WO2001085775A1 - Nouvelle proteine humaine associee aux tumeurs et sa sequence de codage - Google Patents

Nouvelle proteine humaine associee aux tumeurs et sa sequence de codage Download PDF

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Publication number
WO2001085775A1
WO2001085775A1 PCT/CN2001/000559 CN0100559W WO0185775A1 WO 2001085775 A1 WO2001085775 A1 WO 2001085775A1 CN 0100559 W CN0100559 W CN 0100559W WO 0185775 A1 WO0185775 A1 WO 0185775A1
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polypeptide
protein
seq
polynucleotide
dna
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PCT/CN2001/000559
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English (en)
French (fr)
Inventor
Jianren Gu
Xintai Zhao
Dafang Wan
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Shanghai Cancer Institute
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Priority to AU62013/01A priority Critical patent/AU6201301A/en
Priority to US10/257,124 priority patent/US7112419B2/en
Publication of WO2001085775A1 publication Critical patent/WO2001085775A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology, and specifically relates to a novel polynucleotide encoding a human liver cancer-related protein, and a polypeptide encoded by the polynucleotide.
  • the invention also relates to the use and preparation of such polynucleotides and polypeptides.
  • the liver cancer-related protein of the present invention is a hepatocellular carcinoma inhibitory factor.
  • liver cancer is one of the most common tumors in China.
  • the occurrence of liver cancer is a complex process with multiple genes and multiple steps. It is the result of activation of multiple oncogenes and inactivation of tumor suppressor genes. Among them, tumor suppressor genes are even more critical. Therefore, searching for tumor suppressor genes is one of the current research hotspots. If a certain tumor has a higher frequency of loss of heterozygosity in a certain segment of the chromosome, it indicates that this segment exists in the tumor suppressor gene associated with the tumor.
  • the 17pl3.3 region of chromosome has high heterozygosity loss in many tumors. Therefore, many international laboratories are looking for tumor suppressor genes located in the chromosome 17pl3.3 region. So far, three possible tumor suppressor genes (Hic-I, OVCA1 and OVCA2) have been discovered. Hic-I is highly methylated in tumor tissues and has a reduced expression. OVCA1 and OVCA2 are reduced or not expressed in ovarian cancer or cell lines. All three genes are located near the YNZ22 locus of the chromosome.
  • cancer is one of the main diseases that endanger human health.
  • tumors such as liver cancer
  • people have paid more and more attention to the early diagnosis and gene therapy of tumors. Therefore, there is an urgent need in the art to develop new cancer-related and / or cancer suppressing human proteins and their agonists / inhibitors.
  • the object of the present invention is to provide a novel liver cancer-related protein-human c63R protein polypeptide and fragments, analogs and derivatives thereof.
  • Another object of the invention is to provide polynucleotides encoding these polypeptides.
  • Another object of the present invention is to provide a method for producing these polypeptides and the use of the polypeptide and coding sequence.
  • an isolated human c63R protein polypeptide which includes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an activity derivative thereof. Thing.
  • the polypeptide is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • an isolated polynucleotide which comprises a nucleotide sequence that is at least 85% identical to a nucleotide sequence selected from the group consisting of : (A) a polynucleotide encoding the polypeptide of claims 1 and 2; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polypeptide encoded by the polynucleotide has an amino acid sequence shown in SEQ ID NO: 2; more preferably, the polynucleotide has a sequence selected from the group consisting of the coding region shown in SEQ ID NO: 3 Sequence or full-length sequence.
  • a method for preparing a polypeptide that is associated with liver cancer-related C63R protein activity comprises: (a) culturing the transformed or transduced host cell under conditions suitable for protein expression; b) from culture
  • an antibody that specifically binds to the above-mentioned liver cancer-related c63R protein polypeptide.
  • Nucleic acid molecules that can be used for detection are also provided, which contain consecutive 10-800 nucleotides in the above-mentioned polynucleotides.
  • a pharmaceutical composition which contains a safe and effective amount of the liver cancer-related c63R protein polypeptide of the present invention and a pharmaceutically acceptable carrier.
  • These pharmaceutical compositions can treat conditions such as cancer and abnormal cell proliferation.
  • a method for detecting whether hepatocytes are cancerous or susceptible to cancerous changes which includes the steps of: detecting whether a C63R transcript in a liver cell sample is changed from a normal c63R transcript If there is a change, it means that the liver cell is cancerous or susceptible to cancer; or if there is a change in the activity of c63R protein in the liver cell sample compared with normal c63R protein, if there is a change, it means that the liver cell is cancerous or susceptible to cancer .
  • the change is a nucleotide deletion or insertion substitution mutation.
  • the change is selected from the group consisting of deletions of nucleotides 395-481 in SEQ ID NO: 1, deletions of nucleotides 328-1511 in SEQ ID NO: 1, and nucleotides 1143-1231 in SEQ ID NO: 1. Deletion, deletion of nucleotide 1325-1419 in SEQ ID NO: 1, mutation of nucleotide 1106 in SEQ ID NO: 1 from C to T, deletion of nucleotide 1048 in SEQ ID NO: 1.
  • kits for detecting liver cancer which comprises: (1) a primer pair that specifically amplifies the human c63R gene, and (2) detecting whether the amplified product is compared with a normal C63R gene There are reagents required for the change.
  • the markers used for genomic clone screening and the structure of contigs are shown above; the contigs of genomic clones covering HCCS1 are shown in the middle; the exons of the HCCS1 gene are shown below.
  • Figure 2 shows the cellular localization of HCCS1.
  • Figure 2a Fluorescent photograph of MH3T3 cells transfected with pEGF-HCCS1.
  • Figure 2b Immunofluorescent staining of the cells in Figure 2a with anti-mitochondrial antibodies.
  • Figure 2c Overlapping of photos 2a and 2b shows that HCCS1 is located in the mitochondria.
  • FIG. 1 Results of immunohistochemical staining of human liver cancer cells and surrounding non-cancer cells.
  • HCC liver cancer cells; HCC-liver cells.
  • Figure 4 shows the effect of HCCS1 expression on cell growth.
  • Figure 4A Colony formation of SMMC-7721 cells transfected with the vector pcDNA3.1 / V5-His
  • Figure 4B Colony formation of SMMC-7721 cells transfected with the vector containing HCCS1
  • Figure 4C Comparison of colony formation . The data were repeated 3 times, p ⁇ 0.01 compared with the empty vector.
  • Figure 5 shows the effect of HCCS1 on tumor formation in nude mice. Compared to the GFP vector group (control), PO.01. Detailed description of the invention
  • the inventor found that the heterozygous deletion range of the chromosome 17pl3.3 region is located from the YNZ22 site to the D17S34 site of the proximal telomere.
  • the minimum hotspot deletion range was from D17S1574 to D17S849.
  • the inventors performed gene cloning in this region, obtained the C63R gene located at the D17S849 site, and found that the c63R gene has deletions and decoding changes in liver cancer tissues, thereby confirming that the C63R gene is a liver cancer-related gene. Further experiments show that c63R gene is a hepatocellular carcinoma suppressor, so c63R is also called HCCS1 (Hepatocellular Carcinoma suppressor 1).
  • HCCS1 is localized to mitochondria.
  • HCCS1 was negatively expressed in liver cancer cells and positive in normal liver cells.
  • Transfection of HCCS1 into liver cancer cells and nude mice can inhibit tumor cell growth and tumor formation.
  • c63R protein c63R polypeptide
  • HCCS1 protein HCCS1 polypeptide
  • HCCS1 polypeptide hepatocellular carcinoma suppressor HCCS1
  • a protein or polypeptide of the liver cancer-related protein c63R amino acid sequence SEQ ID NO: 2).
  • the term also includes liver cancer-related protein c63R with or without the starting methionine.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. . .
  • isolated liver cancer-related C63R protein or polypeptide and "isolated c63R protein or polypeptide” means that the liver cancer-related c63R protein polypeptide is substantially free of other proteins, lipids, carbohydrates, or other substances naturally associated with it.
  • Those skilled in the art can purify the c63R protein using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of c63R protein polypeptide can be analyzed by amino acid sequence.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • Polypeptides of the invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude initial methionine residues.
  • the invention also includes fragments, derivatives and analogs of human c63R protein associated with liver cancer.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the natural liver cancer-related human c63R protein of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (Hi) a mature polypeptide with another compound (such as a compound that extends the half-life of a polypeptide, such as (Polyethylene glycol), a polypeptide formed by fusion, or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify the polypeptide or a protein sequence).
  • these fragments, derivatives and analogs are within the scope of those skilled in the art.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 3 or a degenerate variant.
  • degenerate variant refers in the present invention to a nucleic acid sequence that encodes a protein having SEQ ID NO: 2, but which differs from the coding region sequence shown in SEQ ID NO: 3.
  • Polynucleotides encoding mature polypeptides include: coding sequences that only encode mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optional additional coding sequences); and non-coding sequences.
  • the term "polynucleotide encoding a polypeptide" may include a polynucleotide that encodes the polypeptide, or a polynucleotide that also includes additional coding and / or non-coding sequences.
  • the present invention also relates to a variant of the above-mentioned polynucleotide, which encodes a polypeptide having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptide.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants ⁇
  • an allelic variant is an alternate form of a polynucleotide, it may be one or more nucleotides Substitutions, deletions, or insertions, but without substantially altering the function of the polypeptide it encodes.
  • the present invention also relates to a polynucleotide that hybridizes to the sequence described above and has at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C; or (2) during hybridization Added denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the identity between the two sequences is at least 95% or more , More preferably, hybridization occurs only when it is above 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragment that hybridizes to the sequence described above.
  • a "nucleic acid fragment” contains at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding the c63R protein.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity. .
  • DNA sequence of the present invention can be obtained by several methods.
  • DNA is isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous nucleotide sequences, and 2) antibody screening of expression libraries to detect cloned DNA fragments with common structural characteristics .
  • the specific DNA fragment sequence encoding the c63R protein can also be obtained by: 1) separating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the desired polypeptide.
  • genomic DNA isolation is the least commonly used.
  • direct chemical synthesis of the DNA sequence is often the method of choice. If the entire sequence of the desired amino acid is unclear, direct chemical synthesis of the DNA sequence is not possible, and the method chosen is the isolation of the cDNA sequence.
  • the standard method for isolating the cDNA of interest is to isolate inRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for mRNA extraction, and kits are also commercially available (Qiagene;).
  • cDNA libraries are also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, even very few expression products can be cloned. .
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2 kb, preferably within lkb.
  • the probe used here is generally a DNA sequence chemically synthesized based on the DNA sequence information of the gene of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of the c63R protein gene expression.
