers corresponding to the 5' and 3' ends of the cDRC1 nucleic acid sequence. Additional nucleotides corresponding to the DRC1 gene were also added to the 5' and 3' end sequences, respectively. The sequence of the 5' end oligonucleotide primer is: 5'-CCCGCATGC CTCAGTTACTGCAAAACA -3' (SEQ ID No.4), which contains a SphI restriction site (italics), followed by 18 cores of the DRC1 coding sequence Nucleotides (underlined part), starting from the 5th nucleotide of cDRC1 open reading frame sequence. The ATG codon is included in the SphI site. In the codon after ATG, the first base is from the SphI site, and the remaining two bases are consistent with the 5th-6th nucleotides of the cDRC1 open reading frame sequence. The 3' end sequence is: 5'-CCCGGATCC GAAGTCATGGCTTGGTGC TTCT -3' (SEQ ID No.5), which contains a BamHI site (italics), followed by 22 nucleotides of the specific sequence comprising the gene stop codon ( underlined part). These restriction sites corresponded to those on the bacterial expression vector pQE-70 (Qiagen, Inc. Chatsworth, CA). pQE-70 encodes antibiotic resistance (amp ^r ), a bacterial origin of replication (ori), an IPTG-regulated promoter operon (P/O), a ribosome binding site (RBS), a 6-His tag and restriction Restriction sites. The PCR product and pQE-70 were digested with SphI and BamHI. The amplified sequence was ligated into pQE-70, inserting in frame the sequence encoding the histidine tag and RBS. E. coli strain M15/rep4 (Qiagen, Inc.) was transformed with the ligation mixture as described in Sambrook et al., A Laboratory Guide to Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. M15/rep4 contains multiple copies of the pREP4 plasmid, which expresses the LacI repressor and also exhibits kanamycin resistance (Kan ^r ). Transformants were identified on LB plates containing ampicillin and kanamycin, and resistant colonies were selected for growth. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired construct were grown overnight (O/N) in LB broth supplemented with Amp (100 μg/ml) and Kan (25 μg/ml). O/N cultures were then inoculated into larger cultures at a ratio of 1:100 to 1:250. Let the cells grow to an optimal density: OD600 between 0.4 and 0.6. Then IPTG (isopropyl-β-D-thiogalactopyranoside) was added at a final concentration of 1 mM. IPTG is induced by inactivating the LacI repressor, clearing P/O leading to increased gene expression. Cells were allowed to grow for an additional 3 to 4 hours. Cells were harvested by centrifugation. The cell pellet was dissolved in 6M guanidine hydrochloride pH 5.0. After clarification, solubilized DRC1 was purified from solution using nickel chelate column chromatography under conditions that allow tight binding of the protein including the 6-His tag (Hochuli et al.: J chromatography 411:177-184, 1984) . DRC1 (>98% pure) was eluted from the column with 6M guanidine hydrochloride. Renaturation of proteins by removal of guanidine hydrochloride can be achieved by several protocols (Jaenicke & Rudolph: Protein Structure-APractical Approach, IRL Press, New York, 1990). First, guanidine hydrochloride was removed by fractional dialysis. Alternatively, the purified protein from the nickel-chelate column is bound to another column with a linearly decreasing gradient of guanidine hydrochloride. When bound to this column, the protein can be refolded and then eluted with a buffer containing 250 mM imidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.5 and 10% glycerol. Finally, the soluble protein was dialyzed against storage buffer containing 5 mM ammonium bicarbonate.

Example 4: Expression of recombinant DRC1 in COS cells

The expression plasmid DRC1 HA is derived from the vector pcDNAI/Amp (Invitrogen, Inc.), which includes: 1) SV40 origin of replication; 2) ampicillin resistance gene; 3) Escherichia coli origin of replication; 4) CMV promoter followed by a Multiple cloning site region, one SV40 intron and polyadenylation site. A HA-tagged DNA fragment encoding the entire DRC1 and its 3'-end in-frame fusion was cloned into the multiple cloning site region of the vector, and the recombinant protein was expressed under the control of the CMV promoter. The HA tag is an epitope of the previously described influenza hemagglutinin protein (Wilson et al.: Cell 37:767, 1984). The HA tag is fused to the target protein to facilitate detection of the recombinant protein with an antibody that recognizes the HA epitope.

