CN1329065A - Noven huamn protein with function of promoting growth of cancer cell and its code sequence - Google Patents

Abstract

The invention discloses a novel human protein with the function of promoting the growth of cancer cells, a polynucleotide encoding the polypeptide and a method for producing the polypeptide through recombinant technology. The invention also discloses a method for using the polypeptide to treat various diseases such as cancer and the like. The invention also discloses an antagonist against the polypeptide and its therapeutic effect. The invention also discloses the application of the polynucleotide encoding the novel human protein with the function of promoting the growth of cancer cells.

Description

Novel human protein with the function of promoting cancer cell growth and its coding sequence

The invention belongs to the field of biotechnology, in particular, the invention relates to a new polynucleotide encoding a human protein with the function of promoting the growth of cancer cells, and a polypeptide encoded by the polynucleotide. The present invention also relates to the use and preparation of such polynucleotides and polypeptides.

Human genomics research is currently a hot spot in the world. In addition to large-scale sequencing of human chromosomal DNA and expressed sequence sequencing (EST), there is still a lack of high-throughput methods for screening functional genes starting from function.

Cancer is one of the major diseases that endanger human health. In order to effectively treat and prevent tumors, people have paid more and more attention to gene therapy of tumors. Therefore, there is an urgent need in this field to develop and study human proteins and their agonists/inhibitors related to cancer cell growth.

The purpose of the present invention is to provide a new class of human protein polypeptides with the function of promoting the growth of cancer cells 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, there is provided a novel isolated protein polypeptide having the function of promoting cancer cell growth, which comprises a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 5; or Its conservative variant polypeptide, or its active fragment, or its active derivative.

Preferably, the polypeptide is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5.

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 above-mentioned protein polypeptide with the function of promoting cancer cell growth; (b) a polynucleotide complementary to the polynucleotide (a). Preferably, the polypeptide encoded by the polynucleotide has an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 5. More preferably, the sequence of the polynucleotide is selected from the following group: coding region sequence or full-length sequence of SEQ ID NO: 3, SEQ ID NO: 6.

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, there is provided a method for preparing a polypeptide having a function of promoting the growth of cancer cells, the method comprising: (a) under conditions suitable for expressing a protein having the function of promoting the growth of cancer cells, culturing the above A transformed or transduced host cell; (b) isolating from the culture a polypeptide having protein activity that promotes the growth of cancer cells.

In the fifth aspect of the present invention, an antibody that specifically binds to the above-mentioned protein polypeptide with the function of promoting cancer cell growth 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 protein polypeptide with the function of promoting the growth of cancer cells of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions can be used to promote the growth of cells. The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of antibodies against the protein polypeptide of the present invention that can promote the growth of cancer cells and a pharmaceutically acceptable carrier. The pharmaceutical composition can treat diseases such as cancer and abnormal cell proliferation.

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

The invention adopts large-scale cDNA clones to transfect cancer cells, and on the basis of obtaining the effect of promoting the growth of cancer cells, it is proved to be new genes by sequencing, and further obtains full-length cDNA clones. The DNA transfection test proves that the protein with the function of promoting the growth of cancer cells has the effect of promoting the formation of clones of cancer cells (liver cancer cells), and the promoting effect is 50% or more.

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 protein or polypeptide having the function of promoting cancer cell growth" means that the protein or polypeptide having the function of promoting cancer cell growth does not substantially contain other proteins, lipids, carbohydrates or other substances associated with it in nature. Those skilled in the art can use standard protein purification techniques to purify proteins that have the function of promoting cancer cell growth. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the protein polypeptide with the function of promoting the growth of cancer cells 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 the human protein having the function of promoting the growth of cancer cells. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially maintains the same biological function or activity of the natural human protein of the present invention that has the function of promoting cancer cell growth. 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.

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. Taking the PP3999 protein (in this application, the protein is named using its clone number) as an example, the coding region sequence encoding the mature polypeptide can be the same as the coding region sequence 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. Taking the PP4534 protein (in this application, the protein is named using its clone number) as an example, the coding region sequence encoding the mature polypeptide can be the same as the coding region sequence shown in SEQ ID NO: 6 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: 5, but differs from the sequence of the coding region shown in SEQ ID NO: 6. For other proteins that have the function of promoting the growth of cancer cells,

A polynucleotide encoding a mature polypeptide includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding sequences) and non-coding sequences.

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 that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% 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 (such as PCR) to identify and/or isolate polynucleotides encoding proteins that promote the growth of cancer cells.

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 production of specific DNA fragment sequences encoding proteins with the function of promoting cancer cell growth can also be obtained by the following methods: 1) isolation of double-stranded DNA sequences from genomic DNA; 2) chemical synthesis of DNA sequences to obtain double-stranded DNA of desired polypeptides.

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) marker gene function appearance or loss; (3) measuring the level of transcripts of proteins with the function of promoting cancer cell growth; (4) Detecting the protein product expressed by the gene by immunological techniques or measuring biological activity. 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 protein gene that has the function of promoting the growth of cancer cells.

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 comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or a protein coding sequence having the function of promoting the growth of cancer cells, and a recombinant technology to produce the polypeptide of the present invention method.