  • ELISA enzyme-linked immunosorbent assay
  • a method for amplifying DNA / RNA using a PCR technique is preferably used to obtain the gene of the present invention.
  • the RACE method ACE-cDNA terminal rapid amplification method
  • the primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein.
  • the amplified DNA / RA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • nucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such nucleotide sequence determination can also be performed using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector or c63R protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant c63R protein polypeptide by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally there are the following steps:
  • a human c63R protein polynucleotide sequence related to liver cancer can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors (Lee and Nathans, J Bio Chem.
  • any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translation control elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing the c63R protein-encoding DNA sequence and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mR A synthesis. Representative examples of these promoters are: lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS or Bowes melanoma cells.
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription.
  • Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the obtained transformants can be cultured by a conventional method and express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide in the above method may be coated intracellularly, extracellularly, or expressed on a cell membrane or secreted extracellularly. If necessary, recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation, treatment with a protein precipitant (salting out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the recombinant liver cancer-related human C63R egg S or polypeptide of the present invention has many uses. These uses include (but are not limited to): direct use as a drug to treat diseases caused by hypofunction or loss of C63R protein (such as liver cancer), and for screening Antibodies, polypeptides or other ligands that promote or counteract c63R protein function. For example, antibodies can be used to activate or inhibit the function of the c63R protein. Screening the peptide library using the expressed recombinant c63R protein can be used to find therapeutic peptide molecules that can inhibit or stimulate the function of c63R protein.
  • the invention also provides methods for screening drugs to identify agents that increase (agonist) or suppress (antagonist) c63R protein.
  • mammalian cells or a membrane preparation expressing c63R protein can be cultured with the labeled c63R protein in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of c63R protein include antibodies, compounds, receptor deletions, and the like that have been screened. C63R protein.
  • the antagonist can bind to c63R protein and eliminate its function, or inhibit the production of c63R protein, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions. Antagonists of the c63R protein are useful for therapeutic use.
  • c63R proteins can be added to bioanalytical assays to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between the c63R protein and its receptor. In the same manner as described above for the screening of compounds, it is possible to screen for receptor deletions and analogs that act as antagonists.
  • the polypeptide of the present invention can be directly used for the treatment of diseases, for example, various malignant tumors and abnormal proliferation of cells, etc., especially for the treatment of liver cancer.
  • polypeptides of the present invention and fragments, derivatives, analogs or cells thereof can be used as antigens to produce antibodies.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • Polyclonal antibodies can be obtained by directly injecting the polypeptide into animals. Techniques for preparing monoclonal antibodies include hybridoma technology, triple tumor technology, human B-cell hybridoma technology, and EBV-hybridoma technology.
  • the polypeptides and antagonists of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • the c63R protein is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of c63R protein administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.
  • the c63R protein polynucleotide can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression of c63R protein or abnormal / inactive C63R protein expression.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated c63R protein to inhibit endogenous c63R protein activity.
  • a mutated c63R protein may be a shortened c63R protein that lacks a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of C63R protein.
  • Viral-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the C63R protein gene into cells.
  • Method c63R construct recombinant viral vectors carrying the gene can be found in existing literature (Sambrook, e t al.) .
  • Recombinant c63R egg White genes can be packaged into liposomes and transferred into cells.
  • Oligonucleotides including antisense RA and DNA
  • ribozymes that inhibit c63R protein mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the solid-phase phosphate amide chemical synthesis of oligonucleotides has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell via a vector (such as a virus, phage, or plasmid) in vitro Then, the cells are transplanted into the body.
  • a vector such as a virus, phage, or plasmid
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis.
  • the invention also provides antibodies against the c63R protein epitope. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Anti-c63R protein antibodies can be used in immunohistochemical techniques to detect c63R protein in biopsy specimens. Monoclonal antibodies that bind to c63R protein can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • the antibodies of the present invention can be used to treat or prevent diseases related to c63R protein.
  • Administration of appropriate doses of antibodies can stimulate or block c63R protein production or activity.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • c63R protein high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill c63R protein-positive cells.
  • Polyclonal antibodies can be produced by immunizing animals such as rabbits, mice, and rats with c63R proteins or peptides.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvants.
  • the c63R protein monoclonal antibody can be produced by hybridoma technology (KoMer and Milstein. Nature, 1975, 256: 495-497). Chimeric antibodies that bind human constant and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). Existing techniques for producing single-chain antibodies (US Pat No. .4946778) can also be used to produce single-chain antibodies against c63R protein.
  • Polypeptide molecules capable of binding to the C63R protein can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, the C63R protein molecule must be labeled.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of c63R protein levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of c63R protein detected in the test can be used to explain the importance of C63R protein in various diseases and to diagnose diseases in which C63R protein plays a role.
  • the c63R protein polynucleotide can be used for the diagnosis and treatment of c63R protein-related diseases, especially liver cancer. In terms of diagnosis, the polynucleotide of c63R protein can be used to detect the expression of c63R protein or to detect the abnormal expression of C63R protein in a disease state.
  • the C63R protein DNA sequence can be used to hybridize biopsy specimens to determine the abnormal expression of c63R protein.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • the c63R protein-specific primers can also be used to detect the c63R protein transcripts by in vitro amplification of RNA-polymerase chain reaction (RT-PCR).
  • Detection of mutations in the c63R protein gene can also be used to diagnose c63R protein-related diseases (especially liver cancer).
  • the c63R protein mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type c63R protein DNA sequence (such as the normal sequence shown in SEQ ID NO: 1). Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the full-length nucleotide sequence of the c63R protein of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequences publicly known in the present invention, especially open reading frame sequences, and prepared using commercially available cDNA libraries or prepared according to conventional methods known to those skilled in the art.
  • the cDNA library was used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then stitch the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • relevant methods can also be synthesized by artificial synthesis, especially when the fragment length is short.
  • long fragments can be obtained by synthesizing multiple small fragments first and then concatenating them.
  • the DNA sequence encoding the protein (or a fragment, or a derivative thereof) of the present invention can be completely synthesized by chemical synthesis. This DNA sequence can then be introduced into various DNA molecules (e.g., vectors) and cells in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the c63R protein of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and / or lower side effects when administered to humans compared to homologous proteins derived from other species (Eg, less or not immunogenic in humans).
  • the present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.
  • the experimental methods without specific conditions in the following examples are usually performed according to the conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions.
  • Example 1 Determination of the range of loss of heterozygosity of the 3 chromosome 17pl3 region in liver cancer:
  • the heterozygous deletion analysis of the liver cancer tissue is performed by selecting a polymorphic marker of the 17pl3.3 region of the chromosome to determine the deletion range and the minimum hotspot deletion range.
  • LOH was detected by Southern blot analysis on VNTR (variable number of tandem repeat) and RFLP probes. After 10ug of DNA from liver cancer and adjacent tissues were digested with appropriate restriction enzymes, electrophoresis was performed at 25V for 13 hours and transferred to hybond-N membrane (Amersham). The YNZ22 probe was donated by Hu Ying from Shanghai Medical University. The 144D6 and YNH37.3 probes were purchased from the American Type Culture Collection (ATCC). cCI17- 708 and cCI17- 680 were donated by Dr. Nakamura. Probes were labeled with [ ⁇ - 32 P] dATP using random primers. The site names of the probes and the restriction enzymes used for digestion are listed in Table 1.
  • the YNZ22.1, 144D6, and YNH37.3 probes were hybridized overnight at 42 V with a conventional hybridization solution containing 50% formamide.
  • the pre-hybridization solution is 50% formamide, 5 X SSC, 0.5% SDS, 10 X Denhardfs solution, 250ug / ml human placental DNA, and pre-hybridize at 42 ° C 24 Hours, except that the 10X Denhardfs solution was replaced with 10% dextran sulfate, and the remaining components were the same as the pre-hybridization solution and hybridized overnight at 42 ° C.
  • Microsatellite marker analysis was performed in a 12.5ul reaction volume.
  • the reaction contained luM PCR primers, 25n g tissue DNA, 200uM dNTP, 50mM KCI, 10mM Tris (pH9. 0), 1.5mM MgCl 2 0.625U Taq DNA. Polymerase (Promega).
  • the microsatellite marker site names and annealing temperatures are listed in Table 1.
  • the PCR reaction was performed according to the procedure of denaturation at 97 ° C for 4 minutes, 94 ° C ⁇ , appropriate annealing temperature 1 ', 72 ° C 1', 30 cycles, and 72 ° C extension 10 '.
  • the products were examined by 2% agarose gel electrophoresis.
  • [r- 32 P] ATP labeled one of the primers In a 5ul PCR reaction volume, labeled lpmol primers, 1ul of the above PCR product, and 0.25U Taq DNA polymerase were used, except that the PCR reaction was performed in 4 cycles, and the remaining components and reaction conditions were the same as above. After the PCR products were denatured, they were separated and compressed on a 6% denatured polyacrylamide gel, and the LOH results were observed.
  • the L0H of 21 samples near the telomere D17S34 was detected, and 8 of the 12 heterozygotes showed L0H with a frequency of 67%, and it was particularly noticeable that all the samples with Y0 at the YNZ22 locus were in The D17S34 site has L0H (except homozygotes). Is it possible that the deletion range of 17pl3.3 in liver cancer samples is at least from the YNZ22 site to the D17S34 site of the proximal telomere. Based on this consideration, 14 markers around two points were selected to further detect the LOH of each marker in liver cancer samples.
  • results The eight markers located between D17S34 and D17S5 loci were higher in liver cancer samples, while the three markers located near the centromere in D17S5 had lower or no L0H. This confirms our speculation that the deletion of the liver cancer sample in the chromosome 17pl3.3 region ranges from the D17S34 to D17S5 sites. Among them, D17S926 mark detected 22 samples, 10 heterozygotes showed L0H, and the frequency was 100%. The sample with L0H at YNZ22 also showed L0H at this site (except homozygotes).
  • liver cancer samples have D17S34 and D17S1866 near the telomere.
  • No LOH, NO. 34 HCC samples also had no L0H at the D17S34, D17S1866 and D17S849 sites in the proximal telomere direction.
  • the two liver cancer samples were homozygous for L0H at all sites between D17S849 and D17S1574. Therefore, the minimum hotspot deletion range for the liver cancer sample in the chromosome 17pl3.3 region is from D17S849 to D17S1574.