The following is the plasmid construction strategy:

The cDNA sequence encoding pDRC1 was constructed by PCR with double primers: the 5'-end primer was 5'-CCCAAGCTT CTTCAAAGATGCCTCAGTTACTGCAAAACA -3' (SEQ ID No.6), which contained a HindIII restriction site (italics), followed by the DEC1 sequence 30 nucleotides (underlined part, ATG codon is its 9-11 bp). The 3' end sequence is 5'- CCCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTATGGCTTGGTGCTTCTCCAAGTAGG -3' (SEQ ID No. 7), which contains an XbaI site (italics), followed by a translation stop codon (bold), HA tag and DRC1 coding sequence (not Last 23 nucleotides (underlined) including stop codon). The PCR product therefore contained a HindIII site, DRC1 coding sequence, followed by an HA tag fused in frame, a translation stop codon after the HA tag, and an XbaI site. The DNA fragment amplified by PCR and the vector pcDNAI/Amp were digested with HindIII and XbaI restriction endonucleases and ligated. The E. coli strain SURE (Stratagene, Inc.) was transformed with the ligation mixture. Spread the transformants on ampicillin plates and select resistant colonies. Plasmid DNA was isolated from transformants and checked by restriction analysis for the presence of the correct fragment. To express recombinant DRC1, COS cells were transfected with the expression vector by the DEAE-dextran method (Sambrook et al.: A Laboratory Guide to Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989). Expression of the DRC1 HA protein can be detected by radiolabeling and immunoprecipitation (Harlow & Lane: A Laboratory Manual for Antibodies, Cold Spring Harbor Laboratory Press, 1988). Two days after transfection, the cells were labeled with ³⁵ S-cysteine for 8 hours, then the culture medium was collected, and the cells were lysed with detergent (RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC , 50mM Tirs, pH7.5). Cell lysates and culture medium were precipitated with an HA-specific monoclonal antibody, and precipitated proteins were analyzed by SDS-PAGE.

Example 5: Cloning and expression of DRC1 with baculovirus expression system

The DNA sequence encoding the full-length DRC1 protein was amplified by PCR, and the oligonucleotide primers were consistent with the 5' and 3' end sequences of the gene: the 5' end primer sequence was 5'-CCCGGATCC TTCAAAGATGCCTCAGTTACT GCAAAACA -3' (SEQ ID No. 8), which contains a BamHI restriction site (italics), followed by 29 nucleotides of the DRC1 sequence (the underlined part, the ATG codon is its 8-10 bp). The 3' end sequence is 5'-CCCGGTACC TCATGGCTTGGTGCTTCTCAAGTAGG -3' (SEQ ID No.9), which contains an Asp781 site (italics), followed by 26 nucleotides of the specific sequence including the gene stop codon (underlined part). The amplified sequence was separated from a 1% agarose gel using a commercially available kit (Qiagen, Inc.). This fragment was digested with endonucleases BamHI and Asp781, and then purified again on 1% agarose gel. This fragment is designated as F2.

The vector pRG1 (a modification of the pVL941 vector) was used for expression of the DRC1 protein in a baculovirus expression system (Sumers & Smith: Handbook of Baculovirus Vectors and Insect Cell Culture Methods, 1987). The expression vector includes the strong polyhedrin promoter of the californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites of restriction endonucleases BamHI and Asp781. The polyadenylation site of monkey virus SV40 was used for efficient polyadenylation. To facilitate selection of recombinant viruses, the E. coli β-galactosidase gene was inserted in the same orientation as the polyhedrin promoter, followed by the polyadenylation signal for the polyhedrin gene. The polyhedrin sequence is flanked by viral sequences for cell-mediated homologous recombination with co-transfected wild-type viral DNA. Many other baculovirus vectors can replace pRG1, such as pAc373, pVL941 and pAcIM1, etc.

The plasmid was digested with restriction enzymes BamHI and Asp781 and then dephosphorylated with calf intestinal phosphatase by methods known in the art. DNA was isolated from 1% agarose gel using a commercially available kit (Qiagen, Inc.). This vector DNA was designated V2.

The F2 fragment and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase, and then transformed into Escherichia coli HB101 cells, and the bacteria containing the plasmid (pBac-DRC1) with the DRC1 gene were identified with BamHI and Asp781 enzymes. The sequence of the cloned fragment was confirmed by DNA sequencing.

5 μg of plasmid pBac-DRC1 was transfected with 1 μg of commercially obtained linearized baculovirus (“BaculoGoldTM baculovirus DNA”, Pharmingen, San Diego, CA.) using lipofection method (Felgner et al.: Proc. Natl Acad Sci USA 84:7413-7417, 1987).

1 μg of BaculoGoldTM viral DNA and 5 μg of plasmid pBac-DRC1 were mixed in the wells of a sterile microtiter plate containing 50 μl of serum-free Grace's medium (LifeTechnologies Inc., Gaithersburg, MD). Then add 10 μl liposome transfection agent and 90 μl Grace's medium, mix well, and incubate at room temperature for 15 minutes. The transfection mixture was then added dropwise to Sf9 insect cells (ATCC CRL1711) seeded on a 35 mm tissue culture plate with 1 ml of serum-free Grace's medium. Shake the plate back and forth to mix the newly added solution and incubate at 27°C for 5 hours. After 5 hours the transfection solution was removed from the culture plate and 1 ml of Grace's insect broth supplemented with 10% fetal bovine serum was added. The culture plate was put back into the incubator and cultured at 27°C for 4 days.