Through conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant protein polypeptides with the function of promoting cancer cell growth (Science, 1984; 224: 1431). Generally speaking, there are the following steps:

(1). Transform or transduce suitable host cells with the polynucleotide (or variant) encoding the human protein encoding the growth-promoting function of cancer cells of the present invention, or 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 human protein polynucleotide sequence with the function of promoting the growth of cancer cells 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 human protein-coding DNA sequences with the function of promoting cancer cell growth 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 recombinant human protein or polypeptide having the function of promoting the growth of cancer cells has many uses. These uses include (but are not limited to): direct use as a drug to treat diseases caused by the hypofunction or loss of protein functions that promote cancer cell growth, and for screening antibodies that promote or resist protein functions that promote cancer cell growth, peptides or other ligands. For example, the antibody can be used to treat cancer or abnormal cell proliferation. Screening the polypeptide library with the expressed recombinant human protein with the function of promoting the growth of cancer cells can be used to find therapeutically valuable polypeptide molecules that can inhibit or stimulate the function of the human protein with the function of promoting the growth of cancer cells.

The invention also provides methods of screening drugs to identify agents that increase (agonists) or repress (antagonists) human proteins that have the function of promoting the growth of cancer cells. Agonists enhance the biological functions of human proteins that promote cancer cell growth, such as stimulating cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or a membrane preparation expressing a human protein capable of promoting cancer cell growth can be cultured together with a labeled human protein capable of promoting cancer cell growth in the presence of a drug. The ability of the drug to enhance or repress this interaction is then determined.

Antagonists of human proteins capable of promoting the growth of cancer cells include screened antibodies, compounds, receptor deletions and analogs. The antagonist of the human protein with the function of promoting the growth of cancer cells can bind to the human protein with the function of promoting the growth of cancer cells and eliminate its function, or inhibit the production of the human protein with the function of promoting the growth of cancer cells, or interact with the activity of the polypeptide Site binding renders the polypeptide incapable of biological function. Antagonists of human proteins that function to promote the growth of cancer cells are useful for therapeutic use.

When screening compounds as antagonists, proteins with the function of promoting cancer cell growth can be added to bioanalytical assays to determine whether the compound affects the interaction between the protein with the function of promoting cancer cell growth and its receptor. is an antagonist. 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, abnormal cell proliferation and the like.

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. The protein with the function of promoting the growth of cancer cells or its specific antibody can be administered in an amount effective for treating and/or preventing specific indications. The amount and dosage range of the protein having the function of promoting the growth of cancer cells 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 human proteins that function to promote the growth of cancer cells may also be used for various therapeutic purposes. Gene therapy technology can be used to treat abnormalities in cell development or metabolism due to non-expression or abnormal/inactive expression of proteins with the function of promoting the growth of cancer cells. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated proteins that can promote the growth of cancer cells, so as to inhibit the activity of endogenous proteins that can promote the growth of cancer cells. For example, a mutated protein with the function of promoting the growth of cancer cells may be a shortened protein with the function of promoting the growth of cancer cells without a signal transduction domain. Although it can bind to downstream substrates, it lacks signal transduction activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of proteins with the function of promoting the growth of cancer cells. Expression vectors derived from viruses such as retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and parvoviruses can be used to transfer protein genes that can promote the growth of cancer cells into cells. The method for constructing a recombinant viral vector carrying a protein gene that promotes the growth of cancer cells can be found in existing literature (Sambrook, et al.). In addition, the recombinant human protein gene with the function of promoting the growth of cancer cells can be packaged into liposomes and transferred into cells.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit the mRNA of human proteins that function to promote cancer cell growth 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. Since the protein of the present invention has the function of promoting the growth of cancer cells, the antisense sequence of the coding sequence of the protein of the present invention can be introduced into cells to inhibit abnormal proliferation of cells (such as canceration).

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 present invention also provides an antibody against the epitope of the human protein with the function of promoting the growth of cancer cells. 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 human proteins that promote cancer cell growth can be used in immunohistochemical techniques to detect human proteins that promote cancer cell growth in biopsy samples.

Monoclonal antibodies that bind to human proteins that promote the growth of cancer cells can also be labeled with radioactive isotopes and injected into the body to track their location and distribution. 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 the human protein having the function of promoting the growth of cancer cells. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human proteins that can promote the growth of cancer cells, thereby inhibiting the growth of cancer cells and/or the abnormal proliferation of cells.

Antibodies can also be used to design immunotoxins that target a particular site in the body. For example, monoclonal antibodies with high affinity to human proteins that have the function of promoting the growth of cancer cells can be covalently bonded to bacteria 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 human proteins that have the function of promoting cancer cell growth. positive cells.

The production of polyclonal antibodies can be used to immunize animals, such as rabbits, mice, rats, etc., with human proteins or polypeptides that can promote the growth of cancer cells. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

Human protein monoclonal antibodies capable of promoting the growth of cancer cells 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 human proteins that have the function of promoting cancer cell growth.

The polypeptide molecule capable of binding to the human protein with the function of promoting the growth of cancer cells can be obtained by screening a random polypeptide library composed of various possible combinations of amino acids bound to solid phases. During screening, it is necessary to label human protein molecules that have the function of promoting the growth of cancer cells.