  • the primer sequence of D17s926 was obtained from Genbank,
  • the phage solution of the cDNA library was plated into 30 plates (diameter of 150 bands) per plate of 50,000 pfu, and incubated at 41 ° C for 3-5 hours until the plaque diameter was 0.5 mm and the adjacent plaques were not combined, Remove the plate from the incubator and let it stand at 4 ° C for at least 1 hour. Take out the plate from the refrigerator and place the nitrocellulose filter (Hybond-C Amersham LIFE Science) on the plate gently at room temperature. Use a syringe with carbon ink to make 4 points to set the direction. (0.5 mol Na0H-1.5 mol NaCl), neutralize for 5 minutes (1 mol Tris-HCl pH 7. 4, 1.5 mol NaCl) and transfer to 6xSSC for a little soaking, put it on 3MM filter paper to dry, and then bake the film at 80 ° C 2 hours.
  • the membrane was hybridized at 65-68 V for 6-8 hours in the pre-hybridization solution.
  • the pre-hybridization solution contains the following components: 6xSSC, 5xDenhart's, 0.5% SDS, denatured fish sperm DNA 200ug / ml, denatured placental DNA 100ug / ml, and mixed DNA ( ⁇ DNA5.8, ⁇ DNA 7.3, and PBR plasmid DNA, (30 ug / ml each).
  • the labeled 300ul probe solution was added with Cot-1 DNA, 1mg mixed DNA, 50ug each, 20xSSC 250ul, boiled at 100 ° C for 15 minutes and placed on ice for 10 minutes, and 60-65 ° C water bath was blocked for 20 minutes, placed on ice for 20 minutes, added In a prehybridization solution with a membrane, hybridize at 65-68 V for 16-24 hours. After the hybridization is completed, the hybridization solution is discarded, and the membrane is rinsed twice with a pre-warmed 2xSSC -0.1% SDS solution at 42 ° C for 10 minutes each time, and washed and detected with an isotope detector while adjusting the time and time of washing After washing, autoradiography was performed.
  • 150ul taken phage expansion board added 1ml lOmM M g S0 4 Y1090 bacteria treated 37 ° C for 15 minutes, add 40ml LB (containing 1/100 0. 5M CaC12) 37 ° C , until the liquid is strongly shaken by a turbid Clear (3 hours or so), add 1ml of chloroform, shake at 37 ° C for 20 minutes, centrifuge at 4000rpm for 10 minutes, add 50ul of RNase A / DNase solution at 37 ° C, incubate for 1 hour at 4 ° C, and then centrifuge at 1000rpm x10 minutes, the supernatant was subjected to ultracentrifugation, 27000rpm x 70 minutes.
  • the c63R clone was dideoxy-terminated, and the sequences at both ends were determined on an ABI 337 DNA sequencer. Primers were designed based on the obtained sequence and sequenced again until a full-length sequence was obtained, totaling 2132bp. Further analysis revealed that the 54Bp at the 5 'end was the sequence on the vector, so the full-length sequence was 2078bp as shown in SEQ ID NO: 1, where the coding reading frame is located at positions 56-2077.
  • the full length of the encoded c63R protein is 673 amino acids, and the sequence is shown in SEQ ID NO: 2. A combined arrangement of a nucleic acid sequence and an amino acid sequence is shown in SEQ ID NO: 1.
  • Example 5 Obtaining gene clones from human liver cDNA by PCR method:
  • LV-RT-Superscript II (GIBCO BRL) was used to perform reverse transcription recording at 42 ° to perform reverse transcription reaction to obtain placental cDNA.
  • Specific primers c63R-1 of the following gene-coding frames were synthesized: atgatggaggaggaggaga (SEQ ID NO: 5); c63R-2: gttgtcacggatgatggg (SEQ ID NO: 6).
  • the c63R fragment was digested and isolated from the c63R recombinant plasmid, and probe labeled with a 32 P-dATP according to the kit (Megaprime DNA Labelling System, Amersham) according to the instructions. Hybridization with a multi-tissue mRNA patch (Biochain) showed three hybridization bands, all of which were expressed in multiple tissues.
  • Example 7 Changes of the C63R gene in liver cancer tissues:
  • C63R-a-PF CGGGTGGCGGAATGATG (SEQ ID NO: 7)
  • C63R- a- PR CTCCACCCCCATCTACCA (SEQ ID NO: 8)
  • C63R-b-PR CAAAACGCTTCTCCGGC (SEQ ID NO: 10)
  • C63R-C-PR ACAACCCCGTGGCTTCC (SEQ ID NO: 12)
  • C63R-d-PR CAAGCAGAACGTCCCCAT (SEQ ID NO: 14)
  • C63R-C-SR CCAAGACAAGAACCTCGGA (SEQ ID NO: 17)
  • C63R-a-PF and C63R-a-PR Using C63R-a-PF and C63R-a-PR, C63R-c-PF and C63R-c-PR as primers, cDNA as template, denaturation at 94 ° C for 3 minutes, one cycle; denaturation at 94 ° C for 30 seconds, Anneal at 60 ° C for 30 seconds, 72 ° C extension for 1 minute, 35 cycles; 72 ° C extension for 10 minutes for the first PCR.
  • C63R- b- PF and C63R- b- PR, C63R- d-PF and C63R- d- PR were used as primers respectively, and the second nested PCR was performed according to the above procedure to obtain PCR products.
  • PCR products using C63R-b-PF and C63R-b-PR as primers were sequenced using C63R-b-PF, C63R-a-SR, C63R-b-SR, and C63R-b-PR as primers.
  • PCR products using C63R-d-PF and C63R-d-PR as primers were sequenced using C63R-d-PF, C63R-C-SR, C63R-d-SR and C63R-d-PR as primers.
  • liver cancer samples 3, 12, 25, 28, 33, and G11 were deleted in nt341-427, while the corresponding adjacent tissues were not deleted.
  • Liver cancer samples 28 were deleted at ntl274-1457. No corresponding cancerous tissue was missing.
  • Liver cancer sample 31 is missing at ntl090-1177.
  • No corresponding cancerous tissue was missing.
  • Liver cancer sample x31 was missing at ntl271_1365.
  • No corresponding cancerous tissue was missing.
  • the liver cancer sample G11 was mutated from C to T at ntl052, and the corresponding amino acid was mutated from Arg to Cys.
  • Liver cancer sample G4 lost T at nt994, causing a frameshift mutation.
  • the liver cancer sample GT12 lacked ntl612—1769, which caused a frameshift mutation.
  • c63R gene is a liver cancer-related gene, and mutations such as deletion, insertion, and replacement of the c63R gene are associated with liver cancer. These changes may cause the c63R protein to be inactive or too low in liver cells, and eventually lead to liver cancer directly or indirectly.
  • Example 8 Genomic Structure and Cell Localization of HCCS1
  • HCCS1 has a total of 18 exons and spans a genome sequence of about 200 kb ( Figure 1).
  • the HCCS1-GFP fusion gene was cloned into the pEGFP-Nl vector, and the HCCS1-GFP plasmid (pEGF-HCCS1) DNA was transferred into the human SMMC7721 cell line and mouse NIH3T3 fibroblasts. Fluorescence analysis showed that there were dot-like distribution regions in the cytoplasm of the two cell types, and And these distribution regions overlap with the staining regions of mitochondrial monoclonal antibody 113-1 (NeoMarkers, Fremont, CA), which indicates that the HCCS1 fusion protein is located in the mitochondria ( Figure 3).
  • Example 9 immunohistochemical analysis
  • HCC and non-cancer tissues were fixed in 10% formalin in 10 mM phosphate buffered saline (PBS) (pH 7.2).
  • Paraffin sections (4 ⁇ m) were mounted on glass slides coated with poly-L-lysine and dried at 50 ° C overnight.
  • the mouse anti-HCCS1 antibody was diluted 1: 200 in 5% normal sheep serum in PBS, and then incubated at room temperature for 30 molecules. After rinsing 3 times (5 minutes each) with PBS, the sections were coated with DAKO EnVision TM + System, HP (DAB), Mouse, Ready-to-use detection system (DAKO Corp., Carpinteria, CA, USA) 30 minutes. Sections were developed in Substrate-Chromogen Solution (DAB), counterstained with Mayer's hematoxylin, and observed.
  • DAB Substrate-Chromogen Solution
  • liver cancer cells HCC
  • HC liver cells
  • the HCCS1 cDNA was inserted into the pcDNA3.1 / V5-His vector (Invitrogen). According to the manufacturer's instructions, this construct was transfected into SMMC-7721 human hepatoma cells (3 X 10 4 ) with LipofectAMINE reagent (repeat 3 times). An empty vector was used as a control. After 14 days of selection with G418 (800 mg / ml), clones were stained and counted.
  • HCCS1 cDNA or empty vector were collected and resuspended in PBS. 200 microliters of cells (3.7xl0 6) subcutaneously in 5-6 week old male BALB / c nude mice on the right side. The experimental group and the control group each had 6 mice. After 5 weeks, the mice were killed and the tumors were excised and weighed.

