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WO2003087362A1 - Nouveau gene recepteur couple a la proteine g et proteine bg8 - Google Patents

Nouveau gene recepteur couple a la proteine g et proteine bg8 Download PDF

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Publication number
WO2003087362A1
WO2003087362A1 PCT/JP2003/004236 JP0304236W WO03087362A1 WO 2003087362 A1 WO2003087362 A1 WO 2003087362A1 JP 0304236 W JP0304236 W JP 0304236W WO 03087362 A1 WO03087362 A1 WO 03087362A1
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Prior art keywords
protein
nucleic acid
expression level
seq
cells
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PCT/JP2003/004236
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English (en)
Japanese (ja)
Inventor
Shinichi Inoue
Tadahiro Nambu
Toshiyasu Shimomura
Hiraku Itadani
Kenichi Tanaka
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MSD KK
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Banyu Phamaceutical Co Ltd
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Priority to AU2003236351A priority Critical patent/AU2003236351A1/en
Publication of WO2003087362A1 publication Critical patent/WO2003087362A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention relates to a novel gene that is specifically expressed in cells that produce hard keratin brightly, and a new use of a novel protein encoded by the gene.
  • G protein-coupled receptor (hereinafter, referred to as G protein-coupled receptor). It is called GPCR.) It is called protein. Further, since the GPCR protein has seven hydrophobic transmembrane domains, it is also called a seven-transmembrane receptor protein.
  • GPCR proteins are involved in the regulation of various functions in living organisms.Therefore, identification of a novel GPCR protein, a gene encoding the protein, and its expression site requires various regulation of biological functions. For compounds related to the regulation of various biological functions such as pharmaceuticals.
  • GPCR proteins are classified into oral dopsin receptor uniform family A, secretin receptor-like family B, and metapotropic receptor uniform family C.
  • Receptors belonging to family C are classified into four groups, groups 1 to 4, based on the physiological functions presumed to be endogenous ligands.
  • Group 1 has eight metapotropic glutamate receptors
  • Group 2 has a calcium-sensitive receptor
  • Group 3 has two aminobutyric acid type B receptors
  • Group 4 has Belongs to several pheromone receptors I know that.
  • RA IG1 which belongs to a new group 5 that does not belong to any of the four groups belonging to Family C above and whose in vivo ligand is unknown, is a clone alone, is RA IG1. It has been described that GPRC5B, GPRC5C, and GPRC5D were cloned by similarity search based on their sequences (for example, see Non-Patent Document 1).
  • Non-Patent Document 1 describes that a protein (GPRC5D) consisting of an amino acid sequence homologous to the novel protein of the present invention is a GPCR. It is expressed in the kidney, small intestine, spleen, testis, lung, colon, leukocytes, prostate, and thymus. However, there is no description that these genes are specifically expressed in hard keratin-producing cells.
  • keratin means a structural protein (intermediate cytoplasmic filament) found in epithelial cells and epithelium-derived cells (skin, nails, hair, etc.) and playing a role in protecting the animal body.
  • keratins are classified as cytokeratins, ie, soft keratins (soft keratins), and hair keratins, ie, hard ⁇ -keratins (81-dokeratins).
  • cytokeratins ie, soft keratins
  • hair keratins ie, hard ⁇ -keratins (81-dokeratins).
  • hard keratin is involved in the formation of hair, nails and claws, and other structures.
  • the soft keratin and hard keratin are classified into acidic type I and basic or neutral type II.
  • nine types of human type I hard keratin type I hair-keratin
  • C nine types of human type I hard keratin
  • Group ⁇ includes hHa l, hHa 3— I, hHa3-II and hHa4 belong, group B belongs to hHa7 and hHa8, and group C belongs to hHa2, hHa5 and hHa6 (for example, See Non-Patent Document 2).
  • Non-Patent Document 2 suggests that hH a2 and hH a5 appear in the initial stage of hair separation. Furthermore, it has been suggested that six type I hair keratins, hHa1, hHa3-I hHa3-II, hHa4, hHa7 and hHa8, are expressed during terminal differentiation of hair.
  • Six types II hard keratins (type II hair-keratins) are known to date, and these six types are further classified into Group A and Group C based on structural similarities. That is, hHbl, 3, 6 belong to group A, and hHb 2, 4, and 5 belong to group C.
  • In situ hybridization and immunohistochemical staining suggest that hHb2 and hHb5 are involved in the early stages of hair differentiation (eg, see Non-Patent Document 3).
  • Patent Document 1 describes low molecular weight compounds such as ferulic acid and ferulic acid ester which have a cell differentiation promoting effect.However, the gene itself involved in the function of a cell that produces hard keratin, or a gene thereof, There is no description regarding the peptide or protein encoded by the gene, and no description suggesting this. On the other hand, there is also a description of a gene encoding a GPCR protein having high homology to the novel gene of the present invention (see, for example, Patent Document 3 and Patent Document 4). However, Patent Literature 3 and Patent Literature 4 do not describe the gene itself according to the present invention, and the nucleotide sequence of the described gene is obtained by bioinformatics using a gene fragment obtained experimentally. It is only predicted by Takes analysis, and there is no description that the gene is involved in the function of hard keratin-producing cells.
  • Non-Patent Document 3 (Non-Patent Document 3)
  • Patent Document 3 Patent Document 3
  • the present invention has been made in view of the above-mentioned problems of the related art, and by specifying a novel GPCR protein, a nucleic acid encoding the protein, and a site where the gene of the present invention is expressed, It is an object of the present invention to provide a new use of the gene expressed specifically in the ⁇ position and a protein encoded thereby.
  • the present inventors have conducted intensive studies in order to solve the above problems, and as a result, have found that a novel protein having an amino acid sequence different from that of the proteins described in Patent Documents 2 and 3 and that encodes the protein.
  • the present inventors have found nucleic acids from mice and humans, and have found that such nucleic acids are expressed in specific cells, thus completing the present invention.
  • the present inventors searched for a novel GPCR sequence (GenBank Accession No. AB030198) described in Biochemical and Biopnysical Research Communication (vol. 268, No. 553-561) for the purpose of searching for a novel GPCR candidate gene. ) was used to perform a similarity search.
  • partial sequences of AA 168673 and AA 791779 were found, and the new candidate GPCR gene was named "BG8".
  • AA164122 IMAGE: 607331 manufactured by KURABO
  • I MAGE clone A full length clone was isolated from Ibrari (# 937313) (manufactured by STRATAGENE) and its entire nucleotide sequence was determined (SEQ ID NO: 1).
  • STRATAGENE STRATAGENE
  • the open reading frame of BG8 a novel mouse GPCR candidate gene, was 903 bp.
  • the protein encoded by the mouse BG8 gene was estimated to be a seven-transmembrane receptor, that is, a GPCR (Fig. 1).
  • the present inventors attempted to isolate a human BG8 gene based on the obtained mouse BG8 sequence.
  • a DNA clone having the sequence shown in SEQ ID NO: 3 and having an open reading frame consisting of 903 bp as in mouse BG8 was obtained.
  • the protein encoded by this DNA clone was estimated to be a seven-transmembrane receptor, or GPCR (Fig. 2).