The present invention also relates to a diagnostic test method for quantitatively and locally detecting the human protein level with the function of promoting the growth of cancer cells. These assays are well known in the art and include FISH assays and radioimmunoassays. The level of human protein that can promote the growth of cancer cells detected in the test can be used to explain the importance of human proteins that can promote the growth of cancer cells in various diseases and to diagnose the role of proteins that can promote the growth of cancer cells. effect of the disease.

The polynucleotide of the protein with the function of promoting the growth of cancer cells can be used for the diagnosis and treatment of diseases related to the protein with the function of promoting the growth of cancer cells. In terms of diagnosis, the polynucleotide of the protein with the function of promoting the growth of cancer cells can be used to detect the expression of the protein with the function of promoting the growth of cancer cells or the abnormal expression of the protein with the function of promoting the growth of cancer cells in a disease state. For example, the DNA sequence of a protein with the function of promoting the growth of cancer cells can be used for hybridization of biopsy specimens to determine the abnormal expression of the protein with the function of promoting the growth of cancer cells. Hybridization techniques include Southern blotting, Northem blotting, in situ hybridization, etc. 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. The RNA-polymerase chain reaction (RT-PCR) in vitro amplification with the specific primers of the protein with the function of promoting the growth of cancer cells can also detect the transcription product of the protein with the function of promoting the growth of cancer cells.

Detecting the mutation of the protein gene with the function of promoting the growth of cancer cells can also be used to diagnose diseases related to the protein with the function of promoting the growth of cancer cells. The form of protein mutation with the function of promoting cancer cell growth includes point mutation, translocation, deletion, recombination and any other abnormality compared with the normal wild type protein DNA sequence with the function of promoting cancer cell growth. 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 sequences of the invention are also valuable for chromosome identification. The sequence will be specific for a particular location on a human chromosome and can hybridize thereto. Currently, there is a need to identify the specific site of each gene on the chromosome. Currently, only a few chromosomal markers based on actual sequence data (repeat polymorphisms) are available to mark chromosomal positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on chromosomes.

In short, PCR primers (preferably 15-35bp) are prepared according to the cDNA, and the sequence can be positioned on the chromosome. These primers were then used for PCR screening of somatic heterozygous cells containing individual human chromosomes. Only those cells heterozygous for the human gene corresponding to the primer will produce an amplified fragment.

The PCR mapping method of somatic heterozygous cells is a quick method to locate DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, sublocalization can be achieved using a set of fragments from a specific chromosome or a large number of genomic clones by a similar method. Other similar strategies that can be used for chromosome mapping include in situ hybridization, chromosome prescreening by flow sorting with markers, and hybridization preselection to construct chromosome-specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of cDNA clones to metaphase chromosomes allows precise chromosomal mapping in a single step. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to an exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with gene map data. These data can be found, for example, in V. Mckusick, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between the gene and the disease that has been mapped to the chromosomal region.

Next, the cDNA or genome sequence differences between affected and non-affected individuals need to be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping techniques, the cDNA that is pinpointed to a disease-associated chromosomal region can be one of 50 to 500 potential disease-causing genes (assuming 1 megabase mapping resolution capacity and each 20kb corresponds to a gene).

The protein nucleotide full-length sequence or its fragments with the function of promoting cancer cell growth of the present invention can usually be obtained by PCR amplification method, recombination method or artificial synthesis method. 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 protein having the function of promoting the growth of cancer cells of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and/or or lower side effects (eg, lower 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: Acquisition of cDNA gene and promotion of cancer cell clone formation

PP3999 and PP4534 were obtained by constructing human placenta cDNA library by conventional methods. Placenta tissues at the age of 3, 6, and 10 months were taken, and total RNA was extracted with Trizol reagent (GIBCO BRL Company) according to the manufacturer's instructions, and mRNA was extracted with an mRNA purification kit (Pharmacia Company). The cDNA library of the above mRNA was constructed using the pCMV-script TMXR cDNA library construction kit (Stratagene). The reverse transcriptase was changed to MMLV-RT-Superscript II (GIBCO BRL), and the reverse transcription reaction was carried out at 42°C. XL 10-Gold competent cells were transformed, and a cDNA library with a titer of 1×10 ⁶ cfu/μg was obtained. In the first round, cDNA clones were randomly selected, and then high-abundance cDNA clones and cDNA clones that had been proven to inhibit the growth of cancer cells were used as probes to hybridize and screen the cDNA library to pick weakly positive and negative clones. Plasmid DNA was extracted using Qiagen 96-well plate plasmid extraction kit according to the manufacturer's instructions. Plasmid DNA and empty vector were transfected into liver cancer cell line 7721 at the same time. After 100 ng of DNA was precipitated and dried by alcohol, 6 μl of H ₂ O was added to dissolve it and wait for transfection. Add 0.7 μl liposome and 9.3 μl serum-free medium to each DNA sample, mix well, and place at room temperature for 10 minutes. Add 150 μl of serum-free culture medium to each tube, add evenly to 7721 cells grown in 96-well plates in 3 wells, place at 37°C for 2 hours, add 50 μl of serum-free culture medium to each well, and keep at 37°C for 24 hours. Change 100 μl of whole culture solution in each well, 37°C for 24 hours, change 100 μl of whole culture solution containing G418, 37°C for 24-48 hours, observe and change the culture solution with different concentrations of G418. After about 2-3 times, until the microscopic examination of the cells has colony formation, count. It was found that the above two clones can promote the formation of cell clones, and the results are shown in the table below.

