CN1155613C - Human tumor associated gene in 1-zone 3-band 3-subband of short arm of human chromosome No.17 and its coding protein - Google Patents

Abstract

The present invention discloses a new human protein HC6 related to tumors, polyribonucleotide for coding the polypeptide and a method for generating the polypeptide by using a recombination technology. The present invention also discloses a method of using the polypeptide for diagnosing and treating diseases such as cancers, etc. The present invention also discloses an antagonist for resisting the polypeptide and the treating effect thereof. The present invention discloses an application of the polyribonucleotide for coding the human protein related to tumors too.

Description

Human tumor-associated genes and encoded proteins in zone 3, zone 3, subband 3, short arm of human chromosome 17

The invention belongs to the field of biotechnology, in particular, the invention relates to a new polynucleotide encoding human tumor-associated protein and the polypeptide encoded by the polynucleotide. The present invention also relates to the use and preparation of such polynucleotides and polypeptides.

The mortality rate of malignant tumors ranks second only to heart and cerebrovascular diseases in my country. Among malignant tumors, the mortality rate of liver cancer ranks third after lung cancer and gastric cancer. At present, there is no effective diagnosis and treatment for liver cancer (except small liver cancer). It is generally believed that carcinogenesis is a multi-factor and multi-step complex event, which is the result of the activation of various oncogenes and the inactivation of tumor suppressor genes. Among them, tumor suppressor genes may be more critical. Therefore, searching for tumor suppressor genes is one of the current research hotspots. A gene often loses a normal trait through the loss of an allele (Loss of Heterozygocity, LOH), and there may be a tumor suppressor gene in the frequent occurrence of LOH.

The State Key Laboratory of Oncogenes and Related Genes of Shanghai Cancer Institute has been engaged in the research of human liver cancer genes for a long time. As early as 1991, it was found that the occurrence of liver cancer is related to the inactivation of p53. The gene not only has 249 codon mutations, but also other codons A point mutation occurs (Li D.Z; Gu JR et al Carcinogenesis 14(2):169, 1993). p53 is located on human chromosome 17p13.1.

In addition, the research on the 17p13.3 segment has also been reported abroad. Nishida et al. (Noashi Nishida et al., Cancer Research 53:368-372, 1993) selected 10 polymorphic probes on 17p to study the relationship between 17pLOH and p53 gene mutations in human liver cancer. He believed that the occurrence of liver cancer Possibly related to an undocumented tumor suppressor gene on 17p13.3. Schultz et al. (David C. Schultz, et al., Cancer Research 56: 1997-2002 1996), using allelic loss mapping and site-directed cloning methods, proved that two new genes, OVCA1 and OVCA2, were located at 17p13.3, which The two novel genes were expressed in normal ovarian epithelial cells, but had reduced or no expression in ovarian tumors or ovarian tumor cell lines. Wales et al. (Michele M.Wales et al., Nature Medicine: 570-577, 1995), through DNA hypermethylation analysis of tumor tissue, found a candidate gene Hic-1 in the 17p13.3 region, which is a zinc finger structure Transcription factor, ubiquitously expressed in normal tissues, with reduced expression in tumor cells.

However, the above reports have the following deficiencies: 1. The minimum range and location of LOH frequency in liver cancer tissues are not reported. 2. The relationship between the above-mentioned candidate genes and liver cancer has not been reported.

Because cancer is one of the main diseases that endanger human health. In order to effectively treat and prevent 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 this field to develop and study new cancer-related and/or human proteins with tumor suppressor function and their agonists/inhibitors.

The purpose of the present invention is to provide a new tumor-associated protein-human HC6 protein polypeptide and fragments, analogs and derivatives thereof.

Another object of the present invention is to provide polynucleotides encoding these polypeptides.

Another object of the present invention is to provide methods for producing these polypeptides and uses of the polypeptides and coding sequences.

In the first aspect of the present invention, an isolated human HC6 protein polypeptide is provided, which includes a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof . Preferably, the polypeptide is a polypeptide having the amino acid sequence shown in SEQ ID NO:2.

In a second aspect of the present invention there is provided an isolated polynucleotide comprising a nucleotide sequence having at least 85% identity 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 polynucleotide (a). Preferably, the polypeptide encoded by the polynucleotide has the 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 (positions 128-532) or full-length sequence.

In the third aspect of the present invention, there are provided vectors containing the above-mentioned polynucleotides, and host cells transformed or transduced by the vectors or host cells directly transformed or transduced by the above-mentioned polynucleotides.

In the fourth aspect of the present invention, a method for preparing a polypeptide with tumor-associated HC6 protein activity is provided, the method comprising: (a) cultivating the above-mentioned transformed or transduced host cells under conditions suitable for expressing the protein; (b ) to isolate a polypeptide having tumor-associated HC6 protein activity from the culture.

In the fifth aspect of the present invention, an antibody specifically binding to the above-mentioned tumor-associated HC6 protein polypeptide is provided. Also provided is a nucleic acid molecule useful for detection, which contains consecutive 10-800 nucleotides of the above-mentioned polynucleotides.

In the sixth aspect of the present invention, a pharmaceutical composition is provided, which contains a safe and effective amount of the tumor-associated HC6 protein polypeptide of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions can treat diseases such as cancer and abnormal proliferation of cells.

In the seventh aspect of the present invention, there is provided a method for detecting whether liver cells undergo canceration or susceptibility to canceration, which includes the steps of: detecting whether the HC6 transcript in the liver cell sample is changed compared with the normal HC6 transcript, If there is a change, it means that the liver cell is cancerous or has a susceptibility to cancer; or if there is a change in the activity of HC6 protein in the liver cell sample compared with the normal HC6 protein, if there is a change, it means that the liver cell is cancerous or has a susceptibility to cancer. . Preferably, the change is a nucleotide deletion, insertion, or substitution mutation.

In the eighth aspect of the present invention, a test kit for detecting liver cancer is provided, which includes: (1) a pair of primers for specifically amplifying the human HC6 gene, (2) detecting whether the amplified product is compared with the normal HC6 gene Reagents required for changes exist.

Other aspects of the invention will be apparent to those skilled in the art from the technical disclosure herein.

In the attached picture,

Figures 1 and 2 show the LOH status of HC6 in liver cancer tissues detected by Southern hybridization, wherein the genomic DNA was digested with BamH I; Q21, Q27, Q28, Q29, Q30, Z11, Z12, and Z14 were liver cancer samples. K: liver cancer tissue; L: paracancerous tissue.

Figure 3 shows the results of transfection of the empty plasmid into the liver cancer cell line SMMC-7721.

Figure 4 shows the results of HC6 transfection of liver cancer cell line SMMC-7721

Figure 5 shows the results of Northern hybridization of multiple tissue membranes of the HC6 gene.