  • the amino acid sequence deduced from this base sequence was compared with the amino acid sequence of mouse BG8 protein, it showed 83% homology (FIG. 3). From this, it was concluded that the DNA clone shown in SEQ ID NO: 3 was a human BG8 gene.
  • the human BG8 of the present invention differs from the proteins described in WO200157085 and WO200107609 in the number of amino acid residues in the amino acid sequence thereof, and the protein described in WO200157085 is different from the protein described in WO200157085. While the protein described in WO200107609 consists of 323 amino acids, the human BG8 of the present invention consists of 300 amino acids, and these three proteins have amino acids 1 to 298. It was found that they had the same amino acid sequence, but differed at amino acids 299 and 300.
  • nucleotide sequences of the genes described in the above-mentioned WO 200157085 and WO200107609 are only those obtained by analyzing experimentally obtained gene fragments by means of bioinformatics, and There is a high possibility that the structure of Ron is also different from the human BG8 gene of the present invention.
  • the BG8 gene is a gene having a function related to hard keratin present in hair, tongue, nails, skin and others.
  • BG8 gene and the protein encoded thereby are specifically expressed in cells that produce hard keratin, phenomena related to the function of cells that produce hard keratin, such as alopecia and hirsutism Abnormalities in hair, such as abnormalities of the hair shaft, aging of the skin, phenomena such as poor growth and formation of hair and nails, or diseases of the skin, tongue, nails, hair, etc., or diseases in which abnormal formation of hard keratin is observed, for example, Therapeutic and / or prophylactic agent and / or diagnostic agent for keratoderma dermatophytosis, piloma, etc. and screening methods for compounds for the purpose of treating and / or prophylactic agent and / or diagnostic agent for these phenomena or diseases. Available.
  • nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3 in the sequence listing
  • nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3 in the sequence listing A nucleic acid that hybridizes under the conditions or a nucleic acid having a base sequence complementary to the nucleic acid; (7) a nucleic acid comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing or a part of the protein; antibody,
  • a compound screening method comprising the following steps (a) to (c):
  • step (c) selecting a test compound for which binding was detected in step (b)
  • a compound screening method comprising the following steps (a) to (c):
  • step (c) selecting a test compound for which a change in activity was detected in step (b)
  • a compound screening method comprising the following steps (a) to (c):
  • step (c) Step of selecting a test compound in which a change in the expression level is detected in step (b) (11)
  • the target disease of the compound is abnormal formation of hard keratin and abnormal function of cells that produce Z or hard keratin.
  • the target disease of the compound is abnormal hair such as alopecia, hirsutism or abnormal hair shaft; aging of the skin; abnormal formation of the tongue, hair or nails;
  • a pharmaceutical composition comprising the compound according to (13) as an active ingredient.
  • a method for diagnosing hard keratin abnormality and dysfunction of cells producing hard or hard keratin which comprises the following steps (a) and (b):
  • step (b) the expression level of the BG8 protein and the expression level of the nucleic acid encoding the Z or BG8 protein measured in step (a) above, and the expression level of the BG8 protein and the Z or BG8 protein level in normal cells Comparing the expression level of the nucleic acid encoding
  • a method for regulating the function of a hard keratin-producing cell which further comprises the following step in addition to the diagnostic method described in the above (15),
  • the test is performed based on the difference in the expression level of the BG8 protein and the expression level of the nucleic acid encoding Z or BG8 protein between the test cell and the normal cell obtained in the step (b). Controlling the expression level of BG8 protein and the expression level of a nucleic acid encoding Z or BG8 protein in cells
  • a method for diagnosing abnormal hard keratin and abnormal function of cells producing Z or 81-keratin comprising the following steps (a) and (b):
  • step (b) a step of comparing the activity of the BG8 protein measured in the above step (a) with the activity of the BG8 protein in cells (normal cells) extracted from a healthy person
  • step (c) a step of regulating the activity of BG8 protein in the test cells based on the difference in the activity of the BG8 protein between the test cells and the normal cells obtained in the step (b)
  • the target disease is hair abnormality such as alopecia, hirsutism or hair shaft abnormality; aging of the skin; malformation of the tongue, hair or nails; abnormal formation of hard keratin such as punctate keratoderma or trichomema.
  • a pharmaceutical composition characterized in that the disease is recognized.
  • the “nucleic acid” in the present invention refers to, for example, DNA and RNA, and preferably refers to DNA and Z or RNA.
  • the kind of the DNA is not particularly limited, and includes, for example, cDNA, genomic DNA and synthetic DNA.
  • the nucleic acid and protein of the present invention are preferably isolated.
  • isolated means substantially free of cellular material or culture medium when produced by recombinant DNA technology, and precursor chemicals or other when chemically synthesized. Refers to a nucleic acid or polypeptide substantially free of the chemical substance
  • hybridizes under stringent conditions means that two nucleic acid fragments are expressed by the expression of cloned genes in Escherichia coli by Sambrook et al. (Sambrook, J.). coli)) (Molecular Cloning: A laboratory manual 2nd. Edition (1989)) Cold Spring Harbor Laboratory Press, New York, USA, 9.47-9.62 and 11.45-11.61. Means to do.
  • hybridization is performed by ⁇ 9xSSC, 40% formamide, 25 ° C ''. , Washing under "0.2 x SSC, 55", more preferably, hybridization at "6 x SSC, 50% formamide, 37 ° (:)", washing under "0.1 x SSC” , 62 ".
  • mice Other organisms that isolate functionally equivalent proteins using the hybridization technique include, but are not limited to, for example, mice, rats, rabbits, rabbits, dogs, monkeys, and the like.
  • protein and peptide according to the present invention also include salts thereof.
  • BG8 protein refers to a mouse BG8 protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and a human BG8 protein having the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing. Not only proteins but also one or more amino acids in the amino acids constituting these proteins can be substituted, deleted, added, And / or imported proteins.
  • the “BG8 protein” includes not only the protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing, but also homologues of the protein in animals such as rat, peacock, dog, etc. Also includes species splicing variants.
  • BG8 gene refers to a mouse BG8 gene having the base sequence of SEQ ID NO: 1 in the sequence listing and a human BG8 gene having the base sequence of SEQ ID NO: 3 in the sequence listing Not only that, but also includes homologues of the gene in animals such as rats, magpies, and dogs.
  • the protein of the present invention includes not only a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing, but also substitution, deletion, insertion, and addition or deletion of one or more amino acids. Also includes proteins. Here, “2 or more” specifically indicates that the number is 2 to 30, preferably 2 to 20, and more preferably 2 to 10.
  • amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing an amino acid that is at least 80%, preferably at least 90%, and more preferably at least 95% identical to the amino acid sequence A protein having a sequence is also included in the protein of the present invention.
  • the protein of the present invention may be composed of a part of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing.
  • Specific examples of such proteins include, for example, partial peptides at the C-terminal region or N-terminal region of the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing, and among them, partial peptides at the C-terminal region It is preferred that Such a peptide can be used, for example, for preparing an antibody. Such peptides can also be used for screening for compounds described below.
