CN1169954C - Polynucleotide encoding a human protein with the function of inhibiting the growth of cancer cells - 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

Polynucleotide encoding a human protein with the function of inhibiting the growth of cancer cells

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.

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.

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, SEQ ID NO: 23, SEQ ID NO: 26; or its conservative variant polypeptide, or its active fragment, or its active derivatives.

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, SEQ ID NO: 23, SEQ ID NO: 26.

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, SEQ ID NO: 23, SEQ ID NO: 26. More preferably, the sequence of the polynucleotide is selected from the group consisting of 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, SEQ ID NO: 24, SEQ ID NO: 27.

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

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.

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 PP6 protein (in this application, the naming of the protein adopts its clone number) (in this application, the naming of the protein adopts its clone number) as an example, the sequence of the coding region encoding 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 PP208 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) function appearance or loss of marker genes; (3) measuring the level of transcripts 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

PP6, PP208, PP451, PP624, PP722, PP902, PP1628, PP1650, PP2672 were obtained by constructing human placenta cDNA library by conventional methods. Placenta tissues at the age of 3, 6, and 10 months were taken, and total RNA was extracted with Trizol reagent (GIBCO BRL Company) according to the manufacturer's instructions, and mRNA was extracted with an mRNA purification kit (Pharmacia Company). The cDNA library of the above mRNA was constructed with the pCMV-script TMXR cDNA library construction kit (Stratagene). The reverse transcriptase was changed to MMLV-RT-SuperscriptII (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 using Qiagen 96-well plate plasmid extraction kit according to the manufacturer's instructions. Plasmid DNA and empty vector were transfected into liver cancer cell line 7721 at the same time. After 100 ng of DNA was precipitated and dried by alcohol, 6 μl of H ₂ O was added to dissolve it and wait for transfection. Add 0.74 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 could inhibit the formation of cell clones, and the results are shown in the table below.

    Colony formation of cDNA clone transfected cells (7721)

cDNA clone name cDNA clone number (three repeats) empty vector clone number (three repeats)

PP6 1 0 1 50 26 31

PP208 37 40 45 27 31 26

PP451 26 39 29 25 23 30

PP624 3 1 1 21 30 42

PP722 18 19 22 21 18 19

PP902 28 26 24 57 54 40

PP1628 26 20 30 22 25 13

PP1650 18 22 24 22 25 13

PP2672 0 0 1 12 23 28

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 is determined to be a new gene clone, and the other end is sequenced, but the full-length cDNA sequence is still not obtained, primers are designed, and sequencing is performed again until the full-length sequence is obtained.

Embodiment 2: PCR obtains gene clone from placenta 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 mRNA purification kit (pharmacia company). MMLV-RT-Superscript II (GIBCO BRL) reverse transcriptase was used to carry out reverse transcription reaction at 42°C to obtain placental cDNA. Utilize the transmutation primers of each gene (as shown in the table below), and perform 3'1 cycles at 90°C. 94°C 30″, 60°C 30″, 72°C 1′ 35 cycles, 72°C 10′ 1 cycle for PCR amplification to obtain the amplified products of each protein gene containing the complete open reading frame sequence. The amplified product was verified by sequencing and was consistent with the sequence measured in Example 1, and then the amplified product was transferred into host cells by conventional techniques to obtain recombinant protein.

                                                                         

Clone Name Specific Primer 1 (5′→3′) Specific Primer 2 (5′→3′)

PP6 CTTCCCCCTCCTGGTGCAGCCATT CCAAAGCAAACCCAACCCTCGTG

PP208 GAACCTCAATGCCAGCACCGTCC GTACCCCAAGTTGCCCAGGAGGC

PP451 TCTTTGTCATCAGCCTGGCTCGC CTGGGCAACATGTCTGCAAGGGT

PP624 GCAAAAGGAGAATGTGCCCCCAG TGACAAAGGGCAGTGGCTCGCTA

PP722 GCTCCCCATCCCACTGACTGCTT TGGCTGAGTGTATCTGGGTGGGC

PP902 TCCGCCCCTCTCCTAAAGCCTGA CACCTGCAGCCGACGGACTAGTTG

PP1628 CGGCACTTGGGGTTTCTGGGAAT AGGCAAGATGGCAGGGGATCACA

PP1650 TCTGTGACAGGGTCCAACAGGGC CCGGGGAAAAGCCCTACTGGTGT

PP2672 AGCCGTTGCCCTGTTCTTGGTGA CACACCAGCAATCAGTGTGGCGA

Embodiment 3: cDNA clone sequence analysis

1.PP6

A: Nucleotide sequence: (SEQ ID NO: 1) Length: 2104bp

1 GTGAAACAGG GTGAGTCTGG ACATTCTGCA GTCAGCCACT GTTCTTGGCT

51 TCCAACCAAA AGCAAAACTA AGGCAAGGCA GAGCACAGAG GGTGCTCAGG

101 CAGAAGCTGC TTCCCCTCCT GGTGCAGCCA TTAGCTGCTG TAGTATCTGT

151 GACCTGTCAG AACCTGCTTC CTTCATTTTG GGAATATTTG ACCAACCTCA

201 GAGCAATTGC TGTTACGAGC TAAGGAGGTC AAAGAGCAAT GTCCAGTCTT

251 CCCATTCTGT CCAAGTCAGA TTTATCGACC ATGTTTCGGA AAAAGGTGAG

301 CCTCAGGGAT AGTTTGTCAA TGGCTGAGCT AATCACAAAG GTGCCTGGGC

351 AGGAATACTG GCACCAGCCA AATTTGCATT ACTTGTTCTG AGCAATTGAG

401 CTTTGTTTGA AGAATGGGAG GGGATAAAGA AGATAACTGA TCATTTTCTC

451 AGGTGACTGA CCTGGTGATT AGGAGCAGCC TTCTTGGATG CAGTTAGGCA

501 AAGTCTGAAT GTCTTCCCTT CTCCCCCCAC CGCCTCTCTCC TGCCACCCCA

551 GGAGCAACAT ATAAAAATGT GTAGCTCCAG GCATGAAAGT AGCTTCTGTC

601 TACACAATGC AGGTCAAAGA GAAGGAACTG ACCAGGTGTC CAGGCACCAA

651 AATACCAGGC TGGTCTAGCC CCAACTCTCC TTCTCACATG CCCACGTTCA

701 CGCAACTAAC TCACAGGGTT TTGGGGAAGA CTAAGACGGA GTGAATGTAA

751 AACCCACTCC CTTCTGCCCA CGTTCACATG GTCCATGCTG AGGGAATTCA

801 GAAAAGGAGA CAGACCCGGG GGGGTGCGTC AGTCAAGGCA AGTTTCTCGA

851 AGGAAGGAAG CAGAACTCAG GAGGACATGG ACTGGAACAG TCAGGGCAAT

901 TTCAGGCTGT GACAAAGCTG GAACGGACGA CTGTAGGAGG AGCAGGAGTC

951 ACTGACATTC TAGGCCAGGC CAGGGCTAAG CCAGAGAACC TATTAATAGT

1001 AATCCCAAA TAGATATGGG GCACCTCCTA GGAACTCTCC TTGTTCCAAG

1051 CGTCGTACCT CGTGTGATCC TTAGCGGCTC TCTGAAGCAG ACAGAAGAGG

1101 GCCAGCCATC TTTCTTCCAC CTTTGAGGCT TGGGAAGGGT GAGACTTGCT

1151 GGTGACTTAC AACTCCATCA AAGGGGCATG GTGAAATAAG GGCCTGGGCT

1201 CCTGACTTCT GGGCTAGGGC TCTTCCAAAG GCAGAGTCTG GAGAGGCCTG

1251 GCTGTGGCCA GACCATGGGG CAAGTGGCTA GAGGGGCGAG TAGACAGCAG

1301 AGGCAGCTGT GGCCCCCGGG ATTAGCACTG GGGGACCGGA TGGGGGAGGG

1351 AGGCCTCACT TTGTTCTATC TGAGCAGCTT CCTCGGCAGT CATGGGACTG

1401 ATTGAGACCA CGCGAGGGCT CCTCCCGGGG GCAGGAGGGA CTCAGAGGCT

1451 GCCCCGTTGT CTGGGGGTGG CCCTGGCGAA GGAGCTCATC TTCACGGGCC

1501 GACGACTGAG TGGAACTGAG GCCCACGTAC TGGGGCTGGT GAATCACGCT

1551 GTGGCCCAGA ACGAGGAGGG GGACGCCGCC TACCAGCGGG CACGAGCACT

1601 GGCCCAGGAG ATCCTGCCCC AGGCCCCCAT TGCCGTGCGG CTGGGCAAAG

1651 TAGCCATTGA CCGAGGAACG GAGGTGGACA TTGCATCTGG GATGGCCATT

1701 GAAGGGATGT GCTATGCCCA GAATATTCCA ACCCGGGACC GGCTAGAGGG

1751 CATGGCAGCC TTCAGGGAGA AGCGGACTCC CAAATTTGTT GGCAAATGAC

1801 CCCCATTTTA ACCTTCAGCA TGGGAGATGC ATGCCCTGAA GAGCAGGATC

1851 CAGAAGGAAG ATTTGTGGCC AGATTGCCTT CATCATTTCA CCTCTCCAGA

1901 CTTCCATTTC TTCACAAGGA TGATGATGGA AATAAAATGA CTGGCGTGAT

1951 GCCTGGAACC AAGGTGCTGA TCCTACCACC TACTGCTACC TTCCTTAGCT

2001 TCACCCTGGC TAGAAATAAT CACGAGGGTT GGGTTTGCTT TGGAAAATGC

2051 CTGTCTCCTCT ACTTGAATGA TAAAGAATTA AATTAGAAAAA AAAAAAAAAA

2101 AAAA

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

1 MGLIETTRGL LPGAGGTQRL PRCLGVALAK ELIFTGRRLS GTEAHVLGLV

51 NHAVAQNEEG DAAYQRARAL AQEILPQAPI AVRLGKVAID RGTEVDIASG

101 MAIEGMCYAQ NIPTRDRLEG MAAFREKRTP KFVGK

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

Clone number and protein name: PP6

Start code: 1392 ATG Stop code: 1799 TGA

Protein molecular weight: 14435.95

1 GT GAA ACA GGG TGA GTC TGG ACA TTC TGC AGT CAG CCA CTG TTC TTG 47

48 GCT TCC AAC CAA AAG CAA AAC TAA GGC AAG GCA GAG CAC AGA GGG TGC 95

96 TCA GGC AGA AGC TGC TTC CCC TCC TGG TGC AGC CAT TAG CTG CTG TAG 143

144 TAT CTG TGA CCT GTC AGA ACC TGC TTC CTT CAT TTT GGG AAT ATT TGA 191

192 CCA ACC TCA GAG CAA TTG CTG TTA CGA GCT AAG GAG GTC AAA GAG CAA 239

240 TGT CCA GTC TTC CCA TTC TGT CCA AGT CAG ATT TAT CGA CCA TGT TTC 287

288 GGA AAA AGG TGA GCC TCA GGG ATA GTT TGT CAA TGG CTG AGC TAA TCA 335

336 CAA AGG TGC CTG GGC AGG AAT ACT GGC ACC AGC CAA ATT TGC ATT ACT 383

384 TGT TCT GAG CAA TTG AGC TTT GTT TGA AGA ATG GGA GGG GAT AAA GAA 431

432 GAT AAC TGA TCA TTT TCT CAG GTG ACT GAC CTG GTG ATT AGG AGC AGC 479

480 CTT CTT GGA TGC AGT TAG GCA AAG TCT GAA TGT CTT CCC TTC TCC CCC 527

528 CAC CGC TCT CTC CTG CCA CCC CAG GAG CAA CAT ATA AAA ATG TGT AGC 575

576 TCC AGG CAT GAA AGT AGC TTC TGT CTA CAC AAT GCA GGT CAA AGA GAA 623

624 GGA ACT GAC CAG GTG TCC AGG CAC CAA AAT ACC AGG CTG GTC TAG CCC 671

672 CAA CTC TCC TTC TCA CAT GCC CAC GTT CAC GCA ACT AAC TCA CAG GGT 719

720 TTT GGG GAA GAC TAA GAC GGA GTG AAT GTA AAA CCC ACT CCC TTC TGC 767

768 CCA CGT TCA CAT GGT CCA TGC TGA GGG AAT TCA GAA AAG GAG ACA GAC 815

816 CCG GGG GGG TGC GTC AGT CAA GGC AAG TTT CTC GAA GGA AGG AAG CAG 863

864 AAC TCA GGA GGA CAT GGA CTG GAA CAG TCA GGG CAA TTT CAG GCT GTG 911

912 ACA AAG CTG GAA CGG ACG ACT GTA GCA GGA GCA GGA GTC ACT GAC ATT 959

960 CTA GGC CAG GCC AGG GCT AAG CCA GAG AAC CTA TTA ATA GTA ATC CAC 1007

1008 AAA TAG ATA TGG GGC ACC TCC TAG GAA CTC TCC TTG TTC CAA GCG TCG 1055

1056 TAC CTC GTG TGA TCC TTA GCG GCT CTC TGA AGC AGA CAG AAG AGG GCC 1103

1104 AGC CAT CTT TCT TCC ACC TTT GAG GCT TGG GAA GGG TGA GAC TTG CTG 1151

1152 GTG ACT TAC AAC TCC ATC AAA GGG GCA TGG TGA AAT AAG GGC CTG GGC 1199

1200 TCC TGA CTT CTG GGC TAG GGC TCT TCC AAA GGC AGA GTC TGG AGA GGC 1247

1248 CTG GCT GTG GCC AGA CCA TGG GGC AAG TGG CTA GAG GGG CGA GTA GAC 1295

1296 AGC AGA GGC AGC TGT GGC CCC CGG GAT TAG CAC TGG GGG ACC GGA TGG 1343

1344 GGG AGG GAG GCC TCA CTT TGT TCT ATC TGA GCA GCT TCC TCG GCA GTC 1391

1392 ATG GGA CTG ATT GAG ACC ACG CGA GGG CTC CTC CCG GGG GCA GGA GGG 1439

1 Met Gly Leu Ile Glu Thr Thr Arg Gly Leu Leu Pro Gly Ala Gly Gly 16

1440 ACT CAG AGG CTG CCC CGT TGT CTG CGG GTG GCC CTG GCG AAG GAG CTC 1487

17 Thr Gln Arg Leu Pro Arg Cys Leu Gly Val Ala Leu Ala Lys Glu Leu 32

1488 ATC TTC ACG GGC CGA CGA CTG AGT GGA ACT GAG GCC CAC GTA CTG GGG 1535

33 Ile Phe Thr Gly Arg Arg Leu Ser Gly Thr Glu Ala His Val Leu Gly 48

1536 CTG GTG AAT CAC GCT GTG GCC CAG AAC GAG GAG GGG GAC GCC GCC TAC 1583

49 Leu Val Asn His Ala Val Ala Gln Asn Glu Glu Gly Asp Ala Ala Tyr 64

1584 CAG CGG GCA CGA GCA CTG GCC CAG GAG ATC CTG CCC CAG GCC CCC ATT 1631

65 Gln Arg Ala Arg Ala Leu Ala Gln Glu Ile Leu Pro Gln Ala Pro Ile 80

1632 GCC GTG CGG CTG GGC AAA GTA GCC ATT GAC CGA GGA ACG GAG GTG GAC 1679

81 Ala Val Arg Leu Gly Lys Val Ala Ile Asp Arg Gly Thr Glu Val Asp 96

1680 ATT GCA TCT GGG ATG GCC ATT GAA GGG ATG TGC TAT GCC CAG AAT ATT 1727

97 Ile Ala Ser Gly Met Ala Ile Glu Gly Met Cys Tyr Ala Gln Asn Ile 112

1728 CCA ACC CGG GAC CGG CTA GAG GGC ATG GCA GCC TTC AGG GAG AAG CGG 1775

113 Pro Thr Arg Asp Arg Leu Glu Gly Met Ala Ala Phe Arg Glu Lys Arg 128

1776 ACT CCC AAA TTT GTT GGC AAA TGA CCC CCA TTT TAA CCT TCA GCA TGG 1823

129 Thr Pro Lys Phe Val Gly Lys *** 136

1824 GAG ATG CAT GCC CTG AAG AGC AGG ATC CAG AAG GAA GAT TTG TGG CCA 1871

1872 GAT TGC CTT CAT CAT TTC ACC TCT CCA GAC TTC CAT TTC TTC ACA AGG 1919

1920 ATG ATG ATG GAA ATA AAA TGA CTG GCG TGA TGC CTG GAA CCA AGG TGC 1967

1968 TGA TCC TAC CAC CTA CTG CTA CCT TCC TTA GCT TCA CCC TGG CTA GAA 2015

2016 ATA ATC ACG AGG GTT GGG TTT GCT TTG GAA AAT GCC TGT CTC TCT ACT 2063

2064 TGA ATG ATA AAG AAT TAA ATT AGA AAA AAA AAA AAA AAA AA 2104

D: Blastp result

Query=PP6 protein (135 amino acids)

