WO2000040609A1 - Novel protein - Google Patents

Novel protein Download PDF

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Publication number
WO2000040609A1
WO2000040609A1 PCT/JP1999/007335 JP9907335W WO0040609A1 WO 2000040609 A1 WO2000040609 A1 WO 2000040609A1 JP 9907335 W JP9907335 W JP 9907335W WO 0040609 A1 WO0040609 A1 WO 0040609A1
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Prior art keywords
protein
dna
gene
oligonucleotide
cell
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PCT/JP1999/007335
Other languages
French (fr)
Japanese (ja)
Inventor
Tetsuo Yoshida
Yoshiyuki Yonetani
Yoshinori Yamashita
Tamio Mizukami
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Kyowa Hakko Kogyo Co., Ltd.
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Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Priority to AU18019/00A priority Critical patent/AU1801900A/en
Publication of WO2000040609A1 publication Critical patent/WO2000040609A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to a novel F-WD protein, a DNA encoding the protein, a vector containing the DNA, a transformant transformed with the vector, a method for producing the F-WD protein, and recognition of the protein.
  • the present invention relates to an antibody, an oligonucleotide comprising a partial sequence of the DNA, or a method for using these.
  • the present invention is useful for prevention, treatment, diagnosis, pathological analysis, and the like of diseases caused by enhanced or decreased protein degradation.
  • proteolysis plays a very important role.
  • selective proteolysis by the eukaryotic energy-dependent proteolysis system the ubiquitin-proteasome system, involves cell cycle, apoptosis, transcriptional regulation, metabolic regulation, signal transduction, stress response,
  • immune initiation Cell Engineering, 15, 888-896 (1996), Trends in Biological Science, 21, 96- 102 (1996), Cell, 92, 367-380 (1998)].
  • TNF—protein IB which regulates the activity of the transcription factor NF cB of inflammatory site-powered proteins such as spleen, is also known to be degraded by this pathway. Have been. Therefore, controlling this ubiquitin-proteasome system is considered to be effective for the treatment, prevention and diagnosis of various diseases.
  • the ubiquitin-proteasome system is the first step of ⁇ ubiquitination, '' in which the ubiquitin protein is covalently bonded to the lysine residue of the target protein via an isopeptide bond to enter the degradation pathway.
  • the ubiquitinated target protein consists of a second step in which it is recognized and degraded by the proteasome complex.
  • the specificity of molecular recognition in the first step is particularly important for degrading specific proteins at specific times [Cell, 84, 813-815 (1996)].
  • Ubiquitination of proteins consists of the following processes. First, the carboxy-terminal glycine residue of ubiquitin is activated by ubiquitin activating enzyme (hereinafter also referred to as E1) and ATP, and the ubiquitin is thioester bonded to a specific cysteine residue of E1. Next, the activated ubiquitin is transferred to a specific cysteine residue of ubiquitin-conjugating enzyme (hereinafter, also referred to as E2), and forms a thioester bond. The ubiquitin bound to E2 is transferred to a specific lysine residue of the target protein, and the target protein is ubiquitinated by isopeptide bond.
  • E1 ubiquitin activating enzyme
  • E2 ubiquitin-conjugating enzyme
  • Ubiquitination of the target protein may occur directly from E2, but in many cases requires the presence of ubiquitin ligase (hereinafter also referred to as E3) [Cell Engineering! ⁇ , 898-904 (1996), Trends in Cell Biology, 5, 428-434 (1995)].
  • E3 ubiquitin ligase
  • E3 is extremely rich in molecular diversity, and is known to consist of a single protein or to form a protein complex, and is thought to determine the substrate specificity of ubiquitination. Have been.
  • E 3 is its subunit structure and primary sequence Based on the similarity, the group consisting of a hect-domain (hect-domein), the group consisting of a protein complex called APC (anaphase promoting complex), and the group consisting of SCF (Skpl-Cdc53 / Cullin-F-box) protein complex There are several groups, such as groups.
  • SCF is a recently discovered group that has received particular attention as E3, which is involved in the degradation of cell cycle-related proteins.
  • the budding yeast S. cerevisiae S. cerevisiae G 1 cyclin (C lnl, C ln2) and the cyclin-dependent quinase zeinhibi Yuichi (CDK I) (S icl, Far 1) It has been reported that it is ubiquitinated and degraded by a specific SCF protein complex [Cell Engineering, 907-916 (1996), Cell, 91, 149-151 (1997), Proc. Natl. Acad. Sci. USA, 92, 2563-2567 (1995), Cell, 81, 279-288 (1995)].
  • the F box protein has a region called F box consisting of 40 to 50 amino acids near the amino terminus of the protein, and when forming an SCF protein complex, binds to the Skp1 protein at this portion. be able to.
  • F box protein has a function as a substrate-specific adapter [Trends in Genetics, 14, 236-243 (1998)].
  • many F-box proteins have a sequence called a WD domain or leucine-rich repeat near the carboxy terminus of the protein, and it is thought that this region interacts with a specific substrate protein.
  • F-box proteins those with a WD domain near the carboxy terminus The loop is called F-WD protein.
  • the WD domain usually has tributophan-aspartic acid (WD) as its last amino acid residue, and has a sequence in which an amino acid sequence consisting of about 20 to 40 residues is repeated 4 to 8 times, Also called WD40 repeat, GH (glycine-histidine) -WD repeat, etc. It is known that the WD domain exists in many proteins [Nature, 371, 297-300 (1994)]. Furthermore, it is known that the WD domain in the trimeric G protein /? — Subunit has a structure in which seven WDs form a propeller-like ring [Nature, 379, 3U-319 & 369-374] (1996)].
  • Genes encoding the F-WD protein include a gene (Cdc4) encoding the F3-WD protein of E3 of CDKI (Sicl, Far1) of Saccharomyces cerevisiae, and a fission yeast gene.
  • F-WD protein (Me t 30), a gene encoding F-WD protein involved in the degradation of N.ch./L in 12 of Caenorhabditis elegans (Se 1-10), A gene (human ⁇ -TRCP) that binds to the Vpu protein of human immunodeficiency virus HIV-1 and encodes the F-WD protein involved in the degradation of the CD4 protein.
  • F-WD proteins are eukaryotic SCF-type proteins. It is ubiquitous as a subunit of E3, and is expected to have many more types [Cell, 86, 263-274 (1996) Cell, 91, 221-230 (1997), Genes & Development, 11, 1548-1560 (1997), Genes & Development, 11, 3182-3193 (1997), Nature, 391, 493-496 (1998), Genes & Development, 12, 2587-2597 (1998), Molecular Cell, 1, 565-574 (1998), Genes & Development, 11,3046-3060 (1997), Cell, 91, 209-218 (1997)].
  • the present invention uses a novel F-WD protein, a DNA encoding the F-WD protein, and an antibody recognizing the protein to produce neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, leukemia, lung cancer, colon cancer, Diseases caused by increased or decreased protein degradation, such as malignant tumors such as breast cancer, autoimmune diseases such as chronic indirect rheumatism, inflammatory diseases such as asthma, and viral diseases such as acquired immunodeficiency syndrome (AIDS). It aims to provide prophylactic, therapeutic and diagnostic agents.
  • neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, leukemia, lung cancer, colon cancer, Diseases caused by increased or decreased protein degradation, such as malignant tumors such as breast cancer, autoimmune diseases such as chronic indirect rheumatism, inflammatory diseases such as asthma, and viral diseases such as acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • the present inventors based on the gene sequence information of fission yeast Pop1 +, based on EST (Expressed Sequence Tag) registered in the gene sequence database Genbank of random human cDNA sequence, BLAST2.0 [Nucleic Acids Research , 25, 3389-3402 (1997)] and frame search [Compugen, Israel] Using homology search software, analysis and analysis of a portion of the human cDNA with homology to the F box region of popl + Sequences (AA252600, AA452542, AA579079, M536071) were found. Using these EST sequences, cDNA of a novel F-WD protein was obtained and its nucleotide sequence was analyzed, thereby completing the present invention. That is, the present invention relates to the following inventions (1) to (49).
  • a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • An F-WD protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
  • the transformant according to any one of (10) to (13) is cultured in a medium, and the protein according to any one of (i) to (4) is produced and accumulated in the culture.
  • a method for screening a compound which comprises bringing the protein according to any one of (1) to (4) into contact with a test sample, and varying the ubiquitin ligase activity of the protein.
  • a method for screening a compound which comprises bringing a protein according to any one of (1) to (4) into contact with a test sample, and varying a proteolysis promoting activity of the protein.
  • (26) varying expression of a gene encoding the protein according to any one of (1) to (4), which comprises contacting a cell expressing the protein according to any one of (1) to (4) with a test sample; Screening method for the compound to be used.
  • a method for detecting mRNA encoding the protein according to any one of (1) to (4).
  • a method for suppressing the expression of a protein according to any one of 1) to (4).
  • (34) A promoter DNA that controls transcription of the gene encoding the protein according to any one of (1) to (4).
  • a test sample is brought into contact with a transformant containing the promoter DNA described in (34) and a plasmid containing the repo overnight gene ligated downstream of the promoter DNA,
  • a method for screening a compound that changes the efficiency of transcription by said promoter comprising measuring the translation product content of said reporter gene.
  • the reporter gene is chloramphenicol A gene selected from the group consisting of a ratase gene, a galactosidase gene, a luciferase gene and a green fluorescein protein gene.
  • a preventive agent for a disease caused by an increase or decrease in protein degradation comprising the protein according to any one of (1) to (4).
  • a therapeutic agent for a disease caused by enhanced or reduced proteolysis comprising the protein according to any one of (1) to (4).
  • a diagnostic agent for a disease caused by enhanced or reduced protein degradation comprising the protein according to any one of (1) to (4).
  • a preventive agent for a disease caused by enhanced or reduced proteolysis comprising the DNA according to any one of (5) to (7).
  • a therapeutic agent for a disease caused by enhanced or reduced proteolysis comprising the DNA according to any one of (5) to (7).
  • a diagnostic agent for a disease caused by an increase or decrease in protein degradation comprising the DNA according to any one of (5) to (7).
  • a preventive agent for a disease caused by enhanced or reduced proteolysis comprising the oligonucleotide according to (15).
  • a therapeutic agent for a disease caused by increased or decreased proteolysis comprising the oligonucleotide according to (15).
  • a diagnostic agent for a disease caused by increased or decreased proteolysis comprising the oligonucleotide according to (15).
  • a preventive agent for a disease caused by an increase or decrease in protein degradation comprising the antibody according to (16).
  • a therapeutic agent for a disease caused by enhanced or reduced proteolysis comprising the antibody according to (16).
  • the protein of the present invention includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in which one or more amino acids have been deleted, replaced or added.
  • the F-WD protein refers to a protein having an F box near the amino terminal of the protein and a WD domain near the carboxy terminal.
  • the F-WD protein can form ubiquitin ligase as a protein complex.
  • the F box is A 1 PX n A 2 A 3 A 4 X n A 5 A 6 A 7 X B A 8 A 9 A 10 X p A u A 12 A 13
  • the L, V, F or M the A 2 is E or D
  • a 3 is I, L or V
  • a 4 is I, L or V
  • a 5 is K, R, N Or Q
  • a 6 is I, V or L
  • a 7 is L
  • a 8 is Y, ⁇ , ⁇ , ⁇ or R
  • ⁇ 9 is I, L, V, ⁇ or F
  • a 1 () is D, E or P
  • a réelleis D, E, S or T A 12 is I, L or V
  • a 13 is I, L or V
  • I is isoleucine
  • L mouth isine
  • V valine
  • F is phenylalanine
  • M methionine
  • E gluamic acid
  • D is aspartic acid
  • K is lysine
  • R is arginine
  • N is asparagine
  • Q is glutamine
  • Y is tyrosine
  • H histidine
  • A is alanine
  • P proline
  • S serine
  • T threonine
  • C cysteine.
  • X represents any amino acid
  • n is 1 to 5
  • m is 1 to 10
  • q is 2 to 10
  • q is 2 to 10
  • q is 2 to 3
  • s is 0 to 2. Represents an individual.
  • the F-WD protein forms, for example, an SCF protein complex
  • it can bind to the Skp1 protein at the F box [Cell, 86, 263-274 (1996), Trends in Genetics, 14, 236-243 (1998)].
  • the WD domain has, as its last amino acid residue, tributophan-aspartic acid (WD) or its similar amino acid residue, and often has glycine-histidine (GH) or a similar amino acid residue at its beginning, and An amino acid sequence consisting of about 20 to 40 residues has a sequence repeated 4 to 8 times.
  • WD tributophan-aspartic acid
  • GH glycine-histidine
  • the number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably one to several tens, particularly preferably one to several amino acids.
  • the protein of the present invention In order for the protein of the present invention to have a function as an F-WD protein, it must have at least 60%, usually at least 80%, and especially at least 95% homology with the amino acid sequence described in SEQ ID NO: l fe. Is preferred. Further, it is preferable that the homology is 98% or more.
  • deletion, substitution or addition of amino acids can be carried out by a site-specific mutagenesis method or the like which is a well-known technique before filing the application.
  • the term “one or several amino acids” means an amino acid of such a number that can be deleted, substituted or added by site-directed mutagenesis.
  • Such an F-WD protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added is known as Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as "Molecular Cloning").
  • Examples of the DNA of the present invention include a DNA encoding the protein of the present invention, for example, a DNA having the nucleotide sequence shown in SEQ ID NO: 2 as an DNA encoding an F-box protein.
  • a DNA having the nucleotide sequence shown in SEQ ID NO: 2 as an DNA encoding an F-box protein since a plurality of kinds of genetic codes generally exist for one amino acid, even if the DNA of the present invention encodes the DNA of the present invention, even if the DNA has a nucleotide sequence different from that of SEQ ID NO: 2, Included in DN A.
  • the DNA of the present invention includes a DNA encoding the protein of the present invention, a DNA having the nucleotide sequence of SEQ ID NO: 2, a DNA that hybridizes with the DNA under stringent conditions, and a sequence complementary to these DNAs. DNA having the following.
  • DNA capable of hybridizing under stringent conditions refers to a DNA encoding the protein of the present invention, a DNA having the nucleotide sequence of SEQ ID NO: 2 as a probe, and colony 'hybridization method, plaque'.
  • a 0.01- to 2-fold concentration of SSC solution (The composition of a 1-fold concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate.) Can be identified by washing the filter under the conditions of 65-80 ° C.
  • Hybridization is performed by the method described in Molecular 'Cloning 2nd edition, Current Protocols' in' Molecular biology, DNA Clonin 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995), etc. It can be performed according to.
  • a hybridizable DNA a DNA having at least 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 2, preferably a DNA having 80% or more homology, more preferably 95% DNAs having the above homology can be mentioned.
  • the stringent condition is particularly preferably a condition that allows hybridization only when 95% or more, and more preferably 98% or more homology exists.
  • Examples of the database include bases such as GenBank, EMBL, and DDBJ, and databases based on amino acid sequences such as GenPept, PIR, and Swiss-Prot.
  • the DNA of the present invention can be obtained from a partial nucleotide sequence of a gene such as EST having homology to pop 1+ by the following method.
  • Methods for preparing total RNA include the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology, 154, 3 (1987)], the acid guanidine thionate “phenol” and “clonal form (AGPC) method” [Analytical Biochemistry , 162, 156 (1987), Experimental Medicine, 1937 (1991)].
  • an oligo (dT) -immobilized cellulose column method molecular cloning second edition
  • a method using oligo dT latex, or the like can be used.
  • Kits such as Fast Track mRNA Isolation Kit [Fast Track mRNA Isolation Kit; Invitrogen] and Quick Prep mRNA Purification Kit [Quick Prep mRNA Purification Kit; Pharmacia] Can be used to prepare mRNA directly from tissues or cells.
  • a suitable cell or tissue a type of cDNA library containing ESTs and the like found from a database is examined, and the cell or tissue used for constructing the library, or a cell derived from the tissue is used. It is preferable to use a cell line or the like.
  • RNA or mRNA Using the obtained total RNA or mRNA, prepare a cDNA library by a conventional method.
  • a phage vector or a plasmid vector can be used as a closing vector for preparing a cDNA library as long as it can replicate autonomously in Escherichia coli K12 strain.
  • any microorganism belonging to Escherichia coli can be used. Specifically, Escherichia coli XLl-Blue MRF '[Strata Gene, Strategies, Mutual, 81 (1992)], Escherichia coli C600 [Genetics, 39, 440 (1954)], Escherichia coli Y1088 [Science, 222, 778 (1983)], Escherichia coli Y1Q90 [Science, 222, 778 (1983)], Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Escherichia coli K802 [J. Mol.
  • a commercially available cDNA library can also be used.
  • cDNAs from human organs such as Clonetech, Lifetech Oriental, etc. You can give a library.
  • a cDNA clone having the DNA of the present invention was cloned by colony hybridization or plaque hybridization using an isotope or a fluorescently labeled probe [Molecular Cloning 2nd edition].
  • a probe using a primer based on the nucleotide sequence that has been partially identified, a method using polymerase 'chain' reaction (PCR) [PCR Protocols, Aca demic Press (1990)] (PCR The method can also be used to amplify a fragment of a part of the cDNA or to use an oligonucleotide based on the nucleotide sequence that is partially identified.
  • a primer prepared based on the nucleotide sequence can be used as the primer.
  • the full-length DNA of the present invention can be obtained.
  • the nucleotide sequence of the DNA obtained by the above method can be obtained by integrating the DNA fragment as it is, or after digestion with an appropriate restriction enzyme or the like, into a vector by a conventional method, and then using a conventional nucleotide sequence analysis method, for example, Sanger (Sanger Natl. Acad. Sci. USA, 74, 5463 (1977)] or Perkin Elmer (373A, DNA Sequencer), Pharmacia, Lycoa (LI- It can be determined by analysis using a base sequence analyzer such as COR).
  • a base sequence analyzer such as COR
  • the plasmid containing the DNA of the present invention obtained by the above method includes, for example, a plasmid phMD6 having a DNA consisting of the base sequence represented by SEQ ID NO: 2.
  • Escherichia coli DH5 / phMD6 containing plasmid phMD6 was designated as FERM BP-66 11 on December 24, 1998 by the Institute of Biotechnology, Institute of Biotechnology, Tsukuba, Ibaraki, Japan. Deposited at Higashi 1-3-1, 1-3 (zip code 305-8566).
  • the desired DNA can also be prepared by chemical synthesis using a DNA synthesizer.
  • the DNA synthesizer include a DNA synthesizer manufactured by Shimadzu Corporation using the thiophosphite method, and a Perkin 'Elma's DNA synthesizer model 1392 using the phosphoramidite method.
  • nucleotide sequence is a novel sequence can be determined by searching a nucleotide sequence database such as GenBank, EMBL and DDBJ using a homology search program such as BLAST. This can be confirmed by the absence of a nucleotide sequence showing obvious homology that is considered to match the nucleotide sequence of an existing gene.
  • the novel DNA sequence obtained by the above method includes, for example, the nucleotide sequence shown in SEQ ID NO: 2.
  • an antibody having a partial sequence of the DNA of the present invention can be obtained by a conventional method or the above-mentioned DNA synthesizer.
  • Oligonucleotides such as sense oligonucleotides and sense oligonucleotides can be prepared.
  • oligonucleotide examples include a DNA having the same sequence as the contiguous 5 to 60 bases in the base sequence of the DNA or a DNA having a sequence complementary to the DNA. And a DNA having the same sequence as the consecutive 5 to 60 bases in the base sequence represented by SEQ ID NO: 2 or a DNA having a sequence complementary to the DNA.
  • Tm melting temperature
  • oligonucleotide examples include an oligonucleotide represented by SEQ ID NO: 3 or 4.
  • derivatives of these oligonucleotides can also be used as the oligonucleotide of the present invention.
  • Derivative oligonucleotides include derivative oligonucleotides in which the phosphate ester bond in the oligonucleotide has been converted to a phosphorothioate bond, and phosphodiester bonds in the oligonucleotide have been converted to N3, -P5 'phosphoramidate bonds.
  • Derived oligonucleotide Derivative oligonucleotide in which ribose and phosphodiester bond in oligonucleotide is converted to peptide nucleic acid bond, Derivative oligonucleotide in which peracyl in oligonucleotide is substituted with C-5 provinyl peroxyl Derivative oligonucleotides in which peracyl in oligonucleotides is substituted with C-5 thiazylperacyl, derivative oligonucleotides in which cytosines in oligonucleotides are substituted with C-5 propynylcytosine, oligonucleotides Derivative oligonucleotides in which cytosine in the oligonucleotide has been replaced with phenoxazine-modified cytosine, derivative oligonucleotides in which the ribose in the oligonucleotide
  • a recombinant vector in which the DNA of the present invention is inserted into a suitable expression vector at one time downstream of a promoter, and introducing the vector into a host cell, transformation for expressing the protein of the present invention is performed.
  • transformation for expressing the protein of the present invention is performed.
  • the protein of the present invention can be produced.
  • any cell that can express the target gene such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.
  • an expression vector a vector capable of autonomously replicating in the above-described host cell, which can be integrated into a chromosome, and which contains a promoter at a position where the DNA of the present invention can be transcribed is used. .
  • the recombinant vector containing the DNA encoding the protein of the present invention is capable of autonomous replication in the prokaryote and has a promoter, a ribosome binding sequence, It is preferably a recombinant vector comprising the DNA of the present invention and a transcription termination sequence.
  • the gene controlling the promotion may be included.
  • expression vectors include, for example, pBTrp2, pBTac1, pBTac2 (both Boehringer Mannheim), PKK233-2 (Pharmacia), pSE280
  • Any promoter may be used as long as it can be expressed in a host cell.
  • - defense Promo Isseki one P ⁇ £
  • promoter evening one P lac
  • P L promoter Isseki one P L promoter Isseki one, ⁇ ⁇ promo Isseki one, ⁇ 7 promoter evening one, etc., to Eshierihi ⁇ coli Ya phage, etc.
  • the origins of Promo One Night, SP01 Promo One Night, SP 02 Promo One Night, pe ⁇ Promo Night, etc. can be listed.
  • two sets of promoters, ⁇ trp are connected in series (P ⁇ x2); ⁇ promoters, lacT7 promoters, and promoters designed artificially like let I promoter. Etc. can also be used.
  • the ribosome binding sequence it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
  • a transcription termination sequence is not necessarily required for expression of the DNA of the present invention, but it is preferable to arrange a transcription termination sequence immediately below a structural gene.
  • Host cells include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., such as Escherichia coli XLl-Blue Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485 ⁇ Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Serratia font Serratia; liquefaciens, Serrat ia marcescens Bacillus subtil is, Bacillus amyloliquefaciens x Brevibacte rium ammmoniageiies, Brevibacterium i thigh ariop
  • any method for introducing DNA into the above host cells can be used, for example, a method using calcium ions
  • yeast If yeast is used as the host cell, use YEpl3 as an expression vector.
  • Any promoter can be used as long as it can be expressed in yeast.
  • ⁇ 05 Promo One PGK Promo One, GAP Promo One, ADH Promo One, gal 1 promoter, gal 10 promoter, heat shock protein promoter, MF al promoter, CUP 1 promoter, etc.
  • host cells include yeasts belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schizinomyces, Pichia, etc., such as Saccharomyces cerevisiae Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastor is and the like.
  • any method for introducing DNA into yeast can be used.
  • electroporation Methods in Enzymology, 194, 182 (1990)
  • lithium acetate method Journal of Bacteriology, 153, 163 (1983)]
  • examples of expression vectors include pc DNA I / Amp (Invitrogen), pcDNAI, pCDM8 [Nature, 329, 840 (1987)], pAGE107 [ -22979, Cytotechnology, 3, 133 (1990)], pREP4 (Invitrogen), pAGE 103 [Journal of Biochemistry, 101, 1307 (1987)], pAMo, pAMoA, pAS3-3 (Japanese Patent Laid-Open No. 2-227075), etc. Is used.
  • Any promoter can be used as long as it can be expressed in animal cells.
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • Host cells include mouse myeloma cells, rat 'myeloma cells', mouse' hybridoma cells, human namalwa cells, Namalwa KJM-1 cells, human fetal kidney cells or human leukemia Cell, African green monkey kidney cell, Chinese hamster cell CH0 cell or HBT5637 (JP-A-63-299).
  • Mouse '' myeloma cells include SP2 / 0, NS0, etc.
  • Rat '' myeoma cells include YB2 / 0, etc.
  • human fetal kidney cells HEK293 ATCC: CRL-1573
  • Human leukemia cells include BALL-1 and the like
  • African green monkey kidney cells include COS-1 and COS-7.
  • Any method for introducing a recombinant vector can be used so long as it is a method for introducing DNA into animal cells.
  • the method include the electroporation method [Cytotechnology, 3, 133 (1990)] and the calcium phosphate method. Kaihei 2-227075), the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], and the methods described in Virology, 52, 456 (1973).
  • Baculovirus 'Expression' Vectors 'Laboratory' Manual Baculovirus Expression Vectors, A Laboratory Manual, WH Freemanana Company, New York (1992)] Molecular Bio Logistic Biology, A Laboratory Manual ⁇ Current-f. Proteins can be expressed by the method described in, for example, "Kores, Cress, in Molecular Biology", Biology, Bio / Technology, 6, 47 (1988).
  • the recombinant gene transfer vector and baculovirus are co-transfected into insect cells to obtain recombinant virus in the culture supernatant of insect cells, and then the recombinant virus is infected with insect cells to express the protein.
  • the recombinant gene transfer vector and baculovirus are co-transfected into insect cells to obtain recombinant virus in the culture supernatant of insect cells, and then the recombinant virus is infected with insect cells to express the protein.
  • Examples of the gene transfer vector used in the method include pVL1392, pVL1393, and pBlueBacII (all from Invitrogen) and the like.
  • a virus that infects night moth insects such as Atographa, Ariformi, Nuclea, Polyhedrosis, and the like (Autographa californica nuclear polyhedrosis virus) can be used.
  • ovary cells of Spodoptera frugiperda As the host cells, ovary cells of Spodoptera frugiperda, ovary cells of Trichoplusia, cultured cells derived from phytococcus ovary and the like can be used.
  • the ovarian cells of Spodoptera frugiperda include Sf9 and Sf21 (baculovirus '' High ovarian cells of Trichoplu ⁇ , BTI-TN-5B1-4 (Invitrogen), etc. Bombyx mori as cultured cells derived from silkworm ovary N4 etc. can be given.
  • Examples of a method for co-transferring the above-described recombinant gene into insect cells and the above baculovirus into an insect cell for preparing a recombinant virus include a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a lipofection method, and the like. [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
  • Ti plasmid When a plant cell is used as a host, for example, Ti plasmid, tobacco mosaic virus vector, or the like can be used as an expression vector.
  • Any promoter can be used as long as it can be expressed in plant cells.
  • cauliflower mosaic virus (CaMV) 35S promoter, inineactin 1 promoter, etc. Can be.
  • Examples of the host cell include plant cells of tobacco, potato, tomato, carrot, soybean, abrana, alfa alfa, rice, wheat, wheat, and the like.
  • any method for introducing DNA into plant cells can be used.
  • Agrobacterium Japanese Patent Application Laid-Open No. 59-140885, 60-70080, W094 / 00977
  • an electroporation method Japanese Patent Application 60-251887
  • a method using a particle gun Patent No. 2606856, Patent No. 2517813
  • Patent No. 2606856, Patent No. 2517813 Patent No. 2606856, Patent No. 2517813
  • sugar or sugar chain-added protein When expressed by yeast, animal cells, insect cells or plant cells, a sugar or sugar chain-added protein can be obtained.
  • the transformant obtained as described above is cultured in a medium, and the culture of the present invention is added to the culture.
  • the protein of the present invention can be produced by producing and accumulating the protein and collecting the protein from the culture.
  • the method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
  • the protein of the present invention is returned to the living body by introducing the protein of the present invention into the patient. It can also be expressed in the body.
  • the method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
  • a culture medium for culturing a transformant obtained using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like that can be used by the organism. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.
  • the carbon source may be any one that can be assimilated by the organism, such as glucose, fructose, sucrose, molasses containing them, carbohydrates such as starch or hydrolyzed starch, and organic acids such as acetic acid and propionic acid. Alcohols such as acid, ethanol, and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other inorganic or organic acid ammonium salts, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, and copper extract.
  • casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.
  • potassium potassium phosphate potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
  • the cultivation is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • Culture The fermentation temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH is maintained at 3.0 to 9.0.
  • the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.
  • an inducer may be added to the medium, if necessary.
  • an inducer may be added to the medium, if necessary.
  • isopropyl-15-D-thiogalactoviranoside or the like is transformed with an expression vector using a ⁇ 2 promoter.
  • indoleacrylic acid or the like may be added to the medium.
  • Culture is carried out usually p H 6 ⁇ 8, 3 0 ⁇ 4 0 ° 5% C 0 2 under the conditions such as the presence 1-7 days.
  • antibiotics such as kanamycin, penicillin, and streptomycin may be added to the medium during the culture.
  • TNM-FH media As culture media for transformants obtained using insect cells as hosts, commonly used TNM-FH media [Pharmingen], Sf-900 II SFM media (Life's Technologies, Inc.) ), ExCell400, ExCell405 [JRH Biosciences, Inc.], Grace's Insect Medium [Nature 195, 788 (1962)] and the like can be used.
  • the cultivation is usually performed under conditions of pH 6 to 7, 25 to 30 ° C, etc. for 1 to 5 days.
  • an antibiotic such as genyumycin may be added to the medium during the culture.
  • a transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a cell organ of a plant.
  • a medium for culturing the transformant commonly used Murashige 'and' squeeg (MS) medium, white (White) medium, or plant medium such as auxin, cytokinin, etc. And the like can be used.
  • the cultivation is usually performed at pH 5 to 9, 20 to 40 ° C for 3 to 60 days.
  • antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.
  • a transformant derived from a prokaryote, yeast, animal cell, insect cell or plant cell having a recombinant vector into which the DNA encoding the protein of the present invention has been incorporated is cultured according to a conventional culture method.
  • the protein can be produced by accumulating and producing the protein, and collecting the protein from the culture.
  • a method for expressing a gene in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular 'Cloning 2nd edition.
  • the method for producing the protein of the present invention includes a method of producing the protein in a host cell, a method of secreting the protein out of the host cell, and a method of producing the protein on the outer membrane of the host cell.
  • the method can be selected by changing the structure.
  • the protein can be actively secreted out of the host cell.
  • the protein of the present invention is expressed in a form in which a signal peptide is added in front of the protein containing the active site of the protein of the present invention using a gene recombination technique, so that the protein of the present invention can be positively extracellularly outside the host cells. It can be secreted.
  • the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
  • the transgenic animal or plant cells are redifferentiated to create transgenic non-human animals or transgenic plants (transgenic plants) into which the gene has been introduced. Can be used to produce the protein of the present invention.
  • the protein is produced by breeding or cultivating according to a usual method to produce and accumulate the protein, and collecting the protein from the animal or plant individual. can do.
  • Methods for producing the protein of the present invention using animal individuals include, for example, known methods (American Journal of Clinical Nutrition, 63, 639S996), American Journal of Clinical Nutrition, 63, 627S (1996), Bio / Technology, 9, 830 (1991)], and a method for producing the protein of the present invention in an animal constructed by introducing a gene.
  • a transgenic non-human animal into which DNA encoding the protein of the present invention has been introduced is bred, the protein is produced and accumulated in the animal, and the protein is collected from the animal. By doing so, the protein can be produced.
  • the place of production and accumulation in the animal include milk of the animal (JP-A-63-309192), eggs, and the like.
  • the promoter used at this time any promoter that can be expressed in animals can be used.
  • the promoter, casein promoter, casein promoter, and lactate, which are breast cell-specific promoters, can be used. Globulin Promo overnight, whey mono-protein Promoter or the like is preferably used.
  • Examples of a method for producing the protein of the present invention using plant individuals include, for example, transgenic plants into which DNA encoding the protein of the present invention has been introduced [Tissue Culture, 20 (1994), 21 (1995), Trends in Biotechnology, 15, 45 (1997)], producing and accumulating the protein in the plant, and collecting the protein from the plant to produce the protein. There is a way to do it. (3) Isolation and purification of expressed protein
  • a conventional enzyme isolation and purification method may be used. Further, the protein can be efficiently purified by affinity chromatography using an antibody against the protein itself.
  • the cells produced by the transformant of the present invention are collected by centrifugation after completion of culturing, etc. After suspending the liquid, the cells are crushed by an ultrasonic crusher, French press, Menton-Gaurin homogenizer, Dynomill, etc. to obtain a cell-free extract.
  • a normal enzyme isolation and purification method that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Getylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (Mitsubishi Kasei) Anion exchange chromatography using resin, cation exchange using S-Sepharose FF (Pharmacia) etc.
  • a purified sample can be obtained using an electrophoresis method such as point electrophoresis alone or in combination.
  • an electrophoresis method such as point electrophoresis alone or in combination.
  • the insoluble form of the recovered protein is solubilized with a protein denaturant or the like. After diluting or dialyzing the solubilized solution to return the protein to a normal three-dimensional structure, a purified sample of the protein can be obtained by the same isolation and purification method as described above.
  • the protein of the present invention or its derivative such as a modified sugar is secreted extracellularly, the protein or its derivative such as a sugar chain adduct can be recovered in the culture supernatant.
  • a purified fraction is obtained by treating the culture by a method such as centrifugation as described above to obtain a soluble fraction, and by using the same isolation and purification method as described above from the soluble fraction. Obtainable.
  • the protein of the present invention can be produced as a fusion protein with another protein, and purified using affinity chromatography using a substance having an affinity for the fused protein.
  • affinity chromatography using a substance having an affinity for the fused protein.
  • the method of Lowe et al. Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)] JP-A-5-336963, JP-A-6-823021.
  • the protein of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using imnoglobulin G.
  • the protein of the present invention can be produced as a fusion protein with an F1ag peptide and purified by affinity chromatography using an anti-F1ag antibody [Proc. Natl. Acad. Sci. USA , 86, 8227 (1989), Genes Develop., 4, 1288 (1990)]. Further, the protein can be purified by affinity chromatography using an antibody against the protein itself.
  • Examples of the protein obtained in this manner include a protein having the amino acid sequence shown in SEQ ID NO: 1.
  • the protein of the present invention can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t_butyloxycarbonyl method). be able to. Also, Advanced ChemTech, Perkin Elma, Pharmacia, and Protein Technology
  • Chemical synthesis can also be performed using a peptide synthesizer such as Synthecell-Vega, PerSeptive, or Shimadzu Corporation.
  • Structural analysis of the purified protein of the present invention is performed by a method generally used in protein chemistry, for example, a method described in Protein Structural Analysis for Gene Cloning (Hisashi Hirano, published by Tokyo Kagaku Dojin, 1993). be able to.
  • a polyclonal antibody can be prepared by using a purified full-length or partial fragment of the protein of the present invention obtained by the method described in the above [2] as an antigen and administering it to an animal.
  • mice As animals to be administered, egrets, goats, rats, mice, hamsters, etc., which are 3 to 20 weeks old can be used.
  • the dose of the antigen is preferably 50 to 100 g per animal.
  • a peptide When a peptide is used, it is preferable to use, as the antigen, a peptide covalently bonded to a carrier protein such as keyhole limp et haemocyanin or bovine thyroglobulin.
  • the peptide serving as the antigen can be synthesized by a peptide synthesizer.
  • the administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the serum reacts with the antigen used for immunization. Five
  • a polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
  • Methods for separation and purification include centrifugation, salting out with 40-50% saturated ammonium sulfate, and prillic acid precipitation [Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)], or DEAE-cephalo.
  • a single column, an anion exchange column, a chromatography using a protein A or G-column or a gel filtration column, etc., may be used alone or in combination.
  • a rat whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody-producing cells.
  • the spleen is removed.
  • the spleen is shredded in MEM medium (Nissui Pharmaceutical), loosened with tweezers, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
  • the spleen cells in the precipitate fraction thus obtained are treated with Tris-ammonium chloride buffer (PH7.65) for 1 to 2 minutes to remove red blood cells, and then washed three times with MEM medium. Is used as an antibody-producing cell.
  • myeloma cells cell lines obtained from mice or rats are used.
  • 8-azaguanine-resistant mouse derived from BALB / c
  • P3-X63Ag8-U1 P 3-Ul
  • SP2 / 0-Agl4 SP2 / 0-Agl4
  • SP-2 SP2 / 0-Agl4
  • P3-X63-Ag8653 6
  • J. Immunol. 123, 1548 (1979)
  • P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)]
  • 8-azaguanine medium [RPMI-1640 medium containing glutamine (1.5 mM), 2-mercaptoethanol (5 ⁇ 10 " 5 M), genyumycin (10 ⁇ g / ml) and fetal calf A medium containing serum (FCS) (CSL, 10%) (hereinafter referred to as a normal medium) and a medium supplemented with 8-azaguanine (15 ⁇ g
  • the suspension was dispensed into a 96-well culture plate at 100 ° C./well, and the suspension was placed at 37 ° C. in a 5% CO 2 incubator. Incubate for ⁇ 14 days.
  • a purified sample of the full-length or partial fragment of the protein of the present invention used as the antigen was coated on an appropriate plate, and the hybridoma culture supernatant or the purified antibody obtained in (2-4) described later was purified.
  • an anti-rat immunoglobulin antibody labeled with a biotin, an enzyme, a chemiluminescent substance, a radioactive compound, or the like was carried out.
  • a reaction corresponding to the labeling substance is carried out.
  • Those that react specifically are selected as hybridomas that produce monoclonal antibodies against the protein of the present invention.
  • hybridoma Using the hybridoma, cloning was repeated twice by the limiting dilution method [the first time using HT medium (medium from which aminopterin was removed from HAT medium), and the second time using normal medium]. Those with a strong antibody titer are selected as hybridoma strains producing a monoclonal antibody against the protein of the present invention.
  • Ascites is collected from the mouse with ascites tumor and centrifuged at 3,000 rpm for 5 minutes to remove solids.
  • a monoclonal antibody can be purified and obtained in the same manner as in the polyclonal method.
  • the antibody subclass is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit.
  • the protein content is calculated by the Lowry method or the absorbance at 280 nm.
  • the ubiquitin ligase activity is determined by reacting a target protein with a ubiquitin protein labeled with a fluorescent label, a biotin label, or an isotope in the presence of the protein of the present invention in a cell or in a test tube, and the ubiquitin protein incorporated into the target protein. Can be determined by measuring the amount of It can also be determined by detecting the incorporated ubiquitin protein using an anti-ubiquitin antibody [Science, 373 (5), 81-83 (1995), FEBS Letters, 377, 193-196 ( 1995), Science, 269, 682-685 (1995)].
  • the proteolysis-promoting activity is determined by reacting a fluorescent-labeled, biotin-labeled, or isotopically-labeled target protein in a cell or a test tube in the presence of the protein of the present invention, and measuring the amount of change in the target protein. Can be obtained by The amount of change in the target protein can also be detected using a specific antibody for the target protein. When a fusion protein is used as the target protein, specificity for the fused protein Detection can also be performed using an antibody. It can also be determined by immunologically detecting ubiquitin incorporated into the target protein using an anti-ubiquitin antibody [Genes & Development, 12, 2587-2597 (1998), Molecular Cell, 1, 565]. -574 (1998), Genes & Development, 11, 3046-3060 (1998), Genes & Development, U, 1548-1560 (1997)].
  • a test sample is added to the protein of the present invention or a tissue, cell, or the like in which the expression of the protein of the present invention has been confirmed by the method of [7] described below, and a test sample is added thereto.
  • Ubiquitin ligase activity or proteolysis promoting activity is measured.
  • a compound that varies the ubiquitin ligase activity or the proteolysis-promoting activity from the test sample is determined. Can be screened.
  • a compound that enhances the activity is also referred to as an agonist
  • a compound that inhibits the activity is also referred to as an antagonist.
  • a compound that changes ubiquitin ligase activity or proteolysis-promoting activity can be screened by the following method.
  • Cells and tissues expressing the protein of the present invention, cell membranes prepared therefrom, purified protein of the present invention or a partial fragment of the protein are contacted with a labeling compound, and the protein of the present invention binds to the labeling compound.
  • test sample is added under the same conditions as above using a labeled compound that has been observed to bind, and the amount of binding of the labeled compound is measured in the same manner as above.
  • Test samples include synthetic compounds, naturally occurring proteins, artificially synthesized proteins, peptides, carbohydrates, lipids, modified forms and derivatives thereof, and mammals (eg, mice, rats, guinea pigs, hamsters, Urine, body fluids, tissue extracts, cell culture supernatants, cell extracts, and other non-peptide compounds, fermentation products And extracts of plants and other organisms.
  • synthetic compounds naturally occurring proteins, artificially synthesized proteins, peptides, carbohydrates, lipids, modified forms and derivatives thereof, and mammals (eg, mice, rats, guinea pigs, hamsters, Urine, body fluids, tissue extracts, cell culture supernatants, cell extracts, and other non-peptide compounds, fermentation products And extracts of plants and other organisms.
  • the agonist or angonist of the protein of the present invention obtained by the above method can be used alone as a prophylactic or therapeutic agent (also referred to as a drug), but usually it is used in pharmacology. It is admixed with one or more commercially acceptable carriers and used as a pharmaceutical preparation produced by any method well known in the pharmaceutical art.
  • the method of administering the therapeutic agent is desirably the most effective method for the treatment, and is orally or parenterally, such as oral, respiratory, rectal, subcutaneous, intramuscular, and intravenous. Can be used.
  • Examples of the dosage form of the therapeutic agent include ointments, sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, tapes and the like.
  • Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  • Liquid preparations such as emulsions and syrups are prepared from water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, and oils such as sesame oil, olive oil and soybean oil. And preservatives such as P-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint as additives.
  • Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl alcohol , Hydroxyprobi It can be produced using additives such as binders such as cellulose and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin as additives.
  • Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
  • the injection can be prepared, for example, using a carrier comprising a salt solution, a glucose solution, or a mixture of both.
  • Suppositories can be prepared using a carrier such as cocoa butter, hydrogenated fat or carboxylic acid.
  • the agonist or angonist obtained above can be directly used as a propellant, but it does not irritate the oral cavity and airway mucosa of the recipient and disperses the compound as fine particles. Sprays prepared using a carrier or the like that facilitates absorption are preferred.
  • the carrier include lactose and glycerin.
  • Formulations such as aerosols and dry powders can be prepared depending on the properties of the agonist or angonist obtained above and the carrier.
  • the dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 mg / kg to 8 mg / kg per day for an adult.
  • an expression regulating compound present in the cell and in a cell culture supernatant can be searched and identified.
  • the cell may be a cell, cell line or tissue expressing the protein of the present invention. Can be used.
  • a cell, a cell line, or a tissue in which the expression of the protein has been observed can be used by the method of immunologically detecting with the antibody described in [7] below.
  • Suitable cell lines include, for example, human epidermoid carcinoma-derived A431 cells (ATCC: CRL-1555).
  • test sample those mentioned in the test sample in the above [5] can be used.
  • Cells expressing the protein of the present invention are suspended in a medium capable of growing the cells, a test sample is added to the medium, and the cells are contacted with the cells.
  • a test sample is added to the medium, and the cells are contacted with the cells.
  • the expressed protein is quantified.
  • a method for quantification for example, a method using the following immune cell staining can be mentioned.
  • the adherent cells in the culture were washed with PBS buffer, and 3 ml of PBS buffer containing 0.05% trypsin and 0.02% EDTA (ethylenediamine tetraacetic acid) was added. After removing excess solution, 37 ° C, Remove cells from flask by incubating for 5 minutes.
  • PBS buffer containing 0.05% trypsin and 0.02% EDTA (ethylenediamine tetraacetic acid) was added. After removing excess solution, 37 ° C, Remove cells from flask by incubating for 5 minutes.
  • Cultured cells can be used as they are for suspension cells.
  • Cells immune cell staining buffer to perform immunocytochemistry were suspended in such (1% BSA, 0. 02% EDTA, PB S containing 0.05% sodium azide), 1 ⁇ 20 X 10 5 cells each round Dispense into the bottom 96-well plate.
  • the monoclonal antibody of the present invention is dispensed on the plate.
  • Examples of the monoclonal antibody include the culture supernatant of a hybridoma producing the monoclonal antibody of the present invention obtained in [3] (2-3), and the purified monoclonal antibody obtained in [3] (2-4). I can give it. Furthermore, an antibody labeled with the monoclonal antibody can also be used.
  • an antibody labeled with a monoclonal antibody is a biotin-labeled antibody.
  • a biotin-labeled antibody can be prepared by a known method (enzyme antibody method: interdisciplinary project, 1985).
  • the above antibody is diluted to a concentration of 0.1 to 50 ⁇ g / ml using an immune cell staining buffer or an immune cell staining buffer containing 10% animal serum.
  • the diluted antibody is dispensed at 20-500 ⁇ 1 / well and left under ice-cooling for 30 minutes.
  • an unlabeled antibody If an unlabeled antibody is used, add an immunocell staining buffer to the plate, wash the cells, and label the anti-mouse with a fluorescent dye such as FITC (fluorescein isothiocyanate) or phycoerythrin. Dispense 50-500 / 1 / well of immunoglobulin antibody or anti-rat immunoglobulin antibody at a concentration of about 0.1 to 5 ⁇ g / ml in an amount of about 50 to 500/1 / well. Leave for 5 minutes in the shade.
  • FITC fluorescein isothiocyanate
  • a fluorescent dye such as FITC or phycoerythrin
  • An expression-regulating compound can be identified by searching for a test sample capable of increasing or decreasing the protein content of the present invention, as compared with a system to which no test sample is added.
  • Cells expressing the protein of the present invention or mRNA encoding the protein are suspended in a medium capable of growing the cells, a test sample is added to the medium, and the cells are contacted.
  • the content of the expressed mRNA was determined by the usual Northern hybridization method: dot blot hybridization of RNA. Method, RT-PCR, etc.
  • the DNA and the oligonucleotide of the present invention are preferably used as a probe used in the hybridization method and the like, and the oligonucleotide of the present invention is suitably used as a primer that can be used in the RT-PCR method and the like.
  • An expression-regulating compound can be identified by searching for a test sample capable of increasing or decreasing the content of mRNA encoding the protein of the present invention, as compared to a system to which no test sample is added.
  • the content of the mRNA is quantified to vary the expression of the gene encoding the protein of the present invention.
  • Compounds can be searched and identified.
  • the above-mentioned [6] (1) can be used as a cell or a test sample which expresses the protein of the present invention or the protein mRNA.
  • Transformation transformed with a plasmid containing DNA linked to a repo overnight gene downstream of a promoter DNA (hereinafter simply referred to as a promoter DNA) that controls transcription of a gene encoding the protein of the present invention.
  • a promoter DNA a promoter DNA that controls transcription of a gene encoding the protein of the present invention.
  • Promoter DNA is usually contained 5 'upstream of the gene.
  • the promoter DNA of the gene encoding the protein of the present invention can be prepared using the DNA or the oligonucleotide of the present invention by a known method such as Genome Walker kits (Clontech).
  • a fragment obtained by cutting this region to an appropriate length using an appropriate restriction enzyme can be used as a transcription control region.
  • any gene can be used as long as the translation product of the gene is stable in cells and the amount of the translation product can be easily quantified. Examples thereof include chloramphenicylacetyltransferase (CAT), galactosidase (5-ga1), luciferase (luc), and green fluorescent protein (GFP).
  • CAT chloramphenicylacetyltransferase
  • 5-ga1 galactosidase
  • luc luciferase
  • GFP green fluorescent protein
  • any cell can be used.
  • the expression of the protein of the present invention or the protein mRNA described in [6] (1) is preferred. Recognized cell lines can be used.
  • test sample those mentioned in the test sample in the above [5] can be used.
  • the repo overnight gene is ligated to the downstream of the transcription control region by an ordinary method, and the prepared cells are used to transform host cells by an ordinary method.
  • a gene targeting vector by linking a marker for positive selection (such as the G418 resistance gene) and a marker for negative selection (such as the gene for thymidine kinase and zediphteria toxin A fragment of simple virus virus).
  • a marker for positive selection such as the G418 resistance gene
  • a marker for negative selection such as the gene for thymidine kinase and zediphteria toxin A fragment of simple virus virus.
  • the transformant is suspended, for example, in a medium in which the cells can grow, a test sample is added to the medium, and the cells are brought into contact with each other, and then the protein encoded by the reporter gene expressed in the cells is removed.
  • the amount is detected or quantified by a method appropriate for the protein.
  • the DNA of the present invention is obtained by using the DNA or the oligonucleotide of the present invention as a probe and extracting RNA from human tissue ⁇ human-derived cells in the same manner as in [1] (1) using Northern. By performing the hybridization, mRNA of the protein gene of the present invention in the tissue or cell can be detected or quantified. By comparing the mRNA expression levels in various tissues, the tissue expression distribution of the protein of the present invention can be known.
  • the oligonucleotide of the present invention was used as a specific primer for the DNA of the present invention to detect RNA extracted from human tissue ⁇ human-derived cells in the same manner as in [1] (1).
  • PCR reverse transcription PCR; PCR Protocols (1990)]
  • the method for quantifying the mRNA can be used for diagnosis of pathological conditions involving the present gene, prediction of the effects of cytotoxic nucleoside derivatives (antitumor agents, antiviral agents), and the like.
  • the importance of the gene product in the disease condition can be clarified. Further, the drug can be evaluated by comparing the expression amount of the mRNA depending on the presence or absence of the drug.
  • the DNA or oligonucleotide of the present invention is used as a probe
  • finer expression distribution such as identification of cells expressing the protein of the present invention in tissues can be achieved. You can know.
  • the oligonucleotide of the present invention can be used as a specific primer for the DNA of the present invention to perform PCR reaction on genomic DNA of human tissue / human-derived cells. Mutations, deletions, amplifications, and polymorphisms in the gene encoding the protein can be detected.
  • Detection of mutations, deletions, amplifications, and polymorphisms may result in mutations, deletions, amplifications, and polymorphisms in the gene, which may be a cause or risk factor, such as malignant tumors and neurodegenerative diseases It is possible to diagnose diseases caused by increased or decreased protein degradation, such as autoimmune diseases and inflammatory diseases.
  • RNA / DNA antisense 'oligonucleotide (RNA / DNA) of the present invention to suppress the transcription of the gene encoding the protein of the present invention or the translation of mRNA, [Chemistry, 46, 681 (1991), Bio / Technology, 9, 358 (1992)], where the gene may be involved in the pathogenesis, such as malignant tumors, neurodegenerative diseases, autoimmune diseases, inflammatory diseases, etc. It can be used for prevention and treatment of diseases caused by.
  • the antisense 'oligonucleotide described above encodes a protein of the present invention.
  • the medicament containing the DNA of the present invention can be prepared by the same method as the above-mentioned [5] for preparing the pharmaceutical preparation of the agonist or angonist of the protein of the present invention.
  • the prepared pharmaceutical preparation can be administered in the same manner as in the above [5].
  • the medicine containing the protein of the present invention can be prepared and prepared by using the same method as the above-mentioned [5] for preparing the pharmaceutical preparation of the agonist or angonisto of the protein of the present invention.
  • the pharmaceutical preparation can be administered in the same manner as in the above [5].
  • the drug containing the antibody of the present invention can be prepared by the same method as the above-mentioned [5], which is the same as the method for preparing the agonist of the protein of the present invention or the pharmaceutical preparation of an gonist, and the prepared drug is used.
  • the preparation can be administered in the same manner as in [5] above.
  • the DNA or oligonucleotide of the present invention may be incorporated as a single-stranded or double-stranded DNA vector such as retrovirus, adenovirus, adeno-associated virus, and other vectors into a vector for gene therapy to produce a gene therapy vector. Can be used for treatment.
  • the protein of the present invention can be immunologically detected or quantified using an antibody against the protein of the present invention.
  • Specific examples include the ELISA method using a micro-tie plate, immunohistochemical staining using an enzyme-labeled antibody method or a fluorescent antibody method, and a detection method using a Western blot method.
  • the epitope is different among the antibodies that react with the protein of the present invention in the liquid phase.
  • Two monoclonal antibodies Sanditsuchi EL I SA method using, be given such radioimmunoprecipitation Adzu Si method are use antibodies that recognize proteins and protein of the present invention of the present invention labeled with a radioisotope such as 125 1 comprising Can be.
  • the antibody of the present invention can also be used for immunohistological staining using a pathological tissue section.
  • the protein of the present invention present in cells or tissues of healthy subjects and subjects is immunologically detected or quantified, and the amount is compared between healthy subjects and subjects.
  • the antibody of the present invention can be used as a diagnostic agent for a disease state of a subject caused by an increase or decrease in protein degradation such as a malignant tumor, a neurodegenerative disease, an autoimmune disease, an inflammatory disease and the like.
  • the antibody of the present invention can be used as an immunohistochemical stain.
  • FIG. 1 is a diagram simply showing the structure of plasmid phMD6.
  • Figure 2 shows the amino acid sequence of the protein encoded by the plasmid phMD6, and the known F-WD proteins mouse MD6 (mMD6), human — TRCP (hbTRCP), Saccharomyces' Celepiche CDC4 (scCDC4), It is a figure which compared the amino acid sequence of Shizosaccharomyces. Bomb pop 1+ (sppopl). An asterisk indicates an amino acid residue that is identical in all sequences (amino acid residues are indicated by one letter). Above the sequence, the positions of the F box (denoted as F-box) and the WD domain (denoted as WD-1 to WD-7) are shown.
  • mMD6 mouse MD6
  • hbTRCP human — TRCP
  • scCDC4 Saccharomyces' Celepiche CDC4
  • Figure 3 shows the amino acid sequence of the protein encoded by the plasmid phMD6, and the known F-WD proteins mouse MD6 (mMD6), human ⁇ TRCP (hbTRCP), and Saccharomyces' Celepiche CDC4 (scCDC4 ) And a comparison of the amino acid sequences of Schizosaccharomyces ′ s bomb pop 1+ (sppopl) (continuation of FIG. 2).
  • FIG. 4 shows primers corresponding to the sequence contained in hMD6 (SEQ ID NO: 5), using RNAs from four human cancer cell lines (Jurkat, HeLa, A431, Saos-2) as type III.
  • FIG. 9 shows the results of RT-PCR experiments performed using the 5′-end DNA primer used and the 3′-end DNA primer shown in SEQ ID NO: 4.
  • the genetic engineering method was performed by the method described in the known Molecular Cloning 2nd edition.
  • SEQ ID NOS: 3 and 5 from human EST sequence (Genbank, ACCESSION AA252 600, AA452542) having homology to Schizosaccharomyces bombi pop 1+ gene [Genes & Development, 11, 1548-1560 (1997)]
  • a 5′-end DNA primer consisting of a base sequence to be used was designed and chemically synthesized.
  • a 3′-end DNA primer consisting of the nucleotide sequence shown in SEQ ID NO: 4 was designed and chemically synthesized. Chemical synthesis of DNA was carried out by a conventional solid phase synthesis method using a DNA synthesizer Model 392 manufactured by Applied Biosystems.
  • Total RNA was prepared from human epidermoid carcinoma-derived cell line A431 (ATCC CRL-1555) using a Total RNA Separator Kit (Clontech) according to the attached protocol. From 5 ⁇ g of total RNA, SuperscriptTM Preamplification System (GIBC0 BRL Using the oligo (dT) primer according to the attached protocol to prepare cDNA.
  • GIBC0 BRL SuperscriptTM Preamplification System
  • the reaction solution (21 ⁇ 1) was diluted 50-fold, and a PCR reaction was performed using 101 of the diluted solution.
  • primers the 5′-end DNA primer shown in SEQ ID NO: 3 and the 3 ′ end DNA primer shown in SEQ ID NO: 4, 10 ⁇ l of ⁇ type cDNA, each of the two types of primers were used. 25 ⁇ M, 400 ⁇ M each component dNTP (dATP, dGTP, dCTP, dTTP) mixture, 2.5 mM magnesium hydrochloride, TaKaRa LA Taq (Takara Shuzo) 2.5 units and 1 x La Taq buffer (PCR was performed under the following conditions using a reaction solution containing magnesium (free) 40 ⁇ 1.
  • a DNA Thermal Cycler 480 Perkin Elmer Cetus
  • a process consisting of 94 ° C for 30 seconds, 55 ° C for 60 seconds, and 72 ° C for 120 seconds is defined as one cycle. 25 cycles were performed and heating was further performed at 72 ° C for 7 minutes.
  • the PCR solution thus obtained was collected in a 5 1-minute fraction and subjected to agarose electrophoresis. As a result, it was confirmed that an expected DNA fragment of about 1.5 kb was amplified. After confirmation, the PCR reaction solution was subjected to agarose electrophoresis, and the DNA fragment of about 1.5 kb was purified using a Gene Clean III Kit (BI0101) according to the attached protocol.
  • the recombinant plasmid DNA obtained by the reaction was used to transform Escherichia coli DH5 strain (Molecular Cloning, 2nd ed., A.10), and the method described in Molecular cloning 2nd ed.
  • plasmid phMD6 was obtained.
  • the nucleotide sequence of the cloned DNA fragment hMD6 was determined as follows. Using phMD6 as type I, a sample for nucleotide sequence determination was prepared using Big Dye Primer Cycle Sequencing Kit (Applied Biosystems) according to the attached protocol, and ABI PRISif M 377 DNA sequencer (Applied Biosystems).
  • PhMD6 contains the cDNA described in SEQ ID NO: 2, and has an open reading frame (hereinafter abbreviated as ORF) of 1362 base pairs.
  • ORF open reading frame
  • hMD6 a protein consisting of 454 amino acid residues shown in SEQ ID NO: 1 (hereinafter abbreviated as hMD6) was encoded.
  • the F-box protein has a region consisting of amino acids of 40 to 50 residues called an F-box near the amino terminal of the protein.
  • the F-WD protein is It has a sequence called the WD domain near the carboxy terminus, which usually has tributophan-aspartic acid (WD) as its last amino acid residue and from about 20 to 40 residues.
  • the amino acid sequence has a structure having a sequence repeated 4 to 8 times.
  • the amino acid sequence was compared with a known F-WD protein using an analysis program [Gap included in the Wisconsin Package (Genetics Computer Group, USA)]. Accession X54352) has a high homology with the protein (mMD 6), and shows 97% homology (Similarity) between hMD 6 and mMD 6. In addition, when the total length of the translation regions of the hMD6 gene and the mMD6 gene was compared using the above-mentioned analysis program, the nucleotide sequences showed 95% homology. Due to this extremely high homology, hMD6 is considered to be the ortholog of mMD6.
  • JP 1 JP 1
  • the F box is considered to correspond to the amino acid sequence consisting of 43 residues shown in SEQ ID NO: 6 (amino acid sequence at positions 59 to 101 in SEQ ID NO: 1).
  • the WD domain has an amino acid sequence consisting of 29 residues represented by SEQ ID NO: 7 (WD-1: the amino acid sequence at positions 146 to 174 of SEQ ID NO: 1), an amino acid consisting of 27 residues represented by SEQ ID NO: 8 Sequence (WD-2: amino acid sequence at positions 186 to 212 of SEQ ID NO: 1), an amino acid sequence consisting of 31 residues represented by SEQ ID NO: 9 (WD-3: amino acid sequence at positions 224 to 254 of SEQ ID NO: 1) ), An amino acid sequence consisting of 30 residues represented by SEQ ID NO: 10 (WD-4: amino acid sequence at positions 266 to 295 of SEQ ID NO: 1); an amino acid sequence consisting of 29 residues represented by SEQ ID NO: 11 (WD- 5: Amino acid sequence consisting of 6 residues shown in SEQ ID NO: 12 (amino acid sequence at positions 307 to 335 of SEQ ID NO: 1) (WD-6: amino acid sequence of positions 346 to 351 in SEQ ID NO: 1), SEQ
  • the F-box shows high homology (43% for human 5-TRCP, 35% for Schizosaccharomyces .bomb 1) and 47% for Saccharomyces' Celepiche CD C4.
  • the WD domain showed high homology of 34% with human TRCP, 28% with Schizosaccharomyces 'bomb pop 1+, and 33% with Saccharomyces' Celepiche CDC4.
  • FIGS. 2 and 3 show a comparison of the sequences of hMD6 and other F-WD proteins.
  • the ORF encoded by the hMD6 gene was It is thought to encode a novel F-WD protein that is a submittal of zE3.
  • RNA Human T-cell leukemia-derived cell line Jurkat (ATCC TIB-152), human uterine cancer-derived cell line HeLa (ATCC CCL-2), human epidermoid carcinoma-derived cell line A431 (ATCC CRL-1555), human osteosarcoma
  • RNA was prepared from the derived cell line Saos-2 (ATCC HTB-85) using a Total RNA Separator Kit (Clontech) according to the attached protocol. From 5 g of each RNA, reverse transcription reaction was performed with oligo (dT) primer using Superscript TM Preamplification System (GIBC0 BRL) according to the attached protocol to prepare cDNA.
  • the reaction solution (21 ⁇ 1) was diluted 50-fold, and a PCR reaction was performed using 101 of the diluted solution.
  • primers a 5′-end DNA primer shown in SEQ ID NO: 5 and a 3′-end DNA primer shown in SEQ ID NO: 4 were used.
  • Type II cDNA 10 ⁇ 1 each of the two primers 0.5 ⁇ M each, each component 400 ⁇ M dNTP (dATP, dGTP, dCTP, dTTP) mixture, 2.5 mM magnesium chloride, TaKaRa LA Taq (PCR was performed under the following conditions using a reaction solution 201 containing 2.5 units and 1 ⁇ La Taq buffer (magnesium-free).
  • FIG. 4 shows the results obtained by collecting 10 ⁇ 1 of the obtained PCR solution and subjecting it to agarose electrophoresis. It was confirmed that in all of the four types of PCR solutions derived from human cancer cells, a DNA fragment having a size of about 1.5 kb predicted from the nucleotide sequence encoding hMD6 was amplified. From the above results, it was shown that cDNA encoding hMD6 was expressed in these four types of cancer cells.
  • M579079 and AI066748 which are ESTs containing a part of the hMD6 gene, using the UniGene server of the American National Center for Biotechnology Information (NCBI), they were assembled to the Hs. 13755 UniGene clone.
  • the clone contained SHGC-15152, an STS (Sequence-tagged site) already mapped on the human chromosome.
  • the STS was located between markers D9S258 and D9S1821 on human chromosome 9. This position corresponds to the 31-32 region (9q31-32) of the long arm of chromosome 9.
  • This area includes esophageal cancer, basal cell carcinoma, non-Hodgkin's lymphoma, (Japanese clinical,, 986-991 (1996), Cancer Res., 56, 1629-1634 (1996), Cancer Lett, 79, 67-72 (1994), Genes Chromosomes Cancer, 12, 32-36 (1995), etc., where deletions are seen, suggesting the presence of a tumor suppressor gene, and t (7; 9) It is also near the breakpoint of the chromosomal locus, as well as the genes responsible for Tangier disease (Tangier disease), which causes low HDL (high density lipoprotein), a risk factor for ischemic heart disease, and familial autonomic dysfunction.
  • the causative gene has been mapped to this region [Nature Genetics, 4, 160-
  • the present invention provides a novel F-WD protein, a DNA encoding the protein, a recombinant vector containing the DNA, and a transformant containing the recombinant vector.
  • a protein obtained by the present invention, a DNA encoding the protein, an antibody recognizing the protein, the protein, and an oligonucleotide comprising a partial sequence of the DNA may be a neurodegenerative disease, a malignant tumor, an autoimmune disease, an inflammatory disease, It is effectively used for the prevention, treatment, diagnosis, etc. of diseases caused by enhanced or reduced protein degradation such as viral diseases.
  • SEQ ID NO: 3 Description of Artificial Sequence: Synthetic DNA
  • SEQ ID NO: 4 Description of Artificial Sequence: Synthetic DNA
  • SEQ ID NO: 5 Description of Artificial Sequence: Synthetic DNA

Abstract

A novel F-WD protein which is useful in, for example, treating, preventing or diagnosing diseases induced by the acceleration or depression of protein decomposition; a DNA encoding this protein; an antibody recognizing this protein; and an oligonucleotide comprising a part of the sequence of the DNA. An F-WD protein having the amino acid sequence represented by SEQ ID NO:1; a process for producing this protein; a DNA encoding this protein; an oligonucleotide comprising a part of the sequence of this DNA; a recombinant vector containing this DNA; a transformant carrying this recombinant vector; an antibody recognizing the above protein; a method for quantitating the protein and an immunostaining method with the use of this antibody; a method for screening a substance capable of changing the expression of a gene encoding the protein; a method for screening a substance capable of changing the activity of the protein; and preventives, remedies and diagnostics for diseases induced by the acceleration or depression of protein decomposition.

Description

明 細  Details
新規蛋白質 技術分野  New protein technology
本発明は、 新規 F— WD蛋白質、該蛋白質をコードする DNA、 該 DNAを含 むベクター、 該ベクタ一で形質転換された形質転換体および該 F—WD蛋白質 の製造方法、 該蛋白質を認識する抗体、 該 DNAの一部配列からなるオリゴヌ クレオチド、 またはこれらの利用方法に関する。  The present invention relates to a novel F-WD protein, a DNA encoding the protein, a vector containing the DNA, a transformant transformed with the vector, a method for producing the F-WD protein, and recognition of the protein. The present invention relates to an antibody, an oligonucleotide comprising a partial sequence of the DNA, or a method for using these.
本発明は、 蛋白質分解の亢進または低下により引き起こされる疾患の予防、 治療、 診断、 病理解析等に有用である。  INDUSTRIAL APPLICABILITY The present invention is useful for prevention, treatment, diagnosis, pathological analysis, and the like of diseases caused by enhanced or decreased protein degradation.
背景技術 Background art
真核細胞における種々の生命現象の様々な局面において、 蛋白質分解は極め て重要な役割を担っている。 特に、 真核細胞のエネルギー依存的蛋白質分解シ ステムであるュビキチン一プロテアソ一ム系による選択的な蛋白質分解は、 細 胞周期、 アポト一シス、 転写調節、 代謝調節、 情報伝達、 ス トレス応答、 免疫 始動、 などの多くの重要な生命現象に関与していることが、 近年、 明らかとな つてきている [細胞工学、 15, 888-896 (1996)、 Trends in Biological Science, 21, 96-102 (1996)、 Cell, 92, 367-380 (1998)]。  In various aspects of various life phenomena in eukaryotic cells, proteolysis plays a very important role. In particular, selective proteolysis by the eukaryotic energy-dependent proteolysis system, the ubiquitin-proteasome system, involves cell cycle, apoptosis, transcriptional regulation, metabolic regulation, signal transduction, stress response, In recent years, it has been clarified that it is involved in many important life phenomena such as immune initiation [Cell Engineering, 15, 888-896 (1996), Trends in Biological Science, 21, 96- 102 (1996), Cell, 92, 367-380 (1998)].
このュビキチン一プロテアソ一ム系による蛋白質分解機構は、 種々の病態に も関連することが明らかになってきている。 例えば、 DNA型癌ウィルスであ るヒトパピローマウィルスの E 6や癌遺伝子産物 MDM 2はこの経路を亢進し、 癌抑制遺伝子産物 P 53の分解を促進する。 さらに、 最近、 サイクリン依存性 キナーゼインヒビ夕一である P 27K i p 1のュビキチン一プロテアソーム系 による分解の亢進と、 乳癌、 直腸癌などにおける悪性度とが相関していること が報告されている。 さらに、 TNF—ひなどの炎症性サイ ト力インの転写因子 NF cBの活性を制御する蛋白質 I Bもまた、 この経路で分解することが知 られている。 従って、 このュビキチン一プロテアソ一ム系を制御することは種 々の疾患の治療、 予防、 診断に有効であると考えられる。 実際、 プロテアソ一 ム阻害剤のラク夕シスチンや M G 1 3 2が、 癌細胞の増殖抑制、 アポトーシス 促進、 抗炎症作用、 神経突起誘導などの作用を有することが報告されている [Cancer Research, 56, 2679- 2654 ( 1996 )、 Nature Medicine, 3, 222-225 ( 1997) 、 Nature Medicine, 3, 227-230 ( 1997)、 Genes & Development, 9, 1586-1597( 1995) 、 細胞工学， 15, 929-939( 1996)]。 It is becoming clear that the mechanism of proteolysis by the ubiquitin-proteasome system is related to various disease states. For example, E6 of human papilloma virus, a DNA-type oncovirus, and the oncogene product MDM2 enhance this pathway and promote the degradation of the tumor suppressor gene product P53. Furthermore, it has recently been reported that the enhanced degradation of the cyclin-dependent kinase inhibitor P27Kip1 by the ubiquitin-proteasome system is correlated with the degree of malignancy in breast cancer, rectal cancer and the like. In addition, TNF—protein IB, which regulates the activity of the transcription factor NF cB of inflammatory site-powered proteins such as spleen, is also known to be degraded by this pathway. Have been. Therefore, controlling this ubiquitin-proteasome system is considered to be effective for the treatment, prevention and diagnosis of various diseases. In fact, it has been reported that the proteasome inhibitors lactate cystine and MG132 have effects such as suppression of cancer cell growth, promotion of apoptosis, anti-inflammatory activity, and neurite induction [Cancer Research, 56 , 2679-2654 (1996), Nature Medicine, 3, 222-225 (1997), Nature Medicine, 3, 227-230 (1997), Genes & Development, 9, 1586-1597 (1995), Cell Engineering, 15, 929-939 (1996)].
ュビキチン一プロテアソ一ム系は、 標的蛋白質のリシン残基のど-アミノ基に ュビキチン蛋白質をィソぺプチド結合で共有結合させることで分解経路に入る 標識とする 「ュビキチン化」 の第一段階と、 ュビキチン化された標的蛋白質が、 プロテアソ一ム複合体によって認識され分解される第二段階からなる。 特定の 時期に特定の蛋白質を分解するためには、 第一段階における分子認識の特異性 が特に重要である [Cell, 84, 813-815( 1996)]。  The ubiquitin-proteasome system is the first step of `` ubiquitination, '' in which the ubiquitin protein is covalently bonded to the lysine residue of the target protein via an isopeptide bond to enter the degradation pathway. The ubiquitinated target protein consists of a second step in which it is recognized and degraded by the proteasome complex. The specificity of molecular recognition in the first step is particularly important for degrading specific proteins at specific times [Cell, 84, 813-815 (1996)].
蛋白質のュビキチン化は、 次のプロセスからなる。 まず、 ュビキチン活性化 酵素（以下、 E 1ともいう）と A T Pによりュビキチンのカルボキシ末端のグリ シン残基が活性化され、 E 1の特定のシスティン残基にチォエステル結合する。 次に、 この活性化されたュビキチンはュビキチン結合酵素（以下、 E 2ともいう )の特定のシスティン残基に転移し、 チォエステル結合する。 E 2と結合したュ ビキチンは、 標的蛋白質の特定のリシン残基に転移し、 イソペプチド結合する ことにより標的蛋白質がュビキチン化される。 標的蛋白質のュビキチン化は、 E 2から直接起こる場合もあるが、 多くの場合、 ュビキチンリガ一ゼ（以下、 E 3ともいう） の存在が必要である [細胞工学、！^，898-904 ( 1996)、 Trends in Cell Biology, 5, 428-434 ( 1995) ]。  Ubiquitination of proteins consists of the following processes. First, the carboxy-terminal glycine residue of ubiquitin is activated by ubiquitin activating enzyme (hereinafter also referred to as E1) and ATP, and the ubiquitin is thioester bonded to a specific cysteine residue of E1. Next, the activated ubiquitin is transferred to a specific cysteine residue of ubiquitin-conjugating enzyme (hereinafter, also referred to as E2), and forms a thioester bond. The ubiquitin bound to E2 is transferred to a specific lysine residue of the target protein, and the target protein is ubiquitinated by isopeptide bond. Ubiquitination of the target protein may occur directly from E2, but in many cases requires the presence of ubiquitin ligase (hereinafter also referred to as E3) [Cell Engineering! ^, 898-904 (1996), Trends in Cell Biology, 5, 428-434 (1995)].
E 3は、 極めて分子多様性に富み、 また単一の蛋白質からなるもの、 蛋白質 複合体を形成してなるもの等が知られており、 ュビキチン化の基質特異性を決 定していると考えられている。 E 3は、 そのサブユニット構造および一次配列 の類似性から、 ヘクトドメイン （hect- domein) を有するグループ、 AP C ( anaphase promoting complex) と呼ばれる蛋白質複合体からなるグループ、 S CF (Skpl-Cdc53/Cullin-F-box) 蛋白質複合体からなるグループ等、 いくつ かのグループに分けられる。 E3 is extremely rich in molecular diversity, and is known to consist of a single protein or to form a protein complex, and is thought to determine the substrate specificity of ubiquitination. Have been. E 3 is its subunit structure and primary sequence Based on the similarity, the group consisting of a hect-domain (hect-domein), the group consisting of a protein complex called APC (anaphase promoting complex), and the group consisting of SCF (Skpl-Cdc53 / Cullin-F-box) protein complex There are several groups, such as groups.
SCFは、 最近見出されたグループであり、 特に細胞周期に関係する蛋白質 の分解に関与する E 3として注目されている。 出芽酵母であるサヅカロマイセ ス 'セレピシェ （S. cerevisiae) の G 1サイクリン （C ln l、 C ln2) 、 サイクリン依存性キナ一ゼインヒビ夕一 （CDK I ) (S i c l、 Far 1) 等は、 いずれも特定の SCF蛋白質複合体によってュビキチン化され、 分解さ れることが報告されている [細胞工学、 ， 907-916 (1996)、 Cell, 91, 149- 151(1997)、 Proc. Natl. Acad. Sci. USA, 92, 2563-2567 (1995)、 Cell, 81, 279-288 (1995)]。  SCF is a recently discovered group that has received particular attention as E3, which is involved in the degradation of cell cycle-related proteins. The budding yeast S. cerevisiae S. cerevisiae G 1 cyclin (C lnl, C ln2) and the cyclin-dependent quinase zeinhibi Yuichi (CDK I) (S icl, Far 1) It has been reported that it is ubiquitinated and degraded by a specific SCF protein complex [Cell Engineering, 907-916 (1996), Cell, 91, 149-151 (1997), Proc. Natl. Acad. Sci. USA, 92, 2563-2567 (1995), Cell, 81, 279-288 (1995)].
S C F蛋白質複合体を構成する蛋白質群の中で、 例えば出芽酵母では S k p 1蛋白質および Cd c 53蛋白質がそれぞれ 1種類しか知られていないのに対 し、 対応する Fボックス （F-box) 蛋白質としては、 Cd c 4蛋白質、 Grr l 蛋白質、 Me t 30蛋白質等、 多数存在することが知られている。 この多様な Fボックス蛋白質が、 S C F蛋白質複合体の基質特異性を決定していると考え られている。 Fボックス蛋白質は、 該蛋白質のァミノ末端近くに Fボックスと 呼ばれる 40〜50残基のアミノ酸からなる領域を有し、 SCF蛋白質複合体 を構成する場合には、 この部分で Skp 1蛋白質と結合することができる。 こ のように、 Fボックス蛋白質は基質特異的なアダプタ一としての機能を有する ことが知られている [Trends in Genetics, 14, 236-243 (1998)] 。 一方、 多 くの Fボックス蛋白質は、 該蛋白質のカルボキシ末端近くに WDドメインまた はロイシンリッチリピートと呼ばれる配列を有し、 この領域で特異的な基質蛋 白質と相互作用すると考えられている。  Among the proteins constituting the SCF protein complex, for example, in S. cerevisiae, only one Skp1 protein and one Cdc53 protein are known, respectively, while the corresponding F-box (F-box) protein It is known that there are many such as Cdc4 protein, Grl protein, and Met30 protein. These various F-box proteins are thought to determine the substrate specificity of the SCF protein complex. The F box protein has a region called F box consisting of 40 to 50 amino acids near the amino terminus of the protein, and when forming an SCF protein complex, binds to the Skp1 protein at this portion. be able to. As described above, it is known that the F box protein has a function as a substrate-specific adapter [Trends in Genetics, 14, 236-243 (1998)]. On the other hand, many F-box proteins have a sequence called a WD domain or leucine-rich repeat near the carboxy terminus of the protein, and it is thought that this region interacts with a specific substrate protein.
Fボックス蛋白質のうち、 カルボキシ末端近くに WDドメインを有するグル ープは、 F— WD蛋白質と呼ばれる。 Among F-box proteins, those with a WD domain near the carboxy terminus The loop is called F-WD protein.
WDドメインは、 通常はトリブトファンーァスパラギン酸 （WD) をその最 後のアミノ酸残基としてもち、 かつ約 20〜 40残基からなるアミノ酸配列が 4〜 8回反復する配列を有し、 WD40リピート、 GH (グリシン一ヒスチジ ン） 一WDリピート等とも呼ばれる。 また、 WDドメインは多くの蛋白質に存 在することが知られている [Nature, 371, 297-300 (1994)]。 さらに、 3量体 G蛋白質の/?—サブュニットにおける WDドメインは、 7個の WDがプロペラ 状の環を構成する構造をとることが知られている [Nature, 379, 3U- 319 & 369-374 (1996)] 。  The WD domain usually has tributophan-aspartic acid (WD) as its last amino acid residue, and has a sequence in which an amino acid sequence consisting of about 20 to 40 residues is repeated 4 to 8 times, Also called WD40 repeat, GH (glycine-histidine) -WD repeat, etc. It is known that the WD domain exists in many proteins [Nature, 371, 297-300 (1994)]. Furthermore, it is known that the WD domain in the trimeric G protein /? — Subunit has a structure in which seven WDs form a propeller-like ring [Nature, 379, 3U-319 & 369-374] (1996)].
F— WD蛋白質をコ一ドする遺伝子としては、 サッカロマイセス ·セレビシ ェの CDKI (Si c l、 F a r 1 ) の E 3の F— WD蛋白質をコードする遺 伝子 （Cdc4)、 分裂酵母であるシゾサッカロマイセス 'ボンべ （ . pombe ) の CDKI (r uml +) の E 3の F—WD蛋白質をコードする遺伝子 （p o 1+)、 出芽酵母の細胞周期に関与するプロテインキナーゼ Swe 1の E 3 の F— WD蛋白質をコ一ドする遺伝子 （Me t 30)、 線虫 (Caenorhabditis elegans)の N o t c h/L i n 12の分解に関与する F—WD蛋白質をコード する遺伝子 （S e 1— 10) 、 ヒト免疫不全ウィルス H I V— 1の Vpu蛋白 質に結合して CD 4蛋白質の分解に関与する F— WD蛋白質をコ一ドする遺伝 子 （ヒト^ー TRCP)、 ショウジヨウバエの — c at en inの 分解に関与する F— WD蛋白質をコードする遺伝子 （S 1 imb)等が、 それ それクローニングされている。 これらの遺伝子は、 いずれも細胞周期やシグナ ル伝達に関与する重要な蛋白質の分解に関与していることが知られている。 また、 これらの分子が、 分裂酵母、 出芽酵母、 線虫、 ショウジヨウバエ、 ァ フリカヅメガエル、 マウス、 ヒ卜まで種を超えて見出されることから、 F— W D蛋白質は、 真核生物の SCFタイプの E 3のサブュニットとして普遍的に存 在し、さらに多くの種類があることが予測されている [Cell, 86， 263-274 (1996) 、 Cell, 91， 221-230 (1997)、 Genes & Development, 11, 1548-1560 (1997)、 Genes & Development, 11, 3182-3193 (1997)、 Nature, 391, 493-496(1998)、 Genes & Development, 12, 2587 - 2597 (1998)、 Molecular Cell, 1, 565-574 (1998) 、 Genes & Development, 11,3046-3060 (1997)、 Cell, 91, 209-218 (1997)]。 したがって、 ヒトの新規 F— WD蛋白質の塩基配列、 アミノ酸配列を解明す ることにより該蛋白質の DNA検出法、 免疫測定法等を確立し、 疾患に起因す る F—WD蛋白質の変化を検出することは、 発癌機構との関係で臨床上重要な 問題であるとされている （実験医学増刊、 細胞周期と癌、 田矢洋一ら編、 10 8— 1 15頁、 1999年羊土社刊） 。 Genes encoding the F-WD protein include a gene (Cdc4) encoding the F3-WD protein of E3 of CDKI (Sicl, Far1) of Saccharomyces cerevisiae, and a fission yeast gene. The gene (po 1+) encoding the E3 F—WD protein of CDKI (ruml +) of Zosaccharomyces' bomb (. Pombe), a protein kinase Swe1 E3 involved in the cell cycle of budding yeast A gene encoding F-WD protein (Me t 30), a gene encoding F-WD protein involved in the degradation of N.ch./L in 12 of Caenorhabditis elegans (Se 1-10), A gene (human ^ -TRCP) that binds to the Vpu protein of human immunodeficiency virus HIV-1 and encodes the F-WD protein involved in the degradation of the CD4 protein. The gene (S1 imb) encoding the F-WD protein involved in the degradation of . All of these genes are known to be involved in degradation of important proteins involved in cell cycle and signal transmission. In addition, since these molecules are found across species from fission yeast, budding yeast, nematodes, Drosophila, African measles, mice, and humans, F-WD proteins are eukaryotic SCF-type proteins. It is ubiquitous as a subunit of E3, and is expected to have many more types [Cell, 86, 263-274 (1996) Cell, 91, 221-230 (1997), Genes & Development, 11, 1548-1560 (1997), Genes & Development, 11, 3182-3193 (1997), Nature, 391, 493-496 (1998), Genes & Development, 12, 2587-2597 (1998), Molecular Cell, 1, 565-574 (1998), Genes & Development, 11,3046-3060 (1997), Cell, 91, 209-218 (1997)]. Therefore, by elucidating the nucleotide sequence and amino acid sequence of a novel human F-WD protein, a DNA detection method, an immunoassay method, and the like for the protein are established, and changes in the F-WD protein caused by disease are detected. This is considered to be a clinically important problem in relation to the carcinogenesis mechanism (Experimental medicine extra edition, Cell cycle and cancer, Yoichi Taya et al., Pp. 108-115, 1999, published by Youdosha, 1999).
発明の開示 Disclosure of the invention
本発明は、 新規 F— WD蛋白質、 該 F— WD蛋白質をコードする DNA、 お よび該蛋白質を認識する抗体を利用し、 アルツハイマー病、 パーキンソン病等 の神経変性疾患、 白血病、 肺癌、 結腸癌、 乳癌等の悪性腫瘍、 慢性間接リウマ チ等の自己免疫疾患、 喘息等の炎症性疾患、 後天性免疫不全症候群（AIDS) 等のウィルス性疾患等、 蛋白質分解の亢進または低下により引き起こされる疾 患の予防薬、 治療薬、 診断薬等を提供することを目的とする。  The present invention uses a novel F-WD protein, a DNA encoding the F-WD protein, and an antibody recognizing the protein to produce neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, leukemia, lung cancer, colon cancer, Diseases caused by increased or decreased protein degradation, such as malignant tumors such as breast cancer, autoimmune diseases such as chronic indirect rheumatism, inflammatory diseases such as asthma, and viral diseases such as acquired immunodeficiency syndrome (AIDS). It aims to provide prophylactic, therapeutic and diagnostic agents.
本発明者らは、 分裂酵母 P o p 1 +の遺伝子配列情報を基に、 ランダムなヒト cDNA配列の遺伝子配列データベース Genbankに登録されている E S T (Expressed Sequence Tag)に関して、 BLAST2.0 [Nucleic Acids Research, 25, 3389-3402 (1997)]およびフレームサーチ [イスラエル、 コンピュジェン （ Compugen)社]相同性検索ソフトウェアを用レ、解析し、 pop l+の Fボックス 領域と相同性の認められるヒト cDNAの部分配列 (AA252600、 AA452542, AA579079, M536071)を見出した。 これらの E S T配列を用いて新規な F— WD 蛋白質の cDN Aを取得して塩基配列を解析し、 本発明を完成するに至った。 即ち、 本発明は以下の （1) 〜 （49) の発明に関する。  The present inventors, based on the gene sequence information of fission yeast Pop1 +, based on EST (Expressed Sequence Tag) registered in the gene sequence database Genbank of random human cDNA sequence, BLAST2.0 [Nucleic Acids Research , 25, 3389-3402 (1997)] and frame search [Compugen, Israel] Using homology search software, analysis and analysis of a portion of the human cDNA with homology to the F box region of popl + Sequences (AA252600, AA452542, AA579079, M536071) were found. Using these EST sequences, cDNA of a novel F-WD protein was obtained and its nucleotide sequence was analyzed, thereby completing the present invention. That is, the present invention relates to the following inventions (1) to (49).
(1)配列番号 1に示されるアミノ酸配列からなる蛋白質。 (2)配列番号 1に示されるァミノ酸配列において 1以上のァミノ酸が欠失、 置換または付加されたァミノ酸配列からなる F— WD蛋白質。 (1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1. (2) An F-WD protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
(3)配列番号 6で示されるアミノ酸配列を有してなる F—WD蛋白質。  (3) An F-WD protein having the amino acid sequence represented by SEQ ID NO: 6.
( 4 ) 配列番号 7〜 13で示されるァミノ酸配列の 1以上の配列を有してなる F— WD蛋白質。  (4) An F-WD protein having one or more sequences of the amino acid sequences shown in SEQ ID NOs: 7 to 13.
(5) ( 1) ~ (4) のいずれかに記載の蛋白質をコードする DNAまたは該 DNAと相補的な配列を有する DNA。  (5) A DNA encoding the protein according to any one of (1) to (4) or a DNA having a sequence complementary to the DNA.
( 6 )配列番号 2記載の塩基配列を有する D N Aまたは該 D N Aと相補的な配 列を有する DNA。  (6) DNA having the nucleotide sequence of SEQ ID NO: 2 or DNA having a sequence complementary to the DNA.
(7) (5) または （6) に記載の DNAとストリンジェントな条件下でハイ ブリダィズし、 かつ F— WD蛋白質をコードする DNAまたは該 DNAと相補 的な配列を有する DNA。  (7) A DNA that hybridizes with the DNA according to (5) or (6) under stringent conditions and encodes a F-WD protein or a DNA having a sequence complementary to the DNA.
(8) (5) 〜 （7) のいずれかに記載の DN Aを含有する組換えべクタ一。 (8) A recombinant vector containing the DNA according to any one of (5) to (7).
(9) プラスミ ド phMD 6である、 （8) に記載の組換えべクタ一。 (9) The recombinant vector according to (8), which is a plasmid phMD6.
(10) (8) または （9) に記載の組換えベクターを保有する形質転換体。 (10) A transformant having the recombinant vector according to (8) or (9).
(11)形質転換体が、 微生物、 動物細胞、 植物細胞および昆虫細胞から選ば れる形質転換体である、 （10) に記載の形質転換体。 (11) The transformant according to (10), wherein the transformant is a transformant selected from a microorganism, an animal cell, a plant cell, and an insect cell.
(12)微生物がェシエリヒア (Escherichia)属に属する微生物である、 （1 1 ) に記載の形質転換体。  (12) The transformant according to (11), wherein the microorganism is a microorganism belonging to the genus Escherichia.
( 13)ェシエリヒア属に属する微生物がェシエリヒア 'コリ (Escherichia coli ) DH 5 α/phMD 6 (FERM BP— 6611) である、 （12) に記載 の形質転換体。  (13) The transformant according to (12), wherein the microorganism belonging to the genus Escherichia is Escherichia coli DH5α / phMD6 (FERM BP-6611).
(14) (10) 〜 （13) のいずれかに記載の形質転換体を培地に培養し、 培養物中に （i) 〜 （4) のいずれかに記載の蛋白質を生成蓄積させ、 該培養 物から該蛋白質を採取することを特徴とする、 （1)〜 （4) のいずれかに記 載の蛋白質の製造方法。 (15) (5) 〜 （7) のいずれかに記載の DN Aの有する塩基配列中の連続 した 5〜60塩基と同じ配列を有するオリゴヌクレオチド、 該オリゴヌクレオ チドと相補的な配列を有するオリゴヌクレオチド、 およびこれらオリゴヌクレ ォチドの誘導体オリゴヌクレオチドから選ばれるオリゴヌクレオチド。 (14) The transformant according to any one of (10) to (13) is cultured in a medium, and the protein according to any one of (i) to (4) is produced and accumulated in the culture. The method for producing a protein according to any one of (1) to (4), wherein the protein is collected from a product. (15) an oligonucleotide having the same sequence as the contiguous 5 to 60 nucleotides in the nucleotide sequence of DNA according to any one of (5) to (7), and an oligonucleotide having a sequence complementary to the oligonucleotide; Oligonucleotides selected from nucleotides and derivative oligonucleotides of these oligonucleotides.
(16) (1) 〜 （4) のいずれかに記載の蛋白質を認識する抗体。  (16) An antibody that recognizes the protein according to any one of (1) to (4).
(17) (16)記載の抗体を用いることを特徴とする、 （1) 〜 （4) のい ずれかに記載の蛋白質の免疫学的検出法。  (17) The method for immunologically detecting a protein according to any one of (1) to (4), comprising using the antibody according to (16).
(18) (16) に記載の抗体を用いることを特徴とする、 （1) 〜 （4) の いずれかに記載の蛋白質の免疫組織染色法。  (18) An immunohistochemical staining method for the protein according to any one of (1) to (4), which comprises using the antibody according to (16).
(19) (16) に記載の抗体を含有する、 免疫組織染色剤。  (19) An immunohistological stain containing the antibody according to (16).
(20) (1) 〜 （4) のいずれかに記載の蛋白質と被験試料とを接触させ、 該蛋白質の有するュビキチンリガーゼ活性を変動させることを特徴とする化合 物のスクリーニング方法。  (20) A method for screening a compound, which comprises bringing the protein according to any one of (1) to (4) into contact with a test sample, and varying the ubiquitin ligase activity of the protein.
(21) (1) 〜 （4) のいずれかに記載の蛋白質を発現する細胞と被験試料 とを接触させ、 該蛋白質の有するュビキチンリガーゼ活性を変動させることを 特徴とする化合物のスクリ一ニング方法。  (21) Screening of a compound characterized by varying the ubiquitin ligase activity of a protein expressing the protein according to any one of (1) to (4) and a test sample by bringing the cell into contact with the test sample. Method.
(22) (20) または （21) に記載の方法により得られる化合物。  (22) A compound obtained by the method according to (20) or (21).
(23) (1) 〜 （4) のいずれかに記載の蛋白質と被験試料とを接触させ、 該蛋白質の有する蛋白質分解促進活性を変動させることを特徴とする化合物の スクリーニング方法。  (23) A method for screening a compound, which comprises bringing a protein according to any one of (1) to (4) into contact with a test sample, and varying a proteolysis promoting activity of the protein.
(24) (1)〜 （4) のいずれか 1項に記載の蛋白質を発現する細胞と被験 試料とを接触させ、 該蛋白質の有する蛋白質分解促進活性を変動させることを 特徴とする化合物のスクリーニング方法。  (24) Screening for a compound characterized in that the cell expressing the protein according to any one of (1) to (4) is brought into contact with a test sample, and the protein degradation promoting activity of the protein is varied. Method.
(25) (23) または （24) に記載の方法により得られる化合物。  (25) A compound obtained by the method according to (23) or (24).
(26) (1)〜 （4) のいずれかに記載の蛋白質を発現する細胞と被験試料 とを接触させることを特徴とする、 該蛋白質をコードする遺伝子の発現を変動 させる化合物のスクリーニング方法。 (26) varying expression of a gene encoding the protein according to any one of (1) to (4), which comprises contacting a cell expressing the protein according to any one of (1) to (4) with a test sample; Screening method for the compound to be used.
(27) (26) に記載の方法により得られる化合物。  (27) A compound obtained by the method according to (26).
(28) (5) 〜 （7) のいずれかに記載の DNAを用いることを特徴とする、 (28) Use of the DNA according to any one of (5) to (7),
(1) 〜 （4) のいずれかに記載の蛋白質をコードする mRNAを検出する方 法。 A method for detecting mRNA encoding the protein according to any one of (1) to (4).
(29) (15) に記載のオリゴヌクレオチドを用いることを特徴とする、 （ 1) 〜 （4) のいずれか 1項に記載の蛋白質をコードする mRNAを検出する 方法。  (29) A method for detecting an mRNA encoding the protein according to any one of (1) to (4), which comprises using the oligonucleotide according to (15).
(30) (5) 〜 （7) のいずれかに記載の DNAを用いることを特徴とする、 (30) Use of the DNA according to any one of (5) to (7),
(1) 〜 （4) のいずれかに記載の蛋白質の発現を抑制する方法。 A method for suppressing the expression of the protein according to any one of (1) to (4).
(31) (15) に記載のオリゴヌクレオチドを用いることを特徴とする、 （ (31) using the oligonucleotide according to (15),
1) 〜 （4) のいずれかに記載の蛋白質の発現を抑制する方法。 A method for suppressing the expression of a protein according to any one of 1) to (4).
(32) (5) 〜 （7)のいずれかに記載の DNAを用いることを特徴とする、 (1) 〜 （4) のいずれかに記載の蛋白質をコードする遺伝子のプロモー夕一 (32) A promoter of a gene encoding the protein according to any one of (1) to (4), wherein the DNA according to any one of (5) to (7) is used.
DN Aを取得する方法。 How to get a DNA.
(33) (15) に記載のオリゴヌクレオチドを用いることを特徴とする、 （ 1) 〜 （4) のいずれかに記載の蛋白質をコードする遺伝子のプロモーター D N Aを取得する方法。  (33) A method for obtaining a promoter DNA of a gene encoding the protein according to any one of (1) to (4), which comprises using the oligonucleotide according to (15).
(34) (1) 〜 （4) のいずれかに記載の蛋白質をコードする遺伝子の転写 を司るプロモーター DNA。  (34) A promoter DNA that controls transcription of the gene encoding the protein according to any one of (1) to (4).
(35) (34) に記載のプロモー夕一 DNAおよび該プロモ一夕一 DNAの 下流に連結させたレポ一夕一遺伝子を含有するプラスミ ドを保有する形質転換 体と被験試料とを接触させ、 該レポーター遺伝子の翻訳産物含量を測定するこ とを特徴とする、 該プロモーターによる転写の効率を変動させる化合物のスク リ一ニング法。  (35) A test sample is brought into contact with a transformant containing the promoter DNA described in (34) and a plasmid containing the repo overnight gene ligated downstream of the promoter DNA, A method for screening a compound that changes the efficiency of transcription by said promoter, comprising measuring the translation product content of said reporter gene.
(36) レポーター遺伝子が、 クロラムフエ二コール 'ァセチルトランスフエ ラ一ゼ遺伝子、 一ガラクトシダーゼ遺伝子、 ルシフェラ一ゼ遺伝子およびグ リーン ·フルォレツセント ·プロテイン遺伝子から選ばれる遺伝子である、 （(36) The reporter gene is chloramphenicol A gene selected from the group consisting of a ratase gene, a galactosidase gene, a luciferase gene and a green fluorescein protein gene.
35) に記載のスクリ一ニング方法。 The screening method described in 35).
(37) (35) または （36) に記載の方法により得られる化合物。  (37) A compound obtained by the method according to (35) or (36).
(38) (1) 〜 （4) のいずれかに記載の蛋白質を含有する、 蛋白質分解の 亢進または低下により引き起こされる疾患の予防薬。  (38) A preventive agent for a disease caused by an increase or decrease in protein degradation, comprising the protein according to any one of (1) to (4).
(39) (1) 〜 （4) のいずれかに記載の蛋白質を含有する、 蛋白質分解の 亢進または低下により引き起こされる疾患の治療薬。  (39) A therapeutic agent for a disease caused by enhanced or reduced proteolysis, comprising the protein according to any one of (1) to (4).
(40) (1) 〜 （4) のいずれか 1項に記載の蛋白質を含有する、 蛋白質分 解の亢進または低下により引き起こされる疾患の診断薬。  (40) A diagnostic agent for a disease caused by enhanced or reduced protein degradation, comprising the protein according to any one of (1) to (4).
(41) (5) 〜 （7) のいずれかに記載の DN Aを含有する、 蛋白質分解の 亢進または低下により引き起こされる疾患の予防薬。  (41) A preventive agent for a disease caused by enhanced or reduced proteolysis, comprising the DNA according to any one of (5) to (7).
(42) (5) 〜 （7) のいずれかに記載の DN Aを含有する、 蛋白質分解の 亢進または低下により引き起こされる疾患の治療薬。  (42) A therapeutic agent for a disease caused by enhanced or reduced proteolysis, comprising the DNA according to any one of (5) to (7).
(43) (5) ~ (7) のいずれか 1項に記載の DNAを含有する、 蛋白質分 解の亢進または低下により引き起こされる疾患の診断薬。  (43) A diagnostic agent for a disease caused by an increase or decrease in protein degradation, comprising the DNA according to any one of (5) to (7).
(44) (15) に記載のオリゴヌクレオチドを含有する、 蛋白質分解の亢進 または低下により引き起こされる疾患の予防薬。  (44) A preventive agent for a disease caused by enhanced or reduced proteolysis, comprising the oligonucleotide according to (15).
(45) (15) に記載のオリゴヌクレオチドを含有する、 蛋白質分解の亢進 または低下により引き起こされる疾患の治療薬。  (45) A therapeutic agent for a disease caused by increased or decreased proteolysis, comprising the oligonucleotide according to (15).
(46) (15) に記載のオリゴヌクレオチドを含有する、 蛋白質分解の亢進 または低下により引き起こされる疾患の診断薬。  (46) A diagnostic agent for a disease caused by increased or decreased proteolysis, comprising the oligonucleotide according to (15).
(47) (16) に記載の抗体を含有する、 蛋白質分解の亢進または低下によ り引き起こされる疾患の予防薬。  (47) A preventive agent for a disease caused by an increase or decrease in protein degradation, comprising the antibody according to (16).
(48) (16) に記載の抗体を含有する、 蛋白質分解の亢進または低下によ り引き起こされる疾患の治療薬。 ( 4 9 ) ( 1 6 ) に記載の抗体を含有する、 蛋白質分解の亢進または低下によ り引き起こされる疾患の診断薬。 (48) A therapeutic agent for a disease caused by enhanced or reduced proteolysis, comprising the antibody according to (16). (49) A diagnostic agent for a disease caused by increased or decreased protein degradation, comprising the antibody according to (16).
本発明の蛋白質としては、 配列番号 1に示されるァミノ酸配列からなる蛋白 質、 配列番号 1に示されるアミノ酸配列において 1以上のアミノ酸が欠失、 置 換または付加されたァミノ酸配列からなる F— WD蛋白質、 配列番号 6で示さ れるアミノ酸配列を有してなる F— WD蛋白質、 および配列番号 7 ~ 1 3で示 されるアミノ酸配列の 1以上の配列を有してなる F—WD蛋白質をあげること ができる。  The protein of the present invention includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in which one or more amino acids have been deleted, replaced or added. — A WD protein, an F-WD protein having the amino acid sequence represented by SEQ ID NO: 6, and an F-WD protein having at least one of the amino acid sequences represented by SEQ ID NOs: 7 to 13. I can give it.
本発明において F—WD蛋白質とは、 該蛋白質のァミノ末端近くに Fボック スを有し、 かつカルボキシ末端近くに WDドメインを有する蛋白質をいう。 F — W D蛋白質は蛋白質複合体としてュビキチンリガーゼを形成することができ る。  In the present invention, the F-WD protein refers to a protein having an F box near the amino terminal of the protein and a WD domain near the carboxy terminal. The F-WD protein can form ubiquitin ligase as a protein complex.
Fボックスは、 A1 P XnA2A3A4XnA5A6A7XBA8A9A10XpAuA12A13 The F box is A 1 PX n A 2 A 3 A 4 X n A 5 A 6 A 7 X B A 8 A 9 A 10 X p A u A 12 A 13
XmA14Xq > Α15Α16 Α17Αι8Α19 Γ ^2θ Α21 Α22 ·^23 -^24 -^25 ^26 -^2 X m A 14 X q > Α 15 Α 16 Α 17 Αι 8 Α 19 Γ ^ 2θ Α21 Α 22・ ^ 23-^ 24-^ 25 ^ 26-^ 2
7XnA28で示されるコンセンサス配列を有する 4 0〜5 0残基のアミノ酸からな る配列を有することを特徴とする。 Characterized in that it has a Ru sequence name from 4 0-5 0 residues of amino acids having a consensus sequence represented by 7 X n A 28.
上記配列中、 は L、 V、 Fまたは Mを、 A2は Eまたは Dを、 A3は I、 L または Vを、 A4は I、 Lまたは Vを、 A5は K、 R、 Nまたは Qを、 A6は I、 Vまたは Lを、 A7は L、 Fまたは Iを、 A8は Y、 Η、 Κ、 Νまたは Rを、 Α9 は I、 L、 V、 Αまたは Fを、 A1()は D、 Eまたは Pを、 A„は D、 E、 Sまた は Tを、 A12は I、 Lまたは Vを、 A13は I、 Lまたは Vを、 A14は L、 V、 I または Aを、 A15は（、 Sまたは Nを、 A16は Kまたは Rを、 A17は Rまたは K を、 Α18は W、 Fまたは Lを、 A19は Y、 R、 Νまたは Κを、 Α2()は I、 Lまた は Vを、 A21は I、 L、 Vまたは Aを、 A22は D、 E、 Sまたは Tを、 Α23は D 、 Eまたは Sを、 A24は D、 E、 Sまたは Tを、 A25は I、 L、 V、 Fまたは Y を、 A26は W、 F、 Yまたは Lを、 A27は K、 Q、 Rまたは Nを、 A28は L、 V 、 A、 Fまたは Mを、 それそれ表す。 また、 Iはイソロイシンを、 Lは口イシ ンを、 Vはバリンを、 Fはフエ二ルァラニンを、 Mはメチォニンを、 Eはグル 夕ミン酸を、 Dはァスパラギン酸を、 Kはリジンを、 Rはアルギニンを、 Nは ァスパラギンを、 Qはグルタミンを、 Yはチロシンを、 Hはヒスチジンを、 A はァラニンを、 Pはプロリンを、 Sはセリンを Tはスレオニンを、 Cはシステ インを Wはトリブトファンを、 それぞれ表す。 さらに、 Xはいずれのアミノ酸 でもよいことを表し、 nは 1〜5個、 mは 1個、 pは 2〜1 0個、 qは 2個、 rは 1〜3個、 sは 0〜2個を表す。 In the above sequence, the L, V, F or M, the A 2 is E or D, A 3 is I, L or V, A 4 is I, L or V, A 5 is K, R, N Or Q, A 6 is I, V or L, A 7 is L, F or I, A 8 is Y, Η, Κ, Ν or R, Α 9 is I, L, V, Α or F A 1 () is D, E or P, A „is D, E, S or T, A 12 is I, L or V, A 13 is I, L or V, A 14 is L, V, I or A, A 15 (, S or N, A 16 K or R, A 17 R or K, Α 18 W, F or L, A 19 Y, R, Ν or 、, Α 2 () for I, L or V, A 21 for I, L, V or A, A 22 for D, E, S or T, Α 23 for D, E Or S, A 24 is D, E, S or T, A 25 is I, L, V, F or Y, A 26 is W, F, Y or L, A 27 is K, Q, R Or N, A 28 is L, V , A, F or M, respectively. Also, I is isoleucine, L is mouth isine, V is valine, F is phenylalanine, M is methionine, E is gluamic acid, D is aspartic acid, K is lysine, R is arginine, N is asparagine, Q is glutamine, Y is tyrosine, H is histidine, A is alanine, P is proline, S is serine, T is threonine, and C is cysteine. Represents tributofan, respectively. X represents any amino acid, n is 1 to 5, m is 1, p is 2 to 10, q is 2, r is 1 to 3, and s is 0 to 2. Represents an individual.
F— WD蛋白質が、 例えば S C F蛋白質複合体を構成する場合には、 Fボッ クスの部分で S k p 1蛋白質と結合することができる [Cell, 86, 263-274 ( 1996) 、 Trends in Genetics, 14, 236-243 ( 1998)] 。  When the F-WD protein forms, for example, an SCF protein complex, it can bind to the Skp1 protein at the F box [Cell, 86, 263-274 (1996), Trends in Genetics, 14, 236-243 (1998)].
WDドメインは、 その最後のアミノ酸残基としてトリブトフアン一ァスパラ ギン酸 （WD ) またはその類似アミノ酸残基をもち、 その最初にグリシン—ヒ スチジン （G H ) または類似アミノ酸残基をもつことが多く、 かつ約 2 0〜4 0残基からなるアミノ酸配列が 4〜 8回反復する配列を有することを特徴とす る。  The WD domain has, as its last amino acid residue, tributophan-aspartic acid (WD) or its similar amino acid residue, and often has glycine-histidine (GH) or a similar amino acid residue at its beginning, and An amino acid sequence consisting of about 20 to 40 residues has a sequence repeated 4 to 8 times.
欠失、 置換または付加されるアミノ酸の数は特に限定されないが、 1個から 数十個、 特に 1個から数個のアミノ酸であることが好ましい。  The number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably one to several tens, particularly preferably one to several amino acids.
本発明の蛋白質が F—WD蛋白質としての機能を有するためには、 配列番号 l fe載のアミノ酸配列と少なくとも 6 0 %以上、 通常は 8 0 %以上、 特に 9 5 %以上の相同性を有していることが好ましい。 さらに、 9 8 %以上の相同性を 有していることが好ましい。  In order for the protein of the present invention to have a function as an F-WD protein, it must have at least 60%, usually at least 80%, and especially at least 95% homology with the amino acid sequence described in SEQ ID NO: l fe. Is preferred. Further, it is preferable that the homology is 98% or more.
上記のアミノ酸の欠失、 置換若しくは付加は、 出願前周知技術である部位特 異的変異誘発法等により実施することができる。 また、 1若しくは数個のアミ ノ酸とは、 部位特異的変異誘発法により欠失、 置換若しくは付加できる程度の 数のアミノ酸を意味する。 かかる 1以上のアミノ酸が欠失、 置換または付加されたアミノ酸配列からな る F—WD蛋白質は、 Molecular Cloning, A Laboratory Manual , Second Edition, Cold Spring Harbor Laboratory Press (1989) (以下、 モレキユラ一 クロー二 ング第 2版と略す) 、 Current Protocols in Molecular Biology, Supplement 1〜38， John Wiley & Sons (1987-1997) (以下、 カレント プロ トコル イン モ レキユラ バイオロジーと略す） 、 Nucleic Acids Research, 10, 6487 (1982)、 Proc. Natl. Acad. Sci. USA, 79, 6409(1982)、 Gene, 34, 315 (1985)、 Nuclei c Acids Research, 13, 4431 (1985)、 Proc. Natl. Acad. Sci. USA, 82, 488 (1985)、 Proc. Natl. Acad. Sci. USA, 81, 5662 (1984)、 Science, 224, 1431 (1984)、 PCT W085/00817(1985)、 Nature, 316, 601 (1985)等に記載の方法に 準じて調製することができる。 The above-mentioned deletion, substitution or addition of amino acids can be carried out by a site-specific mutagenesis method or the like which is a well-known technique before filing the application. The term “one or several amino acids” means an amino acid of such a number that can be deleted, substituted or added by site-directed mutagenesis. Such an F-WD protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added is known as Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as "Molecular Cloning"). Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as current protocol in molecular biology), Nucleic Acids Research, 10, 6487 Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), Proc. Natl. Acad. Sci. USA, 81, 5662 (1984), Science, 224, 1431 (1984), PCT W085 / 00817 (1985), Nature, 316, 601 (1985) ), Etc. can be prepared.
本発明の DN Aは、 本発明の蛋白質をコードする DNA、 例えば Fボックス 蛋白質をコードする DN Aとして、 配列番号 2に示される塩基配列を有する D NAがあげられる。 しかし、 一般に 1つのアミノ酸に対して複数種の遺伝暗号 が存在するため、 配列番号 2とは異なる塩基配列を有する DN Aであっても本 発明の蛋白質をコ一ドしていれば本発明の DN Aに含まれる。  Examples of the DNA of the present invention include a DNA encoding the protein of the present invention, for example, a DNA having the nucleotide sequence shown in SEQ ID NO: 2 as an DNA encoding an F-box protein. However, since a plurality of kinds of genetic codes generally exist for one amino acid, even if the DNA of the present invention encodes the DNA of the present invention, even if the DNA has a nucleotide sequence different from that of SEQ ID NO: 2, Included in DN A.
本発明の DNAは、 本発明の蛋白質をコードする DNA、 配列番号 2で示さ れる塩基配列を有する DNA、 該 DNAとストリンジェントな条件下でハイブ リダイズする DNA、 およびこれらの DNAと相補的な配列を有する DNAを あげることができる。  The DNA of the present invention includes a DNA encoding the protein of the present invention, a DNA having the nucleotide sequence of SEQ ID NO: 2, a DNA that hybridizes with the DNA under stringent conditions, and a sequence complementary to these DNAs. DNA having the following.
ストリンジェントな条件下でハイブリダイズ可能な D N Aとは、 本発明の蛋 白質をコードする DNA、 配列番号 2で表される塩基配列を有する DNAをプ ローブとして、 コロニー 'ハイブリダィゼ一シヨン法、 プラーク 'ハイブリダ ィゼ一シヨン法あるいはサザンプロットハイブリダイゼ一シヨン法等を用いる ことにより得られる DNAを意味し、 具体的には、 コロニーあるいはプラーク 由来の DNAを固定化したフィル夕一を用いて、 0. 7〜1. 0Mの塩化ナト リウム存在下、 65°Cでハイブリダィゼ一シヨンを行った後、 0. 01〜2倍 濃度の S S C溶液 （ 1倍濃度の S S C溶液の組成は、 150 mM塩化ナトリゥ ム、 15mMクェン酸ナトリウムよりなる） を用い、 65〜80°C条件下でフ ィル夕一を洗浄することにより同定できる DN Aをあげることができる。 DNA capable of hybridizing under stringent conditions refers to a DNA encoding the protein of the present invention, a DNA having the nucleotide sequence of SEQ ID NO: 2 as a probe, and colony 'hybridization method, plaque'. A DNA obtained by using the hybridization method or the Southern plot hybridization method.Specifically, the DNA obtained by immobilizing DNA derived from colonies or plaques is referred to as DNA. 7 ~ 1.0M NaCl After performing hybridization at 65 ° C in the presence of lium, a 0.01- to 2-fold concentration of SSC solution (The composition of a 1-fold concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate.) Can be identified by washing the filter under the conditions of 65-80 ° C.
ハイブリダィゼ一シヨンは、 モレキュラー 'クローニング第 2版、 カレント •プロトコールズ 'イン 'モレキュラー ·バイオロジー、 DNA Clonin 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995) 等に記載されている方法に準じて行うことができる。 ハイプリダイズ可能な D N Aとして具体的には、 配列番号 2で表される塩基配列と少なくとも 60%以 上の相同性を有する DNA、 好ましくは 80%以上の相同性を有する DNA、 さらに好ましくは 95%以上の相同性を有する DNAをあげることができる。 本発明において、 ストリンジェントな条件としては、 95%以上、 さらに 98 %以上の相同性が存在する場合にのみハイプリダイズ可能な条件であることが 特に好ましい。  Hybridization is performed by the method described in Molecular 'Cloning 2nd edition, Current Protocols' in' Molecular biology, DNA Clonin 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995), etc. It can be performed according to. Specifically, as a hybridizable DNA, a DNA having at least 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 2, preferably a DNA having 80% or more homology, more preferably 95% DNAs having the above homology can be mentioned. In the present invention, the stringent condition is particularly preferably a condition that allows hybridization only when 95% or more, and more preferably 98% or more homology exists.
以下に、 本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail.
[ 1 ]本発明の DN Aの取得およびオリゴヌクレオチドの