  • Such a partial peptide has at least 10 amino acids, preferably 15 amino acids, more preferably 20 amino acids or more.
  • the present invention relates to a protein comprising the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4 in the above-mentioned sequence listing, and one or more amino acids in the amino acid.
  • a nucleic acid specifically, for example, a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing can be mentioned.
  • the nucleic acid of the present invention may be a nucleic acid that hybridizes under stringent conditions to a nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing.
  • a nucleic acid is preferably one in which the encoded protein functions as a GPCR protein, but is not necessarily limited to such a function, and may be used as a probe capable of detecting the nucleic acid of the present invention or a primer for PCR. It may be possible.
  • the "BG8" gene isolated from the mouse and human of the present invention contains an open reading frame (903 bp) encoding a protein of 300 amino acid residues (SEQ ID NO: 1 and SEQ ID NO: 3). From the results of the hydrophilic plot analysis of such proteins, the mouse and human BG8J proteins encoded by the “BG8” gene both have seven hydrophobic domains characteristic of GPCR proteins (FIG. 1 and FIG. 2) Therefore, it was presumed that the “BG8” gene was a seven-transmembrane receptor, that is, a gene encoding a GPCR.
  • the BG8 gene is specifically expressed in hair, nail and tongue hard keratin-producing cells, which are known to produce hard keratin. ( Figures 4, 5, 6, and 7).
  • BG8 gene was expressed in skin in mouse tissues and in skin and tongue in human tissues (Figs. 8, 9 and Ten ) .
  • tissues expressing hard keratin include: In addition to hair, for example, nails, tongues and the like are included, but not limited to these.
  • the present invention also provides ": BG8 protein".
  • the “BG8 protein” of the present invention includes a mouse BG8 protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing and a human having the amino acid sequence of SEQ ID NO: 4 in the sequence listing.
  • BG8 proteins include proteins in which one or more amino acids have been substituted, deleted, added and Z- or inserted in the amino acids constituting these proteins.
  • Such proteins can be prepared by methods known in the art for modifying amino acids, for example, the Kunke 1 method (Methods in Enzymology, vol. 154, No. 367-382 (1987), the double primer method (Methods in Enzymology, vol. 154, No. 329-350 (1987)), cassette mutation method (Gene, vol. 34, 315-323, (1985), megaprimer method (Biotechniques, vol. 8, 404-407 (1990)), etc. It can be prepared using the method described in (1).
  • a modified protein by performing a modification such as amino acid substitution in a native "BG8" protein (SEQ ID NO: 2 or SEQ ID NO: 4) using a known method. It is possible. Amino acid mutations in proteins can also occur naturally. As described above, the mutant protein in which the amino acid sequence is mutated with respect to the natural protein by amino acid substitution, deletion, addition, insertion, or the like is also included in the protein of the present invention. Amino acids in such a mutant protein are usually at least 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Are 97-99% identical.
  • a protein functionally equivalent to the “BG 8” protein can be obtained by a hybridization technique known to those skilled in the art (Methods in Enzymology, vol. 100, 333-342 (1983), Science, vol. 196, 180-182 (1977), that is, hybridization using the cDNA of BG8 gene (SEQ ID NO: 1 or SEQ ID NO: 3) or a part thereof.
  • a protein having a function equivalent to that of the “BG8” protein can be obtained from the isolated DNA.
  • Detecting a gene expressed in at least hair, tongue, nails or skin by using a part or all of the nucleotide sequence disclosed in the present invention as a hybridization probe. Is possible.
  • the distribution of gene expression can be identified by examining gene expression in various tissues by using a part or all of the nucleotide sequence disclosed in the present invention as a probe for hybridization.
  • the hybridization method itself is not particularly limited. Hybridization, Southern hybridization, Colony hybridization, Dot hybridization, Fluo resce nc einsitu hyb ridization (FI SH), In situ hybridization, DNA chip method, A microarray method and the like can be mentioned.
  • hybridization is to use a part or all of the nucleotide sequence disclosed in the present invention as a probe for Northern hybridization, and to measure the length of mRNA in a sample tested. In addition, it is possible to quantitatively detect gene expression.
  • nucleotide sequence disclosed in the present invention as a probe for FISH, it is possible to identify the position of the gene on the chromosome. Further, by using part or all of the nucleotide sequence disclosed in the present invention as a probe for in situ hybridization, it is possible to identify the tissue distribution of the expression of the gene.
  • the nucleotide sequence of the present invention is used as a probe for hybridization, it is necessary to have a nucleotide length of at least about 20 nucleotides.
  • a nucleic acid having a continuous nucleotide length of 40 nucleotides or more is required.
  • those having a base length of 60 bases or more are used.
  • the probe may be labeled so as to be easily detected.
  • the detectable label may be any type or moiety that can be detected either visually or by using an instrument, and commonly used detectable labels include, for example, 32 Radioactive labels such as P, 14 125 I, 3 H, and 35 S.
  • a biotin-labeled nucleotide can be incorporated into DNA or RNA by nick translation, chemical and enzymatic means, and the like.
  • the probe labeled with biotin is detected after hybridization using a labeling means such as avidin Z streptavidin, a fluorescent labeling agent, an enzyme, or a gold chloride complex.
  • Nucleic acids may be labeled by binding to proteins.
  • a nucleic acid cross-linked to a radioactive or fluorescent histone single-stranded binding protein may be used.
  • RNA can be extracted from a sample to be assayed, and gene expression can be measured semi-quantitatively by the Live tran s cip lion-PCR (hereinafter referred to as RT-PCR) method.
  • RT-PCR Live tran s cip lion-PCR
  • Such a method is performed by a method well known to those skilled in the art, for example, referring to Molecular Cloning A LABORATORY MANUAL 2nd. Edition (1989) (by T. Maniatis: Cold Spring Harbor Laboratory Press). It is a thing.
  • nucleotide length 10 to 60 bases is required.
  • a nucleic acid having a base of 15 to 30 is preferably used, and a nucleic acid having 15 to 30 bases is more preferably used.
  • the GC content in the primer sequence is 40 to 60%. Furthermore, it is preferred that there is no difference in the Tm value between the two primers used for amplification.
  • the nucleotide sequence disclosed in the present invention it is possible to detect the distribution of gene expression expressed in various tissues and cells.
  • the distribution of gene expression can be detected by using a part or all of the nucleotide sequence disclosed in the present invention as a hybridization probe or a PCR primer.
  • the distribution of gene expression can be detected using a DNA chip, a microarray, or the like. That is, a part or all of the nucleotide sequence disclosed by the present invention can be directly pasted on a chip or an array. Then, RNA extracted from the cells is labeled with a fluorescent substance or the like and hybridized, and it is possible to analyze in which cells the gene is highly expressed.
  • the DNA to be attached to the chip or the array may be a PCR reaction product using part or all of the nucleotide sequence disclosed by the present invention.
  • nucleic acids consisting of a part or all of the nucleotide sequence disclosed in the present invention.
  • a particular drug is susceptible or susceptible to a certain disease, or whether or not a particular drug is effective or not, is governed by genetic information possessed by individuals.
  • the causal relationship between the disease and the nucleic acid in the subject becomes clear, and not only can the disease be diagnosed, but also the drug to be administered can be selected. Becomes possible.
  • the expression level of one of the nucleic acids according to the present invention must be examined, but also the expression of two or more nucleic acids.
  • the relative expression level can be compared and examined, and the drug to be administered can be selected, and more accurate judgment can be made.
  • nucleic acid of the present invention By using part or all of the nucleic acid of the present invention, it is possible to clone the nucleic acid of the present invention expressed in at least hair, nails, tongue, and skin.
  • a part or all of the nucleotide sequence disclosed in the present invention is used as a probe for Northern hybridization, a probe for colony hybridization, or a primer for PCR, and is disclosed in the present invention. It is possible to clone a gene containing part or all of the nucleotide sequence.
  • nucleic acid of the present invention and the protein encoded thereby are considered to be effective for the treatment and / or prevention of phenomena specific to hard keratin-producing cells and for Z or diagnosis.
  • the phenomena peculiar to hard keratin-producing cells include, for example, abnormalities of hair such as hair shaft abnormality, alopecia, hirsutism, formation of tongue, hair or nails, poor growth, horny cuticle, hair It means diseases such as mammary tumors and diseases such as skin and tongue.
  • nucleic acid of the present invention and the protein encoded thereby are effective for constructing a screening method described below.
  • the first compound screening method of the present invention is characterized by including the following steps (a) to (c).
  • step (c) selecting a test compound for which binding was detected in step (b).
  • test compound examples include, but are not limited to, proteins, peptides, non-peptide compounds, artificially synthesized compounds, extracts of tissues and cells, and serum.
  • the protein of the present invention used in the screening includes, for example, a form expressed in a desired cell (including a transformant treated so as to express the protein) or on the cell surface, a form as a cell membrane fraction of the cell, and affinity. It may be in the form of a single column.
  • Compounds used for screening are appropriately labeled and used as necessary. Examples of the label include, but are not limited to, a radiolabel and a fluorescent label.
  • the method for bringing the BG8 protein into contact with the test compound is not particularly limited as long as they can be substantially brought into contact with each other.
  • the method may be carried out in an aqueous solution or buffer, or on a membrane. Examples include contact of the test compound with the BG8 protein immobilized on the dish.
  • the binding activity between the protein or peptide of the present invention and the compound is detected by a label attached to the compound bound to the protein / peptide of the present invention (for example, the amount of binding is detected by radioactivity or fluorescence intensity).
  • changes in cells due to binding of the test compound to the protein of the present invention on the cell surface eg, activation of G protein, change in Ca 2+ or cAMP concentration, activation of phospholipase C, change in pH, Arachidonic acid release, reporter gene atssay, etc.
  • the method of Zlokarmik et al. Science vol. 279, 84 (1998) can be used.
  • the literature Cell Calcium, vol.
  • the second compound screening method of the present invention is characterized by including the following steps (a) to (c).
  • step (c) selecting a test compound in which a change in activity was detected in step (b).
  • test compound examples include, but are not limited to, proteins, peptides, non-peptide compounds, artificially synthesized compounds, extracts of tissues and cells, and serum.
  • the protein of the present invention used for screening is, for example, in a desired cell (including a transformant treated so as to express the protein) or in a cell
  • the form may be a form expressed on the surface, a form as a cell membrane fraction of the cell, or a form bound to an affinity column.
  • Compounds used for screening are appropriately labeled and used as necessary. Examples of the label include, but are not limited to, a radiolabel and a fluorescent label.
  • Methods for detecting the activity of the BG8 protein include, for example, changes in cells due to the binding of the test compound to the protein of the present invention on the cell surface (for example, activation of G protein, change in the concentration of Ca 2+ or cAMP, Activation of phospholipase C, change in pH, release of arachidonic acid, repo overnight gene access, etc.) can be used as detection methods.
  • changes in cells due to the binding of the test compound to the protein of the present invention on the cell surface for example, activation of G protein, change in the concentration of Ca 2+ or cAMP, Activation of phospholipase C, change in pH, release of arachidonic acid, repo overnight gene access, etc.
  • a method for detecting such cell functions may be used.
  • Specific examples of such a method for measuring cell function include measuring the expression level of hard keratin and the structural change of Z or hard keratin.
  • the third compound screening method of the present invention is characterized by including the following steps (a) to (c).
  • step (c) selecting a test compound whose expression level has been measured in step (b).
  • test compound examples include, but are not limited to, proteins, peptides, non-peptide compounds, artificially synthesized compounds, extracts of tissues and cells, and serum.
  • the protein of the present invention used for screening includes, for example, a form expressed in a desired cell (including a transformant treated so as to express the protein) or a cell surface, a form as a cell membrane fraction of the cell, and an affinity. It may be in the form of being connected to a two-column.
  • Compounds used for screening are appropriately labeled and used as necessary. Examples of the label include, but are not limited to, a radiolabel and a fluorescent label.
  • the method is not particularly limited, and a known measurement method can be used. Specifically, for example, Western blotting, Northern hybridization, quantitative RT-PCR, DNA chip, repo One Targene Atsushi.
  • the target phenomena of the compounds obtained as a result of the screening method of the present invention include abnormal formation of hard keratin and dysfunction of cells that produce Z or hard keratin. It is preferably a phenomenon that promotes or slows down or inhibits the function, and specifically, such a phenomenon is hair loss such as alopecia, hirsutism, abnormal hair shaft, skin aging, tongue, hair, or nail. The formation and poor growth, or keratoderma punctata, and trichomema are preferred.
  • Compounds obtained by the screening method of the present invention include, for example, hair growth, hair growth, control of hair growth in hirsutism, correction of hair shaft abnormalities, prevention of skin aging, growth of tongue, hair and nails. Possible uses include correction of malformation, treatment and / or prevention and / or diagnosis of keratoderma dermatosis and trichomema. When these compounds are used, in addition to directly administering the isolated compound itself to humans, it is also possible to administer or apply as a compound formulated by a known pharmaceutical method.
  • pharmacologically acceptable carriers or vehicles specifically, sterile water or saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, binders, lubricants, sweeteners, flavors
  • pharmacologically acceptable carriers or vehicles specifically, sterile water or saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, binders, lubricants, sweeteners, flavors
  • Administration to humans is performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by a method known to those skilled in the art. sell.
  • the dose varies depending on the weight and age of the subject, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.
  • the compounds can be prepared and used in, for example, medicated cosmetics (quasi-drugs), ointments, liniments, lotions and emulsions.
  • various basic cosmetic bases generally used conventionally, for example, various alcohols, hydrocarbons, fatty acids, phospholipids, Fats and oils and waxes can be mentioned, and these can be used alone or in combination.
  • the antibody of the present invention has an amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing. For proteins containing a portion.
  • the antibody that binds to the GPCR protein of the present invention can be prepared by a method known to those skilled in the art (for example, see “Shinsei Kagaku Jikken Kozai 1, Protein 389-406, Tokyo Kagaku Dojin”).
  • the preparation of the polyclonal antibody can be performed, for example, as follows. (4) An appropriate amount of the above protein or peptide is administered to immunized animals such as herons, guinea pigs, mice, and chickens. Administration may be with an adjuvant (180 ⁇ ? Jihachi) that promotes antibody production. Administration is usually every few weeks. By performing immunization multiple times, the antibody titer can be increased.
  • polyclonal antibodies can be prepared by subjecting this antiserum to, for example, ammonium sulfate precipitation, fractionation by anion exchange chromatography, and affinity purification using protein A or immobilized antigen. it can.
  • preparation of a monoclonal antibody can be performed, for example, as follows.
  • the immunized animal is immunized with the protein or peptide of the present invention in the same manner as described above, and after the final immunization, the spleen or lymph node is collected from the immunized animal.
  • the antibody-producing cells and myeloma cells contained in the spleen or lymph node are fused using polyethylene glycol or the like to prepare a hybridoma.
  • the desired hybridoma is screened, cultured, and a monoclonal antibody can be prepared from the culture supernatant.
  • the monoclonal antibody can be purified, for example, by ammonium sulfate precipitation or fractionation by anion exchange chromatography, or affinity purification using protein A or immobilized antigen.
  • the antibody thus prepared is used for affinity purification of the protein of the present invention, as well as for inspection of phenomena caused by abnormalities of the protein of the present invention, antibody therapy, and detection of the expression level of the protein of the present invention. And so on.
  • human antibodies or human antibodies are preferred.
  • the human antibody is a mouse-human chimeric antibody
  • an antibody gene is isolated from a mouse cell that produces an antibody against the protein of the present invention, and the H chain constant region is recombined into a human IgE H chain constant region gene. It can be prepared by introducing it into mouse myeloma cells J558L (Neuberger, MS et al., Nature, vol. 314, 268-270 (1985). Human antibodies can be used in mice in which the immune system has been replaced with humans). Free of protein It can be prepared by disinfection.
  • the antibody of the present invention can adjust the function of the BG8 protein by reacting with the BG8 protein. Therefore, it can be used as a therapeutic and / or preventive and / or diagnostic agent for a phenomenon caused by overproduction of BG8 protein.
  • the method for diagnosing abnormalities of hard keratin and abnormal function of cells producing Z or hard keratin according to the present invention will be described.
  • the method of diagnosing the abnormality of 81-keratin and the abnormality of the function of cells producing Z or hard keratin according to the present invention is characterized by comprising the following steps (a) and (b).
  • test cell is not particularly limited as long as it is a cell expressing hard keratin, and specific examples include cells of hair, nails, tongue, skin, and the like. In addition, even if the cells do not express hard keratin, it is determined that abnormal diagnosis of hard keratin and / or dysfunction of cells that produce hard keratin is required to be diagnosed. Becomes
  • the method for measuring the expression level of the protein in the test cells is not particularly limited, and can be performed by a known method. Specifically, for example, a test cell is treated with a buffer containing a surfactant or the like to solubilize the BG8 protein contained in the cell, and then subjected to Western blotting using an antibody against the protein. Can be used to quantify the amount of expression of evening protein.
  • the method for measuring the expression level of the nucleic acid in the test cell is not particularly limited, and can be performed by a known method. Specifically, for example, after extracting all RNAs or mRNAs from test cells, the amount of BG8 gene expression can be determined by performing Northern hybridization using a nucleic acid encoding BG8 protein as a probe. Can be measured.
  • the amount of protein and the expression level of Z or nucleic acid measured in this way are By comparing with normal tissues measured by the method, abnormal expression of the BG8 protein of the present invention can be detected, and abnormalities of hard keratin and / or hard keratin that may be related to the expression of the protein can be detected. It is possible to detect, diagnose, and predict abnormalities in the function of cells that produce E. coli.
  • the method of regulating the dysfunction of a cell producing 81-dokeratin according to the present invention is characterized by further comprising the following steps in addition to the above-mentioned diagnostic method.
  • step (c) based on the difference in the expression level of the BG8 protein and the expression level of the nucleic acid encoding Z or BG8 protein between the test cell and the normal cell obtained in the step (b), A step of regulating the expression level of BG8 protein and / or the expression level of a nucleic acid encoding BG8 protein in the test cells.
  • the method for adjusting the expression level of the protein and the expression level of Z or nucleic acid in the test cells is not particularly limited, and can be performed by a known method.
  • an antisense oligonucleotide to the nucleic acid of the present invention can be used.
  • the antisense nucleotide binds to the RNA corresponding to the nucleic acid, thereby preventing protein synthesis. As a result, expression of the protein can be suppressed.
  • Such antisense nucleotides can be synthesized by those skilled in the art by known methods.
  • the nucleic acid of the present invention can be introduced into a vector, and the transgene can be expressed by an arbitrary expression promoter.
  • the virus vector that can be introduced can be prepared based on a DNA or RNA virus.
  • virus vectors include, for example, Mo MLV vector, herpes virus vector, adenovirus vector, AAV vector, HIV vector, SIV vector, and Sendai virus vector.
  • a virus having a host range other than human can be used as a virus vector.
  • vectors other than the above-mentioned virus vectors are also included in the present invention.
  • a complex of phosphoric acid and nucleic acid, a ribosome, a cationic lipid complex, a Sendai virus ribosome, and a polymer carrier having a polycation as a main chain can be used.
  • a gene transfer system an electroversion, a gene gun and the like can be used.
  • the expression cassette used here is not particularly limited as long as it can express a gene in a target cell.
  • an expression cassette capable of expressing a gene in animal-derived cells The expression cassette is preferably a cassette, more preferably an expression cassette capable of expressing genes in mammalian cells, and particularly preferably an expression cassette capable of expressing genes in human cells.
  • Specific examples of the gene promoter used in the expression cassette include, for example, adenovirus, cytomegalovirus, human immunodeficiency virus, simian virus 40, rous sarcoma virus, simple herpes virus, mouse leukemia virus, and simbis virus.
  • Virus hepatitis A virus, hepatitis B virus, hepatitis C virus, papilloma virus, human T-cell leukemia virus, influenza virus, Japanese encephalitis virus, JC virus, palpovirus B19, poliovirus, etc.
  • Promoters derived from mammals such as motor, albumin, SRQ; heat shock proteins, elongation factors, chimeric promoters such as CAG promoter, and promoters whose expression is induced by tetracycline, steroid, etc. That.
  • the method for diagnosing abnormal hard keratin and abnormal function of cells producing Z or hard keratin according to the present invention is characterized by comprising the following steps (a) and (b).
  • step (b) a step of comparing the activity of the BG8 protein measured in the above step (a) with the activity of the BG8 protein in cells (normal cells) extracted from a healthy person.
  • the type of the cell is not particularly limited as long as it is a cell that produces hard keratin, and specific examples thereof include cells such as hair, nails, tongue, and skin.
  • the method for measuring the activity of the protein in the test cell is not particularly limited, and can be performed by a known method. Specifically, for example, cell changes caused by the binding of a ligand compound to the BG8 protein present on the cell surface (eg, activation of G protein, Ca 2+ or cAMP) Change in the concentration, activation of phospholipase C, change in pH, release of arachidonic acid, reporter gene assembly, etc.).
  • the method of regulating the function of an eighteen-dokeratin-producing cell of the present invention is characterized by further comprising the following steps in addition to the above-mentioned diagnostic method.
  • step (c) a step of regulating the activity of the BG8 protein in the test cell based on the difference in the activity of the BG8 protein between the test cell and the normal cell obtained in the step (b).
  • the method for regulating the activity of the protein in the test cell is not particularly limited, and can be carried out, for example, by regulating the expression level of the BG8 protein or the nucleic acid encoding the protein.
  • the activity of a protein can be reduced by suppressing the expression of a nucleic acid encoding the protein with an antisense nucleotide or the like and suppressing the expression of the protein.
  • antisense nucleotides can be synthesized by those skilled in the art by known methods.
  • a method similar to the method used in gene therapy can be used.
  • the nucleic acid of the present invention can be introduced into a vector, and the introduced gene can be expressed by any expression promoter.
  • the virus vector that can be introduced can be prepared based on a DNA or RNA virus.
  • virus vectors include, for example, MoMLV vector, herpes virus vector, adenovirus vector, AAV vector, HIV vector, SIV vector, Sendai virus vector.
  • a virus having a host range other than human can be used as a virus vector as long as the virus has a therapeutic effect.
  • vectors other than the above-described virus vectors can also be used in the present invention.
  • a complex of calcium phosphate and a nucleic acid, a ribosome, a cationic lipid complex, a Sendai virus liposome, a polymer carrier having a polycation as a main chain, and the like can be used.
  • a gene transfer system elect-portation, a gene gun and the like can be used.
  • the expression cassette used here is not particularly limited as long as it can express a gene in a target cell.
  • an expression cassette capable of expressing a gene in animal-derived cells The expression cassette is preferably a cassette, more preferably an expression cassette capable of expressing genes in mammalian cells, and particularly preferably an expression cassette capable of expressing genes in human cells.
  • the gene promoter used for the expression cassette includes, for example, adenovirus, cytomegalovirus, human immunodeficiency virus, simian virus 40, rous sarcoma virus, simple herpes virus, mouse leukemia virus, Synvisvirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, papilloma virus, human T-cell leukemia virus, influenza virus, Japanese encephalitis virus, JC virus, palpovirus B19, poliovirus, etc.
  • Promoters derived from mammals such as promoter, albumin, SRa, heat shock protein, and elongation factor; chimeric promoters such as CAG promoter; promoters whose expression is induced by tetracycline, steroids, etc. Is You.
  • the pharmaceutical composition of the present invention contains any one of the BG8 protein, the BG8 gene of the present invention, and an antibody against the protein as an active ingredient, and the target disease is alopecia, hirsutism, hair shaft abnormality, or the like. Abnormalities in hair; aging of skin; abnormal formation of tongue, hair or nails; punctate keratosis or hair mas, etc., characterized by abnormal keratin formation.
  • the BG8 protein, the BG8 gene or an antibody against the protein refers to
  • one or two or more amino acids in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing have a substituted, deleted, added and / or inserted amino acid sequence, and produce hard keratin A protein characterized by being specifically expressed in cells,
  • nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3 in the sequence listing
  • nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3 in the sequence listing or a nucleic acid having a nucleotide sequence complementary to the nucleic acid
  • Such a pharmaceutical composition contains, as an active ingredient, a nucleic acid and / or a protein specifically expressed in a cell that produces hard keratin, the phenomenon involved in hard keratin, such as alopecia, hirsutism, and hair shaft Abnormal hair and other abnormalities, skin aging, formation of tongue, hair or nails, poor growth, or diseases in which abnormal formation of hard keratin is observed, for example, therapeutic agents for keratoderma dermatosis, trichomema, etc. and / or Useful for prophylactic and / or diagnostic agents.
  • a nucleic acid and / or a protein specifically expressed in a cell that produces hard keratin the phenomenon involved in hard keratin, such as alopecia, hirsutism, and hair shaft Abnormal hair and other abnormalities, skin aging, formation of tongue, hair or nails, poor growth, or diseases in which abnormal formation of hard keratin is observed, for example, therapeutic agents for keratoderma derma
  • Such pharmaceutical compositions include, for example, hair growth, hair growth, control of hair growth in hirsutism, correction of hair shaft abnormalities, prevention of skin aging, correction of growth and poor formation of tongue, hair and nails, punctate angle
  • Application to treatment or prevention or diagnosis of dermatosis or trichomema is conceivable.
  • these in addition to directly administering the protein and / or the nucleic acid itself to a human, it is also possible to formulate and administer or apply by a known pharmaceutical method.
  • pharmacologically acceptable carriers or vehicles specifically, sterile water or saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, binders, lubricants, sweeteners, Formulation and administration in appropriate combinations with flavoring agents, coloring agents, etc.
  • Administration to patients can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., intranasally, transbronchially, intramuscularly, transdermally, or orally by a method known to those skilled in the art. It can be carried out.
  • the dose varies depending on the weight and age of the subject, the method of administration, and the like, but those skilled in the art can appropriately select an appropriate dose.
  • the protein and Z or nucleic acid described above may be used as, for example, medicated cosmetics (quasi-drugs), ointments, liniments, lotions, and emulsions. it can.
  • various basic cosmetic bases generally used conventionally, for example, various alcohols, hydrocarbons, fatty acids, phospholipids, fats and oils And waxes, which can be used alone or in combination.
  • Example 1 Expression of mouse BG8 and confirmation of nucleotide sequence of full-length clone New GPCR sequence (GenBank) isolated by the present inventors (Biochemical and Biophysical Research Communication, vol.268, 553-561) A similarity search was performed using Accession No. AB 030198). The search algorithm was b1ast. As a result of the search, GenBank Accession Nos. AA 164122, AA 562774, AA 8641 16, A AO 68008, AA 821407, AA 795969, AA 799021, and GN Bank Accession No. A A756761. A partial sequence of AA 168673, AA 79 1 779 was found. This candidate GPCR candidate gene was named "BG8".
  • AA 164122 (IMAGE: 607331) was purchased from KUR AB O as an I MAGE clone, and the clone was used as a probe and a mouse skin cDNA library of STRATA GENE (# 937 313).
  • a clone that was considered to be full length was isolated and its entire nucleotide sequence was determined (SEQ ID NO: 1).
  • SEQ ID NO: 1 A clone that was considered to be full length was isolated and its entire nucleotide sequence was determined.
  • the full-length clone was found to contain a 903 bp open reading frame, and the deduced amino acid sequence was 300 amino acid residues (SEQ ID NO: 2).
  • FIG. 1 the numbers on the figure indicate the amino acid numbers of the mouse “BG8” protein, and the seven hydrophobic regions are indicated by I to V I I.
  • PCR for confirming the expression location of BG8 was performed using Mouse Rap Sid Gene ExpR e s s e i o n P an e l (manufactured by ⁇ Ri Gene) as a ⁇ type.
  • the following primers are included in PCR,
  • the expected PCR product is 545 bp.
  • PCR was performed at 94 ° C for 30 seconds, 55 ° C for 1 minute, 72 ° C for 2 minutes in 35 cycles, and 72 ° C for 5 minutes for 1 cycle. Next, the PCR amplification product was confirmed by agarose gel electrophoresis (FIG. 8).
  • Lane 1 brain (Brain), 2, thymus (Thymus), 3, heart (He art), 4, liver (L iver), 5, kidney (K i dn ey), 6, stomach (Stomach), 7, spleen (S p 1 een), 8, small intestine (Sma 1 1 Intestine) ), 9, muscle (Mu sc 1 e), 10, adrenal gland (Ad renal G 1 and), 11, lung (Lung), 12, ovary ( ⁇ vary), 13, testis (Te stis), 14, Uterus (Uterus), 15, skin (Skin), 16, prostate gland (Prostate G 1 and), 17, embryo (8.5 days after fertilization) (Em bryoe 8.5), 18, breast (Virgin) (Breast Virgin), .19, embryo (9.5 days after fertilization) (Embryo 9.5), 20, breast (pregnant) (Breast Pregnant), 21, embryo (12.5 days after fertilization) (Em br yo 6 12.5), 22, breast (lactating), (B reast
  • the PCR product (mBG8FlRl: 545 bp) amplified by PCR was subcloned into a plasmid vector pCR2.1-TOPO (manufactured by InVitrogen).
  • a sequencing reaction is performed using the BigDy e Terminator Cycling Sequence Reaction Kit (Applied Biosystems ems), and electricity is generated by the DNA sequencer 377 (Applied Biosystems ems).
  • the nucleotide sequence was determined by electrophoresis, and it was confirmed that the desired DNA fragment was amplified.
  • the Northern hybridization was performed as follows. That is, 1 g each of mRNA from mouse skin and brain was separated by electrophoresis on a 1% denaturing agarose gel, and plotted on a Hybond N + membrane (Amersham). After fixing the membrane by treating at 80 ° C for 2 hours, Northern hybridization was performed. MBG 8 F 1 R 1 was used as a probe. Sun of each lane The pulls are lane 1, skin (Skin), and 2, brain (Brain) (Fig. 9).
  • RNA PCR kitver Perform a reverse transcription reaction using 1 g of human adultskintotal RNA (manufactured by Invitrogen) as a template using RNA PCR kitver. 2.1 (manufactured by TAKARA), and then perform PCR using the above primers.
  • a human BG8 clone was isolated (SEQ ID NO: 3).
  • human BG8 like the mouse, contains a 903 bp open reading frame, has a deduced amino acid sequence consisting of 300 amino acid residues (SEQ ID NO: 4), and is a seven-transmembrane receptor. It was presumed that there was (Figure 2).
  • the numbers on the figure indicate the amino acid numbers of the human “BG8” protein, and the seven hydrophobic regions are indicated by I to VII.
  • FIGS. 10A and 10B The results of Northern hybridization using human BG8 gene in human tissues are shown in FIGS. 10A and 10B. As a result, it was confirmed that the human BG8 gene was expressed in the skin and tongue.
  • the samples in each lane in Figs. 10a and 10b are as follows.
  • E.coliimBG8 cDNA mouse BG8 cDNA
  • E.colihBG8 human BG8 cDNA
  • Each mouse BG8 antibody has amino acids 6-16 (VKSTEDYYLFC) of mouse BG8 protein! Two types were prepared: one that was used as a backup (BGN) and one that used a copy of the 286-300th (QRS FRMDTQEPTREC). The preparation of the antibody was requested to the Institute of Medical Biology, Inc. (Confirmation of antibody expression)
  • CHO-M Cell membrane fractions were similarly prepared from OCK and HEK-MOCK.
  • the membrane after the completion of the elect opening transfer was subjected to a blocking treatment at 4 to 18 hours using B1OcC Ace (UK_B25, manufactured by Dainippon Pharmaceutical Co., Ltd.). After completion of the blocking treatment, the membrane was washed three times with PBS—0.1% Tween. Next, the membrane was incubated with the primary antibody diluted 1/1000 in PBS-0.1% Tween at room temperature for 1 hour. After the incubation, the membrane was washed three times with PBS-0.1% Tween, and then diluted with PBS-0.1% Tween to 1/1000 for secondary antibody (Anti-rabbit Ig Ho).
  • B1OcC Ace UK_B25, manufactured by Dainippon Pharmaceutical Co., Ltd.
  • FIG 11 From a and b, mouse BG8 was detected at around 34 kD.
  • the samples in each lane are as follows, and BGN and BGC represent two kinds of antibodies having different epitopes as described above.
  • mouse BG8 sense and antisense cRNA probes For the preparation of mouse BG8 sense and antisense cRNA probes, the mouse BG8 full-length sequence (SEQ ID NO: 1) was subcloned into a pCDNA3 vector (manufactured by Invitrogen), and restriction enzymes XhoI (New Engl and Bio) were used. 1 abs) to obtain linearized DNA. Digitoxin-labeled cRNA probe was prepared from DNA linearized using DIG RNA Lab kit (Roche). The paraffin section was deparaffinized with xylene, washed with ethanol, treated with 3% hydrogen peroxide / methanol for 10 minutes, transferred to distilled water, and subjected to the following steps.
  • washing after hybridization was performed as follows. (1) 55 ° in 2 x SSC (2 times, 15 minutes) (2) 20 g RNase A / ml NTE (50 OmM NaC 1, 1 OmM Tris—HCl, ImM EDTA) At 37 ° C, 30 minutes, (3) 2 mM levamisole added 2 x SSC, 55 ° C, 10 minutes, twice (4) 2 mM levamisole added 0.1 l x SSC, 55 ° C, 15 minutes, 2 Visualization of the hybridized probe was performed as follows: The washed section was (1) containing 30 OmM NaCl, 5 OmM Tris-HCl, 2 mM levamisole, 0.1% Tween 20 Wash with an aqueous solution (hereinafter, washing solution A), (2) 1%) serum albumin 30 minutes with washing solution A, (3) Alkaline phosphatase labeled anti-digoxigenin antibody (1% ⁇ serum albumin / wash solution A 2) for 2 hours, (4) Washing with washing solution A (10 minutes, 3
  • the BG8 antisense cRNA probe strongly hybridized to hair, nails, and tongue.
  • Example 5 Method of mouse hair bu lbcell (hereinafter referred to as HBC) of hair keratin (hard keratin) by overexpression of BG8 described in the literature (For example, a method according to Journal of De rma tol ogical Scienc e. 25, 213-8 (2001), Acta de rma to—Ven ereol og ic a. 78, 428-32 (1998)) Prepared.
  • HBC mouse hair bu lbcell
  • skin was collected from neonatal mice (4 days old, C3H / HeN) and subjected to dispace treatment. (Manufactured by Gibco BRL, 20 gZm 1 in D MEM weight 10% feta 1 bov i n s e r FBS at 4 ° C for 16 hours). After washing with PBS (-), the epidermis was removed and further washed with PBS (1). The dermis portion was treated with Col1agenase (manufactured by Gibco BRL, 0.25% inPBS (1), 37 for 1 hour), and the hair tissue was isolated.
  • Col1agenase manufactured by Gibco BRL, 0.25% inPBS (1), 37 for 1 hour
  • Lipofect AMINE PLUS reagent manufactured by Invitrogen
  • OPTI-MEMI manufactured by GI BCOBRL
  • the culture medium was changed to Hume dia-KG2 and Ep i containing 10 MM-nox idilsulfate and IMA II-Trans-Resin tic Acid (manufactured by WAKO, 188-01113).
  • the culture was started by changing to a 1: 1 blend medium of Life-KG2.
  • ISOGEN-LS manufactured by Futatsu Gene
  • Rn easy 96 purified using Rn easy 96 (manufactured by QIAGEN)
  • RNA PCT kit Ver2.1 manufactured by TAKARA
  • Tacjman TM PCR was performed using the obtained cDNA. After 45 minutes of 50 ° C for 2 minutes and 95 ° C for 10 minutes, 95 ° C for 15 seconds and 62 ° C for 1 minute, the amplification was performed for 45 cycles at ABI PR I SM 7700 Sequence De tecti on System (Applied B). iosytem). In the reaction solution, Tacjman TM R i bo s oma l RNA Con trol R ea ge nts VI C pr ob es the (A plied B iosytem Co.) was added, normalize total RNA amount R Ibos oma 1 RNA levels did.
  • a standard curve was prepared using a known amount of cDNA derived from the isolated hair tissue, and the expression level of each gene in the hair tissue was estimated as one.
  • TAMRA (SEQ ID NO: 9)
  • ReversePrimer (mH a3 247 R): 5 -ACCTGTG CCTCCAGATCAGACT—3, (SEQ ID NO: 11)
  • T a qma ⁇ r ob e (mH a 4 1198 Ta q): 5 '-F AM A G TTGCTGTGGACCTTGCGGCAG—3, 1 TAMRA (SEQ ID NO: 12)
  • Re ve rse Prime r (mH a 4 1198 R): 5 '— GTTCAG TTAACAGCAACGCTTTGA-3, (SEQ ID NO: 14) These sequences were obtained by referring to GenBank Accession No. X 75650 (Ha 3) and NMO 27563 (Ha 4), respectively. The design was performed using 5a (manufactured by Applied Biosystems).
  • the G protein shown in the present invention is specifically expressed in cells that produce hard keratin, so that the gene encoding the protein and the protein produces hard keratin. It can be used for the treatment and Z or prevention of various phenomena related to the cells involved, and for the Z or prediction.

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Abstract

Etant donné qu'il est exprimé spécifiquement dans des cellules produisant de la kératine vieillie, un gène BG8 peut être utilisé dans des remèdes et/ou des produits de prévention et/ou des diagnostiques pour des maladies liées aux fonctions des cellules produisant de la kératine vieillie, par exemples pour des phénomènes tels qu'anormalités des cheveux (perte des cheveux et blessure de la tige du cheveux), croissance anormale ou insuffisante des cheveux, etc., pour des maladies de la peau, de la langue, des ongles ou des cheveux, ou pour des maladies accompagnées d'une formation anormale de kératine vieillie telles que la kératodermie punctiforme. L'invention concerne également des composés de criblage destinés à traiter et/ou prévenir et/ou diagnostiquer ces phénomènes ou maladies.
PCT/JP2003/004236 2002-04-03 2003-04-02 Nouveau gene recepteur couple a la proteine g et proteine bg8 Ceased WO2003087362A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003236351A AU2003236351A1 (en) 2002-04-03 2003-04-02 Novel g protein-coupled receptor gene and protein bg8

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JP2002100843 2002-04-03
JP2002-100843 2002-04-03
JP2002369385 2002-12-20
JP2002-369385 2002-12-20

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055865A1 (fr) * 1998-04-29 1999-11-04 Genesis Research And Development Corporation Limited Polynucleotides isoles de cellules de la peau et leurs procedes d'utilisation
WO2000069884A2 (fr) * 1999-05-14 2000-11-23 Genesis Research & Development Corporation Limited Compositions isolees a partir de cellules cutanees, et leurs procedes d'utilisation
WO2001007609A1 (fr) * 1999-07-27 2001-02-01 Smithkline Beecham Corporation Axor39, un recepteur couple a une proteine-g a segments 7-tm
WO2001057085A2 (fr) * 2000-02-02 2001-08-09 Incyte Genomics, Inc. Récepteurs couplés aux protéines g
WO2001057188A2 (fr) * 2000-02-03 2001-08-09 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
WO2001090357A1 (fr) * 2000-05-24 2001-11-29 Genesis Research & Development Corporation Limited Compositions isolees des cellules cutanees et methodes d'utilisation de ces compositions
WO2002016548A2 (fr) * 2000-08-04 2002-02-28 Japan Science And Technology Corporation Recepteur couple a une proteine g
WO2002061087A2 (fr) * 2000-12-19 2002-08-08 Lifespan Biosciences, Inc. Peptides antigeniques destines a des recepteurs couples a la proteine g (gpcr), anticorps s'y rapportant, et systeme d'identification desdits peptides antigeniques
WO2002063004A2 (fr) * 2001-02-07 2002-08-15 Incyte Genomics, Inc. Recepteurs couples a la proteine g

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055865A1 (fr) * 1998-04-29 1999-11-04 Genesis Research And Development Corporation Limited Polynucleotides isoles de cellules de la peau et leurs procedes d'utilisation
WO2000069884A2 (fr) * 1999-05-14 2000-11-23 Genesis Research & Development Corporation Limited Compositions isolees a partir de cellules cutanees, et leurs procedes d'utilisation
WO2001007609A1 (fr) * 1999-07-27 2001-02-01 Smithkline Beecham Corporation Axor39, un recepteur couple a une proteine-g a segments 7-tm
WO2001057085A2 (fr) * 2000-02-02 2001-08-09 Incyte Genomics, Inc. Récepteurs couplés aux protéines g
WO2001057188A2 (fr) * 2000-02-03 2001-08-09 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
WO2001090357A1 (fr) * 2000-05-24 2001-11-29 Genesis Research & Development Corporation Limited Compositions isolees des cellules cutanees et methodes d'utilisation de ces compositions
WO2002016548A2 (fr) * 2000-08-04 2002-02-28 Japan Science And Technology Corporation Recepteur couple a une proteine g
WO2002061087A2 (fr) * 2000-12-19 2002-08-08 Lifespan Biosciences, Inc. Peptides antigeniques destines a des recepteurs couples a la proteine g (gpcr), anticorps s'y rapportant, et systeme d'identification desdits peptides antigeniques
WO2002063004A2 (fr) * 2001-02-07 2002-08-15 Incyte Genomics, Inc. Recepteurs couples a la proteine g

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Title
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MAEKAWA A. ET AL.: "Identification in mice of two isoforms of the cysteinyl leukotrienel receptor that result from alternative splicing", PROC. NATL. ACAD. SCI. USA, vol. 98, no. 5, 2001, pages 2256 - 2261, XP002970240 *

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