>SP_IN: O45106 O45106 caenorhabditis elegans.f56b3.5 protein (fragment).5/1999

length = 281

score=131bits(326), expected value=3e-30

Identity = 66/128 (51%), Similarity = 93/128 (72%), Gap = 5/128 (3%)

Query: 1 MGLIETTRGLLPGAGGTQRLPRCLGVALAKELIFTGRRLSGTEAHVLGLVNHAVAQNEEG 60

MGL+ET L+PGAGG+QRL R +GVA AKELI+T L+G +A LG+VNH V N

Sbjct: 158 MGLVETKWALIPGAGGSQRLYRIVGVAKAKELIYTAEVLNGADAAKLGVVNHVVEANP-- 215

Query: 61 DAAYQRARALAQEILPQAPIAVRLGKVAIDRGTEVDIASGMAIEGMCYAQNIPTRDRLEG 120

+++ +A++I+P+ PIAV+L K+AI+ G++ DI S +++E CYAQ + ++DRLEG

Sbjct: 216 ---IEKSLEIARKIIPRGPIAVKLAKLAINLGSQTDITSALSVEQQCYAQIVHSKDRLEG 272

Query: 121 MAAFREKR 128

MAAF EKR

Sbjct: 273 MAAFAEKR 280

2. PP208

A: Nucleotide sequence: (SEQ ID NO: 4) Length: 1587bp

1 TGGCTCAACA ATGCCTTCCA GGATGTGGAG TCAGAGAACG TCAACGTGGT

51 GAAGCGGCTG TTCAAAGATCC AGAACCTCAA TGCCAGCACC GTCCGCACGG

101 TGATGGTGGC CGACTGCAGC CGCTTCGACA GCCCTGACCT GCTGCTGGAA

151 GCCGGTGACC CGGCCACGTC CCCCTGCCGC ATCTTTGACC TGGGCAGCGA

201 CAACGAGGAG GTGGTGGCTG GCCCGGCCCC CGCCCACGCC AAGGAGGGCT

251 TGCGGCACTT TCTGGACCGC GTGCTGGAGG GGCGGGCGCA GCACAGCTGT

301 CGGAGCGCAT GCTAGAGACC AAGGTGCCGA GCTGCTGGCC CAGGGCACAC

351 CAAGCCACCC GAGCGCAGTG CCACAGGCGC CAAGAGCAAG TACCTCATCT

401 TCACCACTGG CTGCCTCACC TACTCCCCAC ACCAGATCGG CATCAAGCAG

451 ATCCTGCCAC ACCAGATGAC CACGGCAGGG CCCCGTGCTG GGTGAGGGCC

501 GGGGCTCCGA TGCCTTCTTC GACGCGCTGG ACCACGTCAT AGACATACAC

551 GGACACATCA TCGGCATGGG CCTGTCGCCC GACAACAGGT ACCTGTACGT

601 GAACAGCCGC GCCTGGCCCA ACGGTGCGGT GGTGGCCGAC CCCATGCAGC

651 CGCCACCAAT CGCGGAGGAG ATTGACCTGC TGGTGTTCGA CCTCAAGACC

701 ATGCGGGAGG TGAGGCGGGC TCTGCGTGCG CACCGCGCCT ACACGCCCAA

751 CGACGAGTGC TTCTTCATCT TCCTGGACGT CAGCAGGGAC TTCGTGGCCA

801 GCGGGGCGGA GGACCGGCAC GGCTACATCT GGGACCGCCA CTACAACATC

851 TGTCTGGCCA GGCTGCGGCA CGAGGATGTG GTCAACTCAG TGGTCTTCAG

901 TCCCCAGGAG CAGGAGCTGC TGCTCACGGC CAGCGACGAC GCCACCATCA

951 AAGCCTGGCG CTCCCCACGC ACCATGCGCG TCCTCCAGGC ACCTCGCCCA

1001 CGGCCTCGCA CCTTCTTCTC CTGGCTTGCC AGCCAGAGGC GCTGAGGTGT

1051 GCTGGGTGCA CTGGACCACC GGGACCCCTT GAGGACATCG CCAGGCTCTG

1101 TGGCTTTTTC CCGAGCGGGA GAGGTGGAGA TGCTTATAGC AGTTACGCCT

1151 TAGGAAGGGG ACAACCAGGC CCCGCCACAC GCTCACACAC AAACCTGCTC

1201 ACGCAGCTGT GATGCTTGGC ACGGGGTGGC CAGTGCAGAT GGAGCCCAAG

1251 GCCCCCTCGG CCTCCTGGGC AACTTGGGGT ACACAGGATA CTGGGGGTGC

1301 CGCTCCTCAC TCAACCCCAG GCTAGGGGTA CACCTGACCC AGCTGGCCTC

1351 GGCCCGGGGC ACCTTCGGCT GGTCCTGTGG GGCCCTGGAC GGTGGCCCAG

1401 TGGTGGCAGG GGCTGCTCCT GGCTGTGGTT GTGCGCCCGG GGCTTGGGAG

1451 CGGCCGGTCA CGCTGCTGTG GGCCCGAGTG TGTTGCATGT CCACGCACCA

1501 CCCGTTCAGG GCCCTGAATA AACAGTTGGC AACAGCCAA AAAAAAAAAAA

1551 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAA

B: Amino acid sequence: PP208 (SEQ ID NO: 5) Length: 159 amino acids

1 MGLSPDNRYL YVNSRAWPNG AVVADPMQPP PIAEEIDLLV FDLKTMREVR

51 RALRAHRAYT PNDECFFIFL DVSRDFVASG AEDRHGYIWD RHYNICLARL

101 RHEDVVNSVV FSPQEQELLL TASDATIKA WRSPRTMRVL QAPPRPRTF

151 FSWLASQRR

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

Clone number and protein name: PP208

Start code: 566 ATG Stop code: 1045 TGA

Protein molecular weight: 18544.09

1 T GGC TCA ACA ATG CCT TCC AGG ATG TGG AGT CAG AGA ACG TCA ACG 46

47 TGG TGA AGC GGC TGT TCA AGA TCC AGA ACC TCA ATG CCA GCA CCG TCC 94

95 GCA CGG TGA TGG TGG CCG ACT GCA GCC GCT TCG ACA GCC CTG ACC TGC 142

143 TGC TGG AAG CCG GTG ACC CGG CCA CGT CCC CCT GCC GCA TCT TTG ACC 190

191 TGG GCA GCG ACA ACG AGG AGG TGG TGG CTG GCC CGG CCC CCG CCC ACG 238

239 CCA AGG AGG GCT TGC GGC ACT TTC TGG ACC GCG TGC TGG AGG GGC GGG 286

287 CGC AGC ACA GCT GTC GGA GCG CAT GCT AGA GAC CAA GGT GCC GAG CTG 334

335 CTG GCC CAG GGC ACA CCA AGC CAC CCG AGC GCA GTG CCA CAG GCG CCA 382

383 AGA GCA AGT ACC TCA TCT TCA CCA CTG GCT GCC TCA CCT ACT CCC CAC 430

431 ACC AGA TCG GCA TCA AGC AGA TCC TGC CAC ACC AGA TGA CCA CGG CAG 478

479 GGC CCC GTG CTG GGT GAG GGC CGG GGC TCC GAT GCC TTC TTC GAC GCG 526

527 CTG GAC CAC GTC ATA GAC ATA CAC GGA CAC ATC ATC GGC ATG GGC CTG 574

1 Met Gly Leu 3

575 TCG CCC GAC AAC AGG TAC CTG TAC GTG AAC AGC CGC GCC TGG CCC AAC 622

4 Ser Pro Asp Asn Arg Tyr Leu Tyr Val Asn Ser Arg Ala Trp Pro Asn 19

623 GGT GCG GTG GTG GCC GAC CCC ATG CAG CCG CCA CCA ATC GCG GAG GAG 670

20 Gly Ala Val Val Ala Asp Pro Met Gln Pro Pro Pro Ile Ala Glu Glu 35

671 ATT GAC CTG CTG GTG TTC GAC CTC AAG ACC ATG CGG GAG GTG AGG CGG 718

36 Ile Asp Leu Leu Val Phe Asp Leu Lys Thr Met Arg Glu Val Arg Arg 51

719 GCT CTG CGT GCG CAC CGC GCC TAC ACG CCC AAC GAC GAG TGC TTC TTC 766

52 Ala Leu Arg Ala His Arg Ala Tyr Thr Pro Asn Asp Glu Cys Phe Phe 67

767 ATC TTC CTG GAC GTC AGC AGG GAC TTC GTG GCC AGC GGG GCG GAG GAC 814

68 Ile Phe Leu Asp Val Ser Arg Asp Phe Val Ala Ser Gly Ala Glu Asp 83

815 CGG CAC GGC TAC ATC TGG GAC CGC CAC TAC AAC ATC TGT CTG GCC AGG 862

84 Arg His Gly Tyr Ile Trp Asp Arg His Tyr Asn Ile Cys Leu Ala Arg 99

863 CTG CGG CAC GAG GAT GTG GTC AAC TCA GTG GTC TTC AGT CCC CAG GAG 910

100 Leu Arg His Glu Asp Val Val Asn Ser Val Val Phe Ser Pro Gln Glu 115

911 CAG GAG CTG CTG CTC ACG GCC AGC CAC GAC GCC ACC ATC AAA GCC TGG 958

116 Gln Glu Leu Leu Leu Thr Ala Ser Asp Asp Ala Thr Ile Lys Ala Trp 131

959 CGC TCC CCA CGC ACC ATG CGC GTC CTC CAG GCA CCT CGC CCA CGG CCT 1006

132 Arg Ser Pro Arg Thr Met Arg Val Leu Gln Ala Pro Arg Pro Arg Pro 147

1007 CGC ACC TTC TTC TCC TGG CTT GCC AGC CAG AGG CGC TGA GGT GTG CTG 1054

148 Arg Thr Phe Phe Set Trp Leu Ala Ser Gln Arg Arg *** 160

1055 GGT GCA CTG GAC CAC CGG GAC CCC TTG AGG ACA TCG CCA GGC TCT GTG 1102

1103 GCT TTT TCC CGA GCG GGA GAG GTG GAG ATG CTT ATA GCA GTT ACG CCT 1150

1151 TAG GAA GGG GAC AAC CAG GCC CCG CCA CAC GCT CAC ACA CAA ACC TGC 1198

1199 TCA CGC AGC TGT GAT GCT TGG CAC GGG GTG GCC AGT GCA GAT GGA GCC 1246

1247 CAA GGC CCC CTC GGC CTC CTG GGC AAC TTG GGG TAC ACA GGA TAC TGG 1294

1295 GGG TGC CGC TCC TCA CTC AAC CCC AGG CTA GGG GTA CAC CTG ACC CAG 1342

1343 CTG GCC TCG GCC CGG GGC ACC TTC GGC TGG TCC TGT GGG GCC CTG GAC 1390

1391 GGT GGC CCA GTG GTG GCA GGG GCT GCT CCT GGC TGT GGT TGT GCG CCC 1438

1439 GGG GCT TGG GAG CGG CCG GTC ACG CTG CTG TGG GCC CGA GTG TGT TGC 1486

1487 ATG TCC ACG CAC CAC CCG TTC AGG GCC CTG AAT AAA CAG TTG GCA ACA 1534

1535 GCC CAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1582

1583 AAA AA 1587

D: Blastp results

Query=PP208 protein [gene=PP208] (159 amino acids)

>SP_IN: O96698 O96698 drosophila melanogaster (fruit fly).

Lissencephaly-1(lis1 homolog).8/1999

length = 411

Score = 46.5bits (108), expected value = 1e-04

Identity = 28/70 (40%), Similarity = 41/70 (58%), Gap = 4/70 (5%)

Query: 76 FVASGAEDRHGYIWDRHYNICLARLR-HEDVVNSVVFSPQEQELLLTASDDATIKAW--R 132

F+ASG+ D+ IWD +CL L H++ V + F P + L++ASDD TI+ W R

Sbjct: 311 FLASGSRDKTIRIWDVSVGLCLLTLSGHDNWVRGLAFHP-GGKYLVSASDDKTIRVWDLR 369

Query: 133 SPRTMRVLQA 142

+ R M + L A

Sbjct: 370 NKRCMKTLYA 379

>SW: TUP1_CANAL P56093 candida albicans(yeast).transcriptional

repressor tup1.7/1998

length = 514

Score = 43.8bits (101), predicted value = 9e-04

Identity = 37/125 (29%), Similarity = 60/125 (47%), Gap = 14/125 (11%)

Query: 10 LYVNSRAW-PNGAVVADPMQPPPIAEEIDLLVFDLKTMREVRRALRAHRAYTPNDECFFI 68

                                                                   

Sbjct: 258 LYIRSVCFSPDGKLLATG------AEDKLIRIWDLSTKRIIK-ILRGHE-----QDIYSL 305

Query: 69 FLDVSRDFVASGAEDRHGYIWDRHYNICLARLRHEDVVNSVVFSPQEQELLLTASDDATI 128

D + SG+ DR IWD + C L ED V +V SP + +L+ S D T+

Sbjct: 306 DFFPDGDRLVSGSGDRSVRIWDLRTSQCSLTLSIEDGVTTVAVSP-DGKLIAAGSLDRTV 364

Query: 129 KAWRS 133

+ W S

Sbjct: 365 RVWDS 369

>SW: YA3A_SCHPO Q09715 schizosaccharomyces pombe (fission yeast).