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Description

新的人肝癌相关蛋白及其编码序列 发明领域
本发明属于生物技术领域，具体地说， 本发明涉及新的编码人肝癌相关蛋白的多核苷 酸， 以及此多核苷酸编码的多肽。 本发明还涉及此多核苷酸和多肽的用途和制备。 本发明 的肝癌相关蛋白是一种肝细胞癌抑制因子。
发明背景
恶性肿瘤的死亡率仅次于心脑血管疾病；其中肝癌在我国是高发性肿瘤之一。肝癌的 发生是多基因多步骤的复杂过程， 是多种癌基因激活和抑癌基因失活的结果， 其中抑癌基 因更为关键。 因此， 寻找抑癌基因是目前研究热点之一。 如果某种肿瘤在染色体的某一区 段存在较高频率的杂合性缺失， 则预示着这一区段存在于该种肿瘤发生相关的抑癌基因。
染色体 17pl3.3区在多种肿瘤中存在较高的杂合性缺失。 因此， 国际上有多家实验室 均在寻找位于染色体 17pl3.3区的抑癌基因。 至目前为止， 已发现了三个可能的抑癌基因 (Hic-I, OVCA1 和 OVCA2), Hic-I在肿瘤组织中高度甲基化， 表达降低。 OVCA1 和 OVCA2在卵巢癌或细胞株中表达减少或不表达。这三个基因均位于染色体的 YNZ22位点 附近。
由于癌症是危害人类健康的主要疾病之一。 为了有效地治疗和预防肿瘤 (如肝癌)， 目 前人们已越来越关注肿瘤的早期诊断和基因治疗。 因此， 本领域迫切需要开发研究新的癌 症相关的和 /或具有抑癌功能的人蛋白及其激动剂 /抑制剂。
发明目的
本发明的目的是提供一种新的肝癌相关蛋白-人 c63R蛋白多肽以及其片段、类似物和 衍生物。
本发明的另一目的是提供编码这些多肽的多核苷酸。
本发明的另一目的是提供生产这些多肽的方法以及该多肽和编码序列的用途。
发明概述
在本发明的第一方面， 提供了一种分离的人 c63R蛋白多肽， 它包括具有 SEQ ID NO: 2 所示氨基酸序列的多肽， 或其保守性变异多肽、或其活性片段、或其活性衍生物。较佳地， 该该多肽是具有 SEQ ID NO: 2所示氨基酸序列的多肽。
在本发明的第二方面， 提供了一种分离的多核苷酸，.它包含一核苷酸序列， 该核苷酸 序列与选自下组的一种核苷酸序列有至少 85%相同性： （a)编码如权利要求 1和 2所述多肽的 多核苷酸； （b)与多核苷酸 (a)互补的多核苷酸。较佳地， 该多核苷酸编码的多肽具有 SEQ ID NO: 2所示的氨基酸序列； 更佳地， 该多核苷酸具有选自下组的序列： SEQ ID NO: 3中所示 的编码区序列或全长序列。
在本发明的第三方面，提供了含有上述多核苷酸的载体， 以及被该载体转化或转导的 宿主细胞或者被上述多核苷酸直接转化或转导的宿主细胞。
在本发明的第四方面， 提供了制备肝癌相关的 C63R蛋白活性的多肽的制备方法， 该 方法包含： （a)在适合表达蛋白的条件下， 培养上述被转化或转导的宿主细胞； （b)从培养物
-1- 确 认本 中分离出具有肝癌相关 c63R蛋白活性的多肽。
在本发明的第五方面， 提供了与上述的肝癌相关 c63R蛋白多肽特异性结合的抗体。 还提供了可用于检测的核酸分子， 它含有上述的多核苷酸中连续的 10-800个核苷酸。
在本发明的第六方面，提供了一种药物组合物， 它含有安全有效量的本发明的肝癌相 关 c63R蛋白多肽以及药学上可接受的载体。 这些药物组合物可治疗癌症以及细胞异常增 殖等病症。
在本发明的第七方面， 提供了一种检测肝细胞是否发生癌变或存在癌变易感性的方 法， 它包括步骤： 检测肝细胞样品中 C63R转录本与正常的 c63R转录本相比是否有变化， 有 变化就表示该肝细胞发生癌变或存在癌变易感性； 或者检测肝细胞样品中 c63R蛋白的活性 与正常的 c63R蛋白相比是否有变化， 有变化就表示该肝细胞发生癌变或存在癌变易感性。 较佳地，所述的变化是核苷酸的缺失、插入置换突变。更佳地，所述的变化选自下组： SEQ ID NO: 1中核苷酸 395-481缺失、 SEQ ID NO: 1中核苷酸 328-1511缺失、 SEQ ID NO: l中核 苷酸 1143-1231缺失、 SEQ ID NO: 1中核苷酸 1325-1419缺失、 SEQ ID NO: 1中核苷酸 1106 由 C突变为 T、 SEQ ID NO: 1中核苷酸 1048缺失。
在本发明的第八方面，提供了一种检测肝癌的试剂盒，它包括：（1)特异性扩增人 c63R 基因的引物对， （2)检测扩增产物与正常的 C63R基因相比是否存在变化所需的试剂。
本发明的其它方面由于本文的技术的公开， 对本领域的技术人员而言是显而易见的。 图 1.覆盖 HCCS1的基因组克隆毗连群和 HCCS1的基因结构。
用于基因组克隆筛选和毗连群结构的标记示于上方； 覆盖 HCCS1的基因组克隆毗连 群示于中间； HCCS1基因的外显子示于下方。
图 2显示了 HCCS1的细胞定位。 图 2a， 用 pEGF-HCCSl转染 MH3T3细胞的荧光 照片； 图 2b， 对图 2a中细胞用抗线粒体抗体进行免疫荧光染色的照片； 图 2c， 照片 2a 和 2b的重迭表明 HCCS1位于线粒体。
图 3对人肝癌细胞和周围非癌细胞的免疫组化染色结果。 HCC : 肝癌细胞； HCC - 肝细胞。
图 4显示了 HCCS1表达对细胞生长的影响。 图 4A， 用载体 pcDNA3.1/V5-His转染 的 SMMC-7721细胞的集落形成； 图 4B， 用含 HCCS1的载体转染的 SMMC-7721细胞的 集落形成； 图 4C， 集落形成的比较图。 其中数据重复 3次， 与空载体相比 p<0.01。
图 5显示了 HCCS1对裸鼠中肿瘤形成的影响。 与 GFP载体组 (对照)相比， PO.01。 发明详述
本发明人在肝癌的研究中发现染色体 17pl3.3区的杂合性缺失范围位于 YNZ22位点 至近端粒的 D17S34位点。 最小热点缺失范围为 D17S1574位点至 D17S849位点。 本发明 人在这个区域中进行了基因克隆， 获得了位于 D17S849位点的 C63R基因， 并发现 c63R 基因在肝癌组织中存在缺失和译码等变化， 从而确认了 C63R基因为肝癌相关基因。 进一 步的实验表明， c63R 基因是一种肝细胞癌抑制因子， 故 c63R 也被称为 HCCSl(Hepatocellular Carcinoma suppressor 1)。研究表明， HCCS1定位于线粒体。 此外， HCCS1在肝癌细胞中表达呈阴性， 而在正常肝细胞中呈阳性。 将 HCCS1转入肝癌细胞和 裸鼠， 都可抑制肿瘤细胞的生长和肿瘤形成。 在本发明中，术语 " c63R蛋白"、 " c63R多肽"或"肝癌相关蛋白 c63R "和 " HCCS1 蛋白"、 " HCCS1多肽"或 "肝细胞癌抑制因子 HCCS1 "可互换使用， 都指具有人肝癌 相关蛋白 c63R氨基酸序列 (SEQ ID NO:2)的蛋白或多肽。 该术语还包括含有或不含起始甲 硫氨酸的肝癌相关蛋白 c63R。
如本文所用， "分离的"是指物质从其原始环境中分离出来 (如果是天然的物质， 原 始环境即是天然环境)。 如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化 的， 但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开， 则为分离纯化 的。 .
如本文所用， "分离的肝癌相关 C63R蛋白或多肽"， "分离的 c63R蛋白或多肽"是 指肝癌相关 c63R蛋白多肽基本上不含天然与其相关的其它蛋白、 脂类、 糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯化技术纯化 c63R蛋白。 基本上纯的多肽在非还原 聚丙烯酰胺凝胶上能产生单一的主带。 c63R蛋白多肽的纯度能用氨基酸序列分析。
本发明的多肽可以是重组多肽、 天然多肽、 合成多肽， 优选重组多肽。 本发明的多 肽可以是天然纯化的产物， 或是化学合成的产物， 或使用重组技术从原核或真核宿主 (例 如， 细菌、 酵母、 高等植物、 昆虫和哺乳动物细胞)中产生。 根据重组生产方案所用的宿 主， 本发明的多肽可以是糖基化的， 或可以是非糖基化的。本发明的多肽还可包括或不包 括起始的甲硫氨酸残基。
本发明还包括肝癌相关的人 c63R蛋白的片段、 衍生物和类似物。 如本文所用， 术语 "片段"、 "衍生物"和 "类似物"是指基本上保持本发明的天然肝癌相关人 c63R蛋白 相同的生物学功能或活性的多肽。 本发明的多肽片段、 衍生物或类似物可以是 (i)有一个或 多个保守或非保守性氨基酸残基 (优选保守性氨基酸残基)被取代的多肽， 而这样的取代的 氨基酸残基可以是也可以不是由遗传密码编码的，或 (ii)在一个或多个氨基酸残基中具有取 代基团的多肽， 或 (Hi)成熟多肽与另一个化合物 (比如延长多肽半衰期的化合物， 例如聚乙 二醇)融合所形成的多肽，或 (iv)附加的氨基酸序列融合到此多肽序列而形成的多肽 (如前导 序列或分泌序列或用来纯化此多肽的序列或蛋白原序列)。根据本文的教导， 这些片段、衍 生物和类似物属于本领域熟练技术人员公知的范围。
本发明的多核苷酸可以是 DNA形式或 RNA形式。 DNA形式包括 cDNA、 基因组 DNA或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编码链或非编码 链。编码成熟多肽的编码区序列可以与 SEQ ID NO:3所示的编码区序列相同或者是简并的 变异体。如本文所用， "简并的变异体"在本发明中是指编码具有 SEQ ID NO:2的蛋白质， 但与 SEQ ID NO:3所示的编码区序列有差别的核酸序列。
编码成熟多肽的多核苷酸包括： 只编码成熟多肽的编码序列；成熟多肽的编码序列和 各种附加编码序列； 成熟多肽的编码序列 (和任选的附加编码序列)以及非编码序列。 术语 "编码多肽的多核苷酸"可以是包括编码此多肽的多核苷酸，也可以是还包括附 加编码和 /或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或 多肽的片段、 类似物和衍生物。 此多核苷酸的变异体可以是天然发生的等位变异体或非天 然发生的变异体。 这些核苷酸变异体包括取代变异体、 缺失变异体和插入变异体 ό 如本领 域所知的， 等位变异体是一个多核苷酸的替换形式， 它可能是一个或多个核苷酸的取代、 缺失或插入， 但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少 50%，较佳地至少 70%，更 佳地至少 80%相同性的多核苷酸。 本发明特别涉及在严格条件下与本发明所述多核苷酸可 杂交的多核苷酸。 在本发明中， "严格条件"是指：（1)在较低离子强度和较高温度下的杂 交和洗脱， 如 0.2 X SSC, 0.1%SDS, 60 °C ; 或 (2)杂交时加有变性剂， 如 50%(v/v)甲酰胺， 0.1%小牛血清 /0.1% Ficoll， 42 °C等； 或 (3)仅在两条序列之间的相同性至少在 95%以上, 更好是 97%以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与 SEQ ID NO： 2所 示的成熟多肽有相同的生物学功能和活性。
本发明还涉及与上述的序列杂交的核酸片段。 如本文所用， "核酸片段"的长度至少 含 15个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50个核苷酸， 最好是至少 100个核 苷酸以上。 核酸片段可用于核酸的扩增技术 (如 PCR)以确定和 /或分离编码 c63R蛋白的多 聚核苷酸。
本发明中的多肽和多核苷酸优选以分离的形式提供， 更佳地被纯化至均质。 .