In the study of liver cancer, the inventors first determined that there is a high frequency of LOH (60-100%) in the range of 17p13.3 in liver cancer tissue. Recently, genome-wide scanning of liver cancer also proved that 17p13.3 is the highest region of LOH. The inventors isolated and cloned the cancer-associated expression sequence (EST) at the 13.3 site of the short arm of human chromosome 17. Using the phage artificial chromosome (PAC) No. 579 (P579) clone corresponding to the 926 position in the 17p13.3 segment, its sequence was obtained by nine-fold shotgun sequencing, and a representative new gene was found in it by computer analysis The EST obtained the full-length nucleotide sequence and encoded amino acid by RACE method, named HC6. Through Northern and Southern hybridization, it was proved that they were related to tumors, and in vitro experiments proved that they could inhibit the growth of liver cancer cell 7721. Therefore, HC6 gene can be applied to the diagnosis, treatment and prevention of tumors.

In the present invention, the terms "HC6 protein", "HC6 polypeptide", "tumor-associated HC6 protein" or "tumor-associated protein HC6" are used interchangeably, and all refer to the amino acid sequence of human tumor-associated protein HC6 (SEQ ID NO: 2 ) protein or polypeptide. The term also includes tumor-associated protein HC6 with or without an initial methionine.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "isolated tumor-associated HC6 protein or polypeptide", "isolated HC6 protein or polypeptide" means that the tumor-associated HC6 protein polypeptide does not substantially contain other proteins, lipids, carbohydrates or other substances associated with it in nature. Those skilled in the art can purify HC6 protein using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of HC6 protein polypeptide can be analyzed by amino acid sequence.

The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

The present invention also includes fragments, derivatives and analogs of tumor-associated human HC6 protein. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially maintains the same biological function or activity of the native tumor-associated human HC6 protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides 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 (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

In the present invention, the terms "human tumor-associated protein HC6 polypeptide" or "human NIP2 AP protein polypeptide" are used interchangeably, and both refer to a polypeptide having the sequence of SEQ ID NO.2 having human tumor-associated protein HC6 activity. The term also includes variants of SEQ ID NO. 2 that have the same function as human tumor-associated protein HC6. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the human tumor-associated protein HC6.

Variations of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with human tumor-associated protein HC6 DNA under high or low stringency conditions The encoded protein, and the polypeptide or protein obtained by using the antiserum against human tumor-associated protein HC6 polypeptide. The present invention also provides other polypeptides, such as fusion proteins comprising human tumor-associated protein HC6 polypeptides or fragments thereof (such as fusion proteins comprising the sequence shown in SEQ ID NO: 2). In addition to nearly full-length polypeptides, the present invention also includes soluble fragments of human tumor-associated protein HC6 polypeptides. Usually, the fragment has at least about 10 consecutive amino acids, usually at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 80 consecutive amino acids of the human tumor-associated protein HC6 polypeptide sequence. at least about 100 contiguous amino acids.

The invention also provides analogs of human tumor-associated protein HC6 or polypeptide. The difference between these analogs and the natural human tumor-associated protein HC6 polypeptide may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, the "human tumor-associated protein HC6 conservative variant polypeptide" refers to amino acid sequence compared with SEQ ID NO: 2, there are at most 10, preferably at most 8, more preferably at most 5, and optimally Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.

Table A initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence (positions 618-1445) shown in SEQ ID NO: 3 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein having SEQ ID NO: 2, but differs from the sequence of the coding region shown in SEQ ID NO: 3.

A polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequence.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention. 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. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

The present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only the identity between the two sequences is at least 95%, more Preferably hybridization occurs above 97%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:2.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (eg, PCR) to identify and/or isolate polynucleotides encoding HC6 proteins.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The DNA sequences of the present invention can be obtained in several ways. For example, 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 features .

The generation of specific DNA fragment sequence encoding HC6 protein can also be obtained by the following methods: 1) isolation of double-stranded DNA sequence from genomic DNA; 2) chemical synthesis of DNA sequence to obtain double-stranded DNA of desired polypeptide.

Of the methods mentioned above, isolating genomic DNA is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice when the entire amino acid sequence of the desired polypeptide product is known. If the entire sequence of the desired amino acids is not known, direct chemical synthesis of the DNA sequence is not possible and the method of choice is isolation of the cDNA sequence. The standard method for isolating cDNA of interest is to isolate mRNA from donor cells that highly express the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature technologies for the method of extracting mRNA, and the kit is also available from commercial sources (Qiagene). And constructing a cDNA library is 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 various cDNA libraries from the company Clontech. When combined with polymerase reaction technology, even minimal expression products can be cloned.

These cDNA libraries can be screened for the gene of the present invention by a conventional method. These methods include (but are not limited to): (1) DNA-DNA or DNA-RNA hybridization; (2) appearance or loss of marker gene function; (3) determination of the transcript level of HC6 protein; (4) immunological Technology or measurement of biological activity to detect the protein product of gene expression. The above methods can be used alone or in combination with multiple methods.

In the (1) method, the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2kb, preferably within 1kb. The probes used here are usually DNA sequences chemically synthesized based on the gene DNA sequence information of the present invention. The genes themselves or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioactive isotopes, luciferin or enzymes (such as alkaline phosphatase) and the like.

In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the HC6 protein gene.

A method of amplifying DNA/RNA using the PCR technique (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, And can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

The nucleotide sequence of the gene of the present invention obtained as described above, or various DNA fragments, etc., can be determined by conventional methods such as the dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such nucleotide sequence determination can also use commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones before splicing into a full-length cDNA sequence.

The present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector or HC6 protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention through recombinant technology.

Through conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant HC6 protein polypeptide (Science, 1984; 224:1431). Generally speaking, there are the following steps:

(1). Use the polynucleotide (or variant) encoding the tumor-associated human HC6 protein of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

In the present invention, the tumor-associated human HC6 protein polynucleotide sequence can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Vectors applicable in the present invention include, but are not limited to: T7-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem.263:3521, 1988) and vectors derived from baculovirus expressed in insect cells. In short, 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 an origin of replication, a promoter, marker genes, and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing HC6 protein coding DNA sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage _PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retroviruses and other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, 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 for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may 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. Representative examples are: Escherichia coli, Streptomyces spp; 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, etc.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. An alternative is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be encapsulated inside the cell, outside the cell or expressed on the cell membrane or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, 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 research of the present inventors has shown that in cancerous liver cells, there are almost all mutations in the HC6 gene, and these mutations include deletions, insertions and substitutions, and rearrangements of HC6 coding sequences and non-coding sequences. These changes may lead to inactive or underactive HC6 protein in liver cells, which can lead directly or indirectly to liver cancer.

Therefore, the recombinant human tumor-associated HC6 protein or polypeptide of the present invention has multiple uses. These uses include (but are not limited to): direct use as drugs to treat diseases caused by the hypofunction or loss of HC6 protein (such as cancer, especially liver cancer), and for screening antibodies, polypeptides or other compounds that promote or resist the function of HC6 protein body. For example, antibodies can be used to activate or inhibit the function of the HC6 protein. Screening the polypeptide library with the expressed recombinant HC6 protein can be used to find therapeutically valuable polypeptide molecules that can inhibit or stimulate the function of the HC6 protein.