Supernatants were collected after 4 days and subjected to a plaque assay similar to that described by Summers and Smith. The change was the use of agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg), which allowed for easy isolation of blue phagocytic plaques. (A detailed description of the plaque assay can be found on pages 9-10 of the Insect Cell Culture and Baculovirology User's Guide distributed by Life Technologies Inc., Gaithersburg.)

Four days after the serial dilutions, the virus had entered the cells and blue philogenic plaques were picked up with an Eppendorf pipette tip. Resuspend the agar containing the recombinant virus in an Eppendorf tube containing 200 μl of Grace's medium. The agar was removed by brief centrifugation and the supernatant containing the recombinant baculovirus was used to inoculate Sf9 cells in 35 mm dishes. After 4 days, the supernatants from the dishes were harvested and stored at 4 °C.

Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS. These cells were infected with recombinant baculovirus V-DRC1 at a multiplicity of infection (MOI) of 2. After 6 hours, the original medium was removed and replaced with SF900II medium without methionine and cysteine (Life Technologies Inc., Gaithersburg). After 42 hours 5 μCi of 35S-methionine and 5 μCi of 35S-cysteine (Amersham) were added. The cells were incubated for another 16 hours, and the cells were harvested by centrifugation, and the labeled proteins could be seen by SDS-PAGE and autoradiography.

Example 6: Gene Therapy Expression

Fibroblasts can be obtained from a skin biopsy subject. The resulting tissue was placed in tissue culture medium and divided into small pieces. Small, thick pieces of tissue are placed on the wet side of a tissue culture bottle, approximately ten pieces per bottle. Invert the bottle, cap tightly, and let stand overnight at room temperature. After 24 hours at room temperature, invert the bottle with the tissue pieces still fixed at the bottom of the bottle, and add fresh medium (eg Ham's F12 medium) containing 10% FBS, penicillin and streptomycin. Then culture at 37°C for about one week. At this point, fresh medium was added and changed every few days thereafter. After two more weeks in culture, a monolayer of fibroblasts emerged, which was trypsinized and scraped into larger culture flasks.

pMV-7 (Kirschmeier et al.: DNA 7:219-25, 1988), flanked by the long terminal repeats of Moloney mouse sarcoma virus, was digested with EcoRI and HindIII and subsequently treated with calf intestinal squamous acidase. The linear vector was separated on an agarose gel and purified with glass beads.

The cDNA encoding the polypeptide of the present invention is amplified by PCR, and the primers correspond to the 5' and 3' end sequences respectively. The 5' end primer contains an EcoRI site, and the 3' end primer contains a HindIII site. Add the same amount of EcoRI and HindIII site fragments amplified by the linear backbone of Moloney mouse sarcoma virus together, then add T4 DNA ligase, and keep under conditions suitable for the ligation of the two fragments. The ligation mixture was used to transform bacteria HB101, which were then plated on agar containing kanamycin in order to confirm that the vector had the correct inserted gene.

Amphotropic pA317 or Gp+am12 packaging cells were tissue cultured to confluent density in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% calf serum (CS), penicillin and streptomycin. Add the vector containing the desired gene to the culture medium and use it to transduce the packaging cells. Packaging cells produce infectious virus particles with the desired genes (packaging cells are referred to herein as producer cells).

Fresh medium was added to the transduced producer cells and the medium was subsequently harvested from the 10 cm plate confluent with the producer cells. The depleted culture medium, containing infectious virus particles, is filtered through a microporous membrane to remove dissociated producer cells, and this medium is then used to infect fibroblasts. Remove medium from sub-confluent fibroblasts and quickly replace with medium from producer cells. This medium was again replaced with fresh medium. If the titer of the virus is high, then essentially all fibroblasts will be transfected and selection is not required. If the titer is very low, then a reverse transcription vector with a selectable marker such as neo or his should be used.

The engineered fibroblasts can be injected into the host alone or grown to confluence on Cytodex3 microcarrier beads. At this time, the fibroblasts produce protein products.

Various modifications and variations of the present invention are possible in light of the above teaching, so that within the scope of the appended claims the invention may be practiced otherwise than as specifically described.

Sequence Listing

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 AATCAGACAAA 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'- CCCAAGCTTCTTCAAAGATGCCTCAGTTACTGCAAAACA -3'

39bp

SEQ ID No.7: 5'-CCCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTA

TGGCTTGGTGCTTTCTCAAGTAGG -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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