Cloning formation of cDNA clone transfected cells (7721) cDNA clone name Number of cDNA clones (three replicates) Number of empty vector clones (three replicates) PP3999 35 33 twenty three 16 20 18 PP4534 26 33 48 twenty three twenty one 14

The dideoxy termination method was used for the cDNA clone, and the nucleotide sequence of nearly 500 bp at one end was determined on an ABI377 DNA automatic sequencer. After analysis, it was determined to be a new gene clone, and the other end was sequenced, but the full-length cDNA sequence was not obtained, primers were designed, and sequencing was performed again until the full-length sequence (SEQ ID NO: 1, 4) was obtained.

Embodiment 2: PCR obtains full-length gene from placenta cDNA:

Placenta tissues at 3, 6, and 10 months old were taken, and total RNA was extracted with Trizol reagent (GIBCO BRL company) according to the manufacturer's instructions, and mRNA was extracted with mRNA purification kit (Pharmacia company). MMLV-RT-Superscript II (GIBCO BRL) reverse transcriptase was used to carry out reverse transcription reaction at 42°C to obtain placental cDNA. Using the transmutation primers of each gene (as shown in the table below), follow the cycle of 90°C for 3 minutes; 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, a total of 35 cycles; 72°C for 10 minutes, 1 cycle PCR amplification is carried out in cycles to obtain the amplification products of each protein gene containing the complete open reading frame sequence. The amplified product was verified by sequencing and was consistent with the sequence measured in Example 1, and then the amplified product was transferred into host cells by conventional techniques to obtain recombinant protein.

gene specific primer sequence clone name Specific primer 1 (5'→3') Specific primer 2 (5'→3') PP3999 ACTTGGCCGGAAGGAAAGGTGCT TGATGGAGATGGGATGCCTGTGA PP4534 CAGGGGACACTCGGCAGCATCT CTGCGCTCCTTCAGACTTGCTGC Embodiment 3: cDNA clone sequence analysis

