CN1193040C - Human protein with the function of inhibiting the growth of cancer cells and its coding sequence - Google Patents

Abstract

The present invention discloses a novel human protein with the function of inhibiting cancer, polynucleotide for encoding the polypeptide and a method for preparing the polypeptide by a recombinant technology. The present invention also discloses a method of using the polypeptide to treat various diseases, such as cancers. The present invention also discloses an antagonist of the polypeptide and a therapeutic effect thereof. The present invention also discloses the application of the polynucleotide for encoding the human protein with the function of inhibiting cancer.

Description

Human protein with the function of inhibiting the growth of cancer cells and its coding sequence

technical field

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

Background technique

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 with tumor suppressor functions and their agonists/inhibitors.

Contents of the invention

The object of the present invention is to provide a new class of human protein polypeptides with tumor suppressor function 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, a novel isolated protein polypeptide with tumor suppressor function is provided, which comprises a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO : 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20; 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, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20.

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 a tumor suppressor function; (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, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 14, SEQ ID NO: ID NO: 17, SEQ ID NO: 20. More preferably, the sequence of the polynucleotide is selected from the following group; SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18 , the coding region sequence or full-length sequence of SEQ ID NO: 21.

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 protein activity of a tumor suppressor function, the method comprising: (a) cultivating the above-mentioned transformed or transformed protein under conditions suitable for expressing a protein with a tumor suppressor function Induced host cells: (b) isolating from the culture a polypeptide having protein activity of a tumor suppressor function.

In the fifth aspect of the present invention, an antibody specifically binding to the above-mentioned protein polypeptide with tumor suppressor function 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 tumor suppressor function of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions can treat diseases such as cancer and abnormal proliferation of cells.

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

Detailed ways

The invention adopts large-scale cDNA clones to transfect cancer cells, and on the basis of obtaining tumor-suppressing effects, 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 tumor suppressor function of the present invention has the effect of inhibiting the colony formation of cancer cells (liver cancer cells), and the inhibition rate is 50% or above.

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 with tumor suppressor function" means that the protein or polypeptide with tumor suppressor function 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 with tumor suppressor function. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the protein polypeptide with tumor suppressor function 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 human proteins with tumor suppressor function. 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 with tumor suppressor function 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.

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 PP3895 protein (in this application, the protein is named by its clone number) (in this application, the protein is named by its clone number) as an example, the coding sequence of the mature polypeptide can be shown in SEQ ID NO: 3 The coding region sequences of the same or degenerate variants. 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 PP3993 protein (in this application, the protein is named by its clone number) (in this application, the protein is named by its clone number) as an example, the coding sequence of the mature polypeptide can be shown in SEQ ID NO: 6 The coding region sequences of the same or degenerate variants. 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 with tumor suppressor function, it can be deduced by analogy. For other proteins with tumor suppressor function, it can be deduced by analogy.

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 with tumor suppressor function.

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 tumor suppressor function 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) appearance or loss of function of marker genes: (3) determination of transcript levels of proteins with tumor suppressor function; (4) ) Detection of protein products expressed by genes by immunological techniques or assays of 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 with tumor suppressor function.

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 of the present invention or a protein coding sequence with tumor suppressor function, and a method for producing the polypeptide of the present invention by recombinant technology.

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

(1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding a human protein with tumor suppressor function 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 tumor suppressor function 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 tumor suppressor function 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 with tumor suppressor function 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 proteins with tumor suppressor functions, and screening for antibodies, polypeptides or other ligands that promote or resist the functions of proteins with tumor suppressor functions. body. For example, antibodies can be used to activate or inhibit the function of human proteins that function as tumor suppressors. Screening the polypeptide library with the expressed recombinant human protein with tumor suppressor function can be used to find therapeutically valuable polypeptide molecules that can inhibit or stimulate the function of the human protein with tumor suppressor function.

The invention also provides methods of screening drugs to identify agents that enhance (agonists) or repress (antagonists) human proteins with tumor suppressor function. Agonists enhance biological functions of human proteins with tumor suppressor functions 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 membrane preparations expressing a human protein with tumor suppressor function can be cultured with the labeled human protein with tumor suppressor function in the presence of a drug. The ability of the drug to enhance or repress this interaction is then determined.

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

When screening compounds as antagonists, a protein with tumor suppressor function can be added to a bioanalytical assay, and whether the compound is an antagonist can be determined by measuring the effect of the compound on the interaction between the protein with tumor suppressor function 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, 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. Proteins with tumor suppressor function are administered in amounts effective to treat and/or prevent the particular indication. The amount and dosage range of the protein with tumor suppressor function 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 with tumor suppressor function can also be used for various therapeutic purposes. Gene therapy technology can be used to treat abnormalities in cell proliferation, development or metabolism due to non-expression or abnormal/inactive expression of proteins with tumor suppressor functions. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated proteins with tumor suppressor functions to inhibit the activity of endogenous proteins with tumor suppressor functions. For example, a mutated protein with tumor suppressor function may be a shortened protein with tumor suppressor function that lacks a signal transduction domain, and 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 proteins with tumor suppressor function. Expression vectors derived from viruses such as retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and parvoviruses can be used to transfer protein genes with tumor suppressor functions into cells. The method for constructing a recombinant viral vector carrying a protein gene with tumor suppressor function can be found in existing literature (Sambrook, et al.). In addition, the recombinant human protein gene with tumor suppressor function can be packaged into liposomes and transferred into cells.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human protein mRNA with tumor suppressor function 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 an antibody against the human protein antigenic determinant with tumor suppressor function. 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 tumor suppressor proteins can be used in immunohistochemical techniques to detect human tumor suppressor proteins in biopsy specimens.

Monoclonal antibodies that bind to human proteins with tumor suppressor functions 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 with tumor suppressor function. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human proteins with tumor suppressor functions.

Antibodies can also be used to design immunotoxins that target a particular site in the body. For example, high-affinity monoclonal antibodies to human proteins with tumor suppressor functions can be covalently combined with 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 human proteins with tumor suppressor functions. cell.

The production of polyclonal antibodies can be used to immunize animals, such as rabbits, mice, rats, etc., with human proteins or polypeptides with tumor suppressor functions. 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 with tumor suppressor function 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 with tumor suppressor functions.

Polypeptide molecules capable of binding to human proteins with tumor suppressor functions can be obtained by screening random polypeptide libraries composed of various possible combinations of amino acids bound to solid phases. During screening, human protein molecules with tumor suppressor function must be labeled.

The invention also relates to a diagnostic test method for quantitative and localized detection of the human protein level with tumor suppressor function. These assays are well known in the art and include FISH assays and radioimmunoassays. The level of human protein with tumor suppressor function detected in the test can be used to explain the importance of human protein with tumor suppressor function in various diseases and to diagnose diseases in which the protein with tumor suppressor function plays a role.

The polynucleotide of the protein with tumor suppressor function can be used for the diagnosis and treatment of diseases related to the protein with tumor suppressor function. In terms of diagnosis, the polynucleotide of the protein with tumor suppressor function can be used to detect the expression of the protein with tumor suppressor function or the abnormal expression of the protein with tumor suppressor function in a disease state. For example, DNA sequences of proteins with tumor suppressor function can be used for hybridization of biopsy specimens to determine abnormal expression of proteins with tumor suppressor function. 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 specific primers of the protein with tumor suppressor function can also detect the transcription product of the protein with tumor suppressor function.

The detection of the mutation of the protein gene with tumor suppressor function can also be used to diagnose the diseases related to the protein with tumor suppressor function. The form of protein mutation with tumor suppressor function includes point mutation, translocation, deletion, recombination and any other abnormality compared with the normal wild type protein DNA sequence with tumor suppressor function. 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 tumor suppressor function 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 with tumor suppressor function of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and/or lower Low side effects (eg, 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 method that does not indicate specific conditions in the following examples, 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 manufacturer's instructions suggested conditions.

Example 1: Obtaining of cDNA gene and its inhibitory effect on cancer cell clone formation

PP3895, PP3993, PP4052, PP4068, PP4135, PP4189, PP2500 were obtained by constructing human placenta cDNA library by conventional methods. Placental 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 an mRNA purification kit (Pharmacia Company). The cDNA library of the above mRNA was constructed with 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 cDNA 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 with 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 0.74 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 whole culture medium for each well, 37°C for 24 hours, change 100 μl whole culture medium containing G418, 37°C for 24 to 48 hours, change the culture medium with different concentrations of G418 while observing. After about 2 to 3 times, until the microscopic examination of the cells shows colony formation, count them. It was found that the above clones can inhibit the formation of cell clones, and the results are shown in the table below.

                                            

cDNA clone name Number of cDNA clones (three repetitions) The number of empty vector clones (three replicates) PP3895 3 9 0 33 34 38 PP3993 17 8 12 35 twenty four 32 PP4052 0 1 0 16 20 18 PP4068 0 1 1 27 30 twenty three PP4135 14 12 10 27 30 twenty three PP4189 6 5 9 27 30 twenty three PP2500 0 2 0 28 30 27

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. If the full-length cDNA sequence has not yet been obtained, design primers and perform sequencing again until the full-length sequence (SEQ ID NO: 1, 4, 7, 10, 13, 16, 19) is obtained.

Example 2: Obtaining gene clones by PCR from placental cDNA:

Human placental tissues aged 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). MMLV-RT-SuperscriptH (GIBCO BRL) reverse transcriptase was used to carry out reverse transcription reaction at 42°C to obtain placental cDNA. Utilize the transmutation primers of each gene (as shown in the table below), and perform 3'1 cycles at 90°C. 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 1 minute, a total of 35 cycles, 10'1 cycle at 72°C for PCR amplification 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 the recombinant protein.

gene specific primer sequence clone name Specific primer 1 (5'→3') Specific primer 2 (5'→3') PP3895 GGTTTACTGACACCCCCACCCCA GCGCCCGGCCTCTTTTTATCCTT PP3993 ACTTGCATTTGCCCTGACACCCA GTTCTGCTTGGCCGAGCTGTTGA PP4052 GTGTATGCTGCCCCCCTTTCTGGG ACAGGATGGTAGTGGCGATGGCA PP4068 CTGGGCCCAAGGACAAAGCTCAC ATCATGGGGCATGCACAGCATCT PP4135 TGCCCCTAACCACTGAGACAGCA CAACTGCACATTTTGCTCATGTA PP4189 TCAAGGTTGCTCTCCAAGCTCAAGG GTTATTAGGCCCACCACTAAGAG PP2500 CTTGCTGCTCTTCTCGTTCCCGA GCAGGGTCCTGGAACTTCTTGGC