本发明的 DNA序列能用几种方法获得。 例如， 用本领域熟知的杂交技术分离 DNA。 这些技术包括但不局限于： 1)用探针与基因组或 cDNA文库杂交以检出同源性核苷酸序 列， 和 2)表达文库的抗体筛选以检出具有共同结构特征的克隆的 DNA片段。
编码 c63R蛋白的特异 DNA片段序列产生也能用下列方法获得： 1)从基因组 DNA分 离双链 DNA序列； 2)化学合成 DNA序列以获得所需多肽的双链 DNA。
上述提到的方法中， 分离基因组 DNA最不常用。 当需要的多肽产物的整个氨基酸序 列已知时， DNA序列的直接化学合成是经常选用的方法。 如果所需的氨基酸的整个序列 不清楚时， DNA序列的直接化学合成是不可能的， 选用的方法是 cDNA序列的分离。 分 离感兴趣的 cDNA的标准方法是从高表达该基因的供体细胞分离 inRNA并进行逆转录，形 成质粒或噬菌体 cDNA文库。提取 mRNA的方法已有多种成熟的技术，试剂盒也可从商业 途径获得 (Qiagene；)。而构建 cDNA文库也是通常的方法 (Sambrook, et al.， Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York， 1989)。还可得到商业供应 的 cDNA文库， 如 Clontech公司的不同 cDNA文库。 当结合使用聚合酶反应技术时， 即使 极少的表达产物也能克隆。.
可用常规方法从这些 cDNA文库中筛选本发明的基因。 这些方法包括 (但不限于)： (l)DNA-DNA或 DNA-RNA杂交； （2)标志基因的功能出现或丧失； （3)测定 c63R蛋白的转 录本的水平； （4)通过免疫学技术或测定生物学活性， 来检测基因表达的蛋白产物。 上述方 法可单用， 也可多种方法联合应用。 在第 (1)种方法中，杂交所用的探针是与本发明的多核苷酸的任何一部分同源，其长度 至少 15个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50个核苷酸， 最好是至少 100 个核苷酸。 此外， 探针的长度通常在 2kb之内， 较佳地为 lkb之内。 此处所用的探针通常 是在本发明的基因 DNA序列信息的基础上化学合成的 DNA序列。本发明的基因本身或者 片段当然可以用作探针。 DNA探针的标记可用放射性同位素，荧光素或酶 (如碱性磷酸酶) 等。
在第 (4)种方法中， 检测 c63R蛋白基因表达的蛋白产物可用免疫学技术如 Western印 迹法，放射免疫沉淀法， 酶联免疫吸附法 (ELISA)等。
应用 PCR技术扩增 DNA/RNA的方法 (Saiki, et al. Science 1985;230:1350-1354)被优选 用于获得本发明的基因。特别是很难从文库中得到全长的 cDNA时， 可优选使用 RACE法 ( ACE-cDNA末端快速扩增法)， 用于 PCR的引物可根据本文所公开的本发明的序列信息 适当地选择， 并可用常规方法合成。 可用常规方法如通过凝胶电泳分离和纯化扩增的 DNA/R A片段。
如上所述得到的本发明的基因， 或者各种 DNA片段等的核苷酸序列的测定可用常规 方法如双脱氧链终止法 (Sanger et al. PNAS， 1977 , 74 : 5463-5467)。这类核苷酸序列测 定也可用商业测序试剂盒等。 为了获得全长的 cDNA序列，测序需反复进行。 有时需要测 定多个克隆的 cDNA序列， 才能拼接成全长的 cDNA序列。
本发明也涉及包含本发明的多核苷酸的载体， 以及用本发明的载体或 c63R蛋白编码 序列经基因工程产生的宿主细胞， 以及经重组技术产生本发明所述多肽的方法。
通过常规的重组 DNA技术， 可利用本发明的多聚核苷酸序列可用来表达或生产重组 的 c63R蛋白多肽 (Science， 1984； 224： 1431)。 一般来说有以下步骤：
(1) .用本发明的编码肝癌相关人 C63R蛋白的多核苷酸 (或变异体)， 或用含有该多核苷 酸的重组表达载体转化或转导合适的宿主细胞；
(2) .在合适的培养基中培养的宿主细胞；
3).从培养基或细胞中分离、 纯化蛋白质。
本发明中，肝癌相关的人 c63R蛋白多核苷酸序列可插入到重组表达载体中。术语"重 组表达载体"指本领域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细 胞病毒如腺病毒、 逆转录病毒或其他载体。 在本发明中适用的载体包括但不限于： 在细菌 中表达的基于 T7的表达载体 (Rosenberg, et al. Gene, 1987, 56:125)； 在哺乳动物 胞中表达 的 pMSXND表达载体 (Lee and Nathans, J Bio Chem. 263:3521, 1988)和在昆虫细胞中表达的 来源于杆状病毒的载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。 表达载体的一个重要特征是通常含有复制起点、 启动子、 标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含 c63R蛋白编码 DNA序列和合适的转录 / 翻译控制信号的表达载体。这些方法包括体外重组 DNA技术、 DNA合成技术、体内重组 技术等 (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989)。所述的 DNA序列可有效连接到表达载体中的适当启动子上， 以指导 mR A合成。 这些启动子的代表性例子有： 大肠杆菌的 lac或 trp启动子； λ噬菌 体 PL启动子； 真核启动子包括 CMV立即早期启动子、 HSV胸苷激酶启动子、 早期和晚 期 SV40启动子、 反转录病毒的 LTRs和其他一些已知的可控制基因在原核或真核细胞或 其病毒中表达的启动子。 表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转化的宿主 细胞的表型性状， 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗性以及绿色荧光蛋白 (GFP), 或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当 DNA序列以及适当启动子或者控制序列的载体， 可以用于转化适当 的宿主细胞， 以使其能够表达蛋白质。
宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等 真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 鼠伤寒沙门氏菌的细 菌细胞； 真菌细胞如酵母； 植物细胞； 果蝇 S2或 Sf9的昆虫细胞； CHO、 COS或 Bowes 黑素瘤细胞的动物细胞等。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使 转录得到增强。 增强子是 DNA的顺式作用因子， 通常大约有 10到 300个碱基对， 作用于 启动子以增强基因的转录。 可举的例子包括在复制起始点晚期一侧的 100到 270个碱基对 的 SV40增强子、 在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、 启动子、 增强子和宿主细胞。 用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 当宿主为原核 生物如大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获， 用 CaCl2法处理， 所用的步骤在本领域众所周知。 可供选择的是用 MgCl2。 如果需要， 转化也可用电穿孔的 方法进行。 当宿主是真核生物， 可选用如下的 DNA转染方法： 磷酸钙共沉淀法， 常规机 械方法如显微注射、 电穿孔、 脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿 主细胞， 培养中所用的培养基可选自各种常规培养基。 在适于宿主细胞生长的条件下进行 培养。 当宿主细胞生长到适当的细胞密度后， 用合适的方法 (如温度转换或化学诱导)诱导 选择的启动子， 将细胞再培养一段时间。
在上面的方法中的重组多肽可包被于细胞内、细胞外或在细胞膜上表达或分泌到细胞 外。 如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分离和纯化重组的 蛋白。 这些方法是本领域技术人员所熟知的。 这些方法的例子包括但并不限于： 常规的复 性处理、用蛋白沉淀剂处理 (盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析 (凝 胶过滤)、 吸附层析、 离子交换层析、 高效液相层析 (HPLC)和其它各种液相层析技术及这 些方法的结合。
本发明人研究已表明， 在发生癌变的肝细胞中， C63R基因几乎都存在突变， 这些突 变包括 C63R编码序列和非编码序列的缺失、 插入和置换突变。 这些变化可能导致在肝细 胞中的 c63R蛋白没有活性或活性过低， 并最终直接或间接地导致肝癌。
因此， 本发明重组的肝癌相关人 C63R蛋 S或多肽有多方面的用途。这些用途包括 (但 不限于)： 直接做为药物治疗 C63R蛋白功能低下或丧失所致的疾病 (如肝癌)， 和用于筛选 促进或对抗 c63R蛋白功能的抗体、 多肽或其它配体。 例如， 抗体可用于激活或抑制 c63R 蛋白的功能。 用表达的重组 c63R蛋白筛选多肽库可用于寻找有治疗价值的能抑制或剌激 c63R蛋白功能的多肽分子。
本发明也提供了筛选药物以鉴定提高 (激动剂)或阻遏 (拮抗剂) c63R蛋白的药剂的方 法。 例如， 能在药物的存在下， 将哺乳动物细胞或表达 c63R蛋白的膜制剂与标记的 c63R 蛋白一起培养。 然后测定药物提高或阻遏此相互作用的能力。
c63R蛋白的拮抗剂包括筛选出的抗体、化合物、受体缺失物和类似物等。 C63R蛋白 。的拮抗剂可以与 c63R蛋白结合并消除其功能， 或是抑制 c63R蛋白的产生， 或是与多肽的 活性位点结合使多肽不能发挥生物学功能。 c63R蛋白的拮抗剂可用于治疗用途。
在筛选作为拮抗剂的化合物时， 可以将 c63R蛋白加入生物分析测定中， 通过测定化 合物影响 c63R蛋白和其受体之间的相互作用来确定化合物是否是拮抗剂。 用上述筛选化 合物的同样方法， 可以筛选出起拮抗剂作用的受体缺失物和类似物。
本发明的多肽可直接用于疾病治疗， 例如， 各种恶性肿瘤和细胞异常增殖等， 尤其是 用于肝癌的治疗。