The invention also provides methods of screening drugs to identify agents that increase (agonists) or repress (antagonists) HC6 protein. For example, mammalian cells or membrane preparations expressing HC6 protein can be incubated with labeled HC6 protein in the presence of the drug. The ability of the drug to enhance or repress this interaction is then determined.

Antagonists of HC6 protein include screened antibodies, compounds, receptor deletions and analogs. The antagonist of HC6 protein can combine with HC6 protein and eliminate its function, or inhibit the production of HC6 protein, or combine with the active site of the polypeptide so that the polypeptide cannot perform biological functions. Antagonists of the HC6 protein are useful for therapeutic use.

In screening compounds for antagonists, the HC6 protein can be added to a bioanalytical assay to determine whether the compound is an antagonist by determining the compound's effect on the interaction between the HC6 protein and its receptor. Receptor deletions and analogs that function as antagonists can be screened in the same manner as described above for screening compounds.

The polypeptide of the present invention can be directly used in the treatment of diseases, for example, various malignant tumors and abnormal proliferation of cells, etc., especially for the treatment of liver cancer.

The polypeptides of the present invention, and fragments, derivatives, analogs thereof or their cells can be used as antigens to produce antibodies. These antibodies can be polyclonal or monoclonal. Polyclonal antibodies can be obtained by injecting the polypeptide directly into animals. Techniques for preparing monoclonal antibodies include hybridoma technology, trioma technology, human B-cell hybridoma technology, EBV-hybridoma technology, and the like.

The polypeptides and antagonists of the present invention can be used in combination with suitable pharmaceutical carriers. These carriers can be water, dextrose, ethanol, salts, buffers, glycerol and combinations thereof. The composition contains safe and effective doses of polypeptides or antagonists as well as carriers and excipients that do not affect the drug effect. These compositions can be used as medicine for disease treatment.

The invention also provides kits or kits comprising one or more containers containing one or more ingredients of the pharmaceutical compositions of the invention. Along with these containers, there may be an indicative notice given by the governmental regulatory agency that manufactures, uses or sells the drug or biological product reflecting its approval for human administration by the governmental regulatory agency that manufactures, uses or sells the drug or biological product. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes of administration. HC6 protein is administered in an amount effective to treat and/or prevent the particular indication. The amount and dosage range of HC6 protein administered to a patient will depend on many factors, such as the mode of administration, the health condition of the person to be treated, and the judgment of the diagnosing physician.

Polynucleotides of HC6 protein can also be used for various therapeutic purposes. Gene therapy technology can be used to treat cell proliferation, development or metabolic abnormalities caused by non-expression of HC6 protein or expression of abnormal/inactive HC6 protein. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated HC6 protein to inhibit the activity of endogenous HC6 protein. For example, a mutated HC6 protein may be a shortened HC6 protein lacking a signal transduction domain, although it can bind to a downstream substrate, it lacks signal transduction activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of HC6 protein. Expression vectors derived from viruses such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the HC6 protein gene into cells. The method for constructing a recombinant viral vector carrying the HC6 protein gene can be found in existing literature (Sambrook, et al.). In addition, the recombinant HC6 protein gene can be packaged into liposomes and transferred into cells.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit HC6 protein mRNA are also within the scope of the invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA to perform an endonucleic cut. Antisense RNA, DNA and ribozyme can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoamide chemical synthesis of oligonucleotides, which has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DNA sequence encoding the RNA. This DNA sequence has been integrated into the vector downstream of the RNA polymerase promoter. In order to increase the stability of nucleic acid molecules, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the connection between ribonucleosides should use phosphothioester bonds or peptide bonds instead of phosphodiester bonds.

The methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in the body; or first introducing polynucleotides into cells in vitro through vectors (such as viruses, phages, or plasmids, etc.) , and then transplant the cells into the body, etc.

The polypeptide of the present invention can also be used for peptide spectrum analysis, for example, the polypeptide can be specifically cleaved physically, chemically or enzymatically, and subjected to one-dimensional, two-dimensional or three-dimensional gel electrophoresis analysis.

The invention also provides the antibody against the epitope of HC6 protein. These antibodies include, but are not limited to: polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments and fragments produced by a Fab expression library.

Antibodies against HC6 protein can be used in immunohistochemical techniques to detect HC6 protein in biopsy specimens.

The monoclonal antibody combined with HC6 protein can also be labeled with radioactive isotopes, and its location and distribution can be tracked when injected into the body. This radiolabeled antibody can be used as a non-invasive diagnostic method for localization of tumor cells and judgment of metastasis.

The antibody of the present invention can be used to treat or prevent diseases related to HC6 protein. Administration of appropriate doses of antibodies can stimulate or block the production or activity of the HC6 protein.

Antibodies can also be used to design immunotoxins that target a particular site in the body. For example, monoclonal antibodies with high affinity to HC6 protein can be covalently bonded to bacterial or plant toxins (such as diphtheria toxin, ricin, rhododine, etc.). A common method is to use a sulfhydryl cross-linking agent such as SPDP to attack the amino group of the antibody, and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill HC6 protein-positive cells.

For the production of polyclonal antibodies, HC6 protein or polypeptide can be used to immunize animals, such as rabbits, mice, rats, etc. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

HC6 protein monoclonal antibody can be produced by hybridoma technology (Kohler and Milstein. Nature, 1975, 256:495-497). Chimeric antibodies combining human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81:6851). The existing technology for producing single-chain antibodies (U.S. Pat No. 4946778) can also be used to produce single-chain antibodies against HC6 protein.

Polypeptide molecules that can bind to HC6 protein can be obtained by screening a random polypeptide library composed of various possible combinations of amino acids bound to solid phases. During screening, the HC6 protein molecule must be labeled.

The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of HC6 protein. These assays are well known in the art and include FISH assays and radioimmunoassays. The level of HC6 protein detected in the test can be used to explain the importance of HC6 protein in various diseases and to diagnose diseases in which HC6 protein plays a role.

The polynucleotide of HC6 protein can be used for the diagnosis and treatment of HC6 protein-related diseases (especially liver cancer). In terms of diagnosis, the polynucleotide of HC6 protein can be used to detect the expression of HC6 protein, or detect the abnormal expression of HC6 protein in a disease state. The HC6 protein DNA sequence can be used for hybridization of biopsy specimens to determine the abnormal expression of HC6 protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be used as probes to be immobilized on microarrays (Microarray) or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. RNA-polymerase chain reaction (RT-PCR) in vitro amplification with HC6 protein-specific primers can also detect HC6 protein transcripts.

Detection of mutations in the HC6 protein gene can also be used to diagnose HC6 protein-related diseases (especially liver cancer). The form of HC6 protein mutation includes point mutation, translocation, deletion, recombination and any other abnormality compared with the normal wild-type HC6 protein DNA sequence (such as the normal sequence shown in SEQ ID NO: 1). Mutations can be detected using established 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 has a mutation.