Embodiment 3: cDNA clone sequence analysis

1. PP3895 protein

A: Nucleotide sequence (SEQ ID NO: 1) Length: 1972bp

1 GGTTTAGTGA CACCCCCACC CCACCCCATC TGCATATTTTTTCACCACCC

51 CTCCCTTCTG TATATGATGC TTCTGTAGCT CTGTAACGCC CCCTACATTT

101 ACCTTCCTTA TATCTCCCCC GTCTTCCTCT CCATAGATCT CCTCCCATTT

151 CCCCTTCCAT GGTCCCCATC TTCCTTCTGA AATGTCTACT CCTTCATGTT

201 CCTTTATGTA TGTCTTCCAA TCTTTCCTTC CATAGCTCTC ATCACCTTCA

251 TATATTTCTT CCATCTTTCT CCTCCCACCT GCCTCGCCCT CTGTATATAC

301 CCCCACTCTC CCCCTTTTAT ATCTTCTCCA TCTCCCCCCA TATCTTTCCT

351 CTATGTCCAC ATCTGTGTAT TCCCCCCAAC TTCCCCTCCA TATATCTTTT

401 TTTACTCCCC TTTTCCTCCC TGTATCCTCT GTGTTCCCCC CATCTTGCTC

451 TACATCATTC TTCCCAAGAT CTTTACGTCT CCCATCTTGA TCTCTCCATC

501 TCCACTTTCT CCTAACATTT TCATTTCCGT TCCTTAGTGT CTCTAGAGAG

551 ATCATTCTTG ATAGCCTCAG CTCTTTCTCT GTGTTTTTCA GGTTTGTATT

601 CTGCTCTGCT CTACCTCTCC TCCTTGCCCC TTTCTCTCC CAGGATGTCT

651 CTCCTTTCCA AATCCTTTTTT GTACCTGAAT ACCTTTTGCC CCACCCTGGG

701 CTCTCATTTC CATCTCAGAC CTTAGCCTGG GATCTAAAGG GCTGACAGTG

751 TCCCTTTCTT CATGCAGATG ACAGTCGTCT AGAGGAGCTC AAAGCCACTC

801 TGCCCAGCCC AGACAAGCTC CCTGGATTCA AGATGTACCC CATTGACTTT

851 GAGAAGGTAT GGGGTGGGGC TCAGGACAGG GAAGGAGGAT GGGCAAAGCA

901 TAGACAGGCT GGAGAAAACA GAAGTATCTG GAGCCAGCCC CGGGCCTTTG

951 TGGGGATCAG ATTGTGGGCC TGCCATATGG CTCTGAATGA GTAGGTGTTC

1001 CCAGCCATCC CTTTGTGATC TGGGAGAGTC CAGCAGGCAA TTGCAGTGGA

1051 GGATACACAT CTTCTTTATC TGATCCTCTC CCCACTGCCT TCACACCCTC

1101 CCCACTCATA ACAGGATGAT GACAGCAACT TTCATATGGA TTTCATCGTG

1151 GCTGCATCCA ACCTCCGGGC AGAAAACTAT GACATTCCTT CTGCAGACCG

1201 GCACAAGAGC AAGCTGATTG CAGGGAAGAT CATCCCAGCC ATTGCCACGA

1251 CCACAGCAGC CGTGGTTGGC CTTGTGTGTC TGGAGCTGTA CAAGGTTGTG

1301 CAGGGGCACC GACAGCTTGA CTCCTACAAG AATGGTTTCC TCAACTTGGC

1351 CCTGCCTTTC TTTGGTTTCT CTGAACCCCT TGCCGCACCA CGTCACCAGG

1401 TGGGGGCCTG CATCCGAAGC AGGGTTTGGG TGGGGTGTAT CTGTGTAGAT

1451 CTGGTTCTGA TTCACGTCAT ACCCTGTCAC CAGGGGAGGG TTTCTGTCTG

1501 TGTACCTACC CTTTTTGTGT ATCCTTTTTC ACTTATTCAT TAATCACATT

1551 ATTTGAGTAC GTGCGAAAAG ATGGGATATT TGAATTGTGC CCTGGGAGAT

1601 TATTAGTAAC TACACAATAA TGGCAGCCAA AATTTATTGG ACGCTTCCTA

1651 CACTTAAGTG CTTTGCTTGC TTCATTAATG AATTCACTCA AATATTTATT

1701 GAGCACCTTT TGTGGGCAGG GACTCTTCTA AGTTATGTTC CTCAAGTAGA

1751 TTATATAAAT AACCTATTAA ATGATTTTGG AATCAAAAAA GGATAAAAAG

1801 AGGCCGGGCG CGGTGGCTTA CGCCTGTAAT CCCAGCACTT TGGGAGGCCG

1851 AGGCACGTGG TTCACCTGAA GTCAGGAGTT TGAGACCAGC CTGGCCAACA

1901 TGATGAAACC CTGTCTCTAC TAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

1951 AAAAAAAAAA AAAAAAAAAA AA

B: Amino acid sequence (SEQ ID NO: 2) Length: 135 amino acids

1 MDFIVAASNL RAENYDIPSA DRHKSKLIAG KIIPAIATTT AAVVGLVCLE LYKVVQGHRQ

61 LDSYKNGFLN LALPFFGFSE PLAAPRHQVG ACIRSRVWVG CICVDLVLIH VIPCHQGRVS

121 VCVPTLFVYP FSLIH

C. Nucleotide and amino acid combination sequence (SEQ ID NO: 3)

Clone number: PP3895

Start code: 1136 ATG End code: 1543 TAA

Protein molecular weight: 14799

1 G GTT TAC TGA CAC CCC CAC CCC ACC CCA TCT GCA TAT TTT TTC ACC 46

47 ACC CCT CCC TTC TGT ATA TGA TGC TTC TGT AGC TCT GTA ACG CCC CCT 94

95 ACA TTT ACC TTC CTT ATA TCT CCC CCG TCT TCC TCT CCA TAG ATC TCC 142

143 TCC CAT TTC CCC TTC CAT GGT CCC CAT CTT CCT TCT GAA ATG TCT ACT 190

191 CCT TCA TGT TCC TTT ATG TAT GTC TTC TTC CAA TCT TTC CTT CCA TAG CTC 238

239 TCA TCA CCT TCA TAT ATT TCT TCC ATC TTT CTC CTC CCA CCT GCC TCG 286

287 CCC TCT GTA TAT ACC CCC ACT CTC CCC CTT TTA TAT CTT CTC CAT CTC 334

335 CCC CCA TAT CTT TCC TCT ATG TCC ACA TCT GTG TAT TCC CCC CAA CTT 382

383 CCC CTC CAT ATA TCT TTT TTT ACT CCC CTT TTC CTC CCT GTA TCC TCT 430

431 GTG TTC CCC CCA TCT TGC TCT ACA TCA TTC TTC CCA AGA TCT TTA CGT 478

479 CTC CCA TCT TGA TCT CTC CAT CTC CAC TTT CTC CTA ACA TTT TCA TTT 526

527 CCG TTC CTT AGT GTC TCT AGA GAG ATC ATT CTT GAT AGC CTC AGC TCT 574

575 TTC TCT GTG TTT TTC AGG TTT GTA TTC TGC TCT GCT CTA CCT CTC CTC 622

623 CTT GCC CCT TTT CTC TCC CAG GAT GTC TCT CCT TTC CAA ATC CTT TTT 670

671 GTA CCT GAA TAC CTT TTG CCC CAC CCT GGG CTC TCA TTT CCA TCT CAG 718

719 ACC TTA GCC TGG GAT CTA AAG GGC TGA CAG TGT CCC TTT CTT CAT GCA 766

767 GAT GAC AGT CGT CTA GAG GAG CTC AAA GCC ACT CTG CCC AGC CCA GAC 814

815 AAG CTC CCT GGA TTC AAG ATG TAC CCC ATT GAC TTT GAG AAG GTA TGG 862

863 GGT GGG GCT CAG GAC AGG GAA GGA GGA TGG GCA AAG CAT AGA CAG GCT 910

911 GGA GAA AAC AGA AGT ATC TGG AGC CAG CCC CGG GCC TTT GTG GGG ATC 958

959 AGA TTG TGG GCC TGC CAT ATG GCT CTG AAT GAG TAG GTG TTC CCA GCC 1006

1007 ATC CCT TTG TGA TCT GGG AGA GTC CAG CAG GCA ATT GCA GTG GAG GAT 1054

1055 ACA CAT CTT CTT TAT CTG ATC CTC TCC CCA CTG CCT TCA CAC CCT CCC 1102

1103 CAC TCA TAA CAG GAT GAT GAC AGC AAC TTT CAT ATG GAT TTC ATC GTG 1150

1 Met Asp Phe Ile Val 5

1151 GCT GCA TCC AAC CTC CGG GCA GAA AAC TAT GAC ATT CCT TCT GCA GAC 1198

6 Ala Ala Ser Asn Leu Arg Ala Glu Asn Tyr Asp Ile Pro Ser Ala Asp 21

1199 CGG CAC AAG AGC AAG CTG ATT GCA GGG AAG ATC ATC CCA GCC ATT GCC 1246

22 Arg His Lys Ser Lys Leu Ile Ala Gly Lys Ile Ile Pro Ala Ile Ala 37

1247 ACG ACC ACA GCA GCC GTG GTT GGC CTT GTG TGT CTG GAG CTG TAC AAG 1294

38 Thr Thr Thr Ala Ala Val Val Gly Leu Val Cys Leu Glu Leu Tyr Lys 53

1295 GTT GTG CAG GGG CAC CGA CAG CTT GAC TCC TAC AAG AAT GGT TTC CTC 1342

54 Val Val Gln Gly His Arg Gln Leu Asp Ser Tyr Lys Asn Gly Phe Leu 69

1343 AAC TTG GCC CTG CCT TTC TTT GGT TTC TCT GAA CCC CTT GCC GCA CCA 1390

70 Asn Leu Ala Leu Pro Phe Phe Gly Phe Ser Glu Pro Leu Ala Ala Pro 85

1391 CGT CAC CAG GTG GGG GCC TGC ATC CGA AGC AGG GTT TGG GTG GGG TGT 1438

86 Arg His Gln Val Gly Ala Cys Ile Arg Ser Arg Val Trp Val Gly Cys 101

1439 ATC TGT GTA GAT CTG GTT CTG ATT CAC GTC ATA CCC TGT CAC CAG GGG 1486

102 Ile Cys Val Asp Leu Val Leu Ile His Val Ile Pro Cys His Gln Gly 117

1487 AGG GTT TCT GTC TGT GTA CCT ACC CTT TTT GTG TAT CCT TTT TCA CTT 1534

118 Arg Val Ser Val Cys Val Pro Thr Leu Phe Val Tyr Pro Phe Ser Leu 133

1535 ATT CAT TAA TCA CAT TAT TTG AGT ACG TGC GAA AAG ATG GGA TAT TTG 1582

134 Ile His *** 136

1583 AAT TGT GCC CTG GGA GAT TAT TAG TAA CTA CAC AAT AAT GGC AGC CAA 1630

1631 AAT TTA TTG GAC GCT TCC TAC ACT TAA GTG CTT TGC TTG CTT CAT TAA 1678

1679 TGA ATT CAC TCA AAT ATT TAT TGA GCA CCT TTT GTG GGC AGG GAC TCT 1726

1727 TCT AAG TTA TGT TCC TCA AGT AGA TTA TAT AAA TAA CCT ATT AAA TGA 1774

1775 TTT TGG AAT CAA AAA AGG ATA AAA AGA GGC CGG GCG CGG TGG CTT ACG 1822

1823 CCT GTA ATC CCA GCA CTT TGG GAG GCC GAG GCA CGT GGT TCA CCT GAA 1870

1871 GTC AGG AGT TTG AGA CCA GCC TGG CCA ACA TGA TGA AAC CCT GTC TCT 1918

1919 ACT AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1966

1967 AAA AAA 1972

2. PP3993 protein

A: Nucleotide sequence (SEQ ID NO: 4) Length: 2064bp

1 TTCAGACAAA CCTCAGGTAA GATGGGACTG GGCTTCCCCG CCACTGCGCA

51 GCAGCTCCCG CCCTCCTGGG TGCTGTCCCA GGGGTGAAAG GAAGAGGTGG

101 GGAGGTACAG CTGGGAGTGT GGGGGATGGG GAAGGATGGG GAGGGAACGG

151 GCCCGTGGAC AACTTGCATT TGCCCTGACA CCCACCCTCC CTGCAGCTCT

201 ACACCCTCAC CGTGATCGGC CCAGGACCGC CAGATTGCCA GCCAGCCCAG

251 ATCTCTCGCC GTTACTCGGA CTTTGAGCGG CTGCACCGAA ACCTGCAGCG

301 GCAATTCCGG GGCCCAATGG CTGCCATCTC CTTCCCCCGT AAGCGGCTGC

351 GCCGGAATTT TACTGCAGAG ACCATTGCCC GCCGTAGCCG GGCCTTTGAG

401 CAGTTTTGG GTCACCTGCA GGCAGTGCCT GAGCTGCGCC ATGCCCCGGA

451 CCTGCAGGAC TTCTTCGTGC TGCCGGAGCT GCGGCGGGCA CAGAGCCTCA

501 CCTGTACTGG CCTCTATCGT GAGGCTCTGG CACTCTGGGC CAATGCCTGG

551 CAGCTGCAAG CCCAGCTGGG CACCCCCTCT GGCCCAGACC GCCCCCTGCT

601 GACCCTGGCT GGGCTGGCCG TGTGCCACCA GGAGCTGGAA GACCCTGGAG

651 AGGCCCGGGC ATGCTGTGAG AAGGCCCTGC AGCTGCTTGG GGACAAGAGC

701 CTCCACCCTT TGCTGGCACC CTTTCTGGAG GCCCATGTCC GGCTCTCCTG

751 GCGCCTGGGC CTGGACAAAC GTCAATCAGA GGCTCGGCTC CAAGCCCTGC

801 AGGAGGCAGG CCTTACCCCC ACACCACCCC CCAGTCTCAA AGAATTGCTC