1.PP3999A：核苷酸序列(SEQ ID NO：1)长度：1566bp1  GGCAACCGAA AGGTTTCTGT AGTCAGAGCC CTTCGCCACG CCGTCCCGGC51  AGGGATCCTC TGTTTAGGAC TGACGGTTGC TGTGGACTCT TATTTTTGGC 101  GGCAGCTCAC  TTGGCCGGAA  GGAAAGGTGC  TTTGGTACAA  CACTGTCCTG151  AACAAAAGCT  CCAACTGGGG  GACCTCCCCG  CTGCTGTGGT  ACTTCTACTC201  AGCCCTGCCC  CGCGGCCTGG  GCTGCAGCCT  GCTCTTCATC  CCCCTGGGCT251  TGGTAGACAG  AAGGACGCAC  GCGCCGACGG  TGCTGGCACT  GGGCTTCATG301  GCACTCTACT CCCTCCTGCC  ACACAAGGAG  CTACGCTTCA  TCATCTATGC351  CTTCCCCATG  CTCAACATCA  CGGCTGCCAG  AGGCTGCTCC  TACCTGCTGA401  ATAACTATAA  AAAGTCTTGG  CTGTACAAAG  CGGGGTCTCT  GCTTGTGATC451  GGACACCTCG  TGGTGAATGC  CGCCTACTCA  GCCACGGCCC  TGTATGTGTC501  CCATTTCAAC  TACCCAGGTG  GCGTCGCAAT  GCAGAGGCTG  CACCAGCTGG551  TGCCCCCCCA  GACAGACGTC  CTTCTGCACA  TTGACGTGGC  AGCCGCCCAG601  ACAGGTGTGT  CTCGGTTTCT  CCAAGTCAAC  AGCGCCTGGA  GGTACGACAA651  GAGGGAGGAT  GTGCAGCCGG  GGACAGGCAT  GCTGGCATAC  ACACACATCC701  TCATGGAGGC  GGCCCTGGGC  TCCTGGCCCT  CTACAGGGAC  ACACACCGGG751  TCCTGGCCAG  CGTCGTGGGG  ACCACAGGTG  TGAGTCTGAA  CCTGACCCAA801  CTGCCCCCCT TCAACGTCCA  CCTGCAGACA  AAGCTGGTGC  TTCTGGAGAG851  GCTCCCCCGG  CCGTCCTGAG  GGGGACCAGG  CAGCCCTCAG  CAGCCACAGG901  CCTTCCAGGA  GCTGTTATCA  CTACCAGTTT  CTGGCACAAT  TCCAGCACAA951  TTATGACAAT  TCAGAGAAGC  AAGTCAAAGG  ACTGGGCACC  TGCCTCTGAC1001  AGACACCAGA  CCAGGTCCAG  GGCCTCCTCC  ACAGCCTCAG  CTGGGGCTCT1051  CAGCACCAAA  GAACGAGGGG  CCCAGGTCTT  GTTGGCACCC  CGGGAGCCAC1101  TGCCCAGGGT  GATGGTGGCC  AGCTCAGGGC  TTCCTGCGGG  TGACTGTCGC1151  CCAGACCAGG  TGCCATTCAT  GACTAATCAG  GAGCAGCGGG  CTCACCCAGG1201  CACCTGTCTG  CCAGGAGGCC  ACGTGTGTCC  TGCCCACCCA  GGGGGAGCTG1251  TATTTTGGCA  GCACCCCACG  CTTGCTGCCC  GAGGGCCTCT  TGGGGCACCT1301  AAGACAGCAC CCCCTCTCAG  GGGAGACCAT  GGTGGCCCCG  GCCGCACCCC1351  CCCACCCTGG  TGCCACCACT  GCAACTTTTG  TATTCACAGG  CATCCCATCT1401  CCATCACAGA  TAAAATCTTA  GGAGATAAAC  ACATTCAAAA  AGGAATGAGA1451  TAAAAAGAAT  AAGGCAATAA  ATGTTGATTG  GAACCTCTAA  AAAAAAAAAA1501  AAAAAAAAAA  AAAAAAAAAA  AAAAAAAAAA  AAAAAAAAAA  AAAAAAAAAA1551  AAAAAAAAAA  AAAAAAB：氨基酸序列(SEQ ID NO：2)长度：328个氨基酸1     MPPTQPRPCM CPISTTQVAS QCRGCTSWCP PRQTSFCTLT WQPPRQVCLG FSKSTAPGGT61     TRGRMCSRGQ ACWHTHTSSW RRPWAPGPLQ GHTPGPGQRR GDHRCESEPD PTAPLQRPPA121     DKAGASGEAP PAVLRGTRQP SAATGLPGAV ITTSFWHNSS TIMTIQRSKS KDWAPASDRH181     QTRSRASSTA SAGALSTKER GAQVLLAPRE PLPRVMVASS GLPAGDCRPD QVPFMTNQEQ241     RAHPGTCLPG GHVCPAHPGG AVFWQHPTLA ARGPLGAPKT APPLRGDHGG PGRTPPPWCH301     HCNFCIHRHP ISITDKILGD KHIQKGMRC.核苷酸及氨基酸组合序列(SEQ ID NO：3)克隆号：PP3999起始编码子：467 ATG Term Code: 1453 TAA protein molecular weight: 351251 GC GAA AGG TTT CTG TAG TAG TCA GAG CCC GCC ACG CCG TCC 4647 CGG CAG GGA TCC TGA CGG CTG TGG TGG TGG ACT CTT 9495 TTT 9495 TTT CTT  GGC  CGG  AAG  GAA  AGG  TGC  TTT  GGT  ACA  ACA    142 143  CTG TCC TGA ACA AAA GCT CCA ACT GGG GGA CCT CCC CGC TGC TGT GGT     190191  ACT TCT ACT CAG CCC TGC CCC GCG GCC TGG GCT GCA GCC TGC TCT TCA     238239  TCC CCC TGG GCT TGG TAG ACA GAA GGA CGC ACG CGC CGA CGG TGC TGG     286287  CAC TGG GCT TCA TGG CAC TCT ACT CCC TCC TGC CAC ACA AGG AGC TAC     334335  GCT TCA TCA TCT ATG CCT TCC CCA TGC TCA ACA TCA CGG CTG CCA GAG     382383  GCT GCT CCT ACC TGC TGA ATA ACT ATA AAA AGT CTT GGC TGT ACA AAG     430431  CGG GGT CTC TGC TTG TGA TCG GAC ACC TCG TGG TGA ATG CCG CCT ACT     4781                                                  Met Pro Pro