The HC6 protein nucleotide full-length sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragments, or its derivatives) can be completely chemically synthesized. This DNA sequence can then be introduced into various DNA molecules (such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In addition, since the HC6 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 with homologous proteins derived from other species ( For example less or no immunogenicity in humans).

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., Molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions specified by the manufacturer. suggested conditions.

Example 1. Obtaining of the original expression sequence of HC6

The sequence of the P579 clone corresponding to the highest LOH site D17S926 in the 17p13.3 region was obtained by nine-fold shotgun sequencing, and one of them represented a new gene EST6. PCR experiments showed that HC6 was expressed in normal liver tissue.

Determination of the minimum range (0.5-0.6Mb) of LOH common deletion in the 1.17p13.3 region

Using the sequence of the YNZ22.2 polymorphic site in the 17p13.3 segment as a probe, it was found that more than 30% of liver cancers had LOH. Later, 14 polymorphic site markers in the 17p13.3 region were selected, and the loss of heterozygosity (LOH) in 22 hepatocellular carcinoma samples was studied in detail by PCR method, and the location of LOH in the 17p13.3 region of liver cancer was determined. The smallest range of common deletions (0.5-0.6Mb), the deletion frequency is as high as 60-100%.

2. Obtaining the original sequence of HC6

The insert fragment (100 kb) of human DNA corresponding to the PAC579 (P579) clone (provided by Genome System Company) at position 926 in 17p13.3 has been sequenced by nine-fold shotgun sequencing (completed in Kikon format). Bioinformatics analysis showed that it contained an expressed sequence (EST) representing a new gene, numbered EST6.

3. HC6 is expressed in normal liver tissue

According to EST6 representing a new gene in the PAC579 clone, the following primers were designed for PCR amplification of the normal human liver cDNA library, and it was found that EST6 was expressed in the normal liver cDNA library.

EST6 primers: upstream primer 5′-TTCCAAATATGCACAACCGA-3′ downstream primer 5′-CATAGGTCTGACAGAGAAAG-3′ Fragment length (bp) 220 Annealing temperature (℃) 60

Example 2: Northern hybridization of multiple tissue membranes of HC6

Human multi-tissue Northern hybridization membrane (MTN) was purchased from Clontech, and pre-hybridized at 42°C for 3-4 hours (the pre-hybridization solution contained 50% formamide, 0.05M Tris, 1% SDS, 5'Denhardt's 0.1% coke sodium phosphate, 100mg/ml denatured sperm DNA). The EST6 clone was digested with NotI and HindIII, and the insert fragment was recovered and quantified by electrophoresis. Take 25ng DNA, add 2.5μl random primers and appropriate amount of water to make the total volume reach 13.5μl. Boil for 5 minutes, centrifuge to shake the liquid to the bottom of the tube, add 2.5 μl reaction buffer, 1 μl each of dATP, dTTP, and dGTP, 1 μl Klenow enzyme, and 5 ml ³² Pa-dCTP. Mix by flicking and centrifuge briefly. After incubation at 37°C for 20 minutes, 2 μl of 0.5M EDTA was added to terminate the reaction. Insert glass wool into a 1ml syringe, add TE-saturated Sephadex G-50. 2000rpm for 5 minutes. Repeat once, add G-50 to near the mark of 1ml. Equilibrate three times with 100 μl TE. Add 75 μl TE to the labeling reaction, put it on the column, and recover by centrifugation. Probes were denatured at 100°C for 5 minutes and placed on ice. Then add to the pre-hybridization solution and hybridize at 42°C for 12-16 hours. Take the membrane and wash it twice with 1'SSC-0.05% SDS solution at 42°C for 30 minutes each time, then wash it twice with 0.1'SSC-0.1% SDS at 42°C for 30 minutes each time, and finally the X-ray film is self-developed.

The result is shown in Figure 5. The original length of HC6 is only about 700 bases, and Northern hybridization of multiple tissue membranes shows that the full length of the gene is about 1.8kb. The HC6 gene is expressed in the heart, placenta, liver, kidney, pancreas, skeletal muscle and lung, but not in the brain.

Example 3: Southern hybridization of HC6

10 μg of DNA from human liver cancer and paracancerous tissue was thoroughly digested with BamH I endonuclease, and then made into a Southern membrane according to the method in the book "Molecular Cloning". .

The results are shown in Figures 1, 2 and Table 1. HC6 has two bands in Z11, Q27, Q29 paracancerous tissue (L), but only one band in liver cancer tissue (K), indicating a deletion. The band positions of Q21 and Q28 in liver cancer tissue (K) and paracancerous tissue (L) are different, indicating that there is DNA rearrangement in liver cancer tissue.

Table 1 Loss of heterozygosity (LOH) or DNA rearrangement of HC6 gene in liver cancer tissues cDNA clone Specimen (pair) LOH in HCC Liver cancer DNA rearrangement No difference between cancer and paracancer HC6 11 6(Z ₁₂ Z ₂₁ Q ₂₄ Q ₂₇ Q ₂₉ Q ₃₅ ) 2(Q ₁₂ Q ₂₁ ) 3

*Note: Z ₁₂ , Z ₂₁ , etc. are the serial numbers of samples.

In view of the high frequency of loss of heterozygosity and rearrangement of HC6 in liver cancer tissues (77%), it was renamed as tumor-related (liver cancer-related) novel gene 6 (HC6).

Embodiment 4: HC6 full-length cDNA clone

(1) Main reagents used: cDNA pool (Marathon-Ready cDNAs, Clontech), polymerase system (Advantage cDNA polymerase Mix, Clontech) TA cloning system (TOPO TA cloning).

(2) Primers: Gene-specific primers for RACE (Rapid amplification of cDNA ends) reactions should meet the following conditions: (a) length 23-28nt; (b) GC content 50-70%; (c) Tm value greater than 65 ℃.

Primer for HC6: 3′ RACE: HC6-1 CAACCGAAGAGAGTACGACAGAGCCTT

Nest type: HC6-N1 CCGAAGAGAGTACGACAGAGCCTT

              5′ RACE: HC6-2 TGGACACGCTGAGTCTGATATGTGGT

Nest: HC6-N2 AACATCGGTCTGGACAGAGAAAGTGC

(3) RACE reaction: The PCR amplification reaction can be carried out in a reaction volume of 12.5 μl or 25 μl, and the RACE reaction is set according to the following conditions: Total volume 12.μl Marathon-Ready cDNAs 1.25μl adapter primer 0.25μl 10mM dNTPs 0.25μl 10× PCR reaction buffer 1.25μl 50×Polymerase Mix 0.25μl H ₂ O 9.0μl Gene-specific primers (10pmol/μl) 0.25μl

PCR reaction conditions:

                                                                                                         

                                                                                                                                                                                    ,

                                                                             

                                                                                                                                                                                    ,

                                                      4 minutes

                                                                               

                                                                                     

Subcloning of RACE products: Take 0.5-2.5 μl of the recovered PCR product, add 0.5 μl of PCR-TOPO carrier, mix well and place on ice at room temperature for 5 minutes, then carry out bacterial transformation according to the conventional method, and grow on the plate at 37°C for 12-16 hours , blue, white spot screening.