851 ATCAAGGAGG TGCTGGACTA ACCCTTGCCT AGATTTAAGG CCACTGTGAG

901 GAGAGGGGTT GCCCCAGAAG GCAGGGGAAG GACCTGATGA GAACAGAATA

951 GCTGGGAGGC TGCAGAGGGT GCTGGGAGCC CCTAGAAGTT CCAAAAGAGA

1001 ATGTGAAGCA GATCAAGGAA ACTTCTGTTG AGCTAGGCTC AGGGTGAGCT

1051 TTGGCTGGGG TTGCCCTTGT GTAGTACAGG GAAGTCTGAC ACAGCCTCTC

1101 CAGCCTATAA ACAGCCGGGG GGCTGTGGCA CAGGTTGGGG CAATGTTCCC

1151 TTGTTGGTGG GCCCCCAAGC TGGCAAGGCC TCTTGGCTGA AGGCCAGGGA

1201 CTCTGCCCCT GGAGTCCTGG AGTTAAGGGA TGAAGGCAAG GCTGCAGGTC

1251 TGGCCCAGGG GAATTAAAAG CCAGCCACTC CAGTGGTATC AGTCTCTTTA

1301 TTGGATGTGA GGGCCAAAAG GGACTGTAAC TCCTGTCTCA GGAATGGGGA

1351 TAGATGGGAG GTTCTTGAAG CCCCAGGCGA AGCTGGTACC TCTGGCTACA

1401 GCTTGCTCTC TGAGACCTGG GGCTTCACTC GGATCACGCC CTCCTGGGCA

1451 CAGGTCACAG CTAGGACTCC ATCCTGACGC CACAGCCGCC CATGGACCAG

1501 CCCCCGAGAG CCACCTGTGG GTGAGGTGAA GGGTGATGAT GGCCTGCTTC

1551 AGAACAGCCA AATACACTTT TTTTTTTTTT CCTGAAACAG AGTCCCACTA

1601 AGTTGCCAGG CTGGTCTCAA GCCGCCTGGG TTCAAGGGAT CCTCCCGCCT

1651 CAGCCTCCTG AGCAGCTGGG ATTACAGGCG CACATCACCA TGCCCAACCT

1701 CCAAGTGGAC TTCTTGCAAA GGGTCTGGCC CAGGGCAGGG CTGCCCCACA

1751 CAAGGGTGCA CTGAGTGTCG TGGCTGCTCC AAATGCCCCT TCATGAGCTT

1801 ATTATGGACC GTCATTGAGG GGTAACTCCT CCCACAGGAA CCCCAGTTGA

1851 CAGTTTAAAAA GCACTTTTAC ACCTCTCCTC GCTTCCTCAA AAAGATCACA

1901 GAGGGAGGAG CTCTGAGAAC AGTCTCCTTC AACAGCTCGG CCAAGCAGAA

1951 CTGCTGTACC TCTGACCACT TGTGTTAGGA AAACTATCGG CTCCCTGTAT

2001 AATAAATCAA GCCAGGTCCT CCCCAAAAAA AAAAAAAAAA AAAAAAAAAA

2051 AAAAAAAAAAAAAAA

B: Amino acid sequence (SEQ ID NO: 5) Length: 184 amino acids

1 MAAISFPRKR LRRNFTAETI ARRSRAFEQF LGHLQAVPEL RHAPDLQDFF VLPELRRAQS

61 LTCTGLYREA LALWANAWQL QAQLGTPSGP DRPLLTLAGL AVCHQELEDP GEARACCEKA

121 LQLLGDKSLH PLLAPFLEAH VRLSWRLGLD KRQSEARLQA LQEAGLTPTP PPSLKELLIK

181 EVLD

C. Nucleotide and amino acid combination sequence (SEQ ID NO: 6)

Clone number: PP3993

Start codon: 317 ATG Termination codon: 871 TAA

Protein molecular weight: 20611

1 T TCA GAC AAA CCT CAG GTA AGA TGG GAC TGG GCT TCC CCG CCA CTG 46

47 CGC AGC AGC TCC CGC CCT CCT GGG TGC TGT CCC AGG GGT GAA AGG AAG 94

95 AGG TGG GGA GGT ACA GCT GGG AGT GTG GGG GAT GGG GAA GGA TGG GGA 142

143 GGG AAC GGG CCC GTG GAC AAC TTG CAT TTG CCC TGA CAC CCA CCC TCC 190

191 CTG CAG CTC TAC ACC CTC ACC GTG ATC GGC CCA GGA CCG CCA GAT TGC 238

239 CAG CCA GCC CAG ATC TCT CGC CGT TAC TCG GAC TTT GAG CGG CTG CAC 286

287 CGA AAC CTG CAG CGG CAA TTC CGG GGC CCA ATG GCT GCC ATC TCC TTC 334

1 Met Ala Ala Ile Ser Phe 6

335 CCC CGT AAG CGG CTG CGC CGG AAT TTT ACT GCA GAG ACC ATT GCC CGC 382

7 Pro Arg Lys Arg Leu Arg Arg Asn Phe Thr Ala Glu Thr Ile Ala Arg 22

383 CGT AGC CGG GCC TTT GAG CAG TTT TTG GGT CAC CTG CAG GCA GTG CCT 430

23 Arg Ser Arg Ala Phe Glu Gln Phe Leu Gly His Leu Gln Ala Val Pro 38

431 GAG CTG CGC CAT GCC CCG GAC CTG CAG GAC TTC TTC GTG CTG CCG GAG 478

39 Glu Leu Arg His Ala Pro Asp Leu Gln Asp Phe Phe Val Leu Pro Glu 54

479 CTG CGG CGG GCA CAG AGC CTC ACC TGT ACT GGC CTC TAT CGT GAG GCT 526

55 Leu Arg Arg Ala Gln Ser Leu Thr Cys Thr Gly Leu Tyr Arg Glu Ala 70

527 CTG GCA CTC TGG GCC AAT GCC TGG CAG CTG CAA GCC CAG CTG GGC ACC 574

71 Leu Ala Leu Trp Ala Asn Ala Trp Gln Leu Gln Ala Gln Leu Gly Thr 86

575 CCC TCT GGC CCA GAC CGC CCC CTG CTG ACC CTG GCT GGG CTG GCC GTG 622

87 Pro Ser Gly Pro Asp Arg Pro Leu Leu Thr Leu Ala Gly Leu Ala Val 102

623 TGC CAC CAG GAG GAG CTG GAA GAC CCT GGA GAG GCC CGG GCA TGC TGT GAG 670

103 Cys His Gln Glu Leu Glu Asp Pro Gly Glu Ala Arg Ala Cys Cys Glu 118

671 AAG GCC CTG CAG CTG CTT GGG GAC AAG AGC CTC CAC CCT TTG CTG GCA 718

119 Lys Ala Leu Gln Leu Leu Gly Asp Lys Ser Leu His Pro Leu Leu Ala 134

719 CCC TTT CTG GAG GCC CAT GTC CGG CTC TCC TGG CGC CTG GGC CTG GAC 766

135 Pro Phe Leu Glu Ala His Val Arg Leu Ser Trp Arg Leu Gly Leu Asp 150

767 AAA CGT CAA TCA GAG GCT CGG CTC CAA GCC CTG CAG GAG GCA GGC CTT 814

151 Lys Arg Gln Ser Glu Ala Arg Leu Gln Ala Leu Gln Glu Ala Gly Leu 166

815 ACC CCC ACA CCA CCC CCC AGT CTC AAA GAA TTG CTC ATC AAG GAG GTG 862

167 Thr Pro Thr Pro Pro Pro Ser Leu Lys Glu Leu Leu Ile Lys Glu Val 182

863 CTG GAC TAA CCC TTG CCT AGA TTT AAG GCC ACT GTG AGG AGA GGG GTT 910

183 Leu Asp *** 185

911 GCC CCA GAA GGC AGG GGA AGG ACC TGA TGA GAA CAG AAT AGC TGG GAG 958

959 GCT GCA GAG GGT GCT GGG AGC CCC TAG AAG TTC CAA AAG AGA ATG TGA 1006

1007 AGC AGA TCA AGG AAA CTT CTG TTG AGC TAG GCT CAG GGT GAG CTT TGG 1054

1055 CTG GGG TTG CCC TTG TGT AGT ACA GGG AAG TCT GAC ACA GCC TCT CCA 1102

1103 GCC TAT AAA CAG CCG GGG GGC TGT GGC ACA GGT TGG GGC AAT GTT CCC 1150

1151 TTG TTG GTG GGC CCC CAA GCT GGC AAG GCC TCT TGG CTG AAG GCC AGG 1198

1199 GAC TCT GCC CCT GGA GTC CTG GAG TTA AGG GAT GAA GGC AAG GCT GCA 1246

1247 GGT CTG GCC CAG GGG AAT TAA AAG CCA GCC ACT CCA GTG GTA TCA GTC 1294

1295 TCT TTA TTG GAT GTG AGG GCC AAA AGG GAC TGT AAC TCC TGT CTC AGG 1342

1343 AAT GGG GAT AGA TGG GAG GTT CTT GAA GCC CCA GGC GAA GCT GGT ACC 1390

1391 TCT GGC TAC AGC TTG CTC TCT GAG ACC TGG GGC TTC ACT CGG ATC ACG 1438

1439 CCC TCC TGG GCA CAG GTC ACA GCT AGG ACT CCA TCC TGA CGC CAC AGC 1486

1487 CGC CCA TGG ACC AGC CCC CGA GAG CCA CCT GTG GGT GAG GTG AAG GGT 1534

1535 GAT GAT GGC CTG CTT CAG AAC AGC CAA ATA CAC TTT TTT TTT TTT TCC 1582

1583 TGA AAC AGA GTC CCA CTA AGT TGC CAG GCT GGT CTC AAG CCG CCT GGG 1630

1631 TTC AAG GGA TCC TCC CGC CTC AGC CTC CTG AGC AGC TGG GAT TAC AGG 1678

1679 CGC ACA TCA CCA TGC CCA ACC TCC AAG TGG ACT TCT TGC AAA GGG TCT 1726

1727 GGC CCA GGG CAG GGC TGC CCC ACA CAA GGG TGC ACT GAG TGT CGT GGC 1774

1775 TGC TCC AAA TGC CCC TTC ATG AGC TTA TTA TGG ACC GTC ATT GAG GGG 1822

1823 TAA CTC CTC CCA CAG GAA CCC CAG TTG ACA GTT TAA AAG CAC TTT TAC 1870

1871 ACC TCT CCT CGC TTC CTC AAA AAG ATC ACA GAG GGA GGA GCT CTG AGA 1918

1919 ACA GTC TCC TTC AAC AGC TCG GCC AAG CAG AAC TGC TGT ACC TCT GAC 1966

1967 CAC TTG TGT TAG GAA AAC TAT CGG CTC CCT GTA TAA TAA ATC AAG CCA 2014

2015 GGT CCT CCC CAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 2062

2063 AA 2064

3. PP4052 protein

A: Nucleotide sequence (SEQ ID NO: 7) Length: 1794bp

1 CTAAGAGAGC TTGGAAAGGG ATAGAGAAGT CTGACCCAAA TTTGCGGAGC

51 GACTGAGTGT ATGCTGCCCC CTTTCTGGGC CTTGGCTTCT TCCTCAATCA

101 TCTAGGCACA GTCCTATGAC TGCCTGTTTT TGAGGATGTG GGAAGGGTCT

151 GCAAATACAG TGCTTTTCCCA TTGACACACG CTGGTGAGGA TGCAGGCTCC

201 CTGGCACCAG CAGTGAGGGC TCAGATTGCA AGAGTAAAAA CTTCCATCAC

251 TGGGAAGAGA AGTCTGCAGG GGACTGGAGG TGATCTGAAG ATTCTGAAAT

301 AACTCTTCCT CTCTCTGCAG AGAAGGATGG TGCTTCATCC TGTTTAGGTA

351 AGGGTGATAG CCAAGTGTGT TCAGGGTGGC TGCACCAACC CACCCTGAGT

401 CCATGTGGCT CAAGACTGCT GCTCAGGTGG GGTCAGCTGA GTGGGTAGGA

451 AGTCGGGAGG CACTGCCTAG CAGGTTTCAA CTTTAGTCTG GAGGCTGCAT

501 CTGTCTCCTC TAAACACAGT GGTTTCCATT GGTTATACCC CAAAATCACA

551 CCAGGAAACT TTAAAACTAG TGACGACTGA GTCCCGTCTC CAGAAACTCT

601 GACTTAATTG TGGGGGTGTG GCCTGGACAT CAGGATTTGT AATAACTCCT

651 CAGGTGATTC TCATGTAGTC AAGGTTGAGA ACCACTGCTT TAACTTTCTC

701 CAAATCCTAA TTACTTCTAG TGTGGCCTGA GGACTGCACT TTGAATGGCA

751 AGGGCCAAAT AACCAAGTTT TTGTTTTTTT CCAGGAAGTG GAGAAGAATT

801 TGGGGTTGGG ACTAGACTGG GGGTGAGCTG GGGAGATGGA AGATGGAAGT

851 GGGGTCAGTG GGAGGCAATG ATGGTAGGTA TCTTGGAAGA AGGATGCTTA

901 ATTTTAACAC GGAAAGACTG GAAGAAGGGA AGATACAAAG AGGTGGTCTC

951 CAGAAGACAG TCAGACAAAT AACAGAGCCT TAGAAATAAA ACCTTTTGGG

1001 CTGGGTGGCC CAGGTTTCCA ATTGCTGCCT CTACAGGAAA GTCTTTGGGT

1051 TCGGAGGATT CCAGAAACAT GAAGGAGAAG TTGGAGGACA TGGAGAGTGT

1101 CCTCAAGGAC CTGACAGAGG AGAAGAGAAA AGATGTGCTA AACTCCCTCG

1151 CTAAGTGCCT CGGCAAGGAG GATATTCGGC AGGATCTAGA GCAAAGAGTA

1201 TCTGAGGTCC TGATTTCCGG GGAGCTACAC ATGGAGGACC CAGACAAGCC

1251 TCTCCTAAGC AGCCTTTTTA ATGCTGCTGG GGTCTTGGTA GAAGCGCGTG

1301 CAAAAGCCAT TCTGGACTTC CTGGATGCCC TGCTAGAGCT GTCTGAAGAG

