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WO2002099103A1 - Nouveaux genes et proteines codees par ceux-ci - Google Patents

Nouveaux genes et proteines codees par ceux-ci Download PDF

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Publication number
WO2002099103A1
WO2002099103A1 PCT/JP2002/005134 JP0205134W WO02099103A1 WO 2002099103 A1 WO2002099103 A1 WO 2002099103A1 JP 0205134 W JP0205134 W JP 0205134W WO 02099103 A1 WO02099103 A1 WO 02099103A1
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Prior art keywords
polypeptide
dna
present
amino acid
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/JP2002/005134
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English (en)
Japanese (ja)
Inventor
Osamu Ohara
Takahiro Nagase
Daisuke Nakajima
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Kazusa DNA Research Institute Foundation
ProteinExpress Co Ltd
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Kazusa DNA Research Institute Foundation
ProteinExpress Co Ltd
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Publication of WO2002099103A1 publication Critical patent/WO2002099103A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • 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.
  • 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.
  • 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.
  • the present invention provides the following (a) or (b):
  • 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 present invention also relates to an antisense DNA having a nucleotide sequence substantially complementary to the DNA of the present invention.
  • the present invention relates to a gene construct comprising the DNA of the present invention.
  • 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.
  • the present invention provides the following (a) or (b):
  • the present invention also relates to a recombinant polypeptide encoded by the gene construct of the third aspect of the present invention.
  • polypeptides are also collectively referred to as “the polypeptide of the present invention”.
  • 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.
  • 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.
  • the present invention relates to an antibody against the polypeptide of the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the human-derived gene including the DNA of the present invention can also be obtained. It is also possible to prepare the entire area.
  • the present invention includes the DNA of the present invention or a gene construct containing the DNA of the present invention.
  • 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.
  • 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.
  • 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.
  • ⁇ 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.
  • RT-PCR method 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.
  • nucleic acid sequences having the same action as these antisense 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
  • 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.
  • substantially the same means that their activities (functions) are the same in nature.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the vector include plasmids derived from E.
  • coli e.g., pBR322, pBR325, pUC18, pUC118
  • plasmids derived from Bacillus subtilis e.g., pUB110, pT p5, pC194
  • Yeast-derived plasmids eg, pSH19, pSH15
  • bacteriophages such as input phages
  • 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.
  • SPO1 is used when the host is S. cerevisiae.
  • 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.
  • SR promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
  • 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.
  • 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.
  • host cells examples include Escherichia spp., Bacillus spp., Yeast, insect cells, insects, animal cells, and the like.
  • 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.
  • Bacillus genus for example, Bacillus subtilis (Bacillus subtiHs) MI114 (Gene, 24: 255 (1983), 207-21 [Journal of Biochemistry, 95:87 (1984)] is used.
  • yeast examples include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12 and other Saccharomyces and Shizosacaromyces .
  • Bomb Schott al.
  • Bomb Schizosaccaromyces o mbe
  • NC YC1933, NCYC2036, Pichia pastoris (Pichia pastoris) and the like are used.
  • 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).
  • 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.
  • 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.
  • 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.
  • the culture 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • a C6-12 aryl group such as phenyl or 1-naphthyl
  • a phenyl-1C1-2 alkyl group such as benzyl or phenethyl
  • C6-12 aryl group such as a naphthyl-1-C1-2 alkyl group such as 1-naphthylmethyl.
  • 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.
  • ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • 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).
  • 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.
  • an appropriate protecting group for example, e.g., OH, COOH, NH 2 , SH, etc.
  • 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.
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid
  • 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.
  • a chemical synthesis method known in the art.
  • 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.
  • the polypeptide is cleaved from the resin, and at the same time, various protecting groups are removed.
  • 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.
  • the protected amino acids described above for example, polysaccharides represented by DCCs ⁇ , ⁇ ′-diisopropylcarbodiimide and carbodiimides such as ⁇ -ethyl- ⁇ ′-(3-dimethylaminoprolyl) carbodiimide
  • Various activation reagents that can be used for peptide synthesis can be used.
  • 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.
  • the racemization inhibitor eg, HOBt, HOOBt
  • 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.
  • 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. .
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • the above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the well-known Northern High Predication ⁇ 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.
  • DNA or gene construct of the present invention 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.
  • the DNA of the present invention 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.
  • a gene gun or a catheter such as a hydrogel catheter
  • the function of the polypeptide of the present invention or the like can be exhibited in the patient. It is considered possible.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • clones were randomly picked up from the thus constructed cDNA library and spotted on a membrane.
  • 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”)).
  • nr GeneBank + 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.
  • 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.
  • 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.
  • Name Length is a partial sequence Predicted function Basis for estimation
  • AD7C-NTP protein 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
  • Hs Homo sapiens
  • 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.
  • 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.
  • 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
  • the detected functional domains and transmembrane domains are shown in Table 4 for each clone.
  • Table 5 shows the complete description of each functional domain and its Japanese translation.
  • Testis B. subthalamic nucleus (nucleus of the hypothalamus of the brain) X
  • 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.
  • 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.
  • the constitution and the like of the individual are predicted, and it becomes possible to develop a medicine suitable for each individual.
  • 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.
  • 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.
  • DNA or gene construct of the present invention can be used as an active ingredient of a vaccine.

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Abstract

L'invention concerne de nouveaux ADN contenant un domaine codant des protéines, qui sont directement clonés à partir de librairies de cADN provenant de l'ensemble du cerveau humain adulte, de l'amygdale humaine, de l'hippocampe humain adulte et de l'ensemble du cerveau de foetus humain. En outre, les séquences de base associées sont déterminées et les fonctions correspondantes identifiées. Les ADN contenant une séquence de base codant un polypeptide tel que défini par (a) ou (b) suivant: (a) un polypeptide comprenant une séquence d'acide aminé représentée par l'une ou l'autre des SEQ ID NOS 1 à 7, ou une séquence d'acide aminé sensiblement identique à la séquence d'acide aminé précitée; et (b) un polypeptide comprenant une séquence d'acide aminé dérivée d'une des séquences d'acide aminé représentées par SEQ ID NOS 1 à 7 par délétion, substitution ou addition d'une partie des acides aminés, dont l'activité biologique est sensiblement identique à la fonction du polypeptide (a). L'invention concerne également des polypeptides recombinants codés par les ADN précités, ainsi que des protéines contenant ces polypeptides.
PCT/JP2002/005134 2001-06-04 2002-05-27 Nouveaux genes et proteines codees par ceux-ci Ceased WO2002099103A1 (fr)

Applications Claiming Priority (4)

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JP2001168370 2001-06-04
JP2001-168370 2001-06-04
JP2001-246915 2001-08-16
JP2001246915 2001-08-16

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] Database accession no. (AF229644) *
MEANS G.D. ET AL.: "A transcript map of a 2-Mb BAC contig in the proximal portion of the mouse X chromosome and regional mapping of the scurfy mutation", GENOMICS, vol. 65, no. 3, 2000, pages 213 - 223, XP002956645 *

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