(4) Screening and identification of RACE products:

In a 96-well plate, add 30 μl of LB containing Amp resistance to each well. For each RACE reaction, pick 10-20 white spot recombinants to the LB of the above-mentioned 96-well plate, use the bacterial solution as a template, and directly carry out PCR reaction, preliminary screening out candidate positive RACE clones. A small amount of liquid amplification was performed on the candidate positive clones, plasmid DNA was extracted, endonuclease digestion, electrophoresis analysis, large fragment RACE clones were screened out, and then PCR identification was performed.

(5) Sequencing and sequence analysis of RACE products:

Sequence the candidate large-fragment positive clones, and determine whether the full sequence of the gene has been obtained based on the length of the RACE product and the size of the mRNA of the gene. There is a termination codon within the same reading frame. There is a polyA sequence at the 3' end of the reading frame. It also contains the corresponding 5' and 3' non-coding regions. The HC6 sequence and corresponding coding frame were obtained by RACE method, and the results are shown in SEQ ID NO: 1-3.

Example 5: Sequence analysis of HC6

(1) Nucleotide sequence of HC6: (SEQ ID NO: 1) length: 1771bp

TCAGCTCAGA CAGGTGGTTG TTGTTGATGA GGCATAATTT AGGTAGAATT TAAATGTGTT 60

TTGACGGTTG TGTTTACTCA TACAACCACT ACTCAGAATA CAGACCATTT TCCCAGGAAG 120

TTCCCTCATG CCCATCTCCA GTGAGTTGGC ACCTTCATCT CCCAGATCAG CTGCTATTCT 180

GACTTCTGTC ACTGTAGGTG AGTTCGTTCT TGGACTTCAT ACAAATGGAG TCATTCAGTA 240

TTTTCTTTCC TTTTGTGCCT GGCTGCTTTT ACTCACCATG GCTTTTTTTG TTTTTTGTTT 300

TTTGTTTTTT TTGAGACGGA GTCTCGCTCT GTCACCCAGG CTGGAGTGCA GTGGTACAAT 360

CTCTGCTCAC TGCAAGCTCC GCCTCCCGGG TTCACGCCAT TCTCCTGCCT CAGCCTACCG 420

AGTAGCTGGG ACTACAGGCA CCTGCCACCT CGCCCGGCTA ATTTTTTGTA TTTTTAGTAG 480

AGACGGGTTT CACCGTGTTA GCCAGGAAGG TCTCGATCTC CTGACCTTGT GATCCACCCA 540

CCTTGGCCTC CCAAAGGGCT GGGATTACAG GTGTGAGCCA CCCCGCCCGG CCTACTCAAC 600

ATGGTTTTGA GATTCCACCA TGTAATTGCA TATCACAGCT CTTTTTTTTT AATTGCTGAC 660

TGATACTCAA TTTTATGAAT ATAACAATTT GCTCATTCAT TCTTATAATG GTGGACATTT 720

GGTTTATTTC TAGATTTTTT AATTCTATTAT AAGGAAGACT GCCATGAACA TTCTAGTGTA 780

AATCTTCTTG TAGATAGATG TTTCATTTCT CTTGAGTAAA TACCTAGGAG TAGAACTGCT 840

TTTACTGTAT AAGAAAGTGT CAAACAGTCA TCGAAAGTGA TCGGACCATC GTTTGTCCAC 900

GAGAGGTCCA GTAGGTCTAC ATTCTCACCA ACACTTATTT TGCTTTTTGT GGGTTTTTTA 960

GCTATTCTAG CACATGCGAA ATGGTTTCAT TGGTGCAATC AAGCGATCCT TCTGCCTCAG 1020

CCACCACATA GCTAGGGCTG CAGCACTATG CCCAGCTGAT GTTTTTTATG TTTGTAGAGA 1080

CAGATTCTCA CTGTGTTGCC CAGGCTGGCC TCCAGTGATC CTTCCAGCTC GGCCCCACAG 1140

AGGTTTAATT TTTATTTCCC TGATGACTAA TGATATTGAG CACCTTCTCA TGTGCTTATT 1200

AGCCATTTAC TTATTTTTGT GAATTTTATG TTCAAGTCTT TTGTCCATTC TTTAATAATT 1260

TTTAATATTT CATGATGAAA ATTCCAAATA TGCACAACCG AAGAGAGTAC GACAGAGCCT 1320

TACGTGACTC TTACCTTGAC TCTGTACTGT AGTTTTTCAT CATTCCTTCG TCACTTGCAA 1380

ATTCAAGTAT TTTTCCCCAGC CATTAATTGA GTTTTTGAAG ATGAGGAATT TTTTATTTTG 1440

GTGAAGTCCA GCTTATGAAC TTTTTCTCTT ACATGTAGCA CTTTCTCTGT CCAGACCGAT 1500

GTTATATCCT GAGAACCTCT TCACCTAGGA AACCTACCAC ATATCAGACT CAGCGTGTCC 1560

ATAACTTATC TTACCTCAAA TCCATTCTTC TTCCAGTAAT ATTACATAAA GGCTACTTGG 1620

GAGTGTAGAT TCTGGGAAAC CTGAATCTAT GTTCCCGGGC TGCAGTCTTC AAGCTTGGCC 1680

CAGATGAACT CTCTATTTAT ACTAAAAATA TGTAAATAAA TAAAAATAAA AAGAAATAAA 1740

TGCTACTATA TCGAAAAAAA AAAAAAAAAA A 1771

(2) Amino acid sequence of HC6 (SEQ ID NO: 2): 134 amino acids

MPISSELAPS SPRSAAILTS VTVGEFVLGL HTNGVIQYFL SFCAWLLLLT 50

MAFFVFCFLF FLRRSLALSP RLECSGTISA HCKLRLPGSR HSPASAYRVA 100

GTTGTCHLAR LIFCIFSRDG FHRVSQEGLD LLTL* 134

(3) Merged sequence of HC6 (SEQ ID NO: 3)

Start codon: 128ATG; Stop codon: 532TGA

Protein molecular weight: 14.7KD Protein length: 134 amino acids (excluding stop codons)