1351 CAGCAGTTTG TGGCTGAGGC CCTGGAGAAG GGGACCCTTC CTCTGTTGAA

1401 GGACCAGGTG AAATCTGTCA TGGAGCAGAA CTGGGATGAG CTGGCCAGCA

1451 GTCCTCCTGA CATGGACTAT GACCCTGAGG CACGAATTCT CTGTGCGCTG

1501 TATGTTGTTG TTCTCTATCCT GCTGGAGCTG GCTGAGGGGC CTACCTCTGT

1551 CTCTTCCTAA CTACAAAAGC CCTTTCTCCC CACAAGCCTC TGGGTTTTCC

1601 CTTTACCAGT CTGTCCTCAC TGCCATCGCC ACTACCATCC TGTCACCAGT

1651 GGGACCTCTT TAAAACAAGC AGCCAACCAT TCTTTGATGT ATCCCATTCG

1701 CTCCATGTTA ACATCCAAAA CCAGCCTGGA TTTCATACAT GGACTTCTGA

1751 TTAAAAGTGG CAGGTTGTGC ATGTTAAAAA AAAAAAAAAA AAAA

B: Amino acid sequence (SEQ ID NO: 8) Length: 163 amino acids

1 MKEKLEDMES VLKDLTEEKR KDVLNSLAKC LGKEDIRQDL EQRVSEVLIS GELHMEDPDK

61 PLLSSLFNAA GVLVEARAKA ILDFLDALLE LSEEQQFVAE ALEKGTLPLL KDQVKSVMEQ

121 NWDELASSPP DMDYDPEARI LCALYVVVSI LLELAEGPTS VSS

C. Nucleotide and amino acid combination sequence (SEQ ID NO: 9)

Clone number: PP4052

Start code: 1069 ATG End code: 1560 TAA

Protein molecular weight: 18158

1 CTA AGA GAG CTT GGA AAG GGA TAG AGA AGT CTG ACC CAA ATT TGC GGA 48

49 GCG ACT GAG TGT ATG CTG CCC CCT TTC TGG GCC TTG GCT TCT TCC TCA 96

97 ATC ATC TAG GCA CAG TCC TAT GAC TGC CTG TTT TTG AGG ATG TGG GAA 144

145 GGG TCT GCA AAT ACA GTG CTT TCC CAT TGA CAC ACG CTG GTG AGG ATG 192

193 CAG GCT CCC TGG CAC CAG CAG TGA GGG CTC AGA TTG CAA GAG TAA AAA 240

241 CTT CCA TCA CTG GGA AGA GAA GTC TGC AGG GGA CTG GAG GTG ATC TGA 288

289 AGA TTC TGA AAT AAC TCT TCC TCT CTC TGC AGA GAA GGA TGG TGC TTC 336

337 ATC CTG TTT AGG TAA GGG TGA TAG CCA AGT GTG TTC AGG GTG GCT GCA 384

385 CCA ACC CAC CCT GAG TCC ATG TGG CTC AAG ACT GCT GCT CAG GTG GGG 432

433 TCA GCT GAG TGG GTA GGA AGT CGG GAG GCA CTG CCT AGC AGG TTT CAA 480

481 CTT TAG TCT GGA GGC TGC ATC TGT CTC CTC TAA ACA CAG TGG TTC TCA 528

529 TTG GTT ATA CCC CAA AAT CAC ACC AGG AAA CTT TAA AAC TAG TGA CGA 576

577 CTG AGT CCC GTC TCC AGA AAC TCT GAC TTA ATT GTG GGG GTG TGG CCT 624

625 GGA CAT CAG GAT TTG TAA TAA CTC CTC AGG TGA TTC TCA TGT AGT CAA 672

673 GGT TGA GAA CCA CTG CTT TAA CTT TCT CCA AAT CCT AAT TAC TTC TAG 720

721 TGT GGC CTG AGG ACT GCA CTT TGA ATG GCA AGG GCC AAA TAA CCA AGT 768

769 TTT TGT TTT TTT CCA GGA AGT GGA GAA GAA TTT GGG GTT GGG ACT AGA 816

817 CTG GGG GTG AGC TGG GGA GAT GGA AGA TGG AAG TGG GGT CAG TGG GAG 864

865 GCA ATG ATG GTA GGT ATC TTG GAA GAA GGA TGC TTA ATT TTA ACA CGG 912

913 AAA GAC TGG AAG AAG GGA AGA TAC AAA GAG GTG GTC TCC AGA AGA CAG 960

961 TCA GAC AAA TAA CAG AGC CTT AGA AAT AAA ACC TTT TGG GCT GGG TGG 1008

1009 CCC AGG TTC TCA ATT GCT GCC TCT ACA GGA AAG TCT TTG GGT TCG GAG 1056

1057 GAT TCC AGA AAC ATG AAG GAG AAG TTG GAG GAC ATG GAG AGT GTC CTC 1104

1 Met Lys Glu Lys Leu Glu Asp Met Glu Ser Val Leu 12

1105 AAG GAC CTG ACA GAG GAG AAG AGA AAA GAT GTG CTA AAC TCC CTC GCT 1152

13 Lys Asp Leu Thr Glu Glu Lys Arg Lys Asp Val Leu Asn Ser Leu Ala 28

1153 AAG TGC CTC GGC AAG GAG GAT ATT CGG CAG GAT CTA GAG CAA AGA GTA 1200

29 Lys Cys Leu Gly Lys Glu Asp Ile Arg Gln Asp Leu Glu Gln Arg Val 44

1201 TCT GAG GTC CTG ATT TCC GGG GAG CTA CAC ATG GAG GAC CCA GAC AAG 1248

45 Ser Glu Val Leu Ile Ser Gly Glu Leu His Met Glu Asp Pro Asp Lys 60

1249 CCT CTC CTA AGC AGC CTT TTT AAT GCT GCT GGG GTC TTG GTA GAA GCG 1296

61 Pro Leu Leu Ser Ser Ser Leu Phe Asn Ala Ala Gly Val Leu Val Glu Ala 76

1297 CGT GCA AAA GCC ATT CTG GAC TTC CTG GAT GCC CTG CTA GAG CTG TCT 1344

77 Arg Ala Lys Ala Ile Leu Asp Phe Leu Asp Ala Leu Leu Glu Leu Ser 92

1345 GAA GAG CAG CAG TTT GTG GCT GAG GCC CTG GAG AAG GGG ACC CTT CCT 1392

93 Glu Glu Gln Gln Phe Val Ala Glu Ala Leu Glu Lys Gly Thr Leu Pro 108

1393 CTG TTG AAG GAC CAG GTG AAA TCT GTC ATG GAG CAG AAC TGG GAT GAG 1440

109 Leu Leu Lys Asp Gln Val Lys Ser Val Met Glu Gln Asn Trp Asp Glu 124

1441 CTG GCC AGC AGT CCT CCT GAC ATG GAC TAT GAC CCT GAG GCA CGA ATT 1488

125 Leu Ala Ser Ser Pro Pro Asp Met Asp Tyr Asp Pro Glu Ala Arg Ile 140

1489 CTC TGT GCG CTG TAT GTT GTT GTC TCT ATC CTG CTG GAG CTG GCT GAG 1536

141 Leu Cys Ala Leu Tyr Val Val Val Ser Ile Leu Leu Glu Leu Ala Glu 156

1537 GGG CCT ACC TCT GTC TCT TCC TAA CTA CAA AAG CCC TTT CTC CCC ACA 1584

157 Gly Pro Thr Ser Val Ser Ser *** 164

1585 AGC CTC TGG GTT TTC CCT TTA CCA GTC TGT CCT CAC TGC CAT CGC CAC 1632

1633 TAC CAT CCT GTC ACC AGT GGG ACC TCT TTA AAA CAA GCA GCC AAC CAT 1680

1681 TCT TTG ATG TAT CCC ATT CGC TCC ATG TTA ACA TCC AAA ACC AGC CTG 1728

1729 GAT TTC ATA CAT GGA CTT CTG ATT AAA AGT GGC AGG TTG TGC ATG TTA 1776

1777 AAA AAA AAA AAA AAA AAA 1794

4. PP4068 protein

A: Nucleotide sequence (SEQ ID NO: 10) Length: 1949bp

1 GGAAGGCAAA GGTAGAGCAA CTGGATCTCT GGCTCTCCAC ATAGCTTCTG

51 ATCTCAGACC TTACTAAAAT GCTTTCTGGG CCCAAGGACA AAGCTCACAT

101 GAACAAATGA TTTTGAGTCA TGAATGAAAA ATCTTGCTCT TTCCATAGTA

151 AAGAAGAATT AAGAGATGGA CAGGGTGAAA GATTGTCTGC TGGATATTCT

201 CCATCATATG ACAAGGACAA GAGTGTTCTG GCTTTCAGAG GAATCCCTAT

251 CTCAGAGTTG AAGAACCATG GCATTCTCCA GGCTCTGACC ACAGAAGCTT

301 ATGAATGGGA GCCACGTGTT GTGAGTACAG AGGTGGTCAG AGCCCAAGAA

351 GAATGGGAAG CTGTGGACAC CATCCAGCCA GAGACAGGGA GCCAAGCTAG

401 CTCAGAGCAG CCTGGGCAGC TAATCTCCTT CGGTGAGGCC CTGCAGCACT

451 TCCAGACTGT GGACCTTTTCC CCCTTCAAGA AAAGAATCCA GCCAACTATT

501 CGAAGGACTG GGCTCGCCGC CCTCCGACAC TACCTCTTCG GGCCTCCAAA

551 GCTCCACCAG CGCCTTCGGG AAGAAAGGGA CTTGGTCCTG ACCATTGCTC

601 AGTGAGCGAA TCCAGCCACA GACCTGAGAG GCGCAGGCTT CCTTGCCCTC

651 CTGCATCTGC TCTACCTGGT GATGGACTCA AAGACCTTGC CGATGGCGCA

701 GGAGATTTTC CGCCTGTCTC GTCACCACAT CCAGCAATTC CCTTTCTGTT

751 TGATGTCCGT GAACATCACC CACATTGCCA TCCAGGCCTT GAGAGAGGAG

801 TGTCTCTCCA GAGAGTGTAA TCGGCAGCAG AAGGTCATCC CCGTGGTGAA

851 CAGCTTCTAT GCCGCCACAT TCCTCCACCT CGCACATGTC TGGAGGACAC

901 AGCGGAAGAC CATCTCAGAC TCGGGCTTTG TCCTCAAAGA GTTGGAAGTA

951 TTGGCCAAGA AGAGCCCACG GCGGCTGCTC AAGACCCTGG AGCTGTACTT

1001 GGCCAGGGTG TCAAAGGGAC AGGCCTCCTT GTTGGGAGCA CAGAAGTGCT

1051 ATGGGCCAGA AGCCCCTCCC TTCAAGGATC TCACCTTCAC AGGTGAGAGT

1101 GACCTGCAGT CTCACTCATC CGAAGGCGTA TGGCTGATCT GACCTCCGAG

1151 ATGAATGGAG GCTTAAAGGC TGAGCTGCAG GGGCTTTCAG GGGGTCAGTG

1201 GAGCCATGTC AGGAGCCTGG CCAGGCCGCA CCCCTTGCTG TCTCAGCAGA

1251 TGGGATATAG GAAGCTCCTG GGCTTAGCTG TGGGAAGCCA AGTACCCTCA

1301 CCGGCATGGG ACATGAGGGG CAGCTAGACT TCACCCCCTT CCCGCAGACC

1351 TGCCTCCAGA GCAAGGAGAA TTCTGCCTTA ATCTGTTGGG CTCCAGTCTC

1401 CGGGTTGAAT TCCAGTGTAT CCCACTGGGA GTGAATGGAT CATGAGGTGG

1451 GATGGCCCCA TCTGGATGTT CTGCAGATCC CCACATGGGA GGAGATTCCC

1501 AAGTAGAGGC AGCCAGAATT CTGACTCCCT GGCAGCAGCA GGGCTTTCAG

1551 GCACCAGTGT CTGTGTTGTT AGAACTCAGA GGAAGAGGCA GGGGCAGCAG

1601 CCGACAGGGG GCAGCATGAC CCAAGCAAGG GGCCTGAGGC CTCAGTGGGG

1651 GTAGGGCAAG GAGGTACCTC ACAGGTGGGT GTGAGGCCCC CTCTGGAGTT

1701 TCTGGCCATT CACTTACCCA CTCTCTTCTC CCCCTGACCC CCGCTCCATT

1751 GTTTATGATG GAAAAACGGA CATTTGGCTA GGTGTCTCTC ACGGCTGCTC

1801 CATCCTAGCC CCCACAAGCT GGGGCTTCCT CCTGTAGATG CTGTGCATGC

1851 CCCATGATGA GTTTCTGGCC TAATTGAGGG AAGGAGGAAA TTCATACCAG

1901 CAGTTTTTCAA ATAAAAGAAT TGTTCTAATT AAAAAAAAAA AAAAAAAAA

B: Amino acid sequence (SEQ ID NO: 11) Length: 161 amino acids

1 MNEKSCSFHS KEELRDGQGE RLSAGYSPSY DKDKSVLAFR GIPISELKNH GILQALTTEA

61 YEWEPRVVST EVVRAQEEWE AVDTIQPETG SQASSEQPGQ LISFGEALQH FQTVDLSPFK

121 KRIQPTIRRT GLAALRHYLF GPPKLHQRLR EERDLVLTIA Q

C. Nucleotide and amino acid combination sequence (SEQ ID NO: 12)

Clone number: PP4068

Start code: 120 ATG End code: 605 TGA

Protein molecular weight: 18261

1 GG AAG GCA AAG GTA GAG CAA CTG GAT CTC TGG CTC TCC ACA TAG CTT 47

48 CTG ATC TCA GAC CTT ACT AAA ATG CTT TCT GGG CCC AAG GAC AAA GCT 95

96 CAC ATG AAC AAA TGA TTT TGA GTC ATG AAT GAA AAA TCT TGC TCT TTC 143

1 Met Asn Glu Lys Ser Cys Ser Phe 8

144 CAT AGT AAA GAA GAA TTA AGA GAT GGA CAG GGT GAA AGA TTG TCT GCT 191