本发明的多肽， 及其片段、衍生物、类似物或它们的细胞可以用来作为抗原以生产抗 体。 这些抗体可以是多克隆或单克隆抗体。 多克隆抗体可以通过将此多肽直接注射动物的 方法得到。 制备单克隆抗体的技术包括杂交瘤技术， 三瘤技术， 人 B-细胞杂交瘤技术， EBV-杂交瘤技术等。
可以将本发明的多肽和拮抗剂与合适的药物载体组合后使用。这些载体可以是水、葡 萄糖、 乙醇、 盐类、 缓冲液、 甘油以及它们的组合。 组合物包含安全有效量的多肽或拮抗 剂以及不影响药物效果的载体和赋形剂。 这些组合物可以作为药物用于疾病治疗。
本发明还提供含有一种或多种容器的药盒或试剂盒，容器中装有一种或多种本发明的 药用组合物成分。 与这些容器一起， 可以有由制造、 使用或销售药品或生物制品的政府管 理机构所给出的指示性提示， 该提示反映出生产、 使用或销售的政府管理机构许可其在人 体上施用。 此外， 本发明的多肽可以与其它的治疗化合物结合使用。
药物组合物可以以方便的方式给药， 如通过局部、 静脉内、 腹膜内、 肌内、 皮下、 鼻 内或皮内的给药途径。 c63R蛋白以有效地治疗和 /或预防具体的适应症的量来给药。 施用 于患者的 c63R蛋白的量和剂量范围将取决于许多因素， 如给药方式、 待治疗者的健康条 件和诊断医生的判断。
c63R蛋白的多聚核苷酸也可用于多种治疗目的。 基因治疗技术可用于治疗由于 c63R 蛋白的无表达或异常 /无活性的 C63R蛋白的表达所致的细胞增殖、 发育或代谢异常。 重组 的基因治疗载体 (如病毒载体)可设计成表达变异的 c63R蛋白， 以抑制内源性的 c63R蛋白 活性。 例如， 一种变异的 c63R蛋白可以是缩短的、 缺失了信号传导功能域的 c63R蛋白， 虽可与下游的底物结合，但缺乏信号传导活性。因此重组的基因治疗载体可用于治疗 C63R 蛋白表达或活性异常所致的疾病。 来源于病毒的表达载体如逆转录病毒、 腺病毒、 腺病毒 相关病毒、 单纯疱疹病毒、 细小病毒等可用于将 C63R蛋白基因转移至细胞内。 构建携带 c63R蛋白基因的重组病毒载体的方法可见于已有文献 (Sambrook,et al.)。 另外重组 c63R蛋 白基因可包装到脂质体中转移至细胞内。
抑制 c63R蛋白 mRNA的寡聚核苷酸 (包括反义 R A和 DNA)以及核酶也在本发明的 范围之内。核酶是一种能特异性分解特定 RNA的酶样 RNA分子， 其作用机制是核酶分子 与互补的靶 RNA特异性杂交后进行核酸内切作用。 反义的 RNA和 DNA及核酶可用已有 的任何 RNA或 DNA合成技术获得，如固相磷酸酰胺化学合成法合成寡核苷酸的技术已广 泛应用。反义 RNA分子可通过编码该 RNA的 DNA序列在体外或体内转录获得。这种 DNA 序列已整合到载体的 RNA聚合酶启动子的下游。 为了增加核酸分子的稳定性， 可用多种 方法对其进行修饰， 如增加两侧的序列长度， 核糖核苷之间的连接应用磷酸硫酯键或肽键 而非磷酸二酯键。
多聚核苷酸导入组织或细胞内的方法包括： 将多聚核苷酸直接注入到体内组织中； 或 在体外通过载体 (如病毒、 噬菌体或质粒等)先将多聚核苷酸导入细胞中， 再将细胞移植到 体内等。
本发明的多肽还可用作肽谱分析， 例如， 多肽可用物理的、 化学或酶进行特异性切 割， 并进行一维或二维或三维的凝胶电泳分析。
本发明还提供了针对 c63R蛋白抗原决定簇的抗体。 这些抗体包括 (但不限于)： 多克 隆抗体、 单克隆抗体、 嵌合抗体、 单链抗体、 Fab片段和 Fab表达文库产生的片段。
抗 c63R蛋白的抗体可用于免疫组织化学技术中， 检测活检标本中的 c63R蛋白。 与 c63R蛋白结合的单克隆抗体也可用放射性同位素标记， 注入体内可跟踪其位置和 分布。 这种放射性标记的抗体可作为一种非创伤性诊断方法用于肿瘤细胞的定位和判断是 否有转移。
本发明中的抗体可用于治疗或预防与 c63R蛋白相关的疾病。 给予适当剂量的抗体可 以刺激或阻断 c63R蛋白的产生或活性。
抗体也可用于设计针对体内某一特殊部位的免疫毒素。 如 c63R蛋白高亲和性的单克 隆抗体可与细菌或植物毒素 (如白喉毒素， 蓖麻蛋白， 红豆碱等)共价结合。 一种通常的方 法是用巯基交联剂如 SPDP， 攻击抗体的氨基， 通过二硫键的交换， 将毒素结合于抗体上， 这种杂交抗体可用于杀灭 c63R蛋白阳性的细胞。
多克隆抗体的生产可用 c63R蛋白或多肽免疫动物， 如家兔， 小鼠， 大鼠等。 多种佐 剂可用于增强免疫反应， 包括但不限于弗氏佐剂等。
c63R蛋白单克隆抗体可用杂交瘤技术生产 (KoMer and Milstein. Nature, 1975, 256:495- 497)。 将人恒定区和非人源的可变区结合的嵌合抗体可用已有的技术生产 (Morrison et al ,PNAS,1985,81:6851)o 而已有的生产单链抗体的技术 (U.S. Pat No.4946778)也可用于生产 抗 c63R蛋白的单链抗体。
能与 C63R蛋白结合的多肽分子可通过筛选由各种可能组合的氨基酸结合于固相物组 成的随机多肽库而获得。 筛选时， 必须对 C63R蛋白分子进行标记。
本发明还涉及定量和定位检测 c63R蛋白水平的诊断试验方法。 这些试验是本领域所 熟知的， 且包括 FISH测定和放射免疫测定。试验中所检测的 c63R蛋白水平， 可以用作解 释 C63R蛋白在各种疾病中的重要性和用于诊断 C63R蛋白起作用的疾病。 c63R蛋白的多聚核苷酸可用于 c63R蛋白相关疾病 (尤其肝癌)的诊断和治疗。 在诊断 方面， c63R蛋白的多聚核苷酸可用于检测 c63R蛋白的表达与否， 或检测在疾病状态下 C63R蛋白的异常表达。而 C63R蛋白 DNA序列可用于对活检标本的杂交以判断 c63R蛋白 的表达异常。杂交技术包括 Southern印迹法， Northern印迹法、原位杂交等。这些技术方法 都是公开的成熟技术， 相关的试剂盒都可从商业途径得到。 本发明的多核苷酸的一部分或 全部可作为探针固定在微阵列 (Microarray)或 DNA芯片 (又称为 "基因芯片"）上，用于分析 组织中基因的差异表达分析和基因诊断。用 c63R蛋白特异的引物进行 RNA-聚合酶链反应 (RT-PCR)体外扩增也可检测 c63R蛋白的转录产物。
检测 c63R蛋白基因的突变也可用于诊断 c63R蛋白相关的疾病 (尤其是肝癌)。 c63R 蛋白突变的形式包括与正常野生型 c63R蛋白 DNA序列 (如 SEQ ID NO: 1所示的正常序列) 相比的点突变、易位、缺失、重组和其它任何异常等。可用已有的技术如 Southern印迹法、 DNA序列分析、 PCR和原位杂交检测突变。 另外， 突变有可能影响蛋白的表达， 因此用 Northern印迹法、 Western印迹法可间接判断基因有无突变。
本发明的 c63R蛋白核苷酸全长序列或其片段通常可以用 PCR扩增法、重组法或人工合 成的方法获得。 对于 PCR扩增法， 可根据本发明所公幵的有关核苷酸序列， 尤其是开放阅 读框序列来设计引物， 并用市售的 cDNA库或按本领域技术人员已知的常规方法所制备的 cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次 PCR扩增， 然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。这通常是将其克 隆入载体， 再转入细胞， 然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外， 还可用人工合成的方法来合成有关序列， 尤其是片段长度较短时。通常， 通过 . 先合成多个小片段， 然后再进行连接可获得序列很长的片段。
目前， 已经可以完全通过化学合成来编码本发明蛋白 (或其片段，或其衍生物)的 DNA 序列。 然后可将该 DNA序列引入本领域中的各种 DNA分子 (如载体)和细胞中。 此外， 还 可通过化学合成将突变引入本发明蛋白序列中。
此外， 由于本发明的 c63R蛋白具有源自人的天然氨基酸序列， 因此， 与来源于其他 物种的同族蛋白相比， 预计在施用于人时将具有更高的活性和 /或更低的副作用 (例如在人 体内的免疫原性更低或没有)。 下面结合具体实施例， 进一步阐述本发明。应理解， 这些实施例仅用于说明本发明而 不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规条件 如 Sambrook等人， 分子克隆： 实验室手册 (New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议的条件。 实施例 1. 染色体 17pl3. 3区在肝癌中杂合性缺失范围的确定：
在该实施例中， 通过选取染色体 17pl3.3区的多态性标记 (marker),进行肝癌组织的杂 合性缺失分析， 从而确定缺失范围和最小热点缺失范围。 1. 肝癌及癌旁标本-
54例原发性肝癌及癌旁组织来自启东肝癌研究所，手术中切除的组织速冻后，保存于 -80 °C冰箱中。
2. 组织 DNA的提取：
从低温冰箱中取出组织,置组织碾磨器中碾碎,按 SDS/蛋白酶 K-酚 /氯仿抽提方法 [3] 提取组织 DNA。
3. 杂合性缺失 (L0H)分析：
对 VNTR (variable number of tandem repeat)和 RFLP探针通过 Southern印迹分析 检测 LOH。 10ug的肝癌及癌旁组织 DNA经适当限制性内切酶酶切后， 25V电泳 13小时， 转移至 hybond- N膜（Amersham公司）。 YNZ22 探针由上海医科大学胡英赠送， 144D6 、 YNH37. 3探针购自 American Type Culture Collection (ATCC)。 cCI17- 708、 cCI17- 680 由 Dr. Nakamura赠给。 探针用 [α-32Ρ] dATP按随机引物法标记。 探针的位点名称， 用于酶 切的限制性内切酶列于表 1中， YNZ22. 1、 144D6和 YNH37. 3探针用含 50%甲酰胺的常规 杂交液于 42 V杂交过夜。 CCI17-708和 cCI17- 680探针杂交时，预杂交液为 50%甲酰胺， 5 X SSC, 0. 5%SDS, 10 X Denhardfs液， 250ug/ml人胎盘 DNA， 于 42 °C预杂交 24小时， 杂交液除用 10%硫酸葡聚糖代替 10 X Denhardfs液外， 其余成分同预杂交液， 于 42 °C杂 交过夜。