1 T CAG CTC AGA CAG GTG GTT GTT GTT GAT GAG GCA TAA TTT AGG TAG 46

47 AAT TTA AAT GTG TTT TGA CGG TTG TGT TTA CTC ATA CAA CCA CTA CTC 94

95 AGA ATA CAG ACC ATT TTC CCA GGA AGT TCC CTC ATG CCC ATC TCC AGT 142

1 M P I S S S 5

143 GAG TTG GCA CCT TCA TCT CCC AGA TCA GCT GCT ATT CTG ACT TCT GTC 190

6 E L A P P S S P R S A A I L T S V 21

191 ACT GTA GGT GAG TTC GTT CTT GGA CTT CAT ACA AAT GGA GTC ATT CAG 238

22 T V G E F V V L G L H T N G V I Q 37

239 TAT TTT CTT TCC TTT TGT GCC TGG CTG CTT TTA CTC ACC ATG GCT TTT 286

38 Y F L S F C A W L L L L T M A F 53

287 TTT GTT TTT TGT TTT TTG TTT TTT TTG AGA CGG AGT CTC GCT CTG TCA 334

54 F V F F C F L F F L R R R S L A L S 69

335 CCC AGG CTG GAG TGC AGT GGT ACA ATC TCT GCT CAC TGC AAG CTC CGC 382

70 P R L E C S G T I S A H C K L R 85

383 CTC CCG GGT TCA CGC CAT TCT CCT GCC TCA GCC TAC CGA GTA GCT GGG 430

86 L P G S R H S P A S A Y R V A G 101

431 ACT ACA GGC ACC TGC CAC CTC GCC CGG CTA ATT TTT TGT ATT TTT AGT 478

102 T T T G T C H H L A R L I F C I F S 117

479 AGA GAC GGG TTT CAC CGT GTT AGC CAG GAA GGT CTC GAT CTC CTG ACC 526

118 R D G F H R V S Q E G L D L L T 133

527 TTG TGA TCC ACC CAC CTT GGC CTC CCA AAG GGC TGG GAT TAC AGG TGT 574

134 L * 134

575 GAG CCA CCC CGC CCG GCC TAC TCA ACA TGG TTT TGA GAT TCC ACC ATG 622

623 TAA TTG CAT ATC ACA GCT CTT TTT TTT TAA TTG CTG ACT GAT ACT CAA 670

671 TTT TAT GAA TAT AAC AAT TTG CTC ATT CAT TCT TAT AAT GGT GGA CAT 718

719 TTG GTT TAT TTC TAG ATT TTT TAA TCT ATT ATA AGG AAG ACT GCC ATG 766

767 AAC ATT CTA GTG TAA ATC TTC TTG TAG ATA GAT GTT TCA TTT CTC TTG 814

815 AGT AAA TAC CTA GGA GTA GAA CTG CTT TTA CTG TAT AAG AAA GTG TCA 862

863 AAC AGT CAT CGA AAG TGA TCG GAC CAT CGT TTG TCC ACG AGA GGT CCA 910

911 GTA GGT CTA CAT TCT CAC CAA CAC TTA TTT TGC TTT TTG TGG GTT TTT 958

959 TAG CTA TTC TAG CAC ATG CGA AAT GGT TTC ATT GGT GCA ATC AAG CGA 1006

1007 TCC TTC TGC CTC AGC CAC CAC ATA GCT AGG GCT GCA GCA CTA TGC CCA 1054

1055 GCT GAT GTT TTT TAT GTT TGT AGA GAC AGA TTC TCA CTG TGT TGC CCA 1102

1103 GGC TGG CCT CCA GTG ATC CTT CCA GCT CGG CCC CAC AGA GGT TTA ATT 1150

1151 TTT ATT TCC CTG ATG ACT AAT GAT ATT GAG CAC CTT CTC ATG TGC TTA 1198

1199 TTA GCC ATT TAC TTA TTT TTG TGA ATT TTA TGT TCA AGT CTT TTG TCC 1246

1247 ATT CTT TAA TAA TTT TTA ATA TTT CAT GAT GAA AAT TCC AAA TAT GCA 1294

1295 CAA CCG AAG AGA GTA CGA CAG AGC CTT ACG TGA CTC TTA CCT TGA CTC 1342

1343 TGT ACT GTA GTT TTT CAT CAT TCC TTC GTC ACT TGC AAA TTC AAG TAT 1390

1391 TTT TCC CAG CCA TTA ATT GAG TTT TTG AAG ATG AGG AAT TTT TTA TTT 1438

1439 TGG TGA AGT CCA GCT TAT GAA CTT TTT CTC TTA CAT GTA GCA CTT TCT 1486

1487 CTG TCC AGA CCG ATG TTA TAT CCT GAG AAC CTC TTC ACC TAG GAA ACC 1534

1535 TAC CAC ATA TCA GAC TCA GCG TGT CCA TAA CTT ATC TTA CCT CAA ATC 1582

1583 CAT TCT TCT TCC AGT AAT ATT ACA TAA AGG CTA CTT GGG AGT GTA GAT 1630

1631 TCT GGG AAA CCT GAA TCT ATG TTC CCG GGC TGC AGT CTT CAA GCT TGG 1678

1679 CCC AGA TGA ACT CTC TAT TTA TAC TAA AAA TAT GTA AAT AAA TAA AAA 1726

1727 TAA AAA GAA ATA AAT GCT ACT ATA TCG AAA AAA AAA AAA AAA AAA 1771

(4) Homologous comparison

Homology comparison results showed that 17 of the 44 (4-47) amino acids at the N-terminal of the HC6 protein were identical to yeast trehalose phospholipase (38%).

Example 6: The in vitro test of introducing the cDNA of HC6 gene into liver cancer cells

In this example, the liposome kit was used to conduct the in vitro experiment of transfecting liver cancer cells.

(1) Cell line: primary hepatocellular carcinoma cell line 7721.

(2) DNA: DNA derived from expression plasmid pCMV/script-HC6.

(3) Liposome: LIPOFECT AMINE ^™ Reagent Kit (BRL Company)

(4) Culture medium: serum-free culture medium, referred to as SF-DMEM

     Full Culture Solution (10% Calf Serum)

    Full culture medium containing G418

  T25 culture flask.

(5) Preparation of DNA-liposome complex (DNA-liposome complex):

Add 50 μl of lipofectin and 50 μl of SF-DMEM and mix well. DNA (20μg in 20μl TE) was mixed with 80μl SF-DMEM. Add the diluted DNA to the diluted lipofectin solution, mix well and leave at room temperature for 5-10 minutes. Add 1.3ml SF-DMEM into the DNA-lipofectin complex for a final volume of 1.5ml.

(6) Transfected cells: It is better for the cells to grow to 80% fullness, and the culture medium should be changed once before the experiment. Add 1.5ml lipofectin reagent-DNA complex to the cell surface, shake gently, spread evenly, and incubate at 37°C for 1-3 hours. Add 1.5ml SF-DMEM, mix well, and grow overnight at 37°C. Change the culture medium overnight at 37°C, and when the cells grow to 70% fullness, change the culture medium containing G418 and then change the medium routinely until clones appear, and count the number of clones.

The results are shown in Figure 3-4 and Table 2. In Figure 3, the empty expression vector (pCMV/script) transfected 7721 cells had more clone formation, while in Figure 4, the expression vector containing HC6 gene insert fragment transfected 7721 cells had a few clone formation. This indicates that HC6 has an inhibitory effect on the growth of liver cancer cells in vitro.