9 His Ser Lys Glu Glu Leu Arg Asp Gly Gln Gly Glu Arg Leu Ser Ala 24

192 GGA TAT TCT CCA TCA TAT GAC AAG GAC AAG AGT GTT CTG GCT TTC AGA 239

25 Gly Tyr Ser Pro Ser Tyr Asp Lys Asp Lys Ser Val Leu Ala Phe Arg 40

240 GGA ATC CCT ATC TCA GAG TTG AAG AAC CAT GGC ATT CTC CAG GCT CTG 287

41 Gly Ile Pro Ile Ser Glu Leu Lys Asn His Gly Ile Leu Gln Ala Leu 56

288 ACC ACA GAA GCT TAT GAA TGG GAG CCA CGT GTT GTG AGT ACA GAG GTG 335

57 Thr Thr Glu Ala Tyr Glu Trp Glu Pro Arg Val Val Ser Thr Glu Val 72

336 GTC AGA GCC CAA GAA GAA TGG GAA GCT GTG GAC ACC ATC CAG CCA GAG 383

73 Val Arg Ala Gln Glu Glu Trp Glu Ala Val Asp Thr Ile Gln Pro Glu 88

384 ACA GGG AGC CAA GCT AGC TCA GAG CAG CCT GGG CAG CTA ATC TCC TTC 431

89 Thr Gly Ser Gln Ala Ser Ser Glu Gln Pro Gly Gln Leu Ile Ser Phe 104

432 GGT GAG GCC CTG CAG CAC TTC CAG ACT GTG GAC CTT TCC CCC TTC AAG 479

105 Gly Glu Ala Leu Gln His Phe Gln Thr Val Asp Leu Ser Pro Phe Lys 120

480 AAA AGA ATC CAG CCA ACT ATT CGA AGG ACT GGG CTC GCC GCC CTC CGA 527

121 Lys Arg Ile Gln Pro Thr Ile Arg Arg Thr Gly Leu Ala Ala Leu Arg 136

528 CAC TAC CTC TTC GGG CCT CCA AAG CTC CAC CAG CGC CTT CGG GAA GAA 575

137 His Tyr Leu Phe Gly Pro Pro Lys Leu His Gln Arg Leu Arg Glu Glu 152

576 AGG GAC TTG GTC CTG ACC ATT GCT CAG TGA GCG AAT CCA GCC ACA GAC 623

153 Arg Asp Leu Val Leu Thr Ile Ala Gln *** 162

624 CTG AGA GGC GCA GGC TTC CTT GCC CTC CTG CAT CTG CTC TAC CTG GTG 671

672 ATG GAC TCA AAG ACC TTG CCG ATG GCG CAG GAG ATT TTC CGC CTG TCT 719

720 CGT CAC CAC ATC CAG CAA TTC CCT TTC TGT TTG ATG TCC GTG AAC ATC 767

768 ACC CAC ATT GCC ATC CAG GCC TTG AGA GAG GAG TGT CTC TCC AGA GAG 815

816 TGT AAT CGG CAG CAG AAG GTC ATC CCC GTG GTG AAC AGC TTC TAT GCC 863

864 GCC ACA TTC CTC CAC CTC GCA CAT GTC TGG AGG ACA CAG CGG AAG ACC 911

912 ATC TCA GAC TCG GGC TTT GTC CTC AAA GAG TTG GAA GTA TTG GCC AAG 959

960 AAG AGC CCA CGG CGG CTG CTC AAG ACC CTG GAG CTG TAC TTG GCC AGG 1007

1008 GTG TCA AAG GGA CAG GCC TCC TTG TTG GGA GCA CAG AAG TGC TAT GGG 1055

1056 CCA GAA GCC CCT CCC TTC AAG GAT CTC ACC TTC ACA GGT GAG AGT GAC 1103

1104 CTG CAG TCT CAC TCA TCC GAA GGC GTA TGG CTG ATC TGA CCT CCG AGA 1151

1152 TGA ATG GAG GCT TAA AGG CTG AGC TGC AGG GGC TTT CAG GGG GTC AGT 1199

1200 GGA GCC ATG TCA GGA GCC TGG CCA GGC CGC ACC CCT TGC TGT CTC AGC 1247

1248 AGA TGG GAT ATA GGA AGC TCC TGG GCT TAG CTG TGG GAA GCC AAG TAC 1295

1296 CCT CAC CGG CAT GGG ACA TGA GGG GCA GCT AGA CTT CAC CCC CTT CCC 1343

1344 GCA GAC CTG CCT CCA GAG CAA GGA GAA TTC TGC CTT AAT CTG TTG GGC 1391

1392 TCC AGT CTC CGG GTT GAA TTC CAG TGT ATC CCA CTG GGA GTG AAT GGA 1439

1440 TCA TGA GGT GGG ATG GCC CCA TCT GGA TGT TCT GCA GAT CCC CAC ATG 1487

1488 GGA GGA GAT TCC CAA GTA GAG GCA GCC AGA ATT CTG ACT CCC TGG CAG 1535

1536 CAG CAG GGC TTT CAG GCA CCA GTG TCT GTG TTG TTA GAA CTC AGA GGA 1583

1584 AGA GGC AGG GGC AGC AGC CGA CAG GGG GCA GCA TGA CCC AAG CAA GGG 1631

1632 GCC TGA GGC CTC AGT GGG GGT AGG GCA AGG AGG TAC CTC ACA GGT GGG 1679

1680 TGT GAG GCC CCC TCT GGA GTT TCT GGC CAT TCA CTT ACC CAC TCT CTT 1727

1728 CTC CCC CTG ACC CCC GCT CCA TTG TTT ATG ATG GAA AAA CGG ACA TTT 1775

1776 GGC TAG GTG TCT CTC ACG GCT GCT CCA TCC TAG CCC CCA CAA GCT GGG 1823

1824 GCT TCC TCC TGT AGA TGC TGT GCA TGC CCC ATG ATG AGT TTC TGG CCT 1871

1872 AAT TGA GGG AAG GAG GAA ATT CAT ACC AGC AGT TTT CAA ATA AAA GAA 1919

1920 TTG TTC TAA TTA AAA AAA AAA AAA AAA AAA 1949

5. PP4135 protein

A: Nucleotide sequence (SEQ ID NO: 13) Length: 1585bp

1 CATACTTGGT CTATCTTCTA CTTTGTCTTC TCTTAGGACC CAAGGTCTCT

51 TAGCACAAAC ACTGCCTCTT GAGTTCCCAG TGCATCTGTA TCAATCTGGA

101 GATTACGTCC TCATCAAAAG CTGGAAAGAA GAAAAACTCG AACCAACCTG

151 GGAGACCTTA TCTAGTGCCC CTAACCACTG AGACAGCAGT CTGGACCGTT

201 AAGAAAGGGT AGACCCATCA CACTCAGGTG AAAAAGGCAT GACCCCCTTT

251 GGAGGCATAG GTTGTCACTC CCGGGCCAAC ACCTTCCAAA CTAATATTCA

301 AAAAAACTTA ACCTGTCTAA TTTGCTTCCT CTTTCTTTCG TTAGCTACCC

351 AGGAACATTT TATTTTATCA ATGTAACCTG ATCATCGTTT CCTCAAACAA

401 TTACATTTGA TGCTTGTCTT GTTATGCCCT GTGGGGACCT ACAAACCCAA

451 AGGCAACTAG CCTCTTCAGG CAATAACATA CAGGACATAG GAATGGGCAA

501 AGACTTCATG ACTAAAACAC CAAAAGCAAC AGCAACAAAA GCCAAAATTG

551 ACAAATGGGA TCTAATTAAA CTAAAGAGAT TCTGCGCAGC AAAGGAAACT

601 ATCATCAGAG TGAACAGGCA ACCTACAGAA TGGGAGAAAA TTTTTGCAAT

651 CTATCCATTT GACAAAGGGC TAATATCCAG AATCTATAAA GAACTTAAAT

701 TTACAAGAAA AAAACAACCC CATCAAAAAG CGGGCGAAGG ATATGAACAG

751 ACACTTCTCC AAAGAAGACA TTTATGCAGC CAACAAACAT GAAAAATAGC

801 TGATCATCAC TGGTCATTAT AGAAATGCAA ATCAAAACCA CAGTAAGATA

851 CTAACTCATG CCAGTTAGAA TGGCGATCAT TAAAAAGTCA GGAAACAACA

901 GATGCTGGAG AGGATGTGCA GAAATAGGAA TGCTTTTTAT ACTGTTGGTG

951 AAAGTGTAAA TTAGTTCAAC CATTGTGGAA GACAGTGTGG CGATTCCTCA

1001 AGGATCTATA GAACCAGAAC TACCACATGA CCCAGCAATC CCATTACTGG

1051 GTATATACCC AAAGGATTAT ACATCATTCT GCTATAAAGA CACATGCACA

1101 CGTACGTTTA TTGCAGCACT ATTTACAATA GCAAAGACTT GGAATCAACC

1151 CAAATCCCCG TCAATGATAG ACTGGATAAA GACAATGTGG CACTTATACA

1201 CCATGGAATA CTATGCAGCC AAAAAAAGGA TGAATTCATG TCCTTTGCAG

1251 CGACATGGAT GAAGCTGAAA ACCATCATTC TCAGCAAACT AACACGAGAA

1301 CAGAAAACCA AACACTACAT GTTCTCACTC ATAAGTGGGA GTTGAACAAT

1351 GAGAACACAT GGACACACGG AGGGGAACAC CACACACCAG GGCCTGTCGG

1401 CGGGTGGGGA GGCTAGGGGA GGGATAGCAT TAGGAGAAAT ACCTAATGTA

1451 GATGACAGGT TGATGGGTCT GACAAACCAC CATGACACGT GTATACCTAT

1501 GTAATGCAAC TGCACATTTT GCTCATGTAC CCCAGAACTT AAACTATAAT

1551 TAAAAACACA TAATTTCAAA AAAAAAAAAA AAAAA

B: Amino acid sequence (SEQ ID NO: 14) Length: 122 amino acids

1 MPCGDLQTQR QLASSGNNIQ DIGMGKDFMT KTPKATATKA KIDKWDLIKL KRFCAAKETI

61 IRVNRQPTEW EKIFAIYPFD KGLISRIYKE LKFTRKKQPH QKAGEGYEQT LLQRRHLCSQ

121 QT

C. Nucleotide and amino acid combination sequence (SEQ ID NO: 15)

Clone number: PP424

Start code: 424 ATG End code: 792 TGA

Protein molecular weight: 14172

1 CAT ACT TGG TCT ATC TTC TAC TTT GTC TTC TCT TAG GAC CCA AGG TCT 48

49 CTT AGC ACA AAC ACT GCC TCT TGA GTT CCC AGT GCA TCT GTA TCA ATC 96

97 TGG AGA TTA CGT CCT CAT CAA AAG CTG GAA AGA AGA AAA ACT CGA ACC 144

145 AAC CTG GGA GAC CTT ATC TAG TGC CCC TAA CCA CTG AGA CAG CAG TCT 192

193 GGA CCG TTA AGA AAG GGT AGA CCC ATC ACA CTC AGG TGA AAA AGG CAT 240

241 GAC CCC CTT TGG AGG CAT AGG TTG TCA CTC CCG GGC CAA CAC CTT CCA 288

289 AAC TAA TAT TCA AAA AAA CTT AAC CTG TCT AAT TTG CTT CCT CTT TCT 336

337 TTC GTT AGC TAC CCA GGA ACA TTT TAT TTT ATC AAT GTA ACC TGA TCA 384

385 TCG TTT CCT CAA ACA ATT ACA TTT GAT GCT TGT CTT GTT ATG CCC TGT 432

1 Met Pro Cys 3

433 GGG GAC CTA CAA ACC CAA AGG CAA CTA GCC TCT TCA GGC AAT AAC ATA 480

4 Gly Asp Leu Gln Thr Gln Arg Gln Leu Ala Ser Ser Gly Asn Asn Ile 19

481 CAG GAC ATA GGA ATG GGC AAA GAC TTC ATG ACT AAA ACA CCA AAA GCA 528

20 Gln Asp Ile Gly Met Gly Lys Asp Phe Met Thr Lys Thr Pro Lys Ala 35

529 ACA GCA ACA AAA GCC AAA ATT GAC AAA TGG GAT CTA ATT AAA CTA AAG 576

36 Thr Ala Thr Lys Ala Lys Ile Asp Lys Trp Asp Leu Ile Lys Leu Lys 51

577 AGA TTC TGC GCA GCA AAG GAA ACT ATC ATC AGA GTG AAC AGG CAA CCT 624

52 Arg Phe Cys Ala Ala Lys Glu Thr Ile Ile Arg Val Asn Arg Gln Pro 67

625 ACA GAA TGG GAG AAA ATT TTT GCA ATC TAT CCA TTT GAC AAA GGG CTA 672

68 Thr Glu Trp Glu Lys Ile Phe Ala Ile Tyr Pro Phe Asp Lys Gly Leu 83

673 ATA TCC AGA ATC TAT AAA GAA CTT AAA TTT ACA AGA AAA AAA CAA CCC 720

84 Ile Ser Arg Ile Tyr Lys Glu Leu Lys Phe Thr Arg Lys Lys Gln Pro 99

721 CAT CAA AAA GCG GGC GAA GGA TAT GAA CAG ACA CTT CTC CAA AGA AGA 768

100 His Gln Lys Ala Gly Glu Gly Tyr Glu Gln Thr Leu Leu Gln Arg Arg 115

769 CAT TTA TGC AGC CAA CAA ACA TGA AAA ATA GCT GAT CAT CAC TGG TCA 816

116 His Leu Cys Ser Gln Gln Thr *** 123

817 TTA TAG AAA TGC AAA TCA AAA CCA CAG TAA GAT ACT AAC TCA TGC CAG 864

