WO2002099103A1 - Novel genes and proteins encoded thereby - Google Patents

Abstract

Novel DNAs containing a protein-encoding domain are directly cloned from cDNA libraries of human adult whole brain, human tonsil, human adult hippocampus and human fetal whole brain. Further, the base sequences thereof are determined and functions thereof are identified. DNAs containing a base sequence encoding a polypeptide as defined by the following (a) or (b): (a) a polypeptide comprising an amino acid sequence represented by any of SEQ ID NOS:1 to 7 or an amino acid sequence substantially the same as the above amino acid sequence; and (b) a polypeptide comprising an amino acid sequence derived from an amino acid sequence represented by any of SEQ ID NOS:1 to 7 by deletion, substitution or addition of a part of the amino acids and having a biological activity substantially the same as the function of the polypeptide (a); recombinant polypeptides encoded by the above DNAs, and proteins containing these polypeptides.

Description

 Specification

Novel genes and proteins encoded by them

Technical field

 The present invention relates to a DNA and a gene containing the DNA, a recombinant polypeptide encoded by the DNA and a novel recombinant protein containing the polypeptide.

 With the large-scale seek engine in the Human Genome Project, enormous information on the nucleotide sequence of the human genome has been obtained, and analysis is being conducted every day.

 The ultimate goal of the Human Genome Project is not simply to determine the entire nucleotide sequence of the genome, but to interpret the various life phenomena of the human from its structural information, that is, the DNA sequence information.

 Only a small part of the human genomic sequence encodes a protein, and at present, the coding region and region are predicted using neural networks and information science techniques called hidden Markov models. Has been done. However, their prediction accuracy is not yet sufficient.

Disclosure of the invention

 In order to find a novel gene, the present inventor has found that a region encoding a protein from a cDNA library derived from human whole brain, human tonsils, human adult hippocampus and human fetal whole brain. The present inventors succeeded in directly linking a novel DNA containing, and determined the base sequence thereof, thereby completing the present invention.

 That is, in the first aspect, the present invention provides the following (a) or (b):

 (a) a polypeptide consisting of the same or substantially the same amino acid sequence as the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7,

 (b) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, wherein the amino acid sequence is composed of an amino acid sequence in which some amino acids have been deleted, substituted, or added; Polypeptides having biological activity of the same quality

The present invention relates to a DNA comprising a base sequence encoding Examples of such DNAs include, but are not limited to, DNAs containing the base sequences of SEQ ID NOs: 1-7. Further, in the second aspect, in the second aspect, the DNA of the first aspect of the present invention is hybridized / under stringent conditions, and has substantially the same function as the polypeptide of (a). The present invention relates to DNA encoding a polypeptide having biological activity.

 The above-mentioned first and second embodiments of the present invention are collectively referred to as "the present invention DNA". Furthermore, the present invention also relates to an antisense DNA having a nucleotide sequence substantially complementary to the DNA of the present invention.

 Furthermore, in a third aspect, the present invention relates to a gene construct comprising the DNA of the present invention. As used herein, “gene construct” means any gene that has been artificially manipulated. Examples of the gene construct include, but are not limited to, the DNA of the present invention or a vector containing the antisense DNA of the DNA of the present invention, and the expression vector of the DNA of the present invention.

 In a fourth aspect, the present invention provides the following (a) or (b):

 (a) a polypeptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7,

 (b) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, wherein the amino acid sequence is composed of an amino acid sequence in which some amino acids have been deleted, substituted or added, and the function of the polypeptide of (a) A polypeptide having substantially the same biological activity as

According to.

 In a fifth aspect, the present invention also relates to a recombinant polypeptide encoded by the gene construct of the third aspect of the present invention.

 The above polypeptides are also collectively referred to as “the polypeptide of the present invention”. In the present specification, the polypeptide means "a polymer of amino acids having any molecular weight". The present invention also relates to a recombinant protein comprising the polypeptide of the present invention. As defined above, the term "polypeptide" herein does not assume limitations on molecular weight, so the term "polypeptide of the invention" also includes recombinant proteins comprising a polypeptide of the invention.

 In a sixth aspect, the present invention relates to an antibody against the polypeptide of the present invention.

In a seventh aspect, the present invention relates to a DNA chip on which the DNA of the present invention is arranged. The present invention, in an eighth aspect, relates to a polypeptide chip in which the polypeptide of the present invention is arranged.

 In a ninth aspect, the present invention relates to an antibody chip in which an antibody according to the sixth aspect of the present invention is arranged.

 Table 1 shows the name of the clone having the DNA of the present invention, the length of the polypeptide of the present invention, the predicted function, and the basis for the estimation.

 The DNA of the present invention is a cDNA live library prepared by the present inventors using commercially available mRNAs of whole human brain (manufactured by Clontech), human tonsil, human adult hippocampus and whole human fetal brain. After isolation as a cDNA fragment from the rally, the nucleotide sequence was determined and identified.

 Specifically, human adult whole brain, human tonsil, human adult hippocampus, and human fetal whole brain prepared according to the method of Ohara et al. (DNA Research 4: 53-59 (1997)). A clone is randomly isolated from a DNA library.

 Next, duplicated clones (clones containing the same sequence) were removed by hybridization, followed by in vitro transcription and translation, and clones with products of 50 kDa or more were recognized. Determine the unterminated base sequence.

 Furthermore, a homology search is performed using the thus obtained terminal nucleotide sequence as a query in a database of known genes, and as a result, the entire nucleotide sequence is determined for the clone that has been identified as being novel. .