微卫星标志的分析在 12. 5ul反应体积中进行， 反应中含 luM PCR引物， 25ng组织 DNA， 200uM dNTP， 50mM KCI， 10mM Tris (pH9. 0)， 1. 5mM MgCl2 0. 625U Taq DNA聚合 酶 (Promega)。 微卫星标志的位点名称、 退火温度列于表 1中。 PCR反应按 97 Ό变性 4分 钟， 94 °C Γ、 适当退火温度 1'、 72 °C 1'， 30个循环， 72 °C延伸 10'程序进行。 产物经 2%琼脂糖凝胶电泳检查。 [r- 32P]ATP标记其中一条引物。 在 5ul PCR反应体积中， 含标记 的 lpmol引物， lul上述 PCR产物， 0. 25U Taq DNA聚合酶， 除 PCR反应进行 4个循环外， 其余成份和反应条件同上。 PCR产物变性后，于 6%变性聚丙烯酰胺凝胶上分离、 压片， 观 察 L0H结果。
共检测了染色体 17pl3. 3区的 16个多态性标志的杂合性缺失情况。 这些位点的 L0H 频率列于表 1中，每个样品在每个位点的 L0H情况总结于表 2中。首先检测了 22份样品在 YNZ22位点的 L0H， 发现在 19份杂合子中有 12份呈 L0H , 频率为 63%。 接着又检测了 21 份样品在接近端粒的 D17S34位点的 L0H， 在 12份杂合子中 8份呈 L0H， 频率为 67%, 而 且特别引人注意的是所有 YNZ22位点呈 L0H的样品在 D17S34位点都有 L0H (纯合子除外）， 是否可能在肝癌样品中 17pl3. 3的缺失范围至少为从 YNZ22位点至近端粒的 D17S34位点。 基于此种考虑， 又选用了围绕两位点间的 14个标志进一步检测各个标志在肝癌样品中的 L0H。结果位于 D17S34至 D17S5位点间的 8个标志在肝癌样品中均有较高 L0H ,而位于 D17S5 近着丝粒方向的 3个标志有较低或无 L0H 。 这证实了我们的推测， 即肝癌样品在染色体 17pl3. 3区的缺失范围为从 D17S34至 D17S5位点。 其中 D17S926标志检测了 22份样品， 10份杂合子均呈 L0H， 频率为 100%。 YNZ22位点呈 L0H的样品在这个位点也呈 L0H (纯合 子除外）。 D17S1866， D17S849， D17S643， D17S1840， D17S654和 D17S1574标志在 YNZ22位点呈 LOH的样品中均有 LOH (纯合子除外），频率为 68-86%。 YNH37. 3探针（D17S28) 只检测了 9例在 YNZ22位点有 L0H的样品， 当用 Taq l酶切时， 所有 9份样品均为纯合子。 无法确定此位点的 L0H情况。 值得注意的是 NO. 16肝癌样品和 NO. 34肝癌样品， 在近着丝 粒方向的 YNZ22位点和 D17S1574位点均无 LOH, NO. 16肝癌样品在近端粒方向的 D17S34 和 D17S1866位点均无 LOH, NO. 34肝癌样品也在近端粒方向的 D17S34 ， D17S1866和 D17S849位点无 L0H。但这两份肝癌样品在 D17S849位点和 D17S1574位点间的所有位点上 均有 L0H纯合子。 因此， 肝癌样品在染色体 17pl3. 3区的最小热点缺失范围为从 D17S849 位点至 D17S1574位点。
在分析染色体 17P13. 3区各位点 LOH的同时，也进行了位于染色体 17pl3. 1区的 p53基因 中 TP53位点的 L0H分析。结果发现，该位点在肝癌中只有 31%的 L0H，远远低于染色体 17pl3. 3 区 D17S34至 D17S5位点间各位点的 LOH频率 (表 1和表 2)。
探针及 L0H情况
Figure imgf000012_0001
*微卫星重复序列标志 Tel
端粒 表 2· 染色体 17P13.3区 LOH分祈
木
Figure imgf000013_0001
Cen 書 loss of heterozygosity O retention of both alleles ® non-informative Θ loss of homozygosity @ rearrangement space: hot analyzed 着丝粒 杂合子丟失 无杂合子丟失 纯合子 . 纯 子丢失 ¾排 ' 空格： 未分析 -
实施例 2. c63R的 cDNA克隆：
1、 染色体 17pl3. 3区 D17s926基因组 PAC克隆的筛选：
从 Genbank中获得 D17s926的引物序列，
926-1 ： GCA GTG GGC CAT CAT CA(SEQ ID NO: 3)
926-2 ： CCG CAG AAG GCT GTT GT (SEQ ID NO: 4)
将其送至 Genome systems Inc 由他们筛选 PAC人基因组库， 获得阳性克隆 P579 。
2、从含有 P579 (Genome System公司提供）菌的平板上挑一个 PAC单菌，接种在 5ml LB 中（含 25ug/ml卡那霉素） 37 V 3000rpm振摇过夜，在 75ml LB 中加入 2. 5ml过夜培养物振 摇 1. 5小时，加入 IPTG至终浓度 0. 5mM诱导继续生长 5小时后收获细菌离心 10， OOOglO分 钟，细菌沉淀按"分子克隆"（美 J. Sambrook , E. F. F. ritsch, T. Maniatis.金冬雁，梨孟枫 等译. 1992年第二版,科学出版社)书中抽提质粒 DNA的方法制备 PAC DNA ,约获得 lOug P579 DNA 。
取 P579 DNA ， 用 Not I酶切 37 °C 2 - 3小时， 插入片段约 100Kb ， 用低熔点胶 回收 DNA片段做为探针。
取 200 - 300ng上面纯化的 DNA片段， 用 32P- a dATP或 32Ρ- a dCTP)采用随机引物进 行标记， 操作步骤按试剂盒说明书进行 （Megaprime DNA labelling system.
Amersham公司)。
将 cDNA文库的噬菌体溶液按每块平皿 50， OOOpfu铺制 30块平皿（直径 150匪） ， 41 °C培养 3-5小时， 至噬斑直径为 0. 5mm且相邻噬斑未联合时， 从培养箱取出平皿放 4 °C至 少 1 小时。 从冰箱取出平皿放室温轻轻的将硝酸纤维素滤膜（Hybond-C Amersham LIFE Science)铺于平板上， 用注射器以碳素墨水作 4个点以定方向， 取下滤膜碱变性 5分钟 (0. 5mol Na0H-l. 5mol NaCl) ,中和 5分钟（lmol Tris-HCl pH7. 4, 1. 5mol NaCl)转移至 6xSSC 中稍作浸泡,置 3MM滤纸上干燥，然后 80 °C烘膜 2小时.
膜片在预杂交液中 65-68 V杂交 6-8 小时。 预杂交液含有如下成分： 6xSSC ， 5xDenhart's, 0. 5%SDS,变性的鱼精 DNA200ug/ml，变性的胎盘 DNA100ug/ml和混合 DNA ( γ DNA5. 8 ， γ DNA 7. 3和 PBR质粒 DNA ,各为 30 ug/ml) 。
已标记的 300ul探针溶液加入 Cot- 1 DNA lmg混合 DNA各 50ug 20xSSC 250ul ， 100 °C煮沸 15分钟冰上放置 10分钟， 60- 65 °C水浴封闭杂交 20分钟， 冰上放置 20分钟， 加 入到有膜片的预杂交液中， 65-68 V杂交 16-24小时。杂交完成后弃取杂交液，用 42 °C预 温的 2xSSC -0. 1%SDS溶液漂洗膜片 2次，每次 10分钟，一面洗一面用同位素检测仪检测， 调整洗膜片的时间和温度，洗涤后进行放射自显影。 用同样的方法进行第二，第三轮筛选， 最后得到阳性单克隆，挑取单个的阳性噬菌体斑 (phage)放在 lmlSM缓冲液中加 1滴氯仿， 放在室温或 4 °C作为阳性单克隆的储存液 (phage stock)。 通过筛选得到阳性噬菌体克隆 c63R 。 实施例 3 c63R重组质粒的构建：
取 20ul噬菌体储存液 lOOul Y1090菌 (过夜生长用 10mM MgS04处理过）37 °C温育 20 分钟，加 3. 5ml， 50 °C保温的 0. 7%的 LB琼脂糖，铺在含有 1°/。琼脂胶的平皿上（直径 90mm) 37 °C温育过夜,取出平皿,每块加 3ml SM溶液， 4 V过夜，收集含有噬菌体的溶液。
取 150ul板扩的噬菌体加 1ml lOmM MgS04处理过的 Y1090菌 37 °C温育 15分钟， 加 40ml LB (含 1/100 0. 5M CaC12) 37 °C，强烈振摇直至液体由混浊变清（3小时左右）加 1ml 氯仿 37 °C振摇 20分钟， 4000rpm离心 10分钟，上清液加入 50ul RNase A/DNase溶解液 37 °C温育 1 小时， 4 °C过夜，然后离心 lOOOrpm xlO 分钟，上清液进行超离心， 27000rpmx70分钟。 取沉淀加 0. 5ml SM溶解转移到 1. 5ml的离心管中加入 5ul蛋白酶 K 和 1/5体积的 0. 5MSTE 50 O温育 30分钟，酚 /氯仿抽提最后酒精沉淀得 phage DNA用 EcoR I 酶切， 用胶回收 DNA片段 (胶回收试剂盒购自上海华舜生物工程有限公司）。 表达载体 PBK-CMV(Stratagene公司） 赚同样用 EcoR I酶切， 并用 CIAP酶处理。 DNA插入片段和 载体 PBK- CMV DNA以克分子 3： 1的量用 T4 DNA连接酶在 12 °C连接过夜， 转化感受态细 胞 XJ-blue. 挑取白色阳性克隆制备质粒 DNA， 酶切证明了亚克隆的正确性。 实施例 4. c63R核酸序列测定及分析：
对 c63R克隆采用双脱氧终止法， 在 ABI 337 DNA自动测序仪上测定其两端序列， 依 据所得序列设计引物再次进行测序， 直到获得全长序列，共 2132bp。进一步分析发现 5'端 的 54Bp为载体上的序列， 因此全长序列共 2078bp如 SEQ ID NO: 1所示， 其中编码阅读框 ' 位于 56- 2077位。 所编码的 c63R蛋白全长为 673个氨基酸， 序列如 SEQ ID NO: 2所示。 核酸序列和氨基酸序列的组合排列示于 SEQ ID NO: 1。 实施例 5. 从人肝 cDNA中用 PCR法获得基因克隆：
取人肝组织,用 Trizol试剂（ GIBCO. BRL公司）按其说明书提取总 RNA， 用 mRNA纯化 试剂盒（ Pharmacia公司）提取 mRNA。 用腿 LV-RT- Superscript II (GIBCO BRL公司） 反转 录在 42迸行反转录反应，获得胎盘 cDNA。合成如下的基因编码读框的特异引物 c63R- 1： atgatggaggaggaggaa (SEQ ID NO: 5)； c63R- 2 ： gttgtcacggatgatggg (SEQ ID NO: 6)。 按 90 °C 3分钟 1个循环； 94 Ό 30秒， 60 Ό 30秒， 72 °C 1分钟， 共 35个循环； 72 °C 10分钟。 1个循环进行扩增， 获得含有完整开放阅读框序列的蛋白基因的扩增产物。扩增 产物经测序验证， 与实施例 4测得的序列相符。 实施例 6. c63R基因在各种组织中的表达