Thr       4479  CAG CCA CGG CCC TGT ATG TGT CCC ATT TCA ACT ACC CAG GTG GCG TCG     5265  Gln Pro Arg Pro Cys Met Cys Pro Ile Ser Thr Thr Gln Val Ala Ser      20527  CAA TGC AGA GGC TGC ACC AGC TGG TGC CCC CCC AGA CAG ACG TCC TTC     57421  Gln Cys Arg Gly Cys Thr Ser Trp Cys Pro Pro Arg Gln Thr Ser Phe      36575  TGC ACA TTG ACG TGG CAG CCG CCC AGA CAG GTG TGT CTC GGT TTC TCC     62237  Cys Thr Leu Thr Trp Gln Pro Pro Arg Gln Val Cys Leu Gly Phe Ser      52623  AAG TCA ACA GCG CCT GGA GGT ACG ACA AGA GGG AGG ATG TGC AGC CGG     67053  Lys Ser Thr Ala Pro Gly Gly Thr Thr Arg Gly Arg Met Cys Ser Arg      68671  GGA CAG GCA TGC TGG CAT ACA CAC ACA TCC TCA TGG AGG CGG CCC TGG     71869  Gly Gln Ala Cys Trp His Thr His Thr Ser Ser Trp Arg Arg Pro Trp      84719  GCT CCT GGC CCT CTA CAG GGA CAC ACA CCG GGT CCT GGC CAG CGT CGT     76685  Ala Pro Gly Pro Leu Gln Gly His Thr Pro Gly Pro Gly Gln Arg Arg     100767  GGG GAC CAC AGG TGT GAG TCT GAA CCT GAC CCA ACT GCC CCC CTT CAA     814101  Gly Asp His Arg Cys Glu Ser Glu Pro Asp Pro Thr Ala Pro Leu Gln     116815  CGT CCA CCT GCA GAC AAA GCT GGT GCT TCT GGA GAG GCT CCC CCG GCC     862117  Arg Pro Pro Ala Asp Lys Ala Gly Ala Ser Gly Glu Ala Pro Pro Ala     132853  GTC CTG AGG GGG ACC AGG CAG CCC TCA GCA GCC ACA GGC CTT CCA GGA     910133  Val Leu Arg Gly Thr Arg Gln Pro Ser Ala Ala Thr Gly Leu Pro Gly     148911  GCT GTT ATC ACT ACC AGT TTC TGG CAC AAT TCC AGC ACA ATT ATG ACA     958149  Ala Val Ile Thr Thr Ser Phe Trp His Asn Ser Ser Thr Ile Met Thr     164959  ATT CAG AGA AGC AAG TCA AAG GAC TGG GCA CCT GCC TCT GAC AGA CAC    1006165  Ile Gln Arg Ser Lys Ser Lys Asp Trp Ala Pro Ala Ser Asp Arg His     1801007  CAG ACC AGG TCC AGG GCC TCC TCC ACA GCC TCA GCT GGG GCT CTC AGC    1054181  Gln Thr Arg Ser Arg Ala Ser Ser Thr Ala Ser Ala Gly Ala Leu Ser     1961055  ACC AAA GAA CGA GGG GCC CAG GTC TTG TTG GCA CCC CGG GAG CCA CTG    1102197  Thr Lys Glu Arg Gly Ala Gln Val Leu Leu Ala Pro Arg Glu Pro Leu     2121103  CCC AGG GTG ATG GTG GCC AGC TCA GGG CTT CCT GCG GGT GAC TGT CGC    1150213  Pro Arg Val Met Val Ala Ser Ser Gly Leu Pro Ala Gly Asp Cys Arg     2281151  CCA GAC CAG GTG CCA TTC ATG ACT AAT CAG GAG CAG CGG GCT CAC CCA    1198 229  Pro Asp Gln Val Pro Phe Wet Thr Asn Gln Glu Gln Arg Ala His Pro    2441199  GGC ACC TGT CTG CCA GGA GGC CAC GTG TGT CCT GCC CAC CCA GGG GGA   1246245  Gly Thr Cys Leu Pro Gly Gly His Val Cys Pro Ala His Pro Gly Gly    2601247  GCT GTA TTT TGG CAG CAC CCC ACG CTT GCT GCC CGA GGG CCT CTT GGG   1294261  Ala Val Phe Trp Gln His Pro Thr Leu Ala Ala Arg Gly Pro Leu Gly    2761295  GCA CCT AAG ACA GCA CCC CCT CTC AGG GGA GAC CAT GGT GGC CCC GGC   1342277  Ala Pro Lys Thr Ala Pro Pro Leu Arg Gly Asp His Gly Gly Pro Gly    2921343 CGC ACC CCC CCA CCC TGG TGC CAC CAC TGC AAC TTT TGT ATT CAC AGG   1390293  Arg Thr Pro Pro Pro Trp Cys His His Cys Asn Phe Cys Ile His Arg    3081391  CAT CCC ATC TCC ATC ACA GAT AAA ATC TTA GGA GAT AAA CAC ATT CAA 1438309  His Pro Ile Ser Ile Thr Asp Lys Ile Leu Gly Asp Lys His Ile Gln    3241439  AAA GGA ATG AGA TAA AAA GAA TAA GGC AAT AAA TGT TGA TTG GAA CCT   1486325  Lys Gly Met Arg ***                                                3291487  CTA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 15341535 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AA 1566

2.PP4534A：核苷酸序列(SEQ ID NO：4)长度：1683bp1  AGGAATTTCA  GCCAACATAA  TAAGACATGA  AAATGGCATT  CGAGGTGTAT51  TAGACAGACA  AGGGGATGTT  AGTGTTTGCA  GGAGACTTGG  TCTGCCTCAG101  TGATGTCAGT  CAGCAGTGAT  TGTGATTCCC  CAGGGGACAC  TCGGCAGCAT151  CTGGAGACAT  TTTAGTTTAA  ACTTCCCCAG  TGATCTGTGA  TGTACAGGAG201  ACACTTTCGG  TTGTCACACT  GGGGGAGGAG  GCTGCATGTC  ACTGGCATCT251  GTTGGGTGAC  ACCTACAATG  CACAGGACAA  CCACAACAAA  TAATTCAGGC301  CCAAATGTTG  CTGGTGCTGA GGGTGAGGTC  CTAGTGTTAG  TAACAGGAGG351  AAAACCCAGC  AGTCTGGAGG  AGAGACCTCT  TCCCAGGGCA  GCCCAGGGGC401  CATCAGGAGG  GTTCATCTCA  TGCATTAGAG  GTCTTGGGAA  GAATGAGGCT451  TCCTTTCCTC  CATCAAAGCA  AGCAAATCCT  TTAAAAGCTG  CATCTCCAAG501  GGCTGCTCCG  GGCTCATAGC  AAGCAACGTC  GGAGCCCAGA  GGCAAGGCTG551  TGCTACTCAG  CTGCCCTCTG  GGGTCACAAA  GGCTTCACTT  GGCFTCTAAG601  AGCTGATGAG  GCCTCTCGCA  AGGGACCCTG  TGTGCATGGG  CTGACCCTGA651  AACTTCCCAG  CCTCTCTTCT  TCTCAGAGCA  CCCTCAGGTG  GCCTCTCGGG701  GGTTACCCCT  CATTGATACC  ATGTCTCCTC  GTGTTTTTGT  CCAGACTCCA751  ATTCCAGGGT  TTCAGAACCG  CATCGCAGCA  TCTTTCCTGA  AATGCACTCA801  GACTCAGCCA  GCAAAGACGT GCCTGGCCGC  ATCCTGCTGG  ATATAGACAA851  TGATACCGAG  AGCACTGCCC  TGTGAAGAAA  GCCCTTTCCC  AGCCCTCCAC901  CACTTCCACC  CTGGCGAGTG  GAGCAGGGGC  AGGCGAACCT  CTTTCTTTGC951  AGACCGAACA  GTGAAAAGCT  TTCAGTGGAG  GACAAAGGAG  GGCCTCACTG1001  TGCGGGACCT  GGCCTTCTGC  ACGGCCCAAG  GAGAACCTGG  AGGCCACCAC1051  TAAAGCTGAA  TGACCTGTGT  CTTGAAGAAG  TTGGCTTTCT  TTACATGGGA1101  AGGAAATCAT  GCCAAAAAAA  TCCAAAACAA  AGAAGTACCT  GGAGTGGAGA1151  GAGTATTCCT  GCTGAAACGC  GCATAGGAAG  CTTTTGTCCC  TGCTGTTAAT1201  GCGGGCAGCA  CCTACAGCAA  CTTGGAATGA  GTAAGAAGCA  GTGCGTTAAC1251  TATCTATTTA  ATAAAATGCG  CTCATTATGC  AAGTCGCCTA  CTCTCTGCTA1301  CCTGGACGTT  CATTCTTATG TATTAGGAGG  GAGGCTGCGC  TCCTTCAGAC1351  TTGCTGCAGA  ATCATTTTGT  ATCATGTATG  GTCTGTGTCT  CCCCAGTCCC1401  CTCAGAACCA  TGCCCATGGA  TGGTGACTGC  TGGCTCTGTC  ACCTCATCAA1451  ACTGGATGTG  ACCCATGCCG  CCTCGTTGGA  TTGTCGGAAT  GTAGACAGAA1501  ATGTACTGTT  CTTTTTTTTT  TTTTTTAAAC  AATGTAATTG  CTACTTGATA1551  AGGACCGAAC  ATTATTCTAG  TTTCATGTTT  AATTTGAATT  AAATATATTC1601  TGTGGTTTGT  GTGGAAAAAA  AAAAAAAAAA  AAAAAAAAAA  AAAAAAAAAA1651  AAAAAAAAAA  AAAAAAAAAA  AAAAAAAAAA  AAAB：氨基酸序列(SEQ ID NO：5)长度：121个氨基酸1  MSKKQCVNYL FNKMRSLCKS PTLCYLDVHS YVLGGRLRSF RLAAESFCIM YGLCLPSPLR61  TMPMDGDCWL CHLIKLDVTH AASLDCRNVD RNVLFFFFFF KQCNCYLIRT EHYSSFMFNL121  NC.核苷酸及氨基酸组合序列(SEQ ID NO：6)克隆号：PP4534起始编码子：1227 ATG    终止编码子：1592 TAA蛋白质分子量:14225

1 AG GAA TTT CAG CAG CAG CAA TAA TAA GAC ATG AAA ATG GCA TTC GAG GAG 4748 TAG ACA GAC AAG GGG AAG GGG TTA GCA GCA GAC TGC TGC AG CAG CAG GAGA TG TCG     143144  GCA GCA TCT GGA GAC ATT TTA GTT TAA ACT TCC CCA GTG ATC TGT GAT     191192  GTA CAG GAG ACA CTT TCG GTT GTC ACA CTG GGG GAG GAG GCT GCA TGT     239240  CAC TGG CAT CTG TTG GGT GAC ACC TAC AAT GCA CAG GAC AAC CAC AAC     287288  AAA TAA TTC AGG CCC AAA TGT TGC TGG TGC TGA GGG TGA GGT CCT AGT     335335  GTT AGT AAC AGG AGG AAA ACC CAG CAG TCT GGA GGA GAG ACC TCT TCC     383384  CAG GGC AGC CCA GGG GCC ATC AGG AGG GTT CAT CTC ATG CAT TAG AGG     431432  TCT TGG GAA GAA TGA GGC TTC CTT TCC TCC ATC AAA GCA AGC AAA TCC     479480  TTT AAA AGC TGC ATC TCC AAG GGC TGC TCC GGG CTC ATA GCA AGC AAC     527528  GTC GGA GCC CAG AGG CAA GGC TGT GCT ACT CAG CTG CCC TCT GGG GTC     575576  ACA AAG GCT TCA CTT GGC TTC TAA GAG CTG ATG AGG CCT CTC GCA AGG     623624  GAC CCT GTG TGC ATG GGC TGA CCC TGA AAC TTC CCA GCC TCT CTT CTT     671672  CTC AGA GCA CCC TCA GGT GGC CTC TCG GGG GTT ACC CCT CAT TGA TAC     719720  CAT GTC TCC TCG TGT TTT TGT CCA GAC TCC AAT TCC AGG GTT TCA GAA     767768  CCG CAT CGC AGC ATC TTT CCT GAA ATG CAC TCA GAC TCA GCC AGC AAA     815816  GAC GTG CCT GGC CGC ATC CTG CTG GAT ATA GAC AAT GAT ACC GAG AGC     863864  ACT GCC CTG TGA AGA AAG CCC TTT CCC AGC CCT CCA CCA CTT CCA CCC     911912  TGG CGA GTG GAG CAG GGG CAG GCG AAC CTC TTT CTT TGC AGA CCG AAC     959960  AGT GAA AAG CTT TCA GTG GAG GAC AAA GGA GGG CCT CAC TGT GCG GGA    10071008  CCT GGC CTT CTG CAC GGC CCA AGG AGA ACC TGG AGG CCA CCA CTA AAG    10551056  CTG AAT GAC CTG TGT CTT GAA GAA GTT GGC TTT CTT TAC ATG GGA AGG    11031104  AAA TCA TGC CAA AAA AAT CCA AAA CAA AGA AGT ACC TGG AGT GGA GAG    11511152  AGT ATT CCT GCT GAA ACG CGC ATA GGA AGC TTT TGT CCC TGC TGT TAA    11991200  TGC GGG CAG CAC CTA CAG CAA CTT GGA ATG AGT AAG AAG CAG TGC GTT    1247   1                                      Met Ser Lys Lys Gln Cys Val       71248  AAC TAT CTA TTT AAT AAA ATG CGC TCA TTA TGC AAG TCG CCT ACT CTC    12958  Ash Tyr Leu Phe Asn Lys Met Arg Ser Leu Cys Lys Ser Pro Thr Leu      231296  TGC TAC CTG GAC GTT CAT TCT TAT GTA TTA GGA GGG AGG CTG CGC TCC    134324  Cys Tyr Leu Asp Val His Ser Tyr Val Leu Gly Gly Arg Leu Arg Ser      391344  TTC AGA CTT GCT GCA GAA TCA TTT TGT ATC ATG TAT GGT CTG TGT CTC    139140  Phe Arg Leu Ala Ala Glu Ser Phe Cys Ile Met Tyr Gly Leu Cys Leu      551392  CCC AGT CCC CTC AGA ACC ATG CCC ATG GAT GGT GAC TGC TGG CTC TGT    143956  Pro Ser Pro Leu Arg Thr Met Pro Met Asp Gly Asp Cys Trp Leu Cys      711440  CAC CTC ATC AAA CTG GAT GTG ACC CAT GCC GCC TCG TTG GAT TGT CGG    148772  His Leu Ile Lys Leu Asp Val Thr His Ala Ala Ser Leu Asp Cys Arg      871488  AAT GTA GAC AGA AAT GTA CTG TTC TTT TTT TTT TTT TTT AAA CAA TGT    153588  Asn Val Asp Arg Asn Val Leu Phe Phe Phe Phe Phe Phe Lys Gln Cys     1031536  AAT TGC TAC TTG ATA AGG ACC GAA CAT TAT TCT AGT TTC ATG TTT AAT    1583104  Asn Cys Tyr Leu Ile Arg Thr Glu His Tyr Ser Ser Phe Met Phe Asn     1191584  TTG AAT TAA ATA TAT TCT GTG GTT TGT GTG GAA AAA AAA AAA AAA AAA AAA 1631120 Leu Asn *** 1221632 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 16791680 AAA 16833

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.

Claims (10)

1. isolating people's albumen with promotion growth of cancer cells function is characterized in that it comprises the polypeptide of the aminoacid sequence with the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5;

Or its conservative property variation polypeptide or its active fragments or its reactive derivative.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5.

3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 85% homogeny with a kind of nucleotides sequence that is selected from down group:

(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that, the polypeptide of this polynucleotide encoding has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down group:

Coding region sequence or the full length sequence of SEQ ID NO:3, SEQ ID NO:6.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a genetically engineered host cell is characterized in that, it is a kind of host cell that is selected from down group:

(a) host cell that transforms or transduce with the described carrier of claim 6;

(b) host cell that transforms or transduce with the described polynucleotide of claim 3.

8. preparation method with polypeptide of the people's protein-active that promotes the growth of cancer cells function is characterized in that this method comprises:

(a) be fit to express under the proteic condition of people with promotion growth of cancer cells function, cultivating the described host cell of claim 7:

(b) from culture, isolate polypeptide with the people's protein-active that promotes the growth of cancer cells function.

9. an energy and claim 1 are described has the people's protein-specific bonded antibody that promotes the growth of cancer cells function.

10. nucleic acid molecule, it contains a successive 10-800 Nucleotide in the described polynucleotide of claim 3.