 In addition to the screening method described above, the 5 'and 3' end sequences of the cDNA are made to correspond to the genomic sequence of the human, and if an unknown long-chain gene is found in the region between them, Perform a full-length analysis of the cDNA.

 In this way, unknown genes that cannot be obtained by the conventional closing method relying on known genes can be systematically cloned. In addition, by using PCR methods such as RACE while paying sufficient attention so that artificial errors do not occur in the short fragments and the obtained sequence, the human-derived gene including the DNA of the present invention can also be obtained. It is also possible to prepare the entire area.

Furthermore, the present invention includes the DNA of the present invention or a gene construct containing the DNA of the present invention. A recombinant vector, a transformant carrying the recombinant vector, and culturing the transformant to produce and accumulate a recombinant protein containing the polypeptide of the present invention or the polypeptide, and collect this. A method for producing a polypeptide of the present invention or a recombinant protein containing the polypeptide, or a salt thereof, and a polypeptide of the present invention obtained in this manner or a recombinant protein containing the polypeptide / \ ° Provide quality or its salt.

 The present invention also includes a pharmaceutical comprising the DNA or gene construct of the present invention, a polypeptide of the present invention or a partial polypeptide thereof, or a nucleotide sequence encoding a recombinant protein containing the polypeptide. Polynucleotide (DNA), an antisense nucleotide having a nucleotide sequence substantially complementary to the nucleotide sequence thereof, a medicament containing the polynucleotide or the antisense nucleotide, a polypeptide of the present invention or a polypeptide thereof. The present invention relates to a pharmaceutical comprising a partial polypeptide and a recombinant protein comprising the polypeptide or the polypeptide.

 The present invention also relates to a DNA chip, a peptide chip, and an antibody chip prepared by arranging the DNA of the present invention, the polypeptide of the present invention, and an antibody against the polypeptide of the present invention.

 Further, the present invention provides an antibody against the polypeptide of the present invention or a partial polypeptide thereof or a recombinant protein containing the polypeptide or a salt thereof, and a polypeptide of the present invention, a partial polypeptide thereof or the polypeptide thereof. Using a recombinant protein or a salt thereof, or an antibody against the same, comprising: a screening method for a substance that specifically interacts with the substance; a screening kit; and identification by the screening method. It also relates to the substance (compound) itself.

The DNA of the present invention may be any DNA as long as it comprises a base sequence encoding the polypeptide of the present invention described above. In addition, ^ Or identified from other tissues, such as cDNA libraries derived from cells and tissues such as heart, lung, liver, spleen, kidney, testis, etc.- Isolated cDNA or synthetic DNA Either may be used. The vector used for the construction of the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. In addition, a direct reverse transcriptase-polymerase chain reaction (hereinafter abbreviated as "RT-PCR method") using a total RNA fraction or mRNA fraction prepared from the above-described cell / tissue is used. Can also be amplified.

 The antisense oligonucleotide (DNA) having a base sequence substantially complementary to the DNA encoding the polypeptide of the present invention or a partial polypeptide thereof is substantially the same as the base sequence of the DNA. Any antisense DNA may be used as long as it has a complementary base sequence and has an action capable of suppressing the expression of the DNA. The substantially complementary nucleotide sequence is, for example, preferably about 90% or more, more preferably about 95% or more, and most preferably the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention. Is a nucleotide sequence having 100% homology. Further, nucleic acid sequences having the same action as these antisense DNAs (modified RNAs or DNAs) are also included in the antisense DNAs referred to in the present invention. These antisense DNAs can be produced using a known DNA synthesizer or the like. An amino acid sequence substantially identical to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7 is homologous to the entire amino acid sequence represented by any one of SEQ ID NOs: 1 to 7. O means an amino acid sequence having an average of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and particularly preferably about 95% or more on the whole.o

 Accordingly, the polypeptide having an amino acid sequence substantially identical to the amino acid sequence represented by any one of SEQ ID NOS: 1 to 7 of the present invention includes, for example, the amino acid sequence represented by each of the aforementioned SEQ ID NOs: And polypeptides having the above homology to the above and having substantially the same biological activity (function) as the function of the polypeptide consisting of the amino acid sequence represented by each SEQ ID NO. Here, “substantially the same” means that their activities (functions) are the same in nature.

Further, the polypeptide of the present invention includes, for example, a part (preferably about 1 to 20 amino acids, more preferably 1 to 20 amino acids) in the amino acid sequence represented by any one of SEQ ID NOS: 1 to 7. (About 10 amino acids, more preferably several amino acids) consisting of an amino acid sequence in which amino acids have been deleted, substituted or added, or an amino acid sequence obtained by combining them. Also included are polypeptides having a biological activity (function) that is substantially the same as the function of the polypeptide consisting of the rooster sequence.

 A polypeptide consisting of an amino acid sequence substantially identical to the amino acid sequence represented by any one of the above SEQ ID NOs: 1 to 7, or a polypeptide consisting of an amino acid sequence in which some amino acids have been deleted, substituted or added Can be easily prepared by appropriately combining well-known methods such as site-directed mutagenesis, gene homologous recombination, primer-extension, and PCR.

Incidentally, in this case, in order to have a substantially biologically Tekikatsu 'I ¹ raw homogeneous, among Amino acids constituting the polypeptide, homolog Amino acid (polar - nonpolar Amino acid, IFCK -It is considered that substitution between hydrophilic amino acids, positive and negative charged amino acids, aromatic amino acids, etc.) is possible. In order to maintain substantially the same biological activity, it is desirable that amino acids in the functional domain contained in each polypeptide of the present invention be retained.