从 c63R重组质粒中酶切分离 c63R片段， 用 a 32P-dATP按试剂盒 (Megaprime DNA Labelling System ， Amersham 公司），按说明书进行探针标记。 与多组织 mRNA 膜片 (Biochain公司）进行杂交， 结果显示有三条杂交带， 在多组织中均有表达。 实施例 7 C63R基因在肝癌组织中的变化：
获得染色体 17pl3. 3区 C63R基因， 进一步检测其在肝癌组织中的变化。 从肝癌组织和 癌旁组织抽提总 RNA， 反转录为 cDNA。 合成如下的 C63R基因特异的引物：
C63R-a-PF： CGGGTGGCGGAATGATG (SEQ ID NO : 7)
C63R- a- PR : CTCCACCCCCATCTACCA (SEQ ID NO : 8)
C63R-b-PF： GGTGGCGGAATGATGGA (SEQ ID NO : 9)
C63R-b-PR： CAAAACGCTTCTCCGGC (SEQ ID NO : 10)
C63R-C-PF： TGCATGGCTGAGAGGATTG (SEQ ID NO : 11)
C63R-C-PR： ACAACCCCGTGGCTTCC (SEQ ID NO : 12)
C63R- d-PF : TGCGTACCAGAGCGAAGG (SEQ ID NO: 13)
C63R-d-PR： CAAGCAGAACGTCCCCAT (SEQ ID NO : 14)
C63R- a- SR : AGGATGGACGGCAAGCG (SEQ ID NO : 15)
C63R- b- SR : GGAGGACCCAGCAATGTT (SEQ ID NO : 16)
C63R-C-SR： CCAAGACAAGAACCTCGGA (SEQ ID NO : 17)
C63R-d-SR： AACATCCTGAGCACGGCA (SEQ ID NO : 18)
用 C63R- a- PF和 C63R- a- PR， C63R- c- PF和 C63R- c- PR分别为引物， cDNA为模板，按 94 °C变性 3分钟，一个循环； 94 °C变性 30秒， 60 °C退火 30秒， 72 °C延伸 1分钟， 35 个循环； 72 °C延伸 10分钟的程序进行首次 PCR。 再用 C63R- b- PF和 C63R- b- PR， C63R- d-PF和 C63R- d- PR分别为引物， 按同上程序进行第二次巢式 PCR， 获得 PCR产物。
对以 C63R- b- PF和 C63R- b-PR为引物的 PCR产物， 用 C63R- b- PF， C63R- a- SR ， C63R-b-SR , 和 C63R- b- PR为引物进行序列测定。 对以 C63R- d- PF和 C63R- d- PR为引物的 PCR产物， 用 C63R- d- PF， C63R-C- SR， C63R- d- SR和 C63R- d- PR为引物迸行序列测定。
分析结果发现， 肝癌样品 3， 12， 25， 28 , 33和 G11在 nt341- 427有缺失， 而相 应的癌旁组织无缺失。肝癌样品 28在 ntl274- 1457有缺失。相应的癌旁组织无缺失。肝癌 样品 31在 ntl090-1177有缺失。 相应的癌旁组织无缺失。 肝癌样品 x31在 ntl271_1365 有缺失。相应的癌旁组织无缺失。肝癌样品 G11在 ntl052位 C突变为 T，使相应的氨基酸 从 Arg突变为 Cys。 肝癌样品 G4在 nt994位的 T缺失， 造成了移码突变。 肝癌样品 GT7 缺失 nt743― 887位和 ntl221— 1529， 造成了移码突变。 肝癌样品 GT12缺失 ntl612— 1769 , 造成了移码突变。
这些结果表明， c63R基因是一种肝癌相关基因， c63R基因的缺失、插入、置换等类 型的突变与肝癌相关。 这些变化可能导致在肝细胞中的 c63R蛋白没有活性或活性过低， 并最终直接或间接地导致肝癌。 实施例 8 HCCS1的基因组结构和细胞定位
将 HCCS1的 cDNA序列与基因组序列进行比较， 确定了 HCCS1基因的内含子 /外显 子结构。 HCCS1共有 18个外显子， 横跨的基因组序列约 200kb (图 1)。
为了确定 HCCS1蛋白产物的细胞定位， 将 HCCS1-GFP融合基因克隆入 pEGFP-Nl 载体， 然后将 HCCS1-GFP质粒 (pEGF-HCCSl) DNA转入人 SMMC7721 细胞系和小鼠 NIH3T3 成纤维细胞中， 免疫荧光分析显示， 两种细胞类型的细胞质中有点状分布区， 并 且这些分布区与线粒体单克隆抗体 113-l(NeoMarkers, Fremont, CA)的染色区相重迭， 这表 明， HCCS1融合蛋白位于线粒体中 (图 3)。 实施例 9免疫组化分析
HCC和非癌组织被固定在 10%福尔马林的 10mM磷酸盐缓冲液 (PBS)(pH7.2)中。将石 蜡切片 (4μιη)安在涂有聚 L-赖氨酸的载玻片上， 在 50 °C干燥过夜。 小鼠抗 -HCCS1抗体在 含 5%正常羊血清的 PBS中作 1 ： 200稀释，然后在室温下保温 30分子。在用 PBS漂洗 3 次 (每次 5分钟)后， 在室温下对切片涂覆 DAKO EnVision™ + System, H P (DAB), Mouse, Ready-to-use检测体系 (DAKO Corp., Carpinteria, CA, USA) 30分钟。 切片在 Substrate- Chromogen Solution (DAB)中显影， 用 Mayer's苏木精复染色， 然后观察。
结果如图 3所示， 在肝癌细胞 (HCC)中为阴性， 肝细胞 (HC)中为阳性。 实施例 10 HCCS1对细胞生长的影响
将 HCCS1 cDNA插入 pcDNA3.1/V5-His载体 (Invitrogen)。 安装厂商的说明， 通过 LipofectAMINE试剂将该构建物转染入 SMMC-7721人肝癌细胞 (3 X 104) (重复 3次)。将空 载体作为对照。 在用 G418(800mg/ml)选择 14天后， 对克隆进行染色和计数。
结果如图 4所示， 表明 HCCS1可抑制肿瘤细胞的生长。 实施例 11 HCCS1对裸鼠中肿瘤形成的影响
收集携带 HCCSl cDNA或空载体的细胞，并再悬浮于 PBS中。将 200微升细胞 (3.7xl06) 皮下接种于 5-6周龄的雄性 BALB/c裸鼠的右侧。 试验组和对照组各有 6只小鼠。 在 5周 后， 杀死小鼠， 切下肿瘤进行称重。
结果如图 5所示， 表明 HCCS1可抑制裸鼠中肿瘤细胞的形成。 在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用 作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员可以 对本发明作各种改动或修改， 这些等价形式同样落于本申请所附权利要求书所限定的范 围。

Claims

权 利 要 求 书
I.一种分离的人 c63R蛋白多肽， 其特征在于， 它包括具有 SEQ ID NO: 2所示氨基酸 序列的多肽， 或其保守性变异多肽、 或其活性片段、 或其活性衍生物。
2.如权利要求 1所述的多肽， 其特征在于， 该多肽是具有 SEQ ID NO: 2所示氨基酸序 列的多肽。
3.—种分离的多核苷酸， 其特征在于， 它包含一核苷酸序列， 该核苷酸序列与选自 下组的一种核苷酸序列有至少 85%相同性-
(a)编码如权利要求 1和 2所述多肽的多核苷酸；
(b)与多核苷酸 (a)互补的多核苷酸。
4.如权利要求 3所述的多核苷酸， 其特征在于， 该多核苷酸编码的多肽具有 SEQ ID NO: 2所示的氨基酸序列。
5.如权利要求 3所述的多核苷酸，其特征在于，该多核苷酸具有选自下组的序列： SEQ ID NO: 3中所示的编码区序列或全长序列。
6.—种载体， 其特征在于， 它含有权利要求 3所述的多核苷酸。
7.—种遗传工程化的宿主细胞， 其特征在于， 它是选自下组的一种宿主细胞：
(a)用权利要求 6所述的载体转化或转导的宿主细胞；
(b)用权利要求 3所述的多核苷酸转化或转导的宿主细胞。
8. 一种制备具有人蛋白 c63R活性的多肽的制备方法， 其特征在于， 该方法包含： (a)在适合表达蛋白的条件下， 培养权利要求 7所述的宿主细胞；
(b)从培养物中分离出具有人蛋白 C63R活性的多肽。
9.一种能与权利要求 1所述的入 c63R蛋白特异性结合的抗体。
10.—种核酸分子， 它含有权利要求 3所述的多核苷酸中连续的 10-800个核苷酸。
I I.一种药物组合物， 其特征在于， 它含有安全有效量的权利要求 1所述的多肽以及 药学上可接受的载体。
12.—种检测肝细胞是否发生癌变或存在癌变易感性的方法， 其特征在于， 它包括步 骤：
检测肝细胞样品中 C63R转录本与正常的 C63R转录本相比是否有变化，有变化就表示 该肝细胞发生癌变或存在癌变易感性； 或者
检测肝细胞样品中 c63R蛋白的活性与正常的 c63R蛋白相比是否有变化，有变化就表 示该肝细胞发生癌变或存在癌变易感性。
13.如权利要求 12所述的方法，其特征在于，所述的变化包括：核苷酸的缺失、插入、 和置换突变。
14.如权利要求 13所述的方法， 其特征在于， 所述的变化选自下组： SEQ ID NO: l中 核苷酸 341-427缺失、 SEQ ID NO: 1中核苷酸 1274-1457缺失、 SEQ ID NO: 1中核苷酸
1090-1177缺失、 SEQ ID NO: 1中核苷酸 1271-1365缺失、 SEQ ID NO: 1中核苷酸 1052由 C 突变为 T、 SEQ ID NO: 1中核苷酸 994缺失， SEQ ID NO: 1中核苷酸 743— 887缺失， SEQ ID 中核苷酸 1221— 1529缺失， SEQ ID NO: 1中核苷酸 1612— 1769缺失。
15.—种检测肝癌的试剂盒， 其特征在于， 它包括：
(1)特异性扩增人 c63R基因的引物对，
(2)检测扩增产物与正常的 C63R基因相比是否存在变化所需的试剂。
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