Hypothetical 67.3 kd trp-asp repeats containing protein

c18b11.10 in chromosome i.7/1998

Length = 614

Score = 43.4bits (100), predicted value = 0.001

Identity = 36/136 (26%), Similarity = 62/136 (45%), Gap = 15/136 (11%)

Query: 4 SPD-NRYLYVNSRAW-PNGAVVADPMQPPPIAEEIDLLVFDLKTMREVRRALRAHRAYTP 61

SPD +R LYV + A+ P+G + E+ + ++DL T + VR H

Sbjct: 354 SPDPSRDLYVRTIAFSPDGKYLVTG---TEDRQIKLWDLSTQK-VRYVFSGHE---- 402

Query: 62 NDECFFIFLDVSRDFVASGAEDRHGYIWDRHYNICLARLRHEDVVNSVVFSPQEQELLLT 121

+ + + + + F+ SG+ DR +WD C+ +L E+ V ++ SP +Q +

Sbjct: 403 -QDIYSLDFSHNGRFIVSGSGDRTARLWDVETGQCILKLEIENGVTAIAISPNDQ-FIAV 460

Query: 122 ASDDATIKAWRSPRTM 137

S D I+ W T+

Sbjct: 461 GSLDQIIRVWSVSGTL 476

Score = 37.1bits (84), predicted value = 0.086

Identity = 28/89 (31%), Similarity = 42/89 (46%), Gap = 13/89 (14%)

Query: 58 AYTPNDECFFIFLDVSRDFVASGAEDRHGYIWDRHYNICLARLRHEDVVNSVVFSPQEQE 117

A +PND+ F+A G+ D+ +W + H++ V S+ FSP +

Sbjct: 450 AISPNDQ-----------FIAVGSLDQIIRVWSVSGTLVERLEGHKESVYSIAFSP-DSS 497

Query: 118 LLLTASDDATIKAWRSPRTMRV-LQAPRP 145

+LL+ S D TIK W T V L A +P

Sbjct: 498 ILLSGSLDKTIKVWELQATRSVGLSAIKP 526

>SP_IN: O96995 O96995 drosophila melanogaster(fruit fly).notchless

  protein.5/1999

Length = 480

Score = 43.0bits(99), predicted value = 0.002

Identity = 26/92 (28%), Similarity = 50/92 (54%), Gap = 12/92 (13%)

Query: 43 LKTMREVRRALRAHRAYTPNDECFFIFLDVSRDFVASGAEDRHGYIWDRHYNICLARLR- 101

L T AL+ ++A P++ + + S ++D Y+W + N C+ R+

Sbjct: 316 LSTEELQESALKRYQAVCPDEV----------ESLVSCSDDNTLYLWRNNQNKCVERMTG 365

Query: 102 HEDVVNSVVFSPQEQELLLTASDDATIKAWRS 133

H++VVN V +SP + +L ++AS D +++ WR+

Sbjct: 366 HQNVVNDVKYSP-DVKLIASASFDKSVRLWRA 396

Score = 30.9bits (68), predicted value = 6.5

Identity = 25/97 (25%), Similarity = 44/97 (44%), Gap = 3/97 (3%)