 Further, the DNA of the present invention comprises a DNA comprising a base sequence encoding the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, and a DNA which is hybridized with the DNA under stringent conditions, and And DNA encoding a polypeptide having the same biological activity (function) as the function of the polypeptide comprising the amino acid sequence represented by

 Under such conditions, in the base sequence represented by any one of SEQ ID NOs: 1 to 7, as a DNA capable of hybridizing with a DNA containing a base sequence encoding the amino acid sequence represented by each sequence, for example, A DNA containing a nucleotide sequence having a degree of homology with the entire nucleotide sequence of the DNA of about 80% or more, preferably about 90% or more, more preferably about 95% or more on average. And the like.

Hybridization is performed by a method known in the art or a method similar thereto, such as a method described in “Current Protocols in Molecular Biology” (edited by Frederick M. Ausubel et al., 1987). Can be performed according to When a commercially available library is used, the method can be performed according to the method described in the attached instruction manual.

Here, “stringent conditions” refers to, for example, hybridization in ImM EDTA sodium at 65 ° C., 0.5 M sodium phosphate (pH 7.2), and 7% SDS aqueous solution. The DNA probe of the present invention was subjected to Southern blot hybridization under conditions in which the membrane was washed in an aqueous solution of sodium ImM EDTA and 4 OmM sodium hydrogen phosphate (pH 7.2) _s 1% SDS at 65 ° C. \ It is a condition of the degree of hybridization. The same stringency can be achieved under other conditions.

Ό.

 As a means for cloning the DNA of the present invention, the DNA is amplified by PCR using a synthetic DNA primer having an appropriate nucleotide sequence such as the polypeptide portion of the present invention, or a DNA incorporated into an appropriate vector is used. The polypeptide of the present invention can be selected by hybridization with a DNA fragment coding for a part or the entire region of the polypeptide or labeled with a synthetic DNA.

 The hybridization method can be performed, for example, according to the method described in the above-mentioned “Latest Protocol of Molecular Biology” (edited by Frederick M. Ausubel et al., 1987). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

 The DNA encoding the cloned polypeptide can be used as it is depending on the purpose, or it can be digested with a restriction enzyme or added with a linker if desired. The DNA may have ATG as a translation initiation codon at its 5 'end, and may have TAA, TGA or TAG as a translation stop codon at its 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.

The expression vector of the polypeptide of the present invention can be prepared according to a method known in the art. For example, (1) cutting out a DNA fragment containing the DNA of the present invention or a gene containing the DNA of the present invention, and (2) ligating the DNA fragment downstream of a promoter in an appropriate expression vector Can be manufactured. Examples of the vector include plasmids derived from E. coli (e.g., pBR322, pBR325, pUC18, pUC118), and plasmids derived from Bacillus subtilis (e.g., pUB110, pT p5, pC194) ), Yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as input phages, and animal viruses such as retroviruses, vaccinia viruses, and baculoviruses.

 The promoter used in the present invention may be any promoter as long as it is an appropriate promoter corresponding to the host used for gene expression. For example, when the host is S. cerevisiae, trp promoter, lac promoter, recA promoter, λPL promoter, Ίpp promoter, etc., and when the host is Bacillus subtilis, SPO1 is used. When the host is yeast, such as a promoter, an SPO2 promoter, a penP promoter, etc., a PH05 promoter, a PGK promoter, a GAP promoter, an ADH promoter, etc. are preferred. When animal cells are used as hosts, SR promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.

 In addition to the above, an enhancer, a splicing signal, a poly A-adhered signal, a selection marker, an SV40 replication origin, and the like, which are known in the art, can be added to the expression vector, if desired. Also, if necessary, the protein encoded by the DNA of the present invention can be expressed as a fusion protein with another protein (for example, glutathione S-transferase and protein A). . Such fusion proteins can be cleaved using an appropriate protease and separated into their respective proteins.

 Examples of host cells include Escherichia spp., Bacillus spp., Yeast, insect cells, insects, animal cells, and the like.

Specific examples of Escherichia bacteria include Escherichia coli K12 ■ DH1 (Proc. Natl. Acad. Sci. USA, 60: 160 (1968)) _s JM103 (Nucleic Acids Research, 9). : 309 (1981), JA 221 (Journal of Molecular Biology, 120: 517 (1978), HB101 (Journal of Molecular Biology, 41: 459 (1969)), and the like. As the Bacillus genus, for example, Bacillus subtilis (Bacillus subtiHs) MI114 (Gene, 24: 255 (1983), 207-21 [Journal of Biochemistry, 95:87 (1984)]) is used.

 Examples of yeast include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12 and other Saccharomyces and Shizosacaromyces . Bomb (Schizosaccaromyces o mbe) NC YC1933, NCYC2036, Pichia pastoris (Pichia pastoris) and the like are used.

 As animal cells, for example, monkey cells COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO micromoon), dhfr gene-deficient CHO cells, mouse L cells, mouse AtT — 20, mouse myeloma cells, rat GH3, human FL cells, etc. are used.

 Transformation of these host cells can be performed according to methods known in the art. For example, Proc. Natl. Acad. Sci. USA, 69: 2110 (1972), Gene, 17: 107 (19982), Molecular & General Genetics, 168: 111 (1979), `` Methods in Enzymology '' (Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75: 1929 (1978), Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol, 263-267 (1995) (Issued by Shujunsha); and Virology, 52: 456 (1973).

 The thus obtained transformant transformed with the expression vector containing the DNA of the present invention or the gene containing the DNA of the present invention can be cultured according to a method known in the art.

 For example, when the host is a bacterium belonging to the genus Escherichia, cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring may be added. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, ventilation or stirring can be added.

When culturing a transformant in which the host is yeast, the culture is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours using a medium adjusted to about 5 to 8%. However, if necessary, ventilation or stirring may be added. When culturing a transformant in which the host is an animal cell, the pH is adjusted to about 6 to 8 ± ±, usually at about 30 ° C to 40 ° C for about 15 to 60 hours. This can be done and ventilation or agitation can be added as needed.

 In order to separate and purify the polypeptide or protein of the present invention from the above culture, for example, after culture, cells or cells are collected by a known method, and the cells are suspended in an appropriate buffer, and the cells are suspended. After disrupting the cells or cells by sonication, lysozyme and / or freezing, a crude extract of the protein is obtained by centrifugation or filtration. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (trademark). If the protein is secreted into the nutrient solution, after the nutrient solution is completed, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected. The protein contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods. The polypeptide of the present invention thus obtained can be converted to a salt by a known method or a method analogous thereto.On the other hand, when the polypeptide is obtained as a salt, it can be liberated by a known method or a method analogous thereto. Can be converted to body or other salts. In addition, the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be modified before or after purification by the action of an appropriate protein-modifying enzyme such as tribcine and chymotrypsin. It can also be partially removed.

 The presence of the polypeptide of the present invention or a salt thereof can be measured by various binding assays and enzyme immunoassays using specific antibodies.

したグルタミル基がピログル タミン酸化したもの、分子内のァミノ酸の側鎖上の置換基が適当な保護基で保護 されているもの、あるいは糖鎖が結合したいわゆる糖ペプチドなどの複合べプチ ドなども含まれる。本発明の部分べプチドは、例えば、試薬、実験の際の標準物質、 または免疫原もしくはその一部として使用することができる。 本発明ポリべプチドまたはその部分べプチドの塩としては、 とりわけ生理学的 に許容される酸付加塩が好ましい。この様な塩としては、例えば、無機酸 (例えば、 塩酸、 リン酸、 臭化水素酸、硫酸)との塩、 あるいは有機酸 (例えば、酢酸、ギ酸、 プロピオン酸、 フマル酸、 マレイン酸、 コハク酸、酒石酸、 クェン酸、 リンゴ酸、 シユウ酸、安息香酸、メタンスルホン酸、ベンゼンスルホン酸)との塩などが用い られる。 The present invention Poribe peptide is, C-terminal, usually a carboxyl group (one C 00 H) or is a local Bokishire one Bok (one C◦ 0-), C-terminal Amido (one CONH ₂₎ or ester (-COOR) It may be. Here, R in the ester is, for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C3-8 cycloalkyl group such as cyclopentyl and cyclohexyl, For example, a C6-12 aryl group such as phenyl or 1-naphthyl, for example, a phenyl-1C1-2 alkyl group such as benzyl or phenethyl, or a C6-12 aryl group such as a naphthyl-1-C1-2 alkyl group such as 1-naphthylmethyl. 7-14 aralkyl group When the polypeptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the polypeptide of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Further, polypeptides of the present invention include those in which the amino group of the N-terminal methine nin residue is protected with a protecting group (for example, a C 1-6 acetyl group such as a formyl group and an acetyl group). An N-terminal glutamic acid residue formed by cleavage in the body is pyroglutaminated, or an appropriate protecting group (for example, e.g., OH, COOH, NH ₂ , SH, etc.) on the side chain of an amino acid in the molecule. Examples thereof include those protected with a C1-6 acyl group such as a rumil group and an acetyl group, or complex proteins such as so-called glycoproteins to which sugar chains are bonded. The partial polypeptide of the polypeptide of the present invention may be any of the above-described partial peptides of the polypeptide of the present invention as long as they have substantially the same activity. For example, among the constituent amino acid sequences of the polypeptide of the present invention, at least 10 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 20 or more For example, a peptide having 0 or more amino acid sequences and having a biological activity substantially the same as the function of the polypeptide of the present invention is used. As the partial polypeptide of the present invention, for example, those containing each functional domain are preferable. The C-terminus of the partial peptide of the present invention is usually a carboxyl group (1-COOH) or a carboxylate (1-COO-), but as in the above-described polypeptide of the present invention, the C-terminus is an amide (1-COOH). CONH ₂ ) or an ester (—COOR). Further, the partial peptide of the present invention includes, as in the above-mentioned polypeptide of the present invention, those in which the amino group of the N-terminal methine nin residue is protected by a protecting group;

Glutamyl group is pyroglutamine-oxidized, the amino acid side chain of the amino acid is substituted with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound. Is also included. The partial peptides of the present invention can be used, for example, as reagents, experimental standards, or immunogens or parts thereof. As the salt of the polypeptide of the present invention or its partial peptide, a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.

 The polypeptide of the present invention, its partial peptide or a salt thereof or an amide thereof can also be prepared using a chemical synthesis method known in the art. For example, using a commercially available resin for protein synthesis, amino acids appropriately protected with a amino group and a side chain functional group can be subjected to various condensation methods known in the art according to the sequence of the desired polypeptide. According to the above. At the end of the reaction, the polypeptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the desired polypeptide, its partial peptide, or an amide thereof. To get. Regarding the condensation of the protected amino acids described above, for example, polysaccharides represented by DCCs Ν, Ν′-diisopropylcarbodiimide and carbodiimides such as Ν-ethyl-Ν ′-(3-dimethylaminoprolyl) carbodiimide Various activation reagents that can be used for peptide synthesis can be used. For these activations, the protected amino acid may be added directly to the resin along with the racemization inhibitor (eg, HOBt, HOOBt), or may be pre-protected as a control acid anhydride or HOBt ester or HOOBt ester. After acid activation, it can be added to the resin.

Solvents used for the condensation of protected amino acids with the activity of the diamide are acid amides, halogenated hydrocarbons, alcohols, sulfoxides, and ethers, etc. It can be appropriately selected from solvents known to be usable for the reaction. The reaction temperature is appropriately selected from a range known to be usable for the reaction of forming a polypeptide bond. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, if the condensation is insufficient, it is sufficient to repeat the condensation reaction without removing the protecting group. Condensation can be carried out. When sufficient condensation is not obtained even after repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole so as not to affect subsequent reactions. .

 As a protecting group such as each amino group, carboxyl group, and serine hydroxyl group of the raw material, a group usually used in the technical field can be used.

 The protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.

 The partial peptide of the present invention or a salt thereof can be produced according to a peptide synthesis method known in the art, or by cleaving the polypeptide of the present invention with an appropriate peptide. . As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. Known methods of condensation and elimination of protecting groups include, for example, Nobuo Izumiya et al., Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975), Haruaki Yajima and Shunpei Sakakibara, Biochemical Experimental Lecture 1, Protein Chemistry IV, 205, (1977), supervised by Haruaki Yajima, Development of Continuing Drugs, Vol. 14, Peptide Synthesis, and the method described in Hirokawa Shoten.

 Purification after the reaction can also be performed by a known method, for example, by a combination of solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization to purify and isolate the partial peptide of the present invention. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained as a salt, it can be converted to a free form by a known method. can do.

 Antibodies against the polypeptide of the present invention, its partial peptide, or a salt thereof may be any of a polyclonal antibody and a monoclonal antibody as long as they can recognize them. Antibodies against the polypeptide of the present invention, its partial peptide or a salt thereof can be produced by using the polypeptide of the present invention or its partial peptide as an antigen according to a known method for producing an antibody or antiserum.

The antibody of the present invention can be used for detecting the polypeptide of the present invention present in a subject such as a body fluid or a tissue. Also used to purify these For the detection of the polypeptide of the present invention in each fraction at the time of purification, the analysis of the behavior of the polypeptide of the present invention in the test cells, and the like.

 Hereinafter, the use of the DNA of the present invention, the polypeptide of the present invention, the antibody of the present invention and the like will be described in more detail.

 By using the DNA of the present invention, the antisense DNA of the DNA of the present invention, or a gene construct containing these DNAs as a probe, the DNA or mRNA encoding the polypeptide of the present invention or its partial peptide can be obtained. Abnormality (gene abnormality) can be detected.

 For example, it is useful as a gene diagnostic agent for damage, mutation, or decreased expression of the DNA or mRNA, or increase or excessive expression of the DNA or mRNA. The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the well-known Northern High Predication ゃ PCR-SSCP method (Genomics, 5: 874-879 (1989), Pro Natl. Acad. Sci. USA, 86: 2766). -2770 (1989)). Furthermore, if the DNA or gene of the present invention is abnormal, deficient, or has a reduced expression level, patients who cannot exert normal functions in vivo can be retrovirused according to known means. When the DNA or gene construct of the present invention is introduced into a patient and expressed by gene therapy using an appropriate vector such as a vector, an adenovirus vector, or an adenovirus associated virus vector as a vehicle. Considered to be effective. In addition, when normal function cannot be exhibited due to increased expression, introduction of antisense may be effective.

It is also possible to administer the DNA of the present invention, the antisense DNA of the present invention, or the gene construct by a gene gun or a catheter such as a hydrogel catheter, using the DNA or the construct alone or together with an auxiliary for promoting uptake. By injecting the polypeptide of the present invention or the like into a patient having the above-mentioned disease g or _{c, the function} of the polypeptide of the present invention or the like can be exhibited in the patient. It is considered possible. Further, the antibody of the present invention is used for quantification of the polypeptide of the present invention in a test solution by a known method, particularly for quantification by a sandwich immunoassay using a monoclonal antibody, and detection by tissue staining and the like. be able to. Thereby, for example, a disease associated with the polypeptide of the present invention or the like can be diagnosed. For these purposes, an antibody molecule itself may be used, also, of the antibody molecule F (ab ') 2, F ab' s or may be used F ab fraction. The method for quantifying the polypeptide or the like of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of an antigen (eg, the amount of a protein) in a test solution. Any measurement method may be used as long as the amount of the body is detected by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, a competitive method, an immunometric method, and a sandwich method are suitably used, but from the viewpoint of sensitivity and specificity, it is preferable to use a sandwich method described later. As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like known in the art can be used.

For details on the general technical means related to these measurement and detection methods, refer to reviews and publications. For example, edited by Hiroshi Irie, Raji Saito Imnoassy (Kodansha, published in 1954), Eiji Ishikawa et al., Enzyme-linked immunosorbent assay (3rd edition; Medical Shoin, published by Showa B, 22), Methods, Vol. 70, "Immunochemical Techniques (Part I)", mmunochemical Tecliniques (Part A)), ibid., Vol. 73, "Immunochemical Techniques (Part B)," mmunochemical Techniques (Part B), ibid. Vol. 74, "Immunochemical Techniques (Part C) j ammunochemical 1fechniqes (Part C)", Vol. 84, "Immunochemical Techniques (Part D: Selected Immunoassays)" (Immunochemical ec niques (Part C) D: Selected Immunoassays)), Vol. 92, Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods) _{s s} ed. Vol. 121 “Immunization-Dolling Techniques (Part I: Hybridoma Technology and Monochrome Antibody)) (Immunochemical Techniaues (Part IrHybridoma ecnnology and Monoclonal Antibodies)) (above, published by Academic Press). Further, a DNA chip prepared by arranging the DNA of the present invention is useful for detecting mutation polymorphism of the DNA of the present invention and monitoring the expression level. DNA chip

—See “DNA microarrays and the latest PCR method” for seed DNA arrays, etc. (Cell Engineering Separate Volume Genome Science Series 1, Masaaki Muramatsu, Supervised by Hiroyuki Nami, 2

March 2004, 1st edition, 1st print).

 Furthermore, polypeptides prepared by arranging the polypeptides of the present invention can be a powerful tool for functional analysis such as expression, interaction, and post-translational modification of the polypeptide of the present invention, and for protein identification and purification. .

 An antibody chip prepared by arranging antibodies against the polypeptide of the present invention is very useful for analyzing the correlation between diseases, disorders, and other physiological phenomena and the polypeptide of the present invention.

 Methods and materials for making these chips are known to those skilled in the art.

 Furthermore, the polypeptide of the present invention is useful as a reagent for screening for a compound that specifically interacts with these substances. That is, the present invention provides a method for screening a compound that specifically interacts with the substance or a salt thereof, which comprises using the polypeptide of the present invention, a partial peptide thereof or a salt thereof, or an antibody against the same. And a screening kit therefor.

The compound identified by using the screening method or the screening kit of the present invention or a salt thereof is a compound selected from the aforementioned test conjugates, interacts with the polypeptide of the present invention, and Are compounds that regulate, inhibit, promote, or antagonize biological activity. The compound or a salt thereof may act directly on the activity of the polypeptide of the present invention, or may indirectly act on the expression of the polypeptide of the present invention. A substance that acts on an activity such as a peptide may be used. As the salt of the compound, for example, a pharmaceutically acceptable salt and the like are used. Examples thereof include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids. Polypropylene of the present invention Compounds that inhibit biological activity such as tide may also be used as medicaments such as therapeutic-prophylactic agents for the above-mentioned various diseases.

 In the present specification, bases, amino acids and the like are indicated by abbreviations based on the abbreviations of the IUPAC-IUB nomenclature committee or commonly used abbreviations in the art. Unless otherwise specified, L-form shall be indicated.

BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. Various genetic manipulations in the examples were performed according to the method described in the above-mentioned “Latest Protocol of Molecular Biology” (edited by Frederick M. Ausubel et al., (1987)).

 (1) Construction of the cDNA library from human whole brain, human tonsils, human adult hippocampus and human fetal whole brain

Oligonucleotides having N ot I site (GACTAGTTCTAGATC GCGAGCGGCCGCCCCT ₅₎ (manufactured by Inbi Bok port Gen Corp.) primer - and to, human Bok adult whole brain, human tonsil, human Bok adult hippocampus and human fetal whole brain-derived m RNA (click Double-stranded cDNA was synthesized using a SuperScript II reverse transcriptase kit (manufactured by Invitrogen) in a mirror-like manner using Kokutech. Adapter 1 (manufactured by Invitrogen) having a Sal I site was ligated with cDNA. Thereafter, Not I digestion was performed, and a DNA fragment of 3 kb or more was purified by low-agarose gel electrophoresis at a concentration of 1%. The purified cDNA fragment was ligated with the pBluescript IISK + plasmid that had been treated with the Sal I—Notl restriction enzyme. This recombinant plasmid was introduced into Escherichia coli ElectroMax DH10B strain (manufactured by Invitrogen) by an electoral poration method.

 (2) Screening (1)

Next, clones were randomly picked up from the thus constructed cDNA library and spotted on a membrane. Next, based on the nucleotide sequences of about 1,300 clones that have been subjected to full-length angular analysis by the present inventors, Each 3 'end of the prepared mixture of oligo DNAs (21 bases each) is DIG-labeled with a terminal polymerase, and these are used as probes to perform DNA hybridization ("The latest protocol in molecular biology"). ("Frederick M. Ausubel et al., Eds., 1987")).

 Next, we performed a tiller translation at the entrance (Promega's TNT T7 Quick Coupled Transcription / Translation System cat.no 1107), and selected a clone in which animals of 50 kDa or more were recognized. did.

Next, to determine the terminal base sequence of the selected clones, resulting sequence homology search with a query program BLASTN 2.0.14 and 2.1.3 (Altschul, Stephen R, Th omas L. Madden s Alejandro A. Schaffer , Jinghui Zhang Zheng Zhang _s Webb Miller, and David J. Iipman (1997), “Gear 7¾LAST and TSI-B LAST: Creating a new protein database search program” (Gapped BLAST and PSI-BIJAST: a new generation oi protein dataoase search urograms), Nucleic Acids Res. 25: 3389-3402), nr (GenBank + EMBL + DDBJ + PD B sequence (excluding EST, STS, GSS or HTGS sequence of phase 0, 1 or 2)) A homology search was performed on the database. As a result, the entire nucleotide sequence was determined for the case where no homologous gene was present, that is, for the case of a novel gene.

 Screening (Part 2)

The terminal sequence of the 5 'and 3' of the cDNA, using homologous search program BLASTN2.1.3, Kenomu Hr ¹ arsenide Bok,歹Iltft: 〃 ncbi.nlm.nih.gov/genoines/H sapiens /) to j juice Next, the Genscan program (Burge, C. and KarHn, S. 1997, Prediction oi complete gene structures in human genomic DN A, J Mol. Biol, 268, 78) -94, The extracted genes were extracted using a computer software that predicts genes from the genome, and using this as a query, the homodb search program BLASTN2.1.3 was used to mergedb (Kazusa DNA Research Institute). The newly determined human cDNA sequence and the DNA from GenBank's homo sapiens database, excluding ESTs and genomes, are mixed without duplication. Long chain (Genscan expected cds is over 1200 bp) When the genetic power was confirmed, the full-length analysis of the cDNA subjected to 5 ′ and 3 ′ end sequencing was performed.

 For sequencing, a DNA sequencer (ABI PRISM377) manufactured by PE Applied Biosystems and a reaction kit manufactured by the company were used. Most of the sequences were determined using the short gun clone method using the dye terminator method. For some of the nucleotide sequences, ligated nucleotides were synthesized based on the determined nucleotide sequences and determined by the primer matching method.

 Thus, screening of a novel DNA or gene was performed. As a result, a novel DNA or gene shown in any one of SEQ ID NOs: 1 to 7 in the sequence listing was detected.

The base sequence of these novel DNAs or genes was determined by the above-mentioned sequencing method. Table 1 shows the name of the clone having the DNA or gene of the present invention, the length of the polypeptide encoded by the gene in the clone, the expected function, and the basis for the estimation.

Table 1 Sequence clones

Number Tampa ¾Overall length

 Name Length is a partial sequence Predicted function Basis for estimation

 W i sko-Aldrich syndrome, night blindness,

 May be a causative gene of Dent's disease Because of the possible inheritance of Xp11,23-p22, it may be partially diagnosed with DXImx48e therapy, one of the diseases, or a therapeutic drug screening protein. Homology

Useful for the 1 bm00462 571 subsequence. Estimated from having.

 Netrin induces axons in nerve cells

 Receptor. Involved in nerve axon growth

 And is involved in the control of cell migration, and is useful for the diagnosis and treatment of various neurological disorders of netrin transmembrane receptor U, and in part for NC5H1, which is useful for screening therapeutic drugs. Estimate from having

2 fg02988 176 partial sequence Was.

 Netrin / One netrin G2 that partially induces axons in neurons of the Unc6 family. Involved in the growth of nerve axons and has homology to cells

 Laminin Neterm, laminin EG Diagnosis, treatment, or therapeutic agent for various diseases, related to the control of migration Estimated by having the F motif

3 fk03350 530 τέ all: Useful for screening κ. did.

 Melanoma-associated chondroitin sulfate pro-melanoma-associated chondroitin theodalican. Partial homology to cell growth, cell-proteodarican sulfate 4 interaction with basement membrane, cell adhesion

4 ae00146 445 partial sequence given. It is an indicator of melanoma diagnosis. Estimated.

 Presence of antibodies against this protein The types of neurological diseases associated with various cancers are called paraneoplastic, which is an index of neurological diseases associated with various cancers. Therefore, the diagnosis of cancer and the neuronal antigen MAI associated with cancer and the partial disease, and the fact that there is a partial homology between the therapeutic agents and

5read 059 452 Subsequences Useful for leaning. Estimated.

 Partial homology with coding enzyme on the novel herpesvirus genome encoded by herpesvirus DNA genome U88

6 fk03083 A novel kinase with 273 partial sequences. Estimated from having. Expression in Alzheimer's disease patients

 Is a protein that rises. ALU domain Protein interaction via the brain of Alzheimer's disease patients is confirmed to accumulate in this spinal fluid. It can be a target for the development of AD7C-NTP protein, which is a drug that inhibits protein activity. 41.2% Due to homology of diagnosis, treatment, and therapeutic agents for Alzheimer's disease, and because it belongs to the target and subfamily of ALU drug screening

7 hh15219 201 partial sequence Estimated. (3) homology search of the DNA of the present invention

Next, based on the total nucleotide sequence thus obtained, the amino acid sequence of the clone was analyzed against a known sequence library nr using the analysis programs BLASTP 2.0.14 and 2 丄 3 (“Gear ヅ ^ BLAST and PSI-BLAST: protein database search. Using a new program ”, supra), it was found that they showed homology to the same gene for each of the nematodes shown in Table 2. Table 2 shows information on these homologous genes, that is, their names, database IDs, species and their scientific names, protein lengths, and references.

Table 2

* In Table 2 above, the meanings and scientific names of the abbreviations of each species are as follows: Hs: Homo sapiens; Hh: Herpesvirus = Human herpesvirus 6; Mm: Mouse = Mus musculus Further, Table 3 summarizes various data on the homology between the DNA or gene of the present invention contained in each clone and each homologous gene shown in Table 2. The meaning of each item in these tables is as follows.

 "Score" The higher the value, the higher the reliability.

 "E-value" The closer this value is to 0, the higher the confidence

 Amino acid position that is the origin of the "origin" homology region

 Amino acid position at the end of the "end" homology region

 "Homology" Percentage identity between amino acid residues in the homologous region

"Homologous range ratio" Percentage of homologous regions in homologous genes

Table 3

Homology value

Sequence number Clone II homology

 *? Occupancy Score E-value Homology Homology range ratio Origin End point

 1 2 571 165 735 858 0 76% (445/585) 78%

2 4 142 323 433 673 1.90E-15 78% (Fine 39) 12%

17 528 28 537 690 0 60% (312/515) 95%

3 20 547 1 528 1099 0 95 (503/529) 99%

4 1 445 492 937 745 0 85% (380/446) 19%

5 5 339 1 338 313 5.00E-84 46 (158/339) 96%

6 3 235 87 304 223 1 .00E-57 50% (121/240) 53%

7 144 188 299 343 71 7E-12 75 (34/45) 12%

(4) Search for various domains

Next, Pfam HMM ver 2.1 Search (HMMPFAM) (Sonnhammer ELL, Eddy SR, Birney E, Bateman) from the amino acid sequence encoded by the DNA contained in the clone was included in Pfam 6.0 and 6.4. A, and Durbin R (1 998) "Pfam: multiple sequence alignments ana HMM-profiles of Drotem do mains", Nucleic Acids Res. 26: 320-322 ) Was used to search for functional domains. Furthermore, SOSUI system is a membrane protein prediction program (ver. 1.0 I 10, Ma r., 1996) (Takatsugu Hirokawa, Seah Boon-Chieng and Shigeki Mitaku _s

Using "SOSUI: Class incation and Secondary Structure Prediction System lor Membrane Proteins", Bioinformatics (formerly CABIOS) 1998 May; 14 (4): 378-379 We searched for transmembrane domains.

 The detected functional domains and transmembrane domains are shown in Table 4 for each clone.

 The meaning of each item in these tables is as follows.

 "Function domain" Domain detected by Pfam or SOSUI

 Amino acid position that is the starting point of the "starting point" function domain

 Amino acid position at the end of the "end" functional domain

 "Score (Pfam only)" The higher the value, the higher the reliability

 “Exp (Pfam only)” The closer this value is to 0, the higher the reliability.

Table 5 shows the complete description of each functional domain and its Japanese translation.

ι

(5) Expression site

 Using RT-PCR Coupled ELIS A, the expression in tissues and brain was examined, and those showing the strongest expression in each case are shown in Table 6.

 (6) Chromosome location

 Analysis programs BLASTN 2.0.14 and 2.1.3 (“Gear 7 LAST and PSI-BLAST: evening”) were used to analyze human genomic sequences in the known sequence libraries Genbank release 122 and 123 corresponding to the DNA base sequence of the clone. Creation of a New Protein Database Search Program ”, supra). The DNA sequence of the clone was analyzed using the homology search program BLASTN 2.1.3 and the live sequence of the clone encoding the human genome tf p: //nc¾i.idm.nih.gov/g-enomes/H sapiens Woman Φιι,., Hatsuka

The description of the number of the chromosome from which this clone was derived was extracted from the description of the matched clone (Definition), and is shown in Table 6.

σ>

Sequence Expression site Chromosome position

Number Tissue Brain Location

1 S. muscle (skeletal muscle) B. cerebellum (cerebellum) X

 twenty five

 3 Brain B. hippocampus 9

 4 Ovary (ovary) B. cerebellum (cerebellum) 15

 5 Testis (testis) B. subthalamic nucleus (nucleus of the hypothalamus of the brain) X

 6 Brain B. caudate nucleus (caudate nucleus of brain) 19

7 20

Based on the above information on homologous genes and homologous genes, various domains, expression sites, and chromosome positions, those skilled in the art can use the functions shown in Table 1 according to the present invention based on the basis shown in Table 1. It can be predicted that the DNA or the gene has Industrial applicability

 PCR is performed using chromosomal DNA extracted from human blood or tissue using a synthetic DNA primer prepared based on the DNA or gene of the present invention or a partial sequence thereof, and the nucleotide sequence of the product is obtained. By determining the SNP, a single base mutation, that is, an SNP, which differs depending on individuals in the DNA or gene of the present invention can be found. As a result, the constitution and the like of the individual are predicted, and it becomes possible to develop a medicine suitable for each individual.

 Further, by cross-hybridization, a gene encoding a homologous or counterpart gene for the DNA or gene of the present invention in a model organism such as a mouse is isolated, and, for example, by knocking out these genes, the human is obtained. It is also possible to create a disease model animal and search for and identify genes that cause human disease.

 The novel DNA or gene obtained in the present invention is integrated on a so-called DNA chip or the like, and this chip is prepared from, for example, blood or tissue derived from a normal human as a control for patients with cancer or psychiatric disease and the like. Hybridization of the probe can be useful for diagnosis and treatment of these diseases. In addition, an antibody chip prepared and sequenced with an antibody against the polypeptide of the present invention can be used for proteome analysis, such as detecting differences in protein expression levels between patients and normal persons, to diagnose and treat diseases. It can be useful for such things.

 Further, the DNA or gene construct of the present invention can be used as an active ingredient of a vaccine.

Claims

The scope of the claims 1. The following (a) or (b) polypeptides:  (a) a polypeptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7,  (b) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, wherein the amino acid sequence comprises a deletion, substitution, or addition of a part of the amino acids; Polypeptides having biological activity of the same quality DNA containing nucleotide sequence encoding  2. A polypeptide having substantially the same biological activity as the polypeptide of claim 1 (a), which is / are hybridized with the DNA of claim 1 under stringent conditions. The encoding DNA.  3. A gene construct comprising the DNA of claim 1 or 2. 4. The following polypeptide (a) or (b):  (a) a polypeptide consisting of the same or substantially the same amino acid sequence as the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7,  (b) an amino acid sequence represented by any one of SEQ ID NOS: 1 to 7, wherein the amino acid sequence comprises an amino acid sequence in which some amino acids have been deleted, substituted or added, and Polypeptides having the same biological activity.  5. A recombinant polypeptide encoded by the gene construct of claim 3.  6. An antibody against the polypeptide according to claim 4 or 5.  7. A DNA chip in which the DNA according to claim 1 or 2 is arranged.  8 · A polypeptide chip in which the polypeptides according to claim 4 or 5 are arranged. 9. An antibody chip in which the antibody according to claim 6 is arranged.