The results are as follows:

Table 2 Inhibitory effect of HC6 on the growth of liver cancer cells in vitro cDNA clone empty vector Pcmv/script positive control p53 HC6 Colony formation number (average number) 33 0 5

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing

(1) General information:

(ii) Name of the invention: human tumor-related genes and encoded proteins in the region 3, band 3, and subband 3, short arm of human chromosome 17

(iii) Number of sequences: 3

(2) Information on SEQ ID NO: 1

(i) Sequential features

(A) Length: 1771 bases

(B) type: nucleic acid

(C) chain: double chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 1:

TCAGCTCAGA CAGGTGGTTG TTGTTGATGA GGCATAATTT AGGTAGAATT TAAATGTGTT 60

TTGACGGTTG TGTTTACTCA TACAACCACT ACTCAGAATA CAGACCATTT TCCCAGGAAG 120

TTCCCTCATG CCCATCTCCA GTGAGTTGGC ACCTTCATCT CCCAGATCAG CTGCTATTCT 180

GACTTCTGTC ACTGTAGGTG AGTTCGTTCT TGGACTTCAT ACAAATGGAG TCATTCAGTA 240

TTTTCTTTCC TTTTGTGCCT GGCTGCTTTT ACTCACCATG GCTTTTTTTG TTTTTTGTTT 300

TTTGTTTTTT TTGAGACGGA GTCTCGCTCT GTCACCCAGG CTGGAGTGCA GTGGTACAAT 360

CTCTGCTCAC TGCAAGCTCC GCCTCCCGGG TTCACGCCAT TCTCCTGCCT CAGCCTACCG 420

AGTAGCTGGG ACTACAGGCA CCTGCCACCT CGCCCGGCTA ATTTTTTGTA TTTTTAGTAG 480

AGACGGGTTT CACCGTGTTA GCCAGGAAGG TCTCGATCTC CTGACCTTGT GATCCACCCA 540

CCTTGGCCTC CCAAAGGGCT GGGATTACAG GTGTGAGCCA CCCCGCCCGG CCTACTCAAC 600

ATGGTTTTGA GATTCCACCA TGTAATTGCA TATCACAGCT CTTTTTTTTT AATTGCTGAC 660

TGATACTCAA TTTTATGAAT ATAACAATTT GCTCATTCAT TCTTATAATG GTGGACATTT 720

GGTTTATTTC TAGATTTTTT AATTCTATTAT AAGGAAGACT GCCATGAACA TTCTAGTGTA 780

AATCTTCTTG TAGATAGATG TTTCATTTCT CTTGAGTAAA TACCTAGGAG TAGAACTGCT 840

TTTACTGTAT AAGAAAGTGT CAAACAGTCA TCGAAAGTGA TCGGACCATC GTTTGTCCAC 900

GAGAGGTCCA GTAGGTCTAC ATTCTCACCA ACACTTATTT TGCTTTTTGT GGGTTTTTTA 960

GCTATTCTAG CACATGCGAA ATGGTTTCAT TGGTGCAATC AAGCGATCCT TCTGCCTCAG 1020

CCACCACATA GCTAGGGCTG CAGCACTATG CCCAGCTGAT GTTTTTTATG TTTGTAGAGA 1080

CAGATTCTCA CTGTGTTGCC CAGGCTGGCC TCCAGTGATC CTTCCAGCTC GGCCCCACAG 1140

AGGTTTAATT TTTATTTCCC TGATGACTAA TGATATTGAG CACCTTCTCA TGTGCTTATT 1200

AGCCATTTAC TTATTTTTGT GAATTTTATG TTCAAGTCTT TTGTCCATTC TTTAATAATT 1260

TTTAATATTT CATGATGAAA ATTCCAAATA TGCACAACCG AAGAGAGTAC GACAGAGCCT 1320

TACGTGACTC TTACCTTGAC TCTGTACTGT AGTTTTTCAT CATTCCTTCG TCACTTGCAA 1380

ATTCAAGTAT TTTTCCCCAGC CATTAATTGA GTTTTTGAAG ATGAGGAATT TTTTATTTTG 1440

GTGAAGTCCA GCTTATGAAC TTTTTCTCTT ACATGTAGCA CTTTCTCTGT CCAGACCGAT 1500

GTTATATCCT GAGAACCTCT TCACCTAGGA AACCTACCAC ATATCAGACT CAGCGTGTCC 1560

ATAACTTATC TTACCTCAAA TCCATTCTTC TTCCAGTAAT ATTACATAAA GGCTACTTGG 1620

GAGTGTAGAT TCTGGGAAAC CTGAATCTAT GTTCCCGGGC TGCAGTCTTC AAGCTTGGCC 1680

CAGATGAACT CTCTATTTAT ACTAAAAATA TGTAAATAAA TAAAAATAAA AAGAAATAAA 1740

TGCTACTATA TCGAAAAAAA AAAAAAAAAA A 1771

(2) Information on SEQ ID NO: 2:

(i) Sequence features:

(A) Length: 134 amino acids

(B) type: amino acid

(D) Topology: linear

(ii) Molecular type: polypeptide

(xi) Sequence description: SEQ ID NO: 2:

MPISSELAPS SPRSAAILTS VTVGEFVLGL HTNGVIQYFL SFCAWLLLLT 50

MAFFVFCFLF FLRRSLALSP RLECSGTISA HCKLRLPGSR HSPASAYRVA 100

GTTGTCHLAR LIFCIFSRDG FHRVSQEGLD LLTL* 134

(2) Information on SEQ ID NO: 3

(i) Sequential features

(A) Length: 1771 bases

(B) type: nucleic acid

(C) chain: double chain

(D) Topology: Linear

(xi) Sequence description: SEQ ID NO: 3:

1 T CAG CTC AGA CAG GTG GTT GTT GTT GAT GAG GCA TAA TTT AGG TAG 46

47 AAT TTA AAT GTG TTT TGA CGG TTG TGT TTA CTC ATA CAA CCA CTA CTC 94

95 AGA ATA CAG ACC ATT TTC CCA GGA AGT TCC CTC ATG CCC ATC TCC AGT 142

1 M P I S S S 5

143 GAG TTG GCA CCT TCA TCT CCC AGA TCA GCT GCT ATT CTG ACT TCT GTC 190

6 E L A P P S S P R S A A I L T S V 21

191 ACT GTA GGT GAG TTC GTT CTT GGA CTT CAT ACA AAT GGA GTC ATT CAG 238

22 T V G E F V V L G L H T N G V I Q 37

239 TAT TTT CTT TCC TTT TGT GCC TGG CTG CTT TTA CTC ACC ATG GCT TTT 286

38 Y F L S F F C A W L L L L T M A F 53

287 TTT GTT TTT TGT TTT TTG TTT TTT TTG AGA CGG AGT CTC GCT CTG TCA 334

54 F V F F C F L F F L R R R S L A L S 69

335 CCC AGG CTG GAG TGC AGT GGT ACA ATC TCT GCT CAC TGC AAG CTC CGC 382

70 P R L E C S G T I S A H C K L R 85

383 CTC CCG GGT TCA CGC CAT TCT CCT GCC TCA GCC TAC CGA GTA GCT GGG 430

86 L P G S R H S P A S A Y R V A G 101

431 ACT ACA GGC ACC TGC CAC CTC GCC CGG CTA ATT TTT TGT ATT TTT AGT 478

102 T T T G T C H H L A R L I F C I F S 117

479 AGA GAC GGG TTT CAC CGT GTT AGC CAG GAA GGT CTC GAT CTC CTG ACC 526

118 R D G F H R V S Q E G L D L L T 133

527 TTG TGA TCC ACC CAC CTT GGC CTC CCA AAG GGC TGG GAT TAC AGG TGT 574

134 L * 134

575 GAG CCA CCC CGC CCG GCC TAC TCA ACA TGG TTT TGA GAT TCC ACC ATG 622

623 TAA TTG CAT ATC ACA GCT CTT TTT TTT TAA TTG CTG ACT GAT ACT CAA 670

671 TTT TAT GAA TAT AAC AAT TTG CTC ATT CAT TCT TAT AAT GGT GGA CAT 718

719 TTG GTT TAT TTC TAG ATT TTT TAA TCT ATT ATA AGG AAG ACT GCC ATG 766

767 AAC ATT CTA GTG TAA ATC TTC TTG TAG ATA GAT GTT TCA TTT CTC TTG 814

815 AGT AAA TAC CTA GGA GTA GAA CTG CTT TTA CTG TAT AAG AAA GTG TCA 862

863 AAC AGT CAT CGA AAG TGA TCG GAC CAT CGT TTG TCC ACG AGA GGT CCA 910

911 GTA GGT CTA CAT TCT CAC CAA CAC TTA TTT TGC TTT TTG TGG GTT TTT 958

959 TAG CTA TTC TAG CAC ATG CGA AAT GGT TTC ATT GGT GCA ATC AAG CGA 1006

1007 TCC TTC TGC CTC AGC CAC CAC ATA GCT AGG GCT GCA GCA CTA TGC CCA 1054

1055 GCT GAT GTT TTT TAT GTT TGT AGA GAC AGA TTC TCA CTG TGT TGC CCA 1102

1103 GGC TGG CCT CCA GTG ATC CTT CCA GCT CGG CCC CAC AGA GGT TTA ATT 1150

1151 TTT ATT TCC CTG ATG ACT AAT GAT ATT GAG CAC CTT CTC ATG TGC TTA 1198

1199 TTA GCC ATT TAC TTA TTT TTG TGA ATT TTA TGT TCA AGT CTT TTG TCC 1246

1247 ATT CTT TAA TAA TTT TTA ATA TTT CAT GAT GAA AAT TCC AAA TAT GCA 1294

1295 CAA CCG AAG AGA GTA CGA CAG AGC CTT ACG TGA CTC TTA CCT TGA CTC 1342

1343 TGT ACT GTA GTT TTT CAT CAT TCC TTC GTC ACT TGC AAA TTC AAG TAT 1390

1391 TTT TCC CAG CCA TTA ATT GAG TTT TTG AAG ATG AGG AAT TTT TTA TTT 1438

1439 TGG TGA AGT CCA GCT TAT GAA CTT TTT CTC TTA CAT GTA GCA CTT TCT 1486

1487 CTG TCC AGA CCG ATG TTA TAT CCT GAG AAC CTC TTC ACC TAG GAA ACC 1534

1535 TAC CAC ATA TCA GAC TCA GCG TGT CCA TAA CTT ATC TTA CCT CAA ATC 1582

1583 CAT TCT TCT TCC AGT AAT ATT ACA TAA AGG CTA CTT GGG AGT GTA GAT 1630

1631 TCT GGG AAA CCT GAA TCT ATG TTC CCG GGC TGC AGT CTT CAA GCT TGG 1678

1679 CCC AGA TGA ACT CTC TAT TTA TAC TAA AAA TAT GTA AAT AAA TAA AAA 1726

1727 TAA AAA GAA ATA AAT GCT ACT ATA TCG AAA AAA AAA AAA AAA AAA 1771

Claims (11)

1. An isolated human HC6 protein polypeptide is characterized in that it contains the polypeptide of the amino acid sequence shown in SEQ ID NO: 2. 2. The polypeptide according to claim 1, wherein the polypeptide is a polypeptide having an amino acid sequence shown in SEQ ID NO:2. 3. An isolated polynucleotide, characterized in that it comprises a nucleotide sequence selected from the group consisting of: (a) polynucleotide encoding polypeptide as claimed in claim 1; (b) A polynucleotide complementary to polynucleotide (a). 4. The polynucleotide according to claim 3, wherein the polypeptide encoded by the polynucleotide has the amino acid sequence shown in SEQ ID NO: 2, or the polynucleotide has a sequence selected from the group consisting of: SEQ ID NO: coding region sequence or full-length sequence shown in 3. 5. A vector comprising the polynucleotide according to claim 3. 6. A genetically engineered host cell, characterized in that it is a host cell selected from the group consisting of: (a) host cells transformed or transduced with the vector according to claim 5; (b) A host cell transformed or transduced with the polynucleotide of claim 3. 7. A method for preparing a polypeptide having human protein HC6 activity, characterized in that the method comprises: (a) cultivating the host cell according to claim 6 under conditions suitable for expressing the protein; (b) Isolating a polypeptide having human protein HC6 activity from the culture. 8. An antibody capable of specifically binding to the human HC6 protein according to claim 1. 9. A pharmaceutical composition, characterized in that it contains a safe and effective amount of the polypeptide according to claim 1 and a pharmaceutically acceptable carrier. 10. A method for in vitro detection of canceration or susceptibility to canceration in hepatocytes, characterized in that it comprises the steps of: Detect whether the HC6 transcript in the liver cell sample is changed compared with the normal HC6 transcript. If there is a change, it means that the liver cell is cancerous or has cancer susceptibility, wherein the normal human HC6 transcript code has SEQ ID NO : A polypeptide of the amino acid sequence shown in 2, the change is a nucleotide deletion, insertion, or substitution mutation, or a heterozygosity deletion or DNA rearrangement; or Detect whether the activity of the HC6 protein in the liver cell sample is changed compared with the normal HC6 protein. If there is a change, it means that the liver cell is cancerous or has cancer susceptibility, wherein the normal human HC6 protein has SEQ ID NO: 2 As shown in the amino acid sequence, the change is that the degree of inhibition of the primary hepatocellular carcinoma cell line 7721 is less than that of normal human HC6 protein. 11. A test kit for detecting liver cancer, characterized in that it comprises: (1) a pair of primers for specifically amplifying the human HC6 gene, (2) Reagents required for detecting whether there is a change in the amplified product compared with the normal HC6 gene, wherein the normal human HC6 transcript encodes a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, and the change It is a nucleotide deletion, insertion, or substitution mutation, or a heterozygous deletion or DNA rearrangement.

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