Query: 38 LLVFDLKTMREVRRALRAHRAYTPNDECFFIFLDVSRDFVASGAEDRHGYIWDRHYNICL 97

++++D +T ++ R L H+ + D +AS + D IWD CL

Sbjct: 177 IIIWDPETGQQKGRPLSGHKKHINCLAWEPYHRDPECRKLASASGDGDCRIWDVKLGQCL 236

Query: 98 ARLR-HEDVVNSVVFSPQEQELLLTASDDATIKAWRS 133

+ H + V +V + L+ T+S D T+K WR+

Sbjct: 237 MNIAGHTNAVTAVRWGGAG--LIYTSSKDRTVKMWRA 271

3. PP451

A: Nucleotide sequence: (SEQ ID NO: 7) Length: 1456bp

1 GTCAACTTCA TCCACCTGAT CTTAGAAGCA CTAGTGGACG GCCCCCGCAT

51 GCAGGCCTCA GCTCATGTGA CTCGGCCCTC TAAGAGGCCC AGCAAGATAG

101 GGTTTGACGA GGTCTTTGTC ATCAGCCTGG CTCGCAGGCC TGACCGTCGG

151 GAACGCATGC TCGCCTCGCT CTGGGAGATG GAGATCTCTG GGAGGGTGGT

201 GGACGCTGTG GATGGCTGGA TGCTCAACAG CAGTGCCATC AGGAACCTCG

251 GCGTAGACCT GCTCCCGGGC TACCAGGACC CTTACTCGGG CCGCACTCTG

301 ACCAAGGGCG AGGTGGGCTG CTTCCTCAGC CATTACTCCA TCTGGGAAGA

351 GGTGGTTGCC AGGGGCCTGG CCCGGGTCCT GGTGTTTGAG GATGACGTGC

401 GCTTTGAGAG CAACTTCAGG GGGCGGCTGG AACGGCTGAT GGAGGATGTG

451 GAAGCAGAGA AACTGTCTTG GGACCTGATC TACCTCGGAC GGAAGCAGGT

501 GAACCCCTGA GAAGGAGACG GCCGTGGAGG GGCTGCCGGG CCTGGTGGTG

551 GCTGGGTACT CCTACTGGAC GCTGGCCTAT GCCCTGCGTC TGGCGGGTGC

601 CCGCAAGCTG CTGGCCTCAC AGCCTCTGCG CCGCATGCTG CCCGTGGACG

651 AGTTCCTGCC CATCATGTTC GACCAGCACC CCAACGAGCA GTACAAGGCA

701 CACTTCTGGC CACGGGACCT GGTGGCCTTC TCCGCCCAGC CCCTGCTCGC

751 TGCCCCTACC CACTATGCCG GGGACGCCGA GTGGCTCAGT GACACGGAGA

801 CATCCTCTCC ATGGGATGAT GACAGCGGCC GCCTCATCAG CTGGAGCGGC

851 TCCCAAAAGA CCCTGCGCAG CCCCCGCCTG GACCTGACTG GCAGCAGCGG

901 GCACAGCCTC CAACCCCAGC CCCGAGATGA GCTCTAGGTC CAGGTGATGA

951 CTGCAAAGCA GTGTCCAGGA GCAGGCCACT ACTGCCCAGA GAGCAGAGGA

1001 GGAGGTTGTT GCCAGGGACT GCAGATCCTG TCAGACCTGG CCACCACCTT

1051 GGGCATGGCC ACTCTGCCCT CTGGACCTGT CTTTCATCGG GAGAAACCAC

1101 TCAGAGATGG ATCCATTCC CTAAAGGTCT CACAGCAAAG GAGCAGGACT

1151 CCCAGGCCCC TGTACCCTGC CTGGCCTGAT TCAGGGCCTT GTGGCCCCCA

1201 GCTTCTTGTT TCAAGCTGGG CAGACCCCAG GATCCCTTCC CTCCCTAAGG

1251 ACTCAGCTGA GGGGCCCCTC TTGCCCCCTT CTTACCTCCA CCTCAGCACC

1301 CTCCCCCAGC TTGATGTTTG GGTCTCCCCA GCACCCTCCT CCTTGGCCGG

1351 TGCAAAGTAC AGGGAGGTAA AGCAGGACCC TTGCAGACAT GTTGCCCAGC

1401 ACACAGTAGG CCCTCAATAA AAGCCATTTG CACTTTAAAA AAAAAAAAAA

1451 AAAAAAA

B: Amino acid sequence: (SEQ ID NO: 8) Length: 157 amino acids

1 MTAKQCPGAG HYCPESRGGG CCQGLQILSD LATTLGMATL PSGPVFHREK

51 PLRDGSHSLK VSQQRSRTPR PLYPAWPDSG PCGPQLLVSS WADPRIPSLP

101 KDSAEGPLLP PSYLHLSTLP QLDVWVSPAP SSLAGAKYRE VKQDPCHRHVA

151 QHTVGPQ

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

Clone number and protein name: PP451

Start codon: 947 ATG End codon: 1420 TAA

Protein molecular weight: 16819.29

1 G TCA ACT TCA TCC ACC TGA TCT TAG AAG CAC TAG TGG ACG GCC CCC 46

47 GCA TGC AGG CCT CAG CTC ATG TGA CTC GGC CCT CTA AGA GGC CCA GCA 94

95 AGA TAG GGT TTG ACG AGG TCT TTG TCA TCA GCC TGG CTC GCA GGC CTG 142

143 ACC GTC GGG AAC GCA TGC TCG CCT CGC TCT GGG AGA TGG AGA TCT CTG 190

191 GGA GGG TGG TGG ACG CTG TGG ATG GCT GGA TGC TCA ACA GCA GTG CCA 238

239 TCA GGA ACC TCG GCG TAG ACC TGC TCC CGG GCT ACC AGG ACC CTT ACT 286

287 CGG GCC GCA CTC TGA CCA AGG GCG AGG TGG GCT GCT TCC TCA GCC ATT 334

335 ACT CCA TCT GGG AAG AGG TGG TTG CCA GGG GCC TGG CCC GGG TCC TGG 382

383 TGT TTG AGG ATG ACG TGC GCT TTG AGA GCA ACT TCA GGG GGC GGC TGG 430

431 AAC GGC TGA TGG AGG ATG TGG AAG CAG AGA AAC TGT CTT GGG ACC TGA 478

479 TCT ACC TCG GAC GGA AGC AGG TGA ACC CCT GAG AAG GAG ACG GCC GTG 526

527 GAG GGG CTG CCG GGC CTG GTG GTG GCT GGG TAC TCC TAC TGG ACG CTG 574

575 GCC TAT GCC CTG CGT CTG GCG GGT GCC CGC AAG CTG CTG GCC TCA CAG 622

623 CCT CTG CGC CGC ATG CTG CCC GTG GAC GAG TTC CTG CCC ATC ATG TTC 670

671 GAC CAG CAC CCC AAC GAG CAG TAC AAG GCA CAC TTC TGG CCA CGG GAC 718

719 CTG GTG GCC TTC TCC GCC CAG CCC CTG CTC GCT GCC CCT ACC CAC TAT 766

767 GCC GGG GAC GCC GAG TGG CTC AGT GAC ACG GAG ACA TCC TCT CCA TGG 814

815 GAT GAT GAC AGC GGC CGC CTC ATC AGC TGG AGC GGC TCC CAA AAG ACC 862

863 CTG CGC AGC CCC CGC CTG GAC CTG ACT GGC AGC AGC GGG CAC AGC CTC 910

911 CAA CCC CAG CCC CGA GAT GAG CTC TAG GTC CAG GTG ATG ACT GCA AAG 958

1 Met Thr Ala Lys 4

959 CAG TGT CCA GGA GCA GGC CAC TAC TGC CCA GAG AGC AGA GGA GGA GGT 1006

5 Gln Cys Pro Gly Ala Gly His Tyr Cys Pro Glu Ser Arg Gly Gly Gly 20

1007 TGT TGC CAG GGA CTG CAG ATC CTG TCA GAC CTG GCC ACC ACC TTG GGC 1054

21 Cys Cys Gln Gly Leu Gln Ile Leu Ser Asp Leu Ala Thr Thr Leu Gly 36

1055 ATG GCC ACT CTG CCC TCT GGA CCT GTC TTT CAT CGG GAG AAA CCA CTC 1102

37 Met Ala Thr Leu Pro Ser Gly Pro Val Phe His Arg Glu Lys Pro Leu 52

1103 AGA GAT GGA TCC CAT TCC CTA AAG GTC TCA CAG CAA AGG AGC AGG ACT 1150

53 Arg Asp Gly Ser His Ser Leu Lys Val Ser Gln Gln Arg Ser Arg Thr 68

1151 CCC AGG CCC CTG TAC CCT GCC TGG CCT GAT TCA GGG CCT TGT GGC CCC 1198

69 Pro Arg Pro Leu Tyr Pro Ala Trp Pro Asp Ser Gly Pro Cys Gly Pro 84

1199 CAG CTT CTT GTT TCA AGC TGG GCA GAC CCC AGG ATC CCT TCC CTC CCT 1246

85 Gln Leu Leu Val Ser Ser Trp Ala Asp Pro Arg Ile Pro Ser Leu Pro 100

1247 AAG GAC TCA GCT GAG GGG CCC CTC TTG CCC CCT TCT TAC CTC CAC CTC 1294

101 Lys Asp Ser Ala Glu Gly Pro Leu Leu Pro Pro Ser Tyr Leu His Leu 116

1295 AGC ACC CTC CCC CAG CTT GAT GTT TGG GTC TCC CCA GCA CCC TCC TCC 1342

117 Ser Thr Leu Pro Gln Leu Asp Val Trp Val Ser Pro Ala Pro Ser Ser 132

1343 TTG GCC GGT GCA AAG TAC AGG GAG GTA AAG CAG GAC CCT TGC AGA CAT 1390

133 Leu Ala Gly Ala Lys Tyr Arg Glu Val Lys Gln Asp Pro Cys Arg His 148

1391 GTT GCC CAG CAC ACA GTA GGC CCT CAA TAA AAG CCA TTT GCA CTT TAA 1438

149 Val Ala Gln His Thr Val Gly Pro Gln *** 158

1439 AAA AAA AAA AAA AAA AAA 1456

4.PP624

A: Nucleotide sequence: (SEQ ID NO: 10) Length: 1754bp

1 AGATGACCTG GAAATAGGCC CAGGTCAGTT GTCATCTTCT ACATTTGACT

51 CGGAGAAAAA TGAGAGTAGA CGAAATCTGG AACTTCCACG CCTCTCAGAA

101 ACCTCTATAA AGGATCGAAT GGCCAAGTAC CAGGCAGCTG TGTCCAAACA

151 AAGCAGCTCA ACCAACTATA CAAATGAGCT GAAAGCCAGT GGTGGCGAAA

201 TCAAAATTCA TAAAATGGAG CAAAAGGAGA ATGTGCCCCC AGGTCCTGAG

251 GTCTGCATCA CCCATCAGGA AGGGGAAAAG ATTTCTGCAA ATGAGAATAG

301 CCTGGCAGTC CGTTCCACCC CTGCCGAAGA TGACTCCCGT GACTCCCAGG

351 TTAAGAGTGA GGTTCAACAG CCTGTCCATC CCAAGCCACT AAGTCCAGAT

401 CTCCAGAGCC TCCAGTCTTT CTGAAAGTTC TCCTCCCAAA GCAATGAAGA

451 AGTTTCAGGC ACCTGCAAGA GAGACCTGCG TGGAATGTCA GAAGACAGTC

501 TATRCAATGG AGCGTCTCTT GGCCAACCAG CAGGTGTTTC ACATCAGCTG

551 CTTRCGTTGC TRCTATTGCA KCAACAAACT CAGTCTAGGA ACATATGCAT

601 CTTTACATGG AAGAATCTAT TGTAAGCCTC ACTTCAATCA ACTCTTTAAA

651 TCTAAGGGCA ACTATGATGA AGGCTTTGGG CACAGACCAC ACAAGGATCT

701 ATGGGCAAGC AAAAATGAAA ACGAAGAGAT TTTGGAGAGA CCAGCCCAGC

751 TTGCAAATGC AAGGGAGACC CCTCACAGCC CAGGGGTAGA AGATGCCCCT

801 ATTGCTAAGG TGGGTGTCCT GGCTGCAAGT ATGGAAGCCA AGGCCTCCTC

851 TCAGCAGGAG AAGGAAGACA AGCCAGCTGA AACCAAGAAG CTGAGGATCG

901 CCTGGCCACC CCCCACTGAA CTTGGAAGTT CAGGAAGTGC CTTGGAGGAA

951 GGGATCAAAA TGTCAAAGCC CAAATGGCCT CCTGAAGACG AAATCAGCAA

1001 GCCCGAAGTT CCTGAGGATG TCGATCTAGA TCTGAAGAAG CTAAGACGAT

1051 CTTCTTCACT GAAGGAAAGA AGCCGCCCAT TCACTGTAGC AGCTTCATTT

1101 CAAAGCACCT CTGTCAAGAG CCCAAAAACT GTGTCCCCCAC CTATCAGGAA

1151 AGGCTGGAGC ATGTCAGAGC AGAGTGAAGA GTCTGTGGGT GGAAGAGTTG

1201 CAGAAAGGAA ACAAGTGGAA AATGCCAAGG CTTCTAAGAA GAATGGGAAT

1251 GTGGGAAAAA CAACCTGGCA AAACAAAGAA TCTAAAGGAG AGCAGGGAAG

1301 AGAAGTAAGG AAGGTCATAG TTTGGAGATG GAGAATGAGA ATCTTGTAGA

1351 AAATGGTGCA GACTCCGATG AAGATGATAA CAGCTTCCTC AAACAACAAT

1401 CTCCACAAGA ACCCAAGTCT CTGAATTGGT CGAGTTTTGT AGACAACACC

1451 TTTGCTGAAG AATTCACTAC TCAGAATCAG AAATCCCAGG ATGTGGAACT

1501 CTGGGAGGGA GAAGTGGTCA AAGAGCTCTC TGTGGAAGAA CAGATAAAGA

1551 GAAATCGGTA TTATGATGAG GATGAGGATG AAGAGTGACA AATTGCAATG

1601 ATGCTGGGCC TTAAATTCAT GTTAGTGTTA GCGAGCCACT GCCCTTTGTC

1651 AAAATGTGAT GCACATAAGC AGGTATCCCA GCATGAAATG TAATTTACTT

1701 GGAAGTAACT TTGGAAAAGA ATTCCTTCTT AAAATCAAAA AAAAAAAAAA

1751 AAAA

B: Amino acid sequence: (SEQ ID NO: 11) Length: 301 amino acids

1 MKKFQAPARE TCVECQKTVY XMERLLANQQ VFHISCLRCX YCXNKLSLGT

51 YASLHGRIYC KPHFNQLFKS KGNYDEGFGH RPHKDLWASK NENEEILERP

101 AQLANARETP HSPGVEDAPI AKVGVLAASM EAKASSQQEK EDKPAETKKL

151 RIAWPPPTEL GSSGSALEEG IKMSKPKWPP EDEISKPEVP EDVDLDLKKL

201 RRSSSRKERS RPFTVAASFQ STSVKSPKTV SPPIRKGWSM SEQSEESVGG

251 RVAERKQVEN AKASKKNGNV GKTTWQNKES KGEQGREVRK VIVWRWRMRI

301L

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

Clone number and protein name: PP624

Start code: 444 ATG Stop code: 1349 TAG

Protein molecular weight: 33999.27

1 AG ATG ACC TGG AAA TAG GCC CAG GTC AGT TGT CAT CTT CTA CAT TTG 47

48 ACT CGG AGA AAA ATG AGA GTA GAC GAA ATC TGG AAC TTC CAC GCC TCT 95

96 CAG AAA CCT CTA TAA AGG ATC GAA TGG CCA AGT ACC AGG CAG CTG TGT 143

144 CCA AAC AAA GCA GCT CAA CCA ACT ATA CAA ATG AGC TGA AAG CCA GTG 191

192 GTG GCG AAA TCA AAA TTC ATA AAA TGG AGC AAA AGG AGA ATG TGC CCC 239

240 CAG GTC CTG AGG TCT GCA TCA CCC ATC AGG AAG GGG AAA AGA TTT CTG 287

288 CAA ATG AGA ATA GCC TGG CAG TCC GTT CCA CCC CTG CCG AAG ATG ACT 335

336 CCC GTG ACT CCC AGG TTA AGA GTG AGG TTC AAC AGC CTG TCC ATC CCA 383

384 AGC CAC TAA GTC CAG ATC TCC AGA GCC TCC AGT CTT TCT GAA AGT TCT 431

432 CCT CCC AAA GCA ATG AAG AAG TTT CAG GCA CCT GCA AGA GAG ACC TGC 479

2

480 GTG GAA TGT CAG AAG ACA GTC TAT RCA ATG GAG CGT CTC TTG GCC AAC 527

13 Val Glu Cys Gln Lys Thr Val Tyr Xxx Met Glu Arg Leu Leu Ala Asn 28

528 CAG CAG GTG TTT CAC ATC AGC TGC TTR CGT TGC TRC TAT TGC AKC AAC 575

29 Gln Gln Val Phe His Ile Ser Cys Leu Arg Cys Xxx Tyr Cys Xxx Asn 44

576 AAA CTC AGT CTA GGA ACA TAT GCA TCT TTA CAT GGA AGA ATC TAT TGT 623

45 Lys Leu Ser Leu Gly Thr Tyr Ala Ser Leu His Gly Arg Ile Tyr Cys 60

624 AAG CCT CAC TTC AAT CAA CTC TTT AAA TCT AAG GGC AAC TAT GAT GAA 671

61 Lys Pro His Phe Asn Gln Leu Phe Lys Ser Lys Gly Asn Tyr Asp Glu 76

672 GGC TTT GGG CAC AGA CCA CAC AAG GAT CTA TGG GCA AGC AAA AAT GAA 719

77 Gly Phe Gly His Arg Pro His Lys Asp Leu Trp Ala Ser Lys Asn Glu 92

720 AAC GAA GAG ATT TTG GAG AGA CCA GCC CAG CTT GCA AAT GCA AGG GAG 767

93 Asn Glu Glu Ile Leu Glu Arg Pro Ala Gln Leu Ala Asn Ala Arg Glu 108

768 ACC CCT CAC AGC CCA GGG GTA GAA GAT GCC CCT ATT GCT AAG GTG GGT 815

109 Thr Pro His Ser Pro Gly Val Glu Asp Ala Pro Ile Ala Lys Val Gly 124

816 GTC CTG GCT GCA AGT ATG GAA GCC AAG GCC TCC TCT CAG CAG GAG AAG 863

125 Val Leu Ala Ala Ser Met Glu Ala Lys Ala Ser Ser Gln Gln Glu Lys 140

864 GAA GAC AAG CCA GCT GAA ACC AAG AAG CTG AGG ATC GCC TGG CCA CCC 911

141 Glu Asp Lys Pro Ala Glu Thr Lys Lys Leu Arg Ile Ala Trp Pro Pro 156

912 CCC ACT GAA CTT GGA AGT TCA GGA AGT GCC TTG GAG GAA GGG ATC AAA 959

157 Pro Thr Glu Leu Gly Ser Ser Ser Gly Ser Ala Leu Glu Glu Gly Ile Lys 172

960 ATG TCA AAG CCC AAA TGG CCT CCT GAA GAC GAA ATC AGC AAG CCC GAA 1007

173 Met Ser Lys Pro Lys Trp Pro Pro Glu Asp Glu Ile Ser Lys Pro Glu 188

1008 GTT CCT GAG GAT GTC GAT CTA GAT CTG AAG AAG CTA AGA CGA TCT TCT 1055

189 Val Pro Glu Asp Val Asp Leu Asp Leu Lys Lys Leu Arg Arg Ser Ser 204

1056 TCA CTG AAG GAA AGA AGC CGC CCA TTC ACT GTA GCA GCT TCA TTT CAA 1103

205 Ser Leu Lys Glu Arg Ser Arg Pro Phe Thr Val Ala Ala Ser Phe Gln 220

1104 AGC ACC TCT GTC AAG AGC CCA AAA ACT GTG TCC CCA CCT ATC AGG AAA 1151

221 Ser Thr Ser Val Lys Ser Pro Lys Thr Val Ser Pro Pro Ile Arg Lys 236

1152 GGC TGG AGC ATG TCA GAG CAG AGT GAA GAG TCT GTG GGT GGA AGA GTT 1199

237 Gly Trp Ser Met Ser Glu Gln Ser Glu Glu Ser Val Gly Gly Arg Val 252

1200 GCA GAA AGG AAA CAA GTG GAA AAT GCC AAG GCT TCT AAG AAG AAT GGG 1247

253 Ala Glu Arg Lys Gln Val Glu Asn Ala Lys Ala Ser Lys Lys Asn Gly 268

1248 AAT GTG GGA AAA ACA ACC TGG CAA AAC AAA GAA TCT AAA GGA GAG CAG 1295

269 Asn Val Gly Lys Thr Thr Trp Gln Asn Lys Glu Ser Lys Gly Glu Gln 284

1296 GGA AGA GAA GTA AGG AAG GTC ATA GTT TGG AGA TGG AGA ATG AGA ATC 1343

285 Gly Arg Glu Val Arg Lys Val Ile Val Trp Arg Trp Arg Met Arg Ile 300

1344 TTG TAG AAA ATG GTG CAG ACT CCG ATG AAG ATG ATA ACA GCT TCC TCA 1391

301 Leu *** 302

1392 AAC AAC AAT CTC CAC AAG AAC CCA AGT CTC TGA ATT GGT CGA GTT TTG 1439

1440 TAG ACA ACA CCT TTG CTG AAG AAT TCA CTA CTC AGA ATC AGA AAT CCC 1487

1488 AGG ATG TGG AAC TCT GGG AGG GAG AAG TGG TCA AAG AGC TCT CTG TGG 1535

1536 AAG AAC AGA TAA AGA GAA ATC GGT ATT ATG ATG AGG ATG AGG ATG AAG 1583

1584 AGT GAC AAA TTG CAA TGA TGC TGG GCC TTA AAT TCA TGT TAG TGT TAG 1631

1632 CGA GCC ACT GCC CTT TGT CAA AAT GTG ATG CAC ATA AGC AGG TAT CCC 1679

1680 AGC ATG AAA TGT AAT TTA CTT GGA AGT AAC TTT GGA AAA GAA TTC CTT 1727

1728 CTT AAA ATC AAA AAA AAA AAA AAA AAA 1754

5. PP722

A: Nucleotide sequence: (SEQ ID NO: 13) Length: 1690bp

1 GTCTGACCCA GGCCTGCCTC CCTTCCCTAG GCCTGGCTCC TTCTGTTGAC

51 ATGGGAGATT TTAGCTCATC TTGGGGGCCT CCTTAAACAC CCCCATTTCT

101 TGCGGAAGAT GCTCCCCATC CCACTGACTG CTTGACCTTTT ACCTCCAACC

151 CTTCTGTTCA TCGGGAGGGC TCCACCAATT GAGTCTCTCC CACCATGCAT

201 GCAGGTCACT GTGTGTGTGC ATGTGTGCCT GTGTGAGTGT TGACTGACTG

251 TGTGTGTGTG GAGGGGTGAC TGTCCGTGGA GGGGTGACTG TGTCCGTGGT

301 GTGTATTATG CTGTCATATC AGAGTCAAGT GAACTGTGGT GTATGTGCCA

351 CGGGATTTGA GTGGTTGCCG TGGGCAACAC TGTCAGGGTT TGGCGTGTGT

401 GTCATGTGGC TGTGTGTGAC CTCTGCCTGA AAAAGCAGGT ATTTTCTCAG

451 ACCCCAGAGC AGTATTAATG ATGCAGAGGT TGGAGGAGAG AGGTGGAGAC

501 TGTGGCTCAG ACCCAGGTGT GCGGGCATAG CTGGAGCTGG AATCTGCCTC

551 CGGTGTGAGG GAACCTGTCT CCTACCACTT CGGAGCCATG GGGGCAAGTG

601 TGAAGCAGCC AGTCCCTGGG TCAGCCAGAG GCTTGAACTG TTACAGAAGC

651 CCTCTGCCCT CTGGTGGCCT CTGGGCCTGC TGCATGTACA TATTTTCTGT

701 AAATATACAT GCGCCGGGAG CTTCTTGCAG GAATACTGCT CCGAATCACT

751 TTTAATTTTT TTCTTTTTTT TTTCTTGCCC TTTCCATTAG TTGTATTTTT

801 TATTTATTTT TATTTTTATT TTTTTTTAGA GATGGAGTCT CACTATGTTG

851 CTCAGGCTGG CCTTGAACTC CTGGGCTCAA GCAATCCTCC TGCCTCAGCC

901 TCCCTAGTAG CTGGGACTTT AAGTGTACAC CACTGTGCCT GCTTTGAATC

951 CTTTACGAAG AGAAAAAAAA AATTAAAGAA AGCCTTTAGA TTTATCCAAT

1001 GTTTACTACT GGGATTGCTT AAAGTGAGGC CCCTCCAACA CCAGGGGGTT

1051 AATTCCTGTG ATTGTGAAAG GGGCTACTTC CAAGGCATCT TCATGCAGGC

1101 AGCCCCTTGG GAGGGCACCT GAGAGCTGGT AGAGTCTGAA ATTAGGGATG

1151 TGAGCCTCGT GGTTACTGAG TAAGGTAAAA TTGCATCCAC CATTGTTTGT

1201 GATACCTTAG GGAATTGCTT GGACCTGGTG ACAAGGGCTC CTGTTCAATA

1251 GTGGTGTTGG GGAGAGAGAG AGCAGTGATT ATAGACCGAG AGAGTAGGAG

1301 TTGAGGTGAG GTGAAGGAGG TGCTGGGGGT GAGAATGTCG CCTTTCCCCC

1351 TGGGTTTTGG ATCACTAATT CAAGGCTCTT CTGGATGTTT CTCTGGGTTG

1401 GGGCTGGAGT TCAATGAGGT TTATTTTTAG CTGGCCCACC CAGATACACT

1451 CAGCCAGAAT ACCTAGATTT AGTACCCAAA CTCTTCTTAG TCTGAAATCT

1501 GCTGGATTTC TGGCCTAAGG GAGAGGCTCC CATCCTTCGT TCCCCAGCCA

1551 GCCTAGGACT TCGAATGTGG AGCCTGAAGA TCTAAGATCC TAACATGTAC

1601 ATTTTATGTA AATATGTGCA TATTTGTACA TAAAATGATA TTCTGTTTTT

1651 AAATAAACAG ACAAAACTTG AAAAAAAAAA AAAAAAAAAA

B: Amino acid sequence: PP722 (SEQ ID NO: 14) Length: 137 amino acids

1 MRRELLAGIL LRITFNFFLFFFLPFPLVVF FIYFYFYFFL EMESHYVAQA

51 GLELLGSSNP PASASLVAGT LSVHHCACFE SFTKRKKKLK KAFRFIQCLL

101 LGLLKVRPLQ HQGVNSCDCE RGYFQGIFMQ AAPWEGT

C: Nucleotide and amino acid combination sequence (SEQ ID NO: 14)

Clone number and protein name: PP722

Start code: 709 ATG End code: 1122 TGA

Protein molecular weight: 15824.94

1 GTC TGA CCC AGG CCT GCC TCC CTT CCC TAG GCC TGG CTC CTT CTG TTG 48

49 ACA TGG GAG ATT TTA GCT CAT CTT GGG GGC CTC CTT AAA CAC CCC CAT 96

97 TTC TTG CGG AAG ATG CTC CCC ATC CCA CTG ACT GCT TGA CCT TTA CCT 144

145 CCA ACC CTT CTG TTC ATC GGG AGG GCT CCA CCA ATT GAG TCT CTC CCA 192

193 CCA TGC ATG CAG GTC ACT GTG TGT GTG CAT GTG TGC CTG TGT GAG TGT 240

241 TGA CTG ACT GTG TGT GTG TGG AGG GGT GAC TGT CCG TGG AGG GGT GAC 288

289 TGT GTC CGT GGT GTG TAT TAT GCT GTC ATA TCA GAG TCA AGT GAA CTG 336

337 TGG TGT ATG TGC CAC GGG ATT TGA GTG GTT GCC GTG GGC AAC ACT GTC 384

385 AGG GTT TGG CGT GTG TGT CAT GTG GCT GTG TGT GAC CTC TGC CTG AAA 432

433 AAG CAG GTA TTT TCT CAG ACC CCA GAG CAG TAT TAA TGA TGC AGA GGT 480

481 TGG AGG AGA GAG GTG GAG ACT GTG GCT CAG ACC CAG GTG TGC GGG CAT 528

529 AGC TGG AGC TGG AAT CTG CCT CCG GTG TGA GGG AAC CTG TCT CCT ACC 576

577 ACT TCG GAG CCA TGG GGG CAA GTG TGA AGC AGC CAG TCC CTG GGT CAG 624

625 CCA GAG GCT TGA ACT GTT ACA GAA GCC CTC TGC CCT CTG GTG GCC TCT 672

673 GGG CCT GCT GCA TGT ACA TAT TTT CTG TAA ATA TAC ATG CGC CGG GAG 720

1 Met Arg Arg Glu 4

721 CTT CTT GCA GGA ATA CTG CTC CGA ATC ACT TTT AAT TTT TTT CTT TTT 768

5 Leu Leu Ala Gly Ile Leu Leu Arg Ile Thr Phe Asn Phe Phe Leu Phe 20

769 TTT TTC TTG CCC TTT CCA TTA GTT GTA TTT TTT ATT TAT TTT TAT TTT 816

21 Phe Phe Leu Pro Phe Pro Leu Val Val Phe Phe Ile Tyr Phe Tyr Phe 36

817 TAT TTT TTT TTA GAG ATG GAG TCT CAC TAT GTT GCT CAG GCT GGC CTT 864

37 Tyr Phe Phe Leu Glu Met Glu Ser His Tyr Val Ala Gln Ala Gly Leu 52

865 GAA CTC CTG GGC TCA AGC AAT CCT CCT GCC TCA GCC TCC CTA GTA GCT 912

53 Glu Leu Leu Gly Ser Ser Asn Pro Pro Ala Ser Ala Ser Leu Val Ala 68

913 GGG ACT TTA AGT GTA CAC CAC TGT GCC TGC TTT GAA TCC TTT ACG AAG 960

69 Gly Thr Leu Ser Val His His Cys Ala Cys Phe Glu Ser Phe Thr Lys 84

961 AGA AAA AAA AAA TTA AAG AAA GCC TTT AGA TTT ATC CAA TGT TTA CTA 1008

85 Arg Lys Lys Lys Leu Lys Lys Ala Phe Arg Phe Ile Gln Cys Leu Leu 100

1009 CTG GGA TTG CTT AAA GTG AGG CCC CTC CAA CAC CAG GGG GTT AAT TCC 1056

101 Leu Gly Leu Leu Lys Val Arg Pro Leu Gln His Gln Gly Val Asn Ser 116

1057 TGT GAT TGT GAA AGG GGC TAC TTC CAA GGC ATC TTC ATG CAG GCA GCC 1104

117 Cys Asp Cys Glu Arg Gly Tyr Phe Gln Gly Ile Phe Met Gln Ala Ala 132

1105 CCT TGG GAG GGC ACC TGA GAG CTG GTA GAG TCT GAA ATT AGG GAT GTG 1152

133 Pro Trp Glu Gly Thr *** 138

1153 AGC CTC GTG GTT ACT GAG TAA GGT AAA ATT GCA TCC ACC ATT GTT TGT 1200

1201 GAT ACC TTA GGG AAT TGC TTG GAC CTG GTG ACA AGG GCT CCT GTT CAA 1248

1249 TAG TGG TGT TGG GGA GAG AGA GAG CAG TGA TTA TAG ACC GAG AGA GTA 1296

1297 GGA GTT GAG GTG AGG TGA AGG AGG TGC TGG GGG TGA GAA TGT CGC CTT 1344

1345 TCC CCC TGG GTT TTG GAT CAC TAA TTC AAG GCT CTT CTG GAT GTT TCT 1392

1393 CTG GGT TGG GGC TGG AGT TCA ATG AGG TTT ATT TTT AGC TGG CCC ACC 1440

1441 CAG ATA CAC TCA GCC AGA ATA CCT AGA TTT AGT ACC CAA ACT CTT CTT 1488

1489 AGT CTG AAA TCT GCT GGA TTT CTG GCC TAA GGG AGA GGC TCC CAT CCT 1536

1537 TCG TTC CCC AGC CAG CCT AGG ACT TCG AAT GTG GAG CCT GAA GAT CTA 1584

1585 AGA TCC TAA CAT GTA CAT TTT ATG TAA ATA TGT GCA TAT TTG TAC ATA 1632

1633 AAA TGA TAT TCT GTT TTT AAA TAA ACA GAC AAA ACT TGA AAA AAA AAA 1680

1681 AAA AAA AAA A 1690

6. PP902

A: Nucleotide sequence: (SEQ ID NO: 16) Length: 2136bp

1 TGTCGCTGCC GGAGAGTCTC TGCTTCCCCC TTCCTACGCG CTCCGCGGCG

51 GTAGCTCGGG CTCTCCGGAG GAGGGGGCGA CAGAGAAAAA GAGAGGCTGA

101 CTTAATAAAG TTTCCTGAAA CAACAACTAC CACCGAAAAG GCCCAAGGTG

151 GGGGGAGACC CCCATTTTCC TCCGCCCCTC TCCTAAAGCC TGAGGGACCG

201 TTGCAGGTGG AGCGGCGCTG GTGGGACCCG GGTTGCGGCG GCAGCCGTTG

251 CCCCGGCGAG GGAAGCCTCA GGTCCTGCCC GCTCCGGTTC CGGCTCCTTC

301 GGTGGTGGCG GCGGCAGCTG CTGCCGGGCG GTTCCTCCTG TCAACTGCCT

351 CCGCCCGCGC GCCTCCCGCC TAGCCACACA GCGCGGAGGA CGCCGTGAGC

401 CGCCGCCGCA GTCGTCTCGG CCCGCGGAGG GACGTGGTGA GCTGTGGCGC

451 TTCCGCGTCA GCGCCGGCGG GGACTCTTTG GGAGCGCGTC GGGCCCGAGT

501 CCGGGTTCCC CCGCGTGGTC AGCGGAGGCT GCGCCAGGTG GGGCCCGCAG

551 CCGCCTCCCA CGCCAGGCTG TCCCCGCGGC CGCCCGGCCT GCCTTCCTTC

601 CTTCCGCCCG CCCGCCGGCC CGCCCTAAGA GTTGACCACG CCGCAATGAA

651 GAGCTTCTTA CAGTGAAAGC AAAGTAGGTC GAGTCCACTG AAAATGCCTT

701 TAAGGGACAA ATACTGTCAG ACTGACCACC ATCATCACGG ATGCTGTGAA

751 CCAGTTTATA TCCTGGAACC TGGAGATCCT CCTTTGTTAC AGCAACCACT

801 ACAGACATCC AAATCTGGTA TTCAACAAAT AATTGAGTGC TTTCGATCAG

851 GAACTAAACA ACTTAAGCAT ATTTTATTAA AAGATGTGGA CACTATTTTT

901 GAATGTAAGT TATGCCGCAG TCTCTTCAGA GGATTACCAA ATTTAATTAC

951 CCATAAAAAAA TTCTACTGCC CACCAAGTCT CCAGATGGAT GACAACCTTC

1001 CTGATGTAAA TGATAAACAA AGCGAAGCCA TAAATGATCT CCTAGAAGCC

1051 ATATATCCAA GTGTGGACAA ACGAGAATAT ATTATTAAGC TAGAACCCAT

1101 AGAAACTAAT CAAAATGCAG TATTTCAATA TATTTCGAGG ACTGATAATC

1151 CTATTGAAGT CACAGAGTCA AGCAGTACTC CTGAACAAAC CGAAGTTCAG

1201 ATACAGGAAA CTAGCACTGA ACAGTCAAAA ACAGTACCGG TTACAGATAC

1251 AGAGGTGGAA ACTGTAGAGC CCCCTCCTGT TGAGATTGTT ACAGATGAAG

1301 TTGCACCTAC ATCTGATGAA CAACCTCAGG AGTCGCAGGC TGACTTGGAA

1351 ACTTCTGACA ATTCTGATTT TGGTCACCAG TTGATATGTT GTCTTTGTAG

1401 AAAAGAATTC AATTCTAGAC GAGGTGTTCG CCGTCACATT CGAAAAGTAC

1451 ACAAGAAAAA GATGGAAGAA CTAAAAAGT ACATTGAAAC ACGAAAGAAT

1501 CCAAACCAAT CCTCTAAAGG ACGCAGTAAG AATGTTCTAG TTCCATTAAG

1551 TAGGAGTTGT CCAGTATGTT GTAAATCATT TGCTACAAAA GCGAATGTAA

1601 GGAGGCATTT TGATGAAGTT CATAGAGGAC TAAGGAGGGA TTCAATTACT

1651 CCTGATATAG CAACAAAGCC TGGGCAACCT TTGTTCCTGG ATTCATATTT

1701 CTCCTAAAAA ATCTTTTAAG ACTCGAAAAC AAAAGTCTTC TTCAAAGGCT

1751 GAATACAATT TAACTGCATG CAAATGCCTC CTTTGCAAGA GGAAATATAG

1801 TTCACAAATA ATGCTTAAAA GACATATGCA AATTGTCCAC AAGATAACTC

1851 TTTCTGGAAC AAACTCTAAA AGAGAAAAAG GCCCTAATAA TACTGCCAAC

1901 AGTTCAGAAA TAAAAGTTAA AGTTGAACCA GCAGATTCTG TAGAATCTTC

1951 ACCCCCTTCC ATTACCCATT CTCCACAGAA TGAATTAAAG GGAACAAATC

2001 ATTCAAATGA AAAAAAGAAC ACACCGGCAG CACAGAAAAA TAAAGTTAAA

2051 CAAGACTCTG AAAGCCCTAA ATCAACTAGT CCGTCGGCTG CAGGTGGCCA

2101 GCAAAAATAA AAAAAAAAAA AAAAAAAAAA AAAAAA

B: Amino acid sequence: (SEQ ID NO: 17) Length: 337 amino acids

1 MPLRDKYCQT DHHHHGCCEP VYILEPGDPPP LLQQPLQTSK SGIQQIIECF

51 RSGTKQLKHI LLKDVDTIFE CKLCRSLFRG LPNLITHKKF YCPPSLQMDD

101 NLPDVNDKQS QAINDLLEAI YPSVDKREYI IKLEPIETNQ NAVFQYISRT

151 DNPIEVTESS STPEQTEVQI QETSTEQSKT VPVTDTEVET VEPPPVEIVT

201 DEVAPTSDEQ PQESQADLET SDNSDFGHQL ICCLCRKEFN SRRGVRRHIR

251 KVHKKKMEEL KKYIETRKNP NQSSKGRSKN VLVPLSRSCP VCCKSFATKA

301 NVRRHFDEVH RGLRRDSITP DIATKPGQPL FLDSYFS

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

Clone number and protein name: PP902

Start codon: 694 ATG End codon: 1707 TAA

Protein molecular weight: 38572.81

1 TGT CGC TGC CGG AGA GTC TCT GCT TCC CCC TTC CTA CGC GCT CCG CGG 48

49 CGG TAG CTC GGG CTC TCC GGA GGA GGG GGC GAC AGA GAA AAA GAG AGG 96

97 CTG ACT TAA TAA AGT TTC CTG AAA CAA CAA CTA CCA CCG AAA AGG CCC 144

145 AAG GTG GGG GGA GAC CCC CAT TTT CCT CCG CCC CTC TCC TAA AGC CTG 192

193 AGG GAC CGT TGC AGG TGG AGC GGC GCT GGT GGG ACC CGG GTT GCG GCG 240

241 GCA GCC GTT GCC CCG GCG AGG GAA GCC TCA GGT CCT GCC CGC TCC GGT 288

289 TCC GGC TCC TTC GGT GGT GGC GGC GGC AGC TGC TGC CGG GCG GTT CCT 336

337 CCT GTC AAC TGC CTC CGC CCG CGC GCC TCC CGC CTA GCC ACA CAG CGC 384

385 GGA GGA CGC CGT GAG CCG CCG CCG CAG TCG TCT CGG CCC GCG GAG GGA 432

433 CGT GGT GAG CTG TGG CGC TTC CGC GTC AGC GCC GGC GGG GAC TCT TTG 480

481 GGA GCG CGT CGG GCC CGA GTC CGG GTT CCC CCG CGT GGT CAG CGG AGG 528

529 CTG CGC CAG GTG GGG CCC GCA GCC GCC TCC CAC GCC AGG CTG TCC CCG 576

577 CGG CCG CCC GGC CTG CCT TCC TTC CTT CCG CCC GCC CGC CGG CCC GCC 624

625 CTA AGA GTT GAC CAC GCC GCA ATG AAG AGC TTC TTA CAG TGA AAG CAA 672

673 AGT AGG TCG AGT CCA CTG AAA ATG CCT TTA AGG GAC AAA TAC TGT CAG 720

1                                    

721 ACT GAC CAC CAT CAT CAC GGA TGC TGT GAA CCA GTT TAT ATC CTG GAA 768

10 Thr Asp His His His His Gly Cys Cys Glu Pro Val Tyr Ile Leu Glu 25

769 CCT GGA GAT CCT CCT TTG TTA CAG CAA CCA CTA CAG ACA TCC AAA TCT 816

26 Pro Gly Asp Pro Pro Leu Leu Gln Gln Pro Leu Gln Thr Ser Lys Ser 41

817 GGT ATT CAA CAA ATA ATT GAG TGC TTT CGA TCA GGA ACT AAA CAA CTT 864

42 Gly Ile Gln Gln Ile Ile Glu Cys Phe Arg Ser Gly Thr Lys Gln Leu 57

865 AAG CAT ATT TTA TTA AAA GAT GTG GAC ACT ATT TTT GAA TGT AAG TTA 912

58 Lys His Ile Leu Leu Lys Asp Val Asp Thr Ile Phe Glu Cys Lys Leu 73

913 TGC CGC AGT CTC TTC AGA GGA TTA CCA AAT TTA ATT ACC CAT AAA AAA 960

74 Cys Arg Ser Leu Phe Arg Gly Leu Pro Asn Leu Ile Thr His Lys Lys 89

961 TTC TAC TGC CCA CCA AGT CTC CAG ATG GAT GAC AAC CTT CCT GAT GTA 1008

90 Phe Tyr Cys Pro Pro Ser Leu Gln Met Asp Asp Asn Leu Pro Asp Val 105

1009 AAT GAT AAA CAA AGC CAA GCC ATA AAT GAT CTC CTA GAA GCC ATA TAT 1056

106 Asn Asp Lys Gln Ser Gln Ala Ile Asn Asp Leu Leu Glu Ala Ile Tyr 121

1057 CCA AGT GTG GAC AAA CGA GAA TAT ATT ATT AAG CTA GAA CCC ATA GAA 1104

122 Pro Ser Val Asp Lys Arg Glu Tyr Ile Ile Lys Leu Glu Pro Ile Glu 137

1105 ACT AAT CAA AAT GCA GTA TTT CAA TAT ATT TCG AGG ACT GAT AAT CCT 1152

138 Thr Asn Gln Asn Ala Val Phe Gln Tyr Ile Ser Arg Thr Asp Asn Pro 153

1152 ATT GAA GTC ACA GAG TCA AGC AGT ACT CCT GAA CAA ACC GAA GTT CAG 1200

154 Ile Glu Val Thr Glu Ser Ser Ser Thr Pro Glu Gln Thr Glu Val Gln 169

1201 ATA CAG GAA ACT AGC ACT GAA CAG TCA AAA ACA GTA CCG GTT ACA GAT 1248

170 Ile Gln Glu Thr Ser Thr Glu Gln Ser Lys Thr Val Pro Val Thr Asp 185

1249 ACA GAG GTG GAA ACT GTA GAG CCC CCT CCT GTT GAG ATT GTT ACA GAT 1296

186 Thr Glu Val Glu Thr Val Glu Pro Pro Pro Val Glu Ile Val Thr Asp 201

1297 GAA GTT GCA CCT ACA TCT GAT GAA CAA CCT CAG GAG TCG CAG GCT GAC 1244

202 Glu Val Ala Pro Thr Ser Asp Glu Gln Pro Gln Glu Ser Gln Ala Asp 217

1345 TTG GAA ACT TCT GAC AAT TCT GAT TTT GGT CAC CAG TTG ATA TGT TGT 1392

218 Leu Glu Thr Ser Asp Asn Ser Asp Phe Gly His Gln Leu Ile Cys Cys 233

1393 CTT TGT AGA AAA GAA TTC AAT TCT AGA CGA GGT GTT CGC CGT CAC ATT 1440

234 Leu Cys Arg Lys Glu Phe Asn Ser Arg Arg Gly Val Arg Arg His Ile 249

1441 CGA AAA GTA CAC AAG AAA AAG ATG GAA GAA CTA AAA AAG TAC ATT GAA 1488

250 Arg Lys Val His Lys Lys Lys Met Glu Glu Leu Lys Lys Tyr Ile Glu 265

1489 ACA CGA AAG AAT CCA AAC CAA TCC TCT AAA GGA CGC AGT AAG AAT GTT 1536

266 Thr Arg Lys Asn Pro Asn Gln Ser Ser Lys Gly Arg Ser Lys Asn Val 281

1537 CTA GTT CCA TTA AGT AGG AGT TGT CCA GTA TGT TGT AAA TCA TTT GCT 1584

282 Leu Val Pro Leu Ser Arg Ser Cys Pro Val Cys Cys Lys Ser Phe Ala 297

1585 ACA AAA GCG AAT GTA AGG AGG CAT TTT GAT GAA GTT CAT AGA GGA CTA 1632

298 Thr Lys Ala Asn Val Arg Arg His Phe Asp Glu Val His Arg Gly Leu 313

1633 AGG AGG GAT TCA ATT ACT CCT GAT ATA GCA ACA AAG CCT CGG CAA CCT 1680

314 Arg Arg Asp Ser Ile Thr Pro Asp Ile Ala Thr Lys Pro Gly Gln Pro 329

1681 TTG TTC CTG GAT TCA TAT TTC TCC TAA AAA ATC TTT TAA GAC TCG AAA 1728

330 Leu Phe Leu Asp Ser Tyr Phe Ser *** 338

1729 ACA AAA GTC TTC TTC AAA GGC TGA ATA CAA TTT AAC TGC ATG CAA ATG 1776

1777 CCT CCT TTG CAA GAG GAA ATA TAG TTC ACA AAT AAT GCT TAA AAG ACA 1824

1825 TAT GCA AAT TGT CCA CAA GAT AAC TCT TTC TGG AAC AAA CTC TAA AAG 1872

1873 AGA AAA AGG CCC TAA TAA TAC TGC CAA CAG TTC AGA AAT AAA AGT TAA 1920

1921 AGT TGA ACC AGC AGA TTC TGT AGA ATC TTC ACC CCC TTC CAT TAC CCA 1968

1969 TTC TCC ACA GAA TGA ATT AAA GGG AAC AAA TCA TTC AAA TGA AAA AAA 2016

2017 GAA CAC ACC GGC AGC ACA GAA AAA TAA AGT TAA ACA AGA CTC TGA AAG 2064

2065 CCC TAA ATC AAC TAG TCC GTC GGC TGC AGG TGG CCA GCA AAA ATA AAA 2112

2113 AAA AAA AAA AAA AAA AAA AAA AAA 2136

7. PP1628

A: Nucleotide sequence: (SEQ ID NO: 19) Length: 1913bp

1 GCTGGGAGCA CACACGCTGG GACCTATGTG TTTGTGTGGT CGTTCCAAAC

51 TGCCCCAGGG CTTTGGGGGC GGCACTTGGG GTTTCTGGGA ATGACATCAT

101 CTCTGTTCCC CATCCCCAGT AGTTTACATT CCTGACTTCT GAATACAGCA

151 CAGCTGAGCC CCCTGCAGCT CCGATCTCCA GCTATTCCTA GGCAAAGAGC

201 CTCATGGCTA AGGCAGCCTC AAAGCCAGCC CCTCCTCCCA CCTATTCTGA

251 GTAGCTGCAG AGGCCTTGGG TCCAGGCTCT AGGTTCATCC CTCAGTTGGG

301 GGGAACGTAG GACCCAGCTG GAGCCTCTTG AGGGAGATGA GAGGCCTCTT

351 TGTGAGGAGG ACATTAGCTG TGTGGCCTCT CTCTCTTTGG CCCTGTTTCC

401 TTTTTTGCAA AACAAGGACA TTTTCTGCAG CCCCTTCCTC TCAGTGAGCT

451 ATGATTGGAG GGCTTAGGTC TGGAGGATTC AAGAGTGGGA AGAGGAATTT

501 AAGGGGGTCC CCTAGTCTAG TCTCTGCCCC TGGATAGTGT CCAGCCTTTA

551 TATTTCTGAA GAGGTGGATC CCAGAGTGGC TCTGATGTCC ACATTAGAAA

601 AGCTTACTTG TAATGATCAT GTCAGCCTTC AGAAGAGAAT CCCCACCAAC

651 TTCTGTGCCT CCTCAGATGG GGATTTATCT GGATCTCTGT GGTTCCTTCT

701 CAGCCGAAAC AGGTCCAGTA TCCCAGTCAT TTCTTCAAAT GCTGATAGGG

751 GTATGTTGGA ATCCGAAGCC ACTTCCCCGC CTTCAAGCCC CAGATGGGCT

801 GCTCTCCTGT AACTTTCTAG GAGAAGAGAC ATTTTCTTCT TTCCCTTTCC

851 TGGTCCATCC CTGCACCCTG GTCCTCTCCC AGCCTCTCCC CCACATTGTC

901 CCTGACTCTA GGGGCACATC CAGTCTCCAT CGTGCTGCAG CAGCTGGACT

951 GAGGGCAGAG CCTGTAGGTG CAGAGGCCCT GGCTCCCGAG GTCCAGCCAC

1001 TCTCCCTGGG GCCTCTGGGG TGAGAGCAGC TTCCGATAGG ACCTGCCCAG

1051 ATTTCTGCAT GTGCACTTTT GTTTACTGAA AGAGAGAAAG GGGGGGGTCA

1101 CAGCAACATG CCCTGGCCTT TCTGCCCTGT TCCCCAACCC CACTGAGGCC

1151 TGCTGCACAG GTCAATGCCT TCGTTATCGT TATTGTACTG TCACTTTGTT

1201 CTTGAGGTAG TAGTCAAGGA TCAGGAGGGG CAGATGTCTT CTCTGGGCTG

1251 CGTGGGGCCG GAGCAGAGGT GAGCAGCAAT GCACTGGTTC GGGAGCCCCC

1301 ATCAGCCTCC TTGTGCAAAC TGGGCCCCCA TGCCACAGTC TGGCTTTCCC

1351 TCCATCTGCC CCAGGACAAG AGCAAGAAGG ACATCAGTTG CCCAGTCATG

1401 TGATCCCCTG CCATCTTGCC TTAGGAACAG CCTTCCCCCA CCAGCAGCCA

1451 TGGCTGGCTG GGGCGTTAGC CAAGCCACCT ACTGCCAGGA ATTGGAGCCT

1501 CAGTTCCCTC CTGTGTCAAG TAGCTAACTG CAGCAGCTGG ACTGAGGGCA

1551 GAGTCTGTGG GTGCAGAGAC CCTGCATGTA GGTCACAGGT TGAGGCCCAG

1601 CCACTTCTCCC TGGGGCCTGG TGGGTAGGCA AGTAGCTCTG GGGCCACCTC

1651 AAGTGACCAA ATGCTATTAA TTTCCATCCT TTAGCAGGCT GGGCCCTAGG

1701 CAGGAAGCTG GCTTCTGGGA GAGGAGTGAG AACGTGCAGG GCCTGCCTAG

1751 CTTGCGTGCT TGAGGAAGGT GGCATTCCGT GCTTGCCTCC TTGAGGAGGG

1801 TGGCATTCTG TGTCTTCTGC TTATGAAGCG CCTTTCTTAA AGTTTGGCAA

1851 TAAATCCATT TTTATGGAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

1901 AAAAAAAAAAA AAA

B: Amino acid sequence: (SEQ ID NO: 20) Length: 136 amino acids

1 MIMSAFRRES PPTSVPPQMG IYLDLCGSFS AETGPVSQSF LQMLIGVCWN

51 PKPLPRLQAP DGLLSCNFLG EETFSSFPFL VHPCTLVLSQ PLPHIVPDSR

101 GTSSLHRAAA AGLRAEPVGA EALAPEVQPL SLGPLG

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

Clone number and protein name: PP1628

Start code: 613 ATG End code: 1023 TGA

Protein molecular weight: 14421.90

1 GCT GGG ACC ACA CAC GCT GGG ACC TAT GTG TTT GTG TGG TCG TTC CAA 48

49 ACT GCC CCA GGG CTT TGG GGG CGG CAC TTG GGG TTT CTG GGA ATG ACA 96

97 TCA TCT CTG TTC CCC ATC CCC AGT AGT TTA CAT TCC TGA CTT CTG AAT 144

145 ACA GCA CAG CTG AGC CCC CTG CAG CTC CCA TCT CCA GCT ATT CCT AGG 192

193 CAA AGA GCC TCA TGG CTA AGG CAG CCT CAA AGC CAG CCC CTC CTC CCA 240

241 CCT ATT CTG AGT AGC TGC AGA GGC CTT GGG TCC AGG CTC TAG GTT CAT 288

289 CCC TCA GTT GGG GGG AAC GTA GGA CCC AGC TGG AGC CTC TTG AGG GAG 336

337 ATG AGA GGC CTC TTT GTG AGG AGG ACA TTA GCT GTG TGG CCT CTC TCT 384

385 CTT TGG CCC TGT TTC CTT TTT TGC AAA ACA AGG ACA TTT TCT GCA GCC 432

433 CCT TCC TCT CAG TGA GCT ATG ATT GGA GGG CTT AGG TCT GGA GGA TTC 480

481 AAG AGT GGG AAG AGG AAT TTA AGG GGG TCC CCT AGT CTA GTC TCT GCC 528

529 CCT GGA TAG TGT CCA GCC TTT ATA TTT CTG AAG AGG TGG ATC CCA GAG 576

577 TGG CTC TGA TGT CCA CAT TAG AAA AGC TTA CTT GTA ATG ATC ATG TCA 624

1 Met Ile Met Ser 4

625 GCC TTC AGA AGA GAA TCC CCA CCA ACT TCT GTG CCT CCT CAG ATG GGG 672

5 Ala Phe Arg Arg Glu Ser Pro Pro Thr Ser Val Pro Pro Gln Met Gly 20

673 ATT TAT CTG GAT CTC TGT GGT TCC TTC TCA GCC GAA ACA GGT CCA GTA 720

21 Ile Tyr Leu Asp Leu Cys Gly Ser Phe Ser Ala Glu Thr Gly Pro Val 36

721 TCC CAG TCA TTT CTT CAA ATG CTG ATA GGG GTA TGT TGG AAT CCG AAG 768

37 Ser Gln Ser Phe Leu Gln Met Leu Ile Gly Val Cys Trp Asn Pro Lys 52

769 CCA CTT CCC CGC CTT CAA GCC CCA GAT GGG CTG CTC TCC TGT AAC TTT 816

53 Pro Leu Pro Arg Leu Gln Ala Pro Asp Gly Leu Leu Ser Cys Asn Phe 68

817 CTA GGA GAA GAG ACA TTT TCT TCT TTC CCT TTC CTG GTC CAT CCC TGC 864

69 Leu Gly Glu Glu Thr Phe Ser Ser Phe Pro Phe Leu Val His Pro Cys 84

865 ACC CTG GTC CTC TCC CAG CCT CTC CCC CAC ATT GTC CCT GAC TCT AGG 912

85 Thr Leu Val Leu Ser Gln Pro Leu Pro His Ile Val Pro Asp Ser Arg 100

913 GGC ACA TCC AGT CTC CAT CGT GCT GCA GCA GCT GGA CTG AGG GCA GAG 960

101 Gly Thr Ser Ser Ser Leu His Arg Ala Ala Ala Ala Gly Leu Arg Ala Glu 116

961 CCT GTA GGT GCA GAG GCC CTG GCT CCC GAG GTC CAG CCA CTC TCC CTG 1008

117 Pro Val Gly Ala Glu Ala Leu Ala Pro Glu Val Gln Pro Leu Ser Leu 132

1009 GGG CCT CTG GGG TGA GAG CAG CTT CCG ATA GGA CCT GCC CAG ATT TCT 1056

133 Gly Pro Leu Gly *** 137

1057 GCA TGT GCA CTT TTG TTT ACT GAA AGA GAG AAA GGG GGG GGT CAC AGC 1104

1105 AAC ATG CCC TGG CCT TTC TGC CCT GTT CCC CAA CCC CAC TGA GGC CTG 1152

1153 CTG CAC AGG TCA ATG CCT TCG TTA TCG TTA TTG TAC TGT CAC TTT GTT 1200

1201 CTT GAG GTA GTA GTC AAG GAT CAG GAG GGG CAG ATG TCT TCT CTG GGC 1248

1249 TGC GTG GGG CCG GAG CAG AGG TGA GCA GCA ATG CAC TGG TTC GGG AGC 1296

1297 CCC CAT CAG CCT CCT TGT GCA AAC TGG GCC CCC ATG CCA CAG TCT GGC 1344

1345 TTT CCC TCC ATC TGC CCC AGG ACA AGA GCA AGA AGG ACA TCA GTT GCC 1392

1393 CAG TCA TGT GAT CCC CTG CCA TCT TGC CTT AGG AAC AGC CTT CCC CCA 1440

1441 CCA GCA GCC ATG GCT GGC TGG GGC GTT AGC CAA GCC ACC TAC TGC CAG 1488

1489 GAA TTG GAG CCT CAG TTC CCT CCT GTG TCA AGT AGC TAA CTG CAG CAG 1536

1537 CTG GAC TGA GGG CAG AGT CTG TGG GTG CAG AGA CCC TGC ATG TAG GTC 1584

1585 ACA GGT TGA GGC CCA GCC ACT CTC CCT GGG GCC TGG TGG GTA GGC AAG 1632

1633 TAG CTC TGG GGC CAC CTC AAG TGA CCA AAT GCT ATT AAT TTC CAT CCT 1680

1681 TTA GCA GGC TGG GCC CTA GGC AGG AAG CTG GCT TCT GGG AGA GGA GTG 1728

1729 AGA ACG TGC AGG GCC TGC CTA GCT TGC GTG CTT GAG GAA GGT GGC ATT 1776

1777 CCG TGC TTG CCT CCT TGA GGA GGG TGG CAT TCT GTG TCT TCT GCT TAT 1824

1825 GAA GCG CCT TTC TTA AAG TTT GGC AAT AAA TCC ATT TTT ATG GAA AAA 1872

1873 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AA 1913

D: Blastp results

Query＝PP1628[gene＝PP1628] (136 amino acids)

>SP_IN: Q24146 Q24146 drosophila melanogaster(fruit fly).hlh106.

                                          11/1998

Length = 1113

Score = 30.5bits (67), predicted value = 6.6

Identity = 34/129 (26%), Similarity = 52/129 (39%), Gap = 12/129 (9%)

Query: 7 RRESPPTSV-PPQMGIYLDLCGSFSAETGPVSQSF--LQMLIGVCWNPKP----LPRLQA 59

+R P T+V PP +G + S S T PV + + ++ V P P LP+

Sbjct: 145 QRMPPNTAVYPPSLGSSF-VYQSMSPPTSPVESANQNVNVMQPVAATPAPASAPPLPQQSY 203

Query: 60 PDGLLSCNFLGEETFSSFPFLVHPCTLVLSQPLPHIVPDSRGTSSLHRAAAAGLRAEPVG 119

                                 

Sbjct: 204 PQPFITYNSKAGMTSDEAMYLLLQPTVASPTPSPPVAPPPSTGS----RASKVRVAPLA 259

Query: 120 AEALAPEVQ 128

A EVQ

Sbjct: 260 PSPAAMEVQ 268

8. PP1650

A: Nucleotide sequence: (SEQ ID NO: 22) Length: 1494bp

1 TCTTTGTTCT GTCCCCGGTG TGTGGGTCTG TGACAGGGTC CAACAGGGCC

51 TGGTCCGTGT CCGGTCCCCC AAATCTGTCG TCCCTGCCCC CAGGCATTGG

101 CATCAACAAA AGTCAGAATT CCCGGGAACT TGAACAGAGG CTGCTAAATT

151 CCCAGTAATT GCTCCTTTGG CCTTCTAGGG ACTGACTTCA AAGAAGGAAG

201 GAAAGAATCA GGCAGTGCTT CCTCATTCTC TTTAAAACC CGCTTCCCGC

251 TGAGTCTGCA CCCAGGAGAC CAGAGAGCAC CTTGCCCTTC CATGGAAACT

301 CAGGCTGATC TCGTATCTCA GGAACCTCAG GCCCTGCTTG ACAGTGCTCT

351 TCCTTCAAAA GTTCCTGCCT TTTCCGACAA GGACAGCCTG GGGGATGAGA

401 TGTTGGCGGC TGCGCTCCTA AAGGCCAAGT CCCAGGAGCT GGTAACCTTT

451 GAGGATGTAG CTGTGTACTT CATCCGGAAG GAGTGGAAGC GTTTGGAACC

501 TGCTCAGAGG GACCTCTATA GAGATGTGAT GCTGGAGAAT TACGGGAATG

551 TGTTCTCACT GGACTGGATA CACGTCTGTT TAACTACGCA AAAGGTAATG

601 CTGGCATGGC TTACTGGGAC CCTAAGTGTG GCGAAGGGAC TCTGCTCCAG

651 TGAACTGGCG AGTGTGGAAC CTCCTGACAC CTTCTGAGGA CCTCCTGCCT

701 GCCATGTTGC TGTGGAGCTC GCACTCCTCA GGCATCCCCT GATGTTGAGT

751 GATACAAACT CTATCACCGG AATCGATGCT GCTGCAATGA CAAGACTTCT

801 TTCTGGTTTT CAGATTCTAA AGTTTAAAAC AACGACAACA ACAGGAGCGC

851 TTGAAAGTTA CGGTGCTTCT CCCTCTCCAG TGTGGACTCG CTGATGTTTG

901 GAAAGATTGG ACTTGCTACA GAAGGTCTTT CCACAGTGAT GACACCAGTA

951 GGGCTTTTCC CCGGTGTGGA TTCTGTGGTG TTTATTGAAG TTGGAGCTGT

1001 GGTTGAAGGC TTTCCCACAC TCCTTACACT TATATGGCTT CTCTCCGGTG

1051 TGGAGTCTCT GGTGGGAGCT GAGGCCCGCA TGCTGACTGA AGCTCTTCCC

1101 ACATTCGTTA CACTGATAAG GCTTCTCCCCC AGTGTGGATC CGATAGTGAC

1151 GAATGAGGCT GCCTTTCCCG CTGAAAGCCT TGCCACAATC TTTGCACTGA

1201 TATGGCGCCT CTTCTGTGTG CATTCTCTGA TGTTTAAGAA GGTCTGAGCT

1251 CTGCCCAAAA GCTTTTCCAC ACTTACAGTC ATAGGGCCGG TCCACCAAGT

1301 GTGTTCTGTA GTGGAGGGTG AGGTTTGAGC TGTGGCTGAA AGCTTTCCCA

1351 CACTTGGTGC ACACGTAAGG TTTCTCCCCA GTGTGTGTTC TCCTGTGTTT

1401 GGTGAGATTT GAGCTATTAC TAAAGGCTTT GCCACATTCA GCCCAAAAAA

1451 AAAAAAAAAA AAAAAAAAAA AAAAAAAASS AAAAAAAAAA AAAA

B: Amino acid sequence: (SEQ ID NO: 23) Length: 131 amino acids

1 METQADLVSQ EPQALLDSAL PSKVPAFSDK DSLGDEMLAA ALLKAKSQEL

51 VTFEDVAVYF IRKEWKRLEP AQRDLYRDVM LENYGNVFSL DWIHVCLTTQ

101 KVMLAWLTGT LSVAKGLCSS ELASVEPPDT F

C: Nucleotide and amino acid combination sequence (SEQ ID NO: 24)

Clone number and protein name: PP1650

Start codon: 292 ATG End codon: 687 TGA

Protein molecular weight: 14625.94

1 TCT TTG TTC TGT CCC CGG TGT GTG GGT CTG TGA CAG GGT CCA ACA GGG 48

49 CCT GGT CCG TGT CCG GTC CCC CAA ATC TGT CGT CCC TGC CCC CAG GCA 96

97 TTG GCA TCA ACA AAA GTC AGA ATT CCC GGG AAC TTG AAC AGA GGC TGC 144

145 TAA ATT CCC AGT AAT TGC TCC TTT GGC CTT CTA GGG ACT GAC TTC AAA 192

193 GAA GGA AGG AAA GAA TCA GGC AGT GCT TCC TCA TTC TCT TTT AAA ACC 240

241 CGC TTC CCG CTG AGT CTG CAC CCA GGA GAC CAG AGA GCA CCT TGC CCT 288

289 TCC ATG GAA ACT CAG GCT GAT CTC GTA TCT CAG GAA CCT CAG GCC CTG 336

1 Met Glu Thr Gln Ala Asp Leu Val Ser Gln Glu Pro Gln Ala Leu 15

337 CTT GAC AGT GCT CTT CCT TCA AAA GTT CCT GCC TTT TCC GAC AAG GAC 384

16 Leu Asp Ser Ala Leu Pro Ser Lys Val Pro Ala Phe Ser Asp Lys Asp 31

385 AGC CTG GGG GAT GAG ATG TTG GCG GCT GCG CTC CTA AAG GCC AAG TCC 432

32 Ser Leu Gly Asp Glu Met Leu Ala Ala Ala Leu Leu Lys Ala Lys Ser 47

433 CAG GAG CTG GTA ACC TTT GAG GAT GTA GCT GTG TAC TTC ATC CGG AAG 480

48 Gln Glu Leu Val Thr Phe Glu Asp Val Ala Val Tyr Phe Ile Arg Lys 63

481 GAG TGG AAG CGT TTG GAA CCT GCT CAG AGG GAC CTC TAT AGA GAT GTG 528

64 Glu Trp Lys Arg Leu Glu Pro Ala Gln Arg Asp Leu Tyr Arg Asp Val 79

529 ATG CTG GAG AAT TAC GGG AAT GTG TTC TCA CTG GAC TGG ATA CAC GTC 576

80 Met Leu Glu Asn Tyr Gly Asn Val Phe Ser Leu Asp Trp Ile His Val 95

577 TGT TTA ACT ACG CAA AAG GTA ATG CTG GCA TGG CTT ACT GGG ACC CTA 624

96 Cys Leu Thr Thr Gln Lys Val Met Leu Ala Trp Leu Thr Gly Thr Leu 111

625 AGT GTG GCG AAG GGA CTC TGC TCC AGT GAA CTG GCG AGT GTG GAA CCT 672

112 Ser Val Ala Lys Gly Leu Cys Ser Ser Glu Leu Ala Ser Val Glu Pro 127

673 CCT GAC ACC TTC TGA GGA CCT CCT GCC TGC CAT GTT GCT GTG GAG CTC 720

128 Pro Asp Thr Phe *** 132

721 GCA CTC CTC AGG CAT CCC CTG ATG TTG AGT GAT ACA AAC TCT ATC ACC 768

769 GGA ATC GAT GCT GCT GCA ATG ACA AGA CTT CTT TCT GGT TTT CAG ATT 816

817 CTA AAG TTT AAA ACA ACG ACA ACA ACA GGA GCG CTT GAA AGT TAC GGT 864

865 GCT TCT CCC TCT CCA GTG TGG ACT CGC TGA TGT TTG GAA AGA TTG GAC 912

913 TTG CTA CAG AAG GTC TTT CCA CAG TGA TGA CAC CAG TAG GGC TTT TCC 960

961 CCG GTG TGG ATT CTG TGG TGT TTA TTG AAG TTG GAG CTG TGG TTG AAG 1008

1009 GCT TTC CCA CAC TCC TTA CAC TTA TAT GGC TTC TCT CCG GTG TGG AGT 1056

1057 CTC TGG TGG GAG CTG AGG CCC GCA TGC TGA CTG AAG CTC TTC CCA CAT 1104

1105 TCG TTA CAC TGA TAA GGC TTC TCC CCA GTG TGG ATC CGA TAG TGA CGA 1152

1153 ATG AGG CTG CCT TTC CCG CTG AAA GCC TTG CCA CAA TCT TTG CAC TGA 1200

1201 TAT GGC GCC TCT TCT GTG TGC ATT CTC TGA TGT TTA AGA AGG TCT GAG 1248

1249 CTC TGC CCA AAA GCT TTT CCA CAC TTA CAG TCA TAG GGC CGG TCC ACC 1296

1297 AAG TGT GTT CTG TAG TGG AGG GTG AGG TTT GAG CTG TGG CTG AAA GCT 1344

1345 TTC CCA CAC TTG GTG CAC ACG TAA GGT TTC TCC CCA GTG TGT GTT CTC 1392

1393 CTG TGT TTG GTG AGA TTT GAG CTA TTA CTA AAG GCT TTG CCA CAT TCA 1440

1441 GCC CAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1488

1489 AAA AAA 1494

D: Blastp result

Query＝PP1650[gene＝PP1650] (131 amino acids)

>SP_HUM: O14709 O14709 homo sapiens(human).zinc finger protein.

5/1999

length = 1029

Score = 76.5bits (185), expected value = 8e-14

Identity = 45/90 (50%), Similarity = 60/90 (66%), Gap = 6/90 (6%)

Query: 6 DLVS---QEPQALLDSALPSKVPAFSDKDSLGDEMLAAALLKAKSQELVTFEDVAVYFIR 62

DLV+ Q+PQ DS P + A S +++ ++++A LL A+ QELV FE+V+V F

Sbjct: 172 DLVAFNLQDPQH--DSPAP-EASALSQEENPRNQLMALMLLLTAQPQELVMFEEVSVCFTS 228

Query: 63 KEWKRLEPAQRDLYRDVMLENYGNVFSLDW 92

+EW L P QR LY DVMLENYGNV SL+W

Sbjct: 229 EEWACLGPIQRALYWDVMLENYGNVTSLEW 258

9. PP2672

A: Nucleotide sequence: (SEQ ID NO: 25) Length: 2455bp

1 GGCGTCTGGA CACCCTTCAT CTTTTAGCCG TTGCCCTGTT CTTGGTGAAC

51 TGCTTGTTAG GACCAAAAGT GGCCCACCCC TTTCTCTGGG ACAGTGCAGA

101 GCACCGGGGC CAGAGGCTCA AAGCACCTCC AAGCTGAGAT CAGAGACTTT

151 GGATATCGTT GATCCAGAGG CTTCCAAACT TTTCCTGGTG ATTGAATTCT

201 TACCATGAAA TCCAGTGAGG AGCTCCAATG CCTCAAGCAG ATGGAAGAGG

251 AACTGCTCTT CTTGAAGGCA GGGCAGGGCT CTCAGAGGGC AAGACTCACC

301 CCACCCCTGC CACGGGCTCT CCAGGGTAAC TTTGGAGCCC CAGCACTCTG

351 TGGAATATGG TTCGCAGAAC ACTTGCATCC TGCTGTTGGG ATGCCCCCAA

401 ACTATAATAG CAGCATGCTC AGTCTTTCAC CAGAAAGAAC CATCTTATCT

451 GGAGGATGGT CAGGAAAACA AACTCAACAG CCAGTGCCTC CCCTCAGAAC

501 TCTGCTCCTG AGGTCTCCTT TCAGTCTGCA TAAGTCCTCC CAACCTGGGA

551 GCCCCAAAGC TTCTCAGCGG ATCCATCCCC TCTTCCATTC TATCCCAAGG

601 TCTCAGCTGC ATTCTGTTCT GCTTGGTCTT CCTCTCCTCT TCATACAGAC

651 TAGACCTTCA CCCCCTGCCC AGTATGGCGC GCAAATGCCA TTGAGATACA

701 TTTGTTTTGG TCCCAATATC TTTTGGGGCT CCAAAAAGCC CCAGAAAGAG

751 TAATGCCTTC AGCCATGGCC CATGTGATGA CTTTTCCCAG AGACAGCCCC

801 ATGGGTCAGT GATCGCCGGT GGGAGGTCTC CCCAGCCTCC CTTCAAGCCT

851 TCAAGTCTGT GTAGAGGCGC CTTCTCTTCT CTGGGCATTC CCCGCTTTGT

901 TGCAGAGAAA TTATTTTCTA CTCAGCGGCT TTCAGGTAGA GAGAGAAAGT

951 GCATGCGGTA TGAAGGATGT GAAGATGGAG GGCTTGACGT CGTGGGTCAT

1001 GTTTTGTGGT TAATTCCCCC ACATAACAAA ACAGTGAGCA AATGTTTATA

1051 AACAGGAACT CAGTGTTTCA GTGATGAAAG CCTCTGGCCT CGCCACACTG

1101 ATTGCTGGTG TGCGCGCACA CACACACTCA CACACCCTGTTTTTCTTACA

1151 CACACACAGC TTCATGGAGC AGAGAGCTGG AAAGGGTGTC CAGAATGGTG

1201 GATCAGGCTT ATTATTATTA CTTTTTTTAT CTCCACCAAA CTCTCTCCTT

1251 AAGCCCCCAGC AGCCAACCCCC CCCCCCCCCA CCAGACAGCA TGTCGAAGGA

1301 GTAAGCTGCA GGTCAGCCTA TATTTTAGGC AGAAAATTAG GCAACCTTGG

1351 GAGAGACCGG TTTCCAGCAG ACACGCACGG ACGCGATGGT GAGGTTGGGC

1401 TGAGACGGTC CTTCCTGGAT GCAGTCACCC TCCCCATCCC GAGTTTTACT

1451 CCCTGGGGGG ATTGTGGTGG GTGCCTGGCC TCCTCTCCCA CCAGCGCACT

1501 GCCCCAAGCT TGCCTAGGTA GTATCAGCAC TGGGGTGAGG GATGCAATGC

1551 GGGTTTATAG TTGGCTGTGG GGACTTCTGA TTTCTCCACC CCATAGATGA

1601 TAAACGACCG CGGGGCTTCC TCTCCCAGGT CTTTCCTCAC TCTCCTTCGT

1651 CTTTCCCTCT TTCTCCATGG GAAAGGTAGA ATGAGACTTG CCACCAGGGA

1701 CATCCAGGCA CATTCCTCTT CCCAATCAGC CTCTACCCAT TCTCTTTCTG

1751 TCTCGTGTGT ATCAGTGGCA TTTTCTTGTG TAGCTTCCCA GATTTGCAAA

1801 CCCGCTCCAC ATCCTGGACG CAGACCCCCT TCGCTGGCTC TATGAAGGCT

1851 TCACGTTCCT GGGGCATTCG AGACTTGACT TTCCAGAGCA ACTTTGGATC

1901 CACCCTGTTA CCCTTTTTTT TGTTTTGTTT TGTTTTGAGA GGGAGTCTCG

1951 CTGTGTTGTC CAGGCTGGAG TGCAGTGGTG TGATCTCGGC TTACTGCAAC

2001 CTCCGCCTAC TGGGTTCAAG TGATTCTCTT GCCTCAATCT CCCCAGTACC

2051 TGGGATTACA GGTAGACACC ATCACGCCTG GCTAATTTTT GTATTTTTAG

2101 TAAAGACAGG GTTTCATCAT GTTGGCCTGG CTGGTCTCAA ACTCCTGACC

2151 TCAGCCGATC CGCCCGCCTC AGCCTCCCAA AGTTCTGGGA TTACAAGTGT

2201 GAACCACCGT GCCCAACCTC CCCTCGTTTT TGACCTTTTGT CCCCAAATGT

2251 CTTTCATCCA GTTACTAGGA CTTGTAGCCT TTTTGCAAAT AGGTATCTGT

2301 AAGCATAGAA GAATAGGGTG ATTTGTGAAG TGACCCTTAG TGGCATCTGT

2351 GGTGGTCCTA CTAATCAGAT TTGCAGAAGA AACATGGAAA CAAGGAAACT

2401 CCTCTCTTAT ATTAAAATAA ATAAGGTTTT TAATTTCAAA AAAAAAAAAA

2451 AAAAA

B: Amino acid sequence: (SEQ ID NO: 26) Length: 182 amino acids

1 MKSSEELQCL KQMEEELLFL KAGQGSQRAR LTPPLPRALQ GNFGAPALCG

51 IWFAEHLHPA VGMPPNYNSS MLSLSPERTI LSGGWSGKQT QQPVPPLRTL

101 LLRSPFSLHK SSQPGSPKAS QRIHPLFHSI PRSQLHSVLL GLPLLFIQTR

151 PSPPAQYGAQ MPLRYICFGP NIFWGSKKPQ KE

C: Nucleotide and amino acid combination sequence (SEQ ID NO: 27)

Clone number and protein name: PP2672

Start codon: 205 ATG Stop codon: 753 TAA

Protein molecular weight: 20179.43

1 GGC GTC TGG ACA CCC TTC ATC TTT TAG CCG TTG CCC TGT TCT TGG TGA 48

49 ACT GCT TGT TAG GAC CAA AAG TGG CCC ACC CCT TTC TCT GGG ACA GTG 96

97 CAG AGC ACC GGG GCC AGA GGC TCA AAG CAC CTC CAA GCT GAG ATC AGA 144

145 GAC TTT GGA TAT CGT TGA TCC AGA GGC TTC CAA ACT TTT CCT GGT GAT 192

193 TGA ATT CTT ACC ATG AAA TCC AGT GAG GAG CTC CAA TGC CTC AAG CAG 240

1 Met Lys Ser Ser Glu Glu Leu Gln Cys Leu Lys Gln 12

241 ATG GAA GAG GAA CTG CTC TTC TTG AAG GCA GGG CAG GGC TCT CAG AGG 288

13 Met Glu Glu Glu Leu Leu Phe Leu Lys Ala Gly Gln Gly Ser Gln Arg 28

289 GCA AGA CTC ACC CCA CCC CTG CCA CGG GCT CTC CAG GGT AAC TTT GGA 336

29 Ala Arg Leu Thr Pro Pro Leu Pro Arg Ala Leu Gln Gly Asn Phe Gly 44

337 GCC CCA GCA CTC TGT GGA ATA TGG TTC GCA GAA CAC TTG CAT CCT GCT 384

45 Ala Pro Ala Leu Cys Gly Ile Trp Phe Ala Glu His Leu His Pro Ala 60

385 GTT GGG ATG CCC CCA AAC TAT AAT AGC AGC ATG CTC AGT CTT TCA CCA 432

61 Val Gly Met Pro Pro Asn Tyr Asn Ser Ser Met Leu Ser Leu Ser Pro 76

433 GAA AGA ACC ATC TTA TCT GGA GGA TGG TCA GGA AAA CAA ACT CAA CAG 480

77 Glu Arg Thr Ile Leu Ser Gly Gly Trp Ser Gly Lys Gln Thr Gln Gln 92

481 CCA GTG CCT CCC CTC AGA ACT CTG CTC CTG AGG TCT CCT TTC AGT CTG 528

93 Pro Val Pro Pro Leu Arg Thr Leu Leu Leu Arg Ser Pro Phe Ser Leu 108

529 CAT AAG TCC TCC CAA CCT GGG AGC CCC AAA GCT TCT CAG CGG ATC CAT 576

109 His Lys Ser Ser Gln Pro Gly Ser Pro Lys Ala Ser Gln Arg Ile His 124

577 CCC CTC TTC CAT TCT ATC CCA AGG TCT CAG CTG CAT TCT GTT CTG CTT 624

125 Pro Leu Phe His Ser Ile Pro Arg Ser Gln Leu His Ser Val Leu Leu 140

625 GGT CTT CCT CTC CTC TTC ATA CAG ACT AGA CCT TCA CCC CCT GCC CAG 672

141 Gly Leu Pro Leu Leu Phe Ile Gln Thr Arg Pro Ser Pro Pro Ala Gln 156

673 TAT GGC GCG CAA ATG CCA TTG AGA TAC ATT TGT TTT GGT CCC AAT ATC 720

157 Tyr Gly Ala Gln Met Pro Leu Arg Tyr Ile Cys Phe Gly Pro Asn Ile 172

721 TTT TGG GGC TCC AAA AAG CCC CAG AAA GAG TAA TGC CTT CAG CCA TGG 768

173 Phe Trp Gly Ser Lys Lys Pro Gln Lys Glu*** 183

769 CCC ATG TGA TGA CTT TTC CCA GAG ACA GCC CCA TGG GTC AGT GAT CGC 816

817 CGG TGG GAG GTC TCC CCA GCC TCC CTT CAA GCC TTC AAG TCT GTG TAG 864

865 AGG CGC CTT CTC TTC TCT GGG CAT TCC CCG CTT TGT TGC AGA GAA ATT 912

913 ATT TTC TAC TCA GCG GCT TTC AGG TAG AGA GAG AAA GTG CAT GCG GTA 960

961 TGA AGG ATG TGA AGA TGG AGG GCT TGA CGT CGT GGG TCA TGT TTT GTG 1008

1009 GTT AAT TCC CCC ACA TAA CAA AAC AGT GAG CAA ATG TTT ATA AAC AGG 1056

1057 AAC TCA GTG TTT CAG TGA TGA AAG CCT CTG GCC TCG CCA CAC TGA TTG 1104

1105 CTG GTG TGC GCG CAC ACA CAC ACT CAC ACA CCC TGT TTT TCT TAC ACA 1152

1153 CAC ACA GCT TCA TGG AGC AGA GAG CTG GAA AGG GTG TCC AGA ATG GTG 1200

1201 GAT CAG GCT TAT TAT TAT TAC TTT TTT TAT CTC CAC CAA ACT CTC TCC 1248

1249 TTA AGC CCC AGC AGC CAA CCC CCC CCC CCC CAC CAG ACA GCA TGT CGA 1296

1297 AGG AGT AAG CTG CAG GTC AGC CTA TAT TTT AGG CAG AAA ATT AGG CAA 1344

1345 CCT TGG GAG AGA CCG GTT TCC AGC AGA CAC GCA CGG ACG CGA TGG TGA 1392

1393 GGT TGG GCT GAG ACG GTC CTT CCT GGA TGC AGT CAC CCT CCC CAT CCC 1440

1441 GAG TTT TAC TCC CTG GGG GGA TTG TGG TGG GTG CCT GGC CTC CTC TCC 1488

1489 CAC CAG CGC ACT GCC CCA AGC TTG CCT AGG TAG TAT CAG CAC TGG GGT 1536

1537 GAG GGA TGC AAT GCG GGT TTA TAG TTG GCT GTG GGG ACT TCT GAT TTC 1584

1585 TCC ACC CCA TAG ATG ATA AAC GAC CGC GGG GCT TCC TCT CCC AGG TCT 1632

1633 TTC CTC ACT CTC CTT CGT CTT TCC CTC TTT CTC CAT GGG AAA GGT AGA 1680

1681 ATG AGA CTT GCC ACC AGG GAC ATC CAG GCA CAT TCC TCT TCC CAA TCA 1728

1729 GCC TCT ACC CAT TCT CTT TCT GTC TCG TGT GTA TCA GTG GCA TTT TCT 1776

1777 TGT GTA GCT TCC CAG ATT TGC AAA CCC GCT CCA CAT CCT GGA CGC AGA 1824

1825 CCC CCT TCG CTG GCT CTA TGA AGG CTT CAC GTT CCT GGG GCA TTC GAG 1872

1873 ACT TGA CTT TCC AGA GCA ACT TTG GAT CCA CCC TGT TAC CCT TTT TTT 1920

1921 TGT TTT GTT TTG TTT TGA GAG GGA GTC TCG CTG TGT TGT CCA GGC TGG 1968

1969 AGT GCA GTG GTG TGA TCT CGG CTT ACT GCA ACC TCC GCC TAC TGG GTT 2016

2017 CAA GTG ATT CTC TTG CCT CAA TCT CCC CAG TAC CTG GGA TTA CAG GTA 2064

2065 GAC ACC ATC ACG CCT GGC TAA TTT TTG TAT TTT TAG TAA AGA CAG GGT 2112

2113 TTC ATC ATG TTG GCC TGG CTG GTC TCA AAC TCC TGA CCT CAG CCG ATC 2160

2161 CGC CCG CCT CAG CCT CCC AAA GTT CTG GGA TTA CAA GTG TGA ACC ACC 2208

2209 GTG CCC AAC CTC CCC TCG TTT TTG ACC TTT GTC CCC AAA TGT CTT TCA 2256

2257 TCC AGT TAC TAG GAC TTG TAG CCT TTT TGC AAA TAG GTA TCT GTA AGC 2304

2305 ATA GAA GAA AAG GGT GAT TTG TGA AGT GAC CCT TAG TGG CAT CTG TGG 2352

2353 TGG TCC TAC TAA TCA GAT TTG CAG AAG AAA CAT GGA AAC AAG GAA ACT 2400

2401 CCT CTC TTA TAT TAA AAT AAA TAA GGT TTT TAA TTT CAA AAA AAA AAA 2448

2449 AAA AAA A 2455

D: Blastp result

Query=PP2672[gene=PP2672]

  (182 amino acids)

>SP_RO: O70132 O70132 rattus norvegicus(rat).ets domain

Transcription factor pet-1.5/1999

Length = 340

Score = 37.1bits (84), predicted value = 0.10

Identity = 25/78 (32%), Similarity = 37/78 (47%), Gap = 8/78 (10%)

Query: 84 GWSGKQTQQPVPPLRTLLLRSPFSLHKSSQPGSPKASQRIHPLFHSIPRSQLHSVLLGLP 143

GW+G Q Q P+PP TL RS + + P ++ ++ HP PR+ + + L

Sbjct: 35 GWAGMQLQDPLPPHHTLAARS-----RQALPDPAASTLPCHP---QSPRAGIGTPSAKLT 86

Query: 144 LLFIQTRPSPPAQYGAQM 161

+++ PSP AQ A M

Sbjct: 87 CPPVRSPSPTAQSPAAM 104

Claims (5)

1. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:

(a) coding has the proteic polynucleotide of people of cancer suppressing function, and described albumen has the aminoacid sequence of the group of being selected from down:

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、SEQ?ID?NO：23、SEQ?ID?NO：26；

(b) with polynucleotide (a) complementary polynucleotide.

2. polynucleotide as claimed in claim 1, it is characterized in that the polypeptide of this polynucleotide encoding has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26.

3. polynucleotide as claimed in claim 1 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, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27.

4. a carrier is characterized in that, it contains the described polynucleotide of claim 1.

5. 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 4;

(b) host cell that transforms or transduce with the described polynucleotide of claim 1.