865 TTA GAA TGG CGA TCA TTA AAA AGT CAG GAA ACA ACA GAT GCT GGA GAG 912

913 GAT GTG CAG AAA TAG GAA TGC TTT TTA TAC TGT TGG TGA AAG TGT AAA 960

961 TTA GTT CAA CCA TTG TGG AAG ACA GTG TGG CGA TTC CTC AAG GAT CTA 1008

1009 TAG AAC CAG AAC TAC CAC ATG ACC CAG CAA TCC CAT TAC TGG GTA TAT 1056

1057 ACC CAA AGG ATT ATA CAT CAT TCT GCT ATA AAG ACA CAT GCA CAC GTA 1104

1105 CGT TTA TTG CAG CAC TAT TTA CAA TAG CAA AGA CTT GGA ATC AAC CCA 1152

1153 AAT CCC CGT CAA TGA TAG ACT GGA TAA AGA CAA TGT GGC ACT TAT ACA 1200

1201 CCA TGG AAT ACT ATG CAG CCA AAA AAA GGA TGA ATT CAT GTC CTT TGC 1248

1249 AGC GAC ATG GAT GAA GCT GAA AAC CAT CAT TCT CAG CAA ACT AAC ACG 1296

1297 AGA ACA GAA AAC CAA ACA CTA CAT GTT CTC ACT CAT AAG TGG GAG TTG 1344

1345 AAC AAT GAG AAC ACA TGG ACA CAC GGA GGG GAA CAC CAC ACA CCA GGG 1392

1393 CCT GTC GGC GGG TGG GGA GGC TAG GGG AGG GAT AGC ATT AGG AGA AAT 1440

1441 ACC TAA TGT AGA TGA CAG GTT GAT GGG TCT GAC AAA CCA CCA TGA CAC 1488

1489 GTG TAT ACC TAT GTA ATG CAA CTG CAC ATT TTG CTC ATG TAC CCC AGA 1536

1537 ACT TAA ACT ATA ATT AAA AAC ACA TTT TTT CAA AAA AAA AAA AAA AAA 1584

1585 A 1585

6. PP4189 protein

A: Nucleotide sequence (SEQ ID NO: 16) Length: 1762bp

1 GAAAAAGGTA GAATTCTTAT GAATGTTTCA TGATCATGTA TGTGTGGTTA

51 ACGCAGATAC ATTTGGAAGT CGGTTATCAA GGTTGCTCTC CAGCTCAAGG

101 CCTTTGCCCT TTTTGTCAAA ACCAAAGAAG TTCCAACAAA AAGGAGTTTT

151 GAATGTAAAG AAAAATTGTG GAAATGCTGT CAGCAGCTAT TCACAGACCA

201 AACCAGCATC CATAGACATG TGGCAACACA ACATGCTGAT GAAATTTATC

251 ACCAGACAGC TTCTATTTTA AAGCAACTGG CTGTGACATT GAGCACCTCA

301 AAGAGTCTTT CGTCTGCAGA TGAAAAGAAC CCTTTAAAAG AGTGCCTTCC

351 ACATAGCCAT GACGTGTCTG CTTGGCTCCC TGATATAAGC TGCTTTAACC

401 CTGATGAGCT GATAAGTGGC CAGGGCAGTG AAGAAGGGGA GGTGCTCCTT

451 TATTACTGCT ACCATGACCT GGAGGATCCC CAATGGATCT GTGCCTGGCA

501 GACAGCTCTG TGTCAGCACC TGCACCTCAC AGGCAAGGAA TCCATTTATC

551 CCCAGGTGAA TTTCATAAAG AAGTAGAAAAA GTTTTTATCT CAGGCAAATC

601 AAGAACAAAG TGATACTATC CTTCTTGATT GCAGAAACTT CTATGAAAGC

651 AAAATAGGAC GATTCCAAGG CTGCTTAGCC CCAGACATCA GGAAATTCAG

701 TTACTTCCCT AGCTACGTTG ACAAAAATCTAGAACTTTTC AGAGAGAAGA

751 GAGTGCTGAT GTACTGTACC GGGGGCATCC GCTGTGAGCG GGGTTCAGCC

801 TACCTCAAAG CCAAGGGAGT GTGCAAGGAG GTGTTCCAGC TCAAGGGTGG

851 CATCCACAAG TACCTGGAAG AGTTTCCTGA TGGCTTTTAC AAAGGGAAGT

901 TGTTTGTTTT TGATGAACGC TATGCTCTGT CCTACAACAG TGATGTGGTG

951 TCAGGTAGGT CAGCACAGGC TCAGAGCCCA AACTGAAATG AAGCACATTG

1001 TCAGTTCACC ATTCTAGAAA AATGACACAG GGAAGACAGG CCAGTGCTCA

1051 TTACTGAGCA CTGAATAAGC AGGGAAAATA AGTACATTGT GCCACCATTT

1101 TCCCAGCTGT GGAGCTGAGA GAACCCTAGC CCAGGAGTCA GGAGGCCTGG

1151 GTTGGGATCC TGGCTTCACC ATTGCTAGCT GGACAAGCCC ATTAACATGG

1201 GGATCATCTC ACCTGCCCTG CCTGCCTGTC TACCTGCCAA GAGCTGTACT

1251 ACTGGGCTAA TTCAGGGCTC TTAACCTGGA ATTGGTACAT AGATTTCAGG

1301 GATTCTGTGA ATTTGGATGG AAAAATAATT GTATCTTTGT TTTCAATAAC

1351 ACCTCACTAA AATGAAGCAT TTCCTTTAGT TATGAATGTA GGCAACAAAG

1401 TACCAGTTGT ATTAATGTAC CTGTGACTTT GTCTTCAGTA GGATTCACAA

1451 TACTTTCATA TCATGTTCTA GTTGCCTCAG ATATCTCAAA ATAGTATTTA

1501 TACTCATCAC TGCTTCAAAA TGAAAATAGT TATTAGGCCC ACCACTAAGA

1551 GTTGATATAT AATGTGTTAA TAAATGGCAC GTCTTATTAT ATATTACAGA

1601 TTTTGAAAAA GAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAACAAAAAA

1651 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

1701 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

1751 AAAAAAAAAAA AA

B: Amino acid sequence (SEQ ID NO: 17) Length: 167 amino acids

1 MDLCLADSSV SAPAPHRQGI HLSPGEFHKE VEKFLSQANQ EQSDTILLDC RNFYESKIGR

61 FQGCLAPDIR KFSYFPSYVD KNLELFREKR VLMYCTGGIR CERGSAYLKA KGVCKEVFQL

121 KGGIHKYLEE FPDGFYKGKL FVFDERYALS YNSDVVSGRS AQAQSPN

C. Nucleotide and amino acid combination sequence (SEQ ID NO: 18)

Clone number: PP4198

Start code: 483 ATG End code: 986 TGA

Protein molecular weight: 19000

1 GA AAA AGG TAG AAT TCT TAT GAA TGT TTC ATG ATC ATG TAT GTG TGG 47

48 TTA ACG CAG ATA CAT TTG GAA GTC GGT TAT CAA GGT TGC TCT CCA GCT 95

96 CAA GGC CTT TGC CCT TTT TGT CAA AAC CAA AGA AGT TCC AAC AAA AAG 143

144 GAG TTT TGA ATG TAA AGA AAA ATT GTG GAA ATG CTG TCA GCA GCT ATT 191

192 CAC AGA CCA AAC CAG CAT CCA TAG ACA TGT GGC AAC ACA ACA TGC TGA 239

240 TGA AAT TTA TCA CCA GAC AGC TTC TAT TTT AAA GCA ACT GGC TGT GAC 287

288 ATT GAG CAC CTC AAA GAG TCT TTC GTC TGC AGA TGA AAA GAA CCC TTT 335

336 AAA AGA GTG CCT TCC ACA TAG CCA TGA CGT GTC TGC TTG GCT CCC TGA 383

384 TAT AAG CTG CTT TAA CCC TGA TGA GCT GAT AAG TGG CCA GGG CAG TGA 431

432 AGA AGG GGA GGT GCT CCT TTA TTA CTG CTA CCA TGA CCT GGA GGA TCC 479

480 CCA ATG GAT CTG TGC CTG GCA GAC AGC TCT GTG TCA GCA CCT GCA CCT 527

1 Met Asp Leu Cys Leu Ala Asp Ser Ser Val Ser Ala Pro Ala Pro 15

528 CAC AGG CAA GGA ATC CAT TTA TCC CCA GGT GAA TTT CAT AAA GAA GTA 575

16 His Arg Gln Gly Ile His Leu Ser Pro Gly Glu Phe His Lys Glu Val 31

576 GAA AAG TTT TTA TCT CAG GCA AAT CAA GAA CAA AGT GAT ACT ATC CTT 623

32 Glu Lys Phe Leu Ser Gln Ala Ash Gln Glu Gln Ser Asp Thr Ile Leu 47

624 CTT GAT TGC AGA AAC TTC TAT GAA AGC AAA ATA GGA CGA TTC CAA GGC 671

48 Leu Asp Cys Arg Asn Phe Tyr Glu Ser Lys Ile Gly Arg Phe Gln Gly 63

672 TGC TTA GCC CCA GAC ATC AGG AAA TTC AGT TAG TTC CCT AGC TAC GTT 719

64 Cys Leu Ala Pro Asp Ile Arg Lys Phe Ser Tyr Phe Pro Ser Tyr Val 79

720 GAC AAA AAT CTA GAA CTT TTC AGA GAG AAG AGA GTG CTG ATG TAC TGT 767

80 Asp Lys Asn Leu Glu Leu Phe Arg Glu Lys Arg Val Leu Met Tyr Cys 95

768 ACC GGG GGC ATC CGC TGT GAG CGG GGT TCA GCC TAC CTC AAA GCC AAG 815

96 Thr Gly Gly Ile Arg Cys Glu Arg Gly Ser Ala Tyr Leu Lys Ala Lys 111

816 GGA GTG TGC AAG GAG GTG TTC CAG CTC AAG GGT GGC ATC CAC AAG TAC 863

112 Gly Val Cys Lys Glu Val Phe Gln Leu Lys Gly Gly Ile His Lys Tyr 127

864 CTG GAA GAG TTT CCT GAT GGC TTT TAC AAA GGG AAG TTG TTT GTT TTT 911

128 Leu Glu Glu Phe Pro Asp Gly Phe Tyr Lys Gly Lys Leu Phe Val Phe 143

912 GAT GAA CGC TAT GCT CTG TCC TAC AAC AGT GAT GTG GTG TCA GGT AGG 959

144 Asp Glu Arg Tyr Ala Leu Ser Tyr Asn Ser Asp Val Val Ser Gly Arg 159

960 TCA GCA CAG GCT CAG AGC CCA AAC TGA AAT GAA GCA CAT TGT CAG TTC 1007

160 Ser Ala Gln Ala Gln Ser Pro Asn *** 168

1008 ACC ATT CTA GAA AAA TGA CAC AGG GAA GAC AGG CCA GTG CTC ATT ACT 1055

1056 GAG CAC TGA ATA AGC AGG GAA AAT AAG TAC ATT GTG CCA CCA TTT TCC 1103

1104 CAG CTG TGG AGC TGA GAG AAC CCT AGC CCA GGA GTC AGG AGG CCT GGG 1151

1152 TTG GGA TCC TGG CTT CAC CAT TGC TAG CTG GAC AAG CCC ATT AAC ATG 1199

1200 GGG ATC ATC TCA CCT GCC CTG CCT GCC TGT CTA CCT GCC AAG AGC TGT 1247

1248 ACT ACT GGG CTA ATT CAG GGC TCT TAA CCT GGA ATT GGT ACA TAG ATT 1295

1296 TCA GGG ATT CTG TGA ATT TGG ATG GAA AAA TAA TTG TAT CTT TGT TTT 1343

1344 CAA TAA CAC CTC ACT AAA ATG AAG CAT TTC CTT TAG TTA TGA ATG TAG 1391

1392 GCA ACA AAG TAC CAG TTG TAT TAA TGT ACC TGT GAC TTT GTC TTC AGT 1439

1440 AGG ATT CAC AAT ACT TTC ATA TCA TGT TCT AGT TGC CTC AGA TAT CTC 1487

1488 AAA ATA GTA TTT ATA CTC ATC ACT GCT TCA AAA TGA AAA TAG TTA TTA 1535

1536 GGC CCA CCA CTA AGA GTT GAT ATA TAA TGT GTT AAT AAA TGG CAC GTC 1583

1584 TTA TTA TAT ATT ACA GAT TTT GAA AAA GAA AAA AAA AAA AAA AAA AAA 1631

1632 AAA AAA AAA AAA CAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1679

1680 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1727

1728 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AA 1762

7. PP2500 protein

A: Nucleotide sequence (SEQ ID NO: 19) Length: 2153bp

1 CTGGGACGGG GGAAAGGAGA CGCTTCTTCC TCTTGCTGCT CTTCTCGTTC

51 CCGAGATCAG CGGCGGCGGT GACCGCGAGT GGGTCGGCAC CGTCTCCGGC

101 TCCGGGTGCG AACAATGCTG ACTGATAGCG GAGGCGGCGG CACCTCCTTT

151 GAGGAGGACC TGGACTCTGT GGCTCCGCGA TCCGCCCCAG CTGGGGCCTC

201 GGAGCCGCCT CCGCCGGGAG GGGTCGGTCT GGGGATCCGC ACCGTGAGGC

251 TCTTTGGGGA GGCCGGGCCA GCGTCGGGAG TCGGCAGCAG CGGCGGCGGC

301 GGCAGCGGCA GCGGTACGGG CGGAGGGGAC GCGGCGCTGG ATTTCAAGTT

351 GGCGGCTGCC GTGCTGAGGA CCGGGGGTGG AGGTGGTGCC TCTGGCAGTG

401 ACGAGGACGA AGTGTCCGAG GTTGAATCAT TTATTTTGGA CCAAGAAGAT

451 CTGGATAACC CAGTGCTTAA AACAACATCA GAGATATTCT TATCAAGTAC

501 TGCAGAAGGA GCAGACTTAC GCACTGTGGA TCCAGAGACA CAGGCACGAC

551 TAGAAGCATT GCTAGAAGCA GCAGGAATTG GCAAATTGTC AACTGCTGAT

601 GGTAAAGCTT TTGCAGATCC TGAGGTACTC CGGAGACTGA CATCCTCAGT

651 TAGTTGTGCA CTGGATGAAG CTGCTGCTGC ACTGACACGG ATGAAAGCAG

701 AAAACAGCCA CAATGCAGGA CAAGTGGACA CTCGCAGTCT AGCAGAAGCT

751 TGTTCAGATG GGGATGTTAA TGCTGTTCGT AAATTGCTAG ATGAAGGCAG

801 AAGTGTAAAT GAACATACAG AAGAAGGAGA AAGCCTGCTG TGTTTGGCTT

851 GTTCAGCAGG GTATTATGAA TTAGCACAAG TATTGCTTGC TATGCATGCT

901 AATGTTGAAG ATCGAGGGAA TAAAGGAGAC ATAACTCCCC TGATGGCAGC

951 TTCCAGTGGA GGTTACTTAG ATATTGTGAA ATTATTACTT CTTCATGATG

1001 CTGATGTCAA CTCCCAGTCT GCAACAGGAA ACACTGCGCT AACTTATGCA

1051 TGTGCTGGAG GATTTGTTGA CATTGTTAAA GTGCTCCTTA ATGAAGGTGC

1101 AAATATAGAA GATCATAATG AAAATGGACA TACTCCCTTA ATGGAAGCAG

1151 CCAGTGCAGG TCATGTGGAA GTTGCAAGAG TTCTTTTAGA TCATGGTGCA

1201 GGCATCAACA CTCATTCTAA TGAATTCAAA GAAAGTGCTC TAACACTTGC

1251 TTGCTACAAA GGCCATTTGG ATATGGTTCG CTTTCTACTT GAAGCTGGTG

1301 GAGATCAAGA GCACAAAACA GATGAGATGC ACACTGCCTT AATGGAGGCC

1351 TGCATGGATG GACATGTAGA GGTGGCACGT TTGCTTTTGG ATAGTGGTGC

1401 TCAAGTGAAC ATGCCTGCAG ATTCATTTGA ATCTCCATTG ACGCTAGCTG

1451 CCTGTGGAGG ACATGTTGAA TTGGCAGCTC TACTTATTGA AAGGGGAGCA

1501 AATCTTGAAG AAGTTAATGA TGAAGGATAC AGTCCCTTGA TGGAAGCTGC

1551 CCGGGAAGGA CATGAAGAAA TGGTGGCACT ACCTTTAGCA CAAGGAGCAA

1601 ATATAAATGC CCAGACAGAA GAAACTCAAG AAACTGCTCT TACCTTGGCT

1651 TGCTGTGGAG GATTTTCTGA AGTTGCAGAC TTTCTTATTA AGGCAGGGGC

1701 TGATATAGAA CTTGGCTGCT CCACACCTCT GATGGAGGCA TCTCAGGAGG

1751 GACACCTGGA ATTGGTTAAA TATTTGCTGG CTTCTGGCGC TAATGTGCAT

1801 GCTACAACAG CAACAGGAGA CACAGCCTTA ACCTATGCTT GTGAAAATGG

1851 ACATACGGAT GTTGCAGATG TTTTACTTCA AGCAGGGGCT GATTTAGACA

1901 AGCAGGAGGA CATGAAGACT ATTTTGGAGG GCATAGATCC GGCCAAGCAT

1951 CAGGTGAGGG TGGCCTTTGA TGCTTGTAAG CTACTACGTA AAGAATAGAT

2001 GTTGTAGGTA ACCAGAACTC TGGATATCTG AATTCCAGCC AAGAAGTTCC

2051 AGGACCCTGC TGGGTGACAA AGGAAATCCT CTTCAATTGA AAAAGATTAT

2101 GAAGTCCCAA TAAAAAGAGA TTTGTATTGC TAAAAAAAAA AAAAAAAAAA

2151 AAA

B: amino acid sequence (SEQ ID NO: 20) length: 627 amino acids

1 MLTDSGGGGT SFEEDLDSVA PRSAPAGASE PPPPGGVGLG IRTVRLFGEA GPASGVGSSG

61 GGGSGSGTGG GDAALDFKLA AAVLRTGGGG GASGSDEDEV SEVESFILDQ EDLDNPVLKT

121 TSEIFLSSTA EGADLRTVDP ETQARLEALL EAAGIGKLST ADGKAFADPE VLRRLTSSVS

181 CALDEAAAAL TRMKAENSHN AGQVDTRSLA EACSDGDVNA VRKLLDEGRS VNEHTEEGES

241 LLCLACSAGY YELAQVLLAM HANVEDRGNK GDITPLMAAS SGGYLDIVKL LLLHDADVNS

301 QSATGNTALT YACAGGFVDI VKVLLNEGAN IEDHNENGHT PLMEAASAGH VEVARVLLDH

361 GAGINTHSNE FKESALTLAC YKGHLDMVRF LLEAGADQEH KTDEMHTALM EACMDGHVEV

421 ARLLLDSGAQ VNMPADSFES PLTLAACGGH VELAALLIER GANLEEVNDE GYTPLMEAAR

481 EGHEEMVALL LAQGANINAQ TEETQETALT LACCGGFSEV ADFLIKAGAD IELGCSTPLM

541 EASQEGHLEL VKYLLASGAN VHATTATGDT ALTYACENGH TDVADVLLQA GADLDKQEDM

601 KTILEGIDPA KHQVRVAFDA CKLLRKE

C. Nucleotide and amino acid combination sequence (SEQ ID NO: 21)

Clone number: PP2500

Start code: 115 ATG End code: 1998 TAG

Protein molecular weight: 64908

1 CTG GGA CGG GGG AAA GGA GAC GCT TCT TCC TCT TGC TGC TCT TCT CGT 48

49 TCC CGA GAT CAG CGG CGG CGG TGA CCG CGA GTG GGT CGG CAC CGT CTC 96

97 CGG CTC CGG GTG CGA ACA ATG CTG ACT GAT AGC GGA GGC GGC GGC ACC 144

1 Met Leu Thr Asp Ser Gly Gly Gly Gly Thr 10

145 TCC TTT GAG GAG GAC CTG GAC TCT GTG GCT CCG CGA TCC GCC CCA GCT 192

11 Ser Phe Glu Glu Asp Leu Asp Ser Val Ala Pro Arg Ser Ala Pro Ala 26

193 GGG GCC TCG GAG CCG CCT CCG CCG GGA GGG GTC GGT CTG GGG ATC CGC 240

27 Gly Ala Ser Glu Pro Pro Pro Pro Gly Gly Val Gly Leu Gly Ile Arg 42

241 ACC GTG AGG CTC TTT GGG GAG GCC GGG CCA GCG TCG GGA GTC GGC AGC 288

43 Thr Val Arg Leu Phe Gly Glu Ala Gly Pro Ala Ser Gly Val Gly Ser 58

289 AGC GGC GGC GGC GGC AGC GGC AGC GGT ACG GGC GGA GGG GAC GCG GCG 336

59 Ser Gly Gly Gly Gly Ser Gly Ser Gly Thr Gly Gly Gly Asp Ala Ala 74

337 CTG GAT TTC AAG TTG GCG GCT GCC GTG CTG AGG ACC GGG GGT GGA GGT 384

75 Leu Asp Phe Lys Leu Ala Ala Ala Val Leu Arg Thr Gly Gly Gly Gly 90

385 GGT GCC TCT GGC AGT GAC GAG GAC GAA GTG TCC GAG GTT GAA TCA TTT 432

91 Gly Ala Ser Gly Ser Asp Glu Asp Glu Val Ser Glu Val Glu Ser Phe 106

433 ATT TTG GAC CAA GAA GAT CTG GAT AAC CCA GTG CTT AAA ACA ACA TCA 480

107 Ile Leu Asp Gln Glu Asp Leu Asp Asn Pro Val Leu Lys Thr Thr Ser 122

481 GAG ATA TTC TTA TCA AGT ACT GCA GAA GGA GCA GAC TTA CGC ACT GTG 528

123 Glu Ile Phe Leu Ser Ser Thr Ala Glu Gly Ala Asp Leu Arg Thr Val 138

529 GAT CCA GAG ACA CAG GCA CGA CTA GAA GCA TTG CTA GAA GCA GCA GGA 576

139 Asp Pro Glu Thr Gln Ala Arg Leu Glu Ala Leu Leu Glu Ala Ala Gly 154

577 ATT GGC AAA TTG TCA ACT GCT GAT GGT AAA GCT TTT GCA GAT CCT GAG 624

155 Ile Gly Lys Leu Ser Thr Ala Asp Gly Lys Ala Phe ALa Asp Pro Glu 170

625 GTA CTC CGG AGA CTG ACA TCC TCA GTT AGT TGT GCA CTG GAT GAA GCT 672

171 Val Leu Arg Arg Leu Thr Ser Ser Val Ser Cys Ala Leu Asp Glu Ala 186

573 GCT GCT GCA CTG ACA CGG ATG AAA GCA GAA AAC AGC CAC AAT GCA GGA 720

187 Ala Ala Ala Leu Thr Arg Met Lys Ala Glu Asn Ser His Asn Ala Gly 202

721 CAA GTG GAC ACT CGC AGT CTA GCA GAA GCT TGT TCA GAT GGG GAT GTT 768

203 Gln Val Asp Thr Arg Ser Leu Ala Glu Ala Cys Ser Asp Gly Asp Val 218

769 AAT GCT GTT CGT AAA TTG CTA GAT GAA GGC AGA AGT GTA AAT GAA CAT 816

219 Asn Ala Val Arg Lys Leu Leu Asp Glu Gly Arg Ser Val Asn Glu His 234

817 ACA GAA GAA GGA GAA AGC CTG CTG TGT TTG GCT TGT TCA GCA GGG TAT 864

235 Thr Glu Glu Gly Glu Ser Leu Leu Cys Leu Ala Cys Ser Ala Gly Tyr 250

865 TAT GAA TTA GCA CAA GTA TTG CTT GCT ATG CAT GCT AAT GTT GAA GAT 912

251 Tyr Glu Leu Ala Gln Val Leu Leu Ala Met His Ala Asn Val Glu Asp 266

913 CGA GGG AAT AAA GGA GAC ATA ACT CCC CTG ATG GCA GCT TCC AGT GGA 960

267 Arg Gly Asn Lys Gly Asp Ile Thr Pro Leu Met Ala Ala Ser Ser Gly 282

961 GGT TAC TTA GAT ATT GTG AAA TTA TTA CTT CTT CAT GAT GCT GAT GTC 1008

283 Gly Tyr Leu Asp Ile Val Lys Leu Leu Leu Leu His Asp Ala Asp Val 298

1009 AAC TCC CAG TCT GCA ACA GGA AAC ACT GCG CTA ACT TAT GCA TGT GCT 1056

299 Asn Ser Gln Ser Ala Thr Gly Asn Thr Ala Leu Thr Tyr Ala Cys Ala 314

1057 GGA GGA TTT GTT GAC ATT GTT AAA GTG CTC CTT AAT GAA GGT GCA AAT 1104

315 Gly Gly Phe Val Asp Ile Val Lys Val Leu Leu Asn Glu Gly Ala Asn 330

1105 ATA GAA GAT CAT AAT GAA AAT GGA CAT ACT CCC TTA ATG GAA GCA GCC 1152

331 Ile Glu Asp His Asn Glu Asn Gly His Thr Pro Leu Met Glu Ala Ala 346

1153 AGT GCA GGT CAT GTG GAA GTT GCA AGA GTT CTT TTA GAT CAT GGT GCA 1200

347 Ser Ala Gly His Val Glu Val Ala Arg Val Leu Leu Asp His Gly Ala 362

1201 GGC ATC AAC ACT CAT TCT AAT GAA TTC AAA GAA AGT GCT CTA ACA CTT 1248

363 Gly Ile Asn Thr His Ser Asn Glu Phe Lys Glu Ser Ala Leu Thr Leu 378

1249 GCT TGC TAC AAA GGC CAT TTG GAT ATG GTT CGC TTT CTA CTT GAA GCT 1296

379 Ala Cys Tyr Lys Gly His Leu Asp Met Val Arg Phe Leu Leu Glu Ala 394

1297 GGT GCA GAT CAA GAG CAC AAA ACA GAT GAG ATG CAC ACT GCC TTA ATG 1344

395 Gly Ala Asp Gln Glu His Lys Thr Asp Glu Met His Thr Ala Leu Met 410

1345 GAG GCC TGC ATG GAT GGA CAT GTA GAG GTG GCA CGT TTG CTT TTG GAT 1392

411 Glu Ala Cys Met Asp Gly His Val Glu Val Ala Arg Leu Leu Leu Asp 426

1393 AGT GGT GCT CAA GTG AAC ATG CCT GCA GAT TCA TTT GAA TCT CCA TTG 1440

427 Ser Gly Ala Gln Val Asn Met Pro Ala Asp Ser Phe Glu Ser Pro Leu 442

1441 ACG CTA GCT GCC TGT GGA GGA CAT GTT GAA TTG GCA GCT CTA CTT ATT 1488

443 Thr Leu Ala Ala Cys Gly Gly His Val Glu Leu Ala Ala Leu Leu Ile 458

1489 GAA AGG GGA GCA AAT CTT GAA GAA GTT AAT GAT GAA GGA TAC ACT CCC 1536

459 Glu Arg Gly Ala Asn Leu Glu Glu Val Asn Asp Glu Gly Tyr Thr Pro 474

1537 TTG ATG GAA GCT GCC CGG GAA GGA CAT GAA GAA ATG GTG GCA CTA CTC 1584

475 Leu Met Glu Ala Ala Arg Glu Gly His Glu Glu Met Val Ala Leu Leu 490

1585 TTA GCA CAA GGA GCA AAT ATA AAT GCC CAG ACA GAA GAA ACT CAA GAA 1632

491 Leu Ala Gln Gly Ala Asn Ile Asn Ala Gln Thr Glu Glu Thr Gln Glu 506

1633 ACT GCT CTT ACT TTG GCT TGC TGT GGA GGA TTT TCT GAA GTT GCA GAC 1680

507 Thr Ala Leu Thr Leu Ala Cys Cys Gly Gly Phe Ser Glu Val Ala Asp 522

1681 TTT CTT ATT AAG GCA GGG GCT GAT ATA GAA CTT GGC TGC TCC ACA CCT 1728

523 Phe Leu Ile Lys Ala Gly Ala Asp Ile Glu Leu Gly Cys Ser Thr Pro 538

1729 CTG ATG GAG GCA TCT CAG GAG GGA CAC CTG GAA TTG GTT AAA TAT TTG 1776

539 Leu Met Glu Ala Ser Gln Glu Gly His Leu Glu Leu Val Lys Tyr Leu 554

1777 CTG GCT TCT GGC GCT AAT GTG CAT GCT ACA ACA GCA ACA GGA GAC ACA 1824

555 Leu Ala Ser Gly Ala Asn Val His Ala Thr Thr Ala Thr Gly Asp Thr 570

1825 GCC TTA ACC TAT GCT TGT GAA AAT GGA CAT ACG GAT GTT GCA GAT GTT 1872

571 Ala Leu Thr Tyr Ala Cys Glu Asn Gly His Thr Asp Val Ala Asp Val 586

1873 TTA CTT CAA GCA GGG GCT GAT TTA GAC AAG CAG GAG GAC ATG AAG ACT 1920

587 Leu Leu Gln AAa Gly Ala Asp Leu Asp Lys Gln Glu Asp Met Lys Thr 602

1921 ATT TTG GAG GGC ATA GAT CCG GCC AAG CAT CAG GTG AGG GTG GCC TTT 1968

603 Ile Leu Glu Gly Ile Asp Pro Ala Lys His Gln Val Arg Val Ala Phe 618

1969 GAT GCT TGT AAG CTA CTA CGT AAA GAA TAG ATG TTG TAG GTA ACC AGA 2016

619 Asp Ala Cys Lys Leu Leu Arg Lys Glu *** 628

2017 ACT CTG GAT ATC TGA ATT CCA GCC AAG AAG TTC CAG GAC CCT GCT GGG 2064

2065 TGA CAA AGG AAA TCC TCT TCA ATT GAA AAA GAT TAT GAA GTC CCA ATA 2112

2113 AAA AGA GAT TTG TAT TGC TAA AAA AAA AAA AAA AAA AAA AA 2153

D. Blastp results

Query=PP2500 (627 amino acids)

>SP_IN: Q21920 Q21920 caenorhabditis elegans.r11a8.7 protein.11/1999

     Length = 2606 amino acids

Score = 184bits (462), expected value = 1e-45

Identity = 115/306 (37%), Similarity = 168/306 (54%), Gap = 9/306 (2%)

Query: 304 TGNTALTYACAGGFVDIVKVLLNEGANIEDHNENGHTPLMEAASAGHVEVARVLLDHGAG 363

T T LT ACA G DIV++LL EGANIE ++ G +PL+ AA+AGH V VLL + A

Sbjct: 1220 TLETPLTIACANGHKDIVELLLKEGANIEHRDKKGFSPLIIAATAGHSSSVVEVLLKNHAA 1279

Query: 364 INTHSNEFKESALTLACYKGHLDMVRFLLEAGADQEHKTDEMHTALMEACMDGHVEVARL 423

I S+ K++AL+LAC G D+V LL GA++EH+ +T L A G++E+ +

Sbjct: 1280 IEAQSDRTKDTALSLACSGGRKDVVELLLAHGANKEHRNVSDYTPLSLASSGGYIEIVNM 1339

Query: 424 LLDSGAQVNMPADS--FESPLTLAACGGHVELAALLIERGANLE-EVNDEGYTPLMEAAR 480

LL +G+++N S SPL LA+ GH E +L+E+G+++ ++ T L A+

Sbjct: 1340 LLTAGSEINSRTGSKLGISPLMLASMNGHREATRVLLEKGSDINAQIETNRNTALTLASF 1399

Query: 481 EGHEEMVALLLAQGANINXXXXXXXXXXXXXXCCGGFSEVADFLIKAGAD-----IELGC 535

+G E+V LLLA AN+ GG+ +V +LI AGAD ++

Sbjct: 1400 QGRTEVVKLLLAYNANVE-HRAKTGLTPLMECASGGYVDVGNLLIAAGADTNASPVQQTK 1458

Query: 536 STPLMEASQEGHLELVKYLLASGANVHATTATGDTALTYACENGHTDVADVLLQAGADLD 595

T L ++++GH + V+ LL A V G TAL AC G+ A LL+GAD D

Sbjct: 1459 DTALTISAEKGHEKFVRMLLNGDAAVDVRNKKGCTALWLACNGGYLSTAQALLEKGADPD 1518

Query: 596 KQEDMK 601

          ++ K

Sbjct: 1519 MFDNRK 1524

Score = 183bits (460), expected value = 2e-45

Identity = 129/397 (32%), Similarity = 214/397 (53%), Gap = 18/397 (4%)

Query: 170 EVLRRLTSSVSCALDEAAAALTRMKAENSHNAGQVDTRSLAEACSDGDVNAVRKLLDEGR 229

E+++ +S + +++ +L ++ GQ L S+GD +

Sbjct: 1159 EIQKKGKTSSGTLISTSSKSLMAKSVQSQQQQGQ-----LRRTHSEGD--GAERAKSRSN 1211

Query: 230 SVNEHTEEG-ESLLCLACSAGYYELAQVLLAMHANVEDRGNKGDITPLMAASSGGYLDIV 288

++++ TE E+ L +AC+ G+ ++ ++LL AN+E R KG +PL+ A++ G+ +V

Sbjct: 1212 AIDKATETTLETPLTIACANGHKDIVELLLKEGANIEHRDKKG-FSPLIIAATAGHSSVV 1270

Query: 289 KLLLLHDADVNSQS-ATGNTALTYACAGGFVDIVKVLLNEGANIEDHNENGHTPLMEAAS 347

++LL +A + +QS T +TAL+ AC+GG D+V++LL GAN E N+ +TPL A+S

Sbjct: 1271 EVLLKNHAAIEAQSDRTKDTALSLACSGGRKDVVELLLAHGANKEHRNVSDYTPLSLASS 1330

Query: 348 AGHVEVARVLLDHGAGINTHS-NEFKESALTLACYKGHLDMVRFLLEAGADQEHKTD-EM 405

G++E+ +LL G+ IN+ + ++ S L LA GH + R LLE G+D + +

Sbjct: 1331 GGYIEIVNMLLTAGSEINSRTGSKLGISPLMLASMNGHREATRVLLEKGSDINAQIETNR 1390

Query: 406 HTALMEACMDGHVEVARLLLDSGAQVNMPADSFESPLTLAACGGHVELAALLIERGA--N 463

+TAL A G EV+LLL A V A + +PL A GG+V++ LLI GA N

Sbjct: 1391 NTALTLASFQGRTEVVKLLLAYNANVEHRAKTGLTPLMECASGGYVDVGNLLIAAGADTN 1450

Query: 464 LEEVNDEGYTPLMEAAREGHEEMVALLLAQGANINXXXXXXXXXXXXXXCCGGFSEVADF 523

V T L +A +GHE+ V +LL A ++ C GG+ A

Sbjct: 1451 ASPVQQTKDTALTISAEKGHEKFVRMLLNGDAAVD-VRNKKGCTALWLACNGGYLSTAQA 1509

Query: 524 LIKAGADIELGCS---TPLMEASQEGHLELVKYLLAS 557

L++ GAD ++ + +P+M A ++GH+E+VKY++ S

Sbjct: 1510 LLEKGADPDMFDNRKISPMMAAFRKGHVEIVKYMVNS 1546

Score = 160bits (400), expected value = 3e-38

Identity = 120/400 (30%), Similarity = 206/400 (51%), Gap = 32/400 (8%)

Query: 230 SVNEHTEEGESLLCLACSAGYYELAQVLLAMHANVEDRGNK-GDITPLMAASSGGYLDIV 288

S++ + + ++L LA G + + + + ++ RG+K ITPLM A++ IV

Sbjct: 199 SIDSQIVQQNAMLLLAARVGIEQFVEYSHEIGV-MQFRGDKLSKITPLMEAAASSSETIV 257

Query: 289 KLLLLHDADVNSQSATG-NTALTYACAGGFVDIVK-VLLNEGANIEDH---NENGHTPLM 343

+LL +LL AD N S NTAL YA+ D+V+ +L +EG D N + H +M

Sbjct: 258 RRLLELGADPNVASINPNCNTALIYAASTDGRDVVREILMTEGPKKPDVYLINNHYHDAMM 317

Query: 344 EAAGHVEVARVLLDHGAG---INTHSNEFKESALTLACYKGHLDMVRFLLEAGADQEH 400

E A G + + L+ G +N E ++SALTL+ KGH+ +V +++

Sbjct: 318 EVALVGGTDTLKEFLEMGYRPRFLNLRQQE-RDSALTLSAQKGHIKIVTAIMDYYEKNPP 376

Query: 401 KTDE--------MHTALMEACMDGHVEVARLLLDSGAQVNMPADSF--ESPLLTLAACGGH 450

  +T+E   ++ALMEA M+GH++V +L+L G ++ + SPL +A+ GG+

Sbjct: 377 QTEEEKQELCLERYSALMEAAMEGHIDVCKLMLSRGTPADLCTEVTIEPSPLIVASAGGY 436

Query: 451 VELAALLIERGANLEEVNDEGYTPLMEAAREGHEE---MVALLLAQGANINXXXXXXXXXX 507

E+ +L+ GA +EE++++ TPLMEA + +V LLL++ A ++

Sbjct: 437 PEVVEVLLAAGAKIEELSNKKNTPLMEACAGDQGDQAGVVKLLLSKHAEVDVSNPDTGDT 496

Query: 508 XXXXXCCGGFSEVADFLIKAGADIELGCSTPLMEASQEGHLELVKYLLASGANVHATTAT 567

G+ + LI+ G D+ G++P++EA++ GHLE ++++LA H T

Sbjct: 497 PLSLAARNGYIAIMKMLIEKGGDLTAGKTSPIVEAARNGHLECIQFILA-----HCKTIP 551

Query: 568 GD---TALTYACENGHTDVADVLLQAGADLDKQEDMKTIL 604

D AL A+G + + +++AGADL+ ++D +T L

Sbjct: 552 QDQLSRALVSAADFGSLLIVEEVIRAGADLNFEQDERTAL 591

Claims (10)

1. isolating human polypeptides with cancer suppressing function, it is characterized in that, it contains the aminoacid sequence that is selected from down group: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ IDNO:17, SEQ ID NO:20, described polypeptide forms inhibited to the clone of the hepatoma cell line 7721 of cultivation.

2. polypeptide as claimed in claim 1 is characterized in that, this amino acid sequence of polypeptide is selected from down group: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:20.

3. isolating polynucleotide is characterized in that, it is selected from down group:

(a) polynucleotide of polypeptide according to claim 1 of encoding;

(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:8, SEQ ID NO:11, SEQ ID NO:17, SEQ IDNO:20.

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, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQID NO:18, SEQ ID NO:21.

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

(a) have under the proteic condition of people of cancer suppressing function suitable the expression, cultivate the described host cell of claim 7;

(b) isolate the polypeptide of the people's protein-active with cancer suppressing function from culture, described polypeptide forms inhibited to the clone of the hepatoma cell line 7721 of cultivating.

9. energy and the described human polypeptides specificity bonded antibody with cancer suppressing function of claim 1, wherein said polypeptide forms inhibited to the clone of the hepatoma cell line 7721 of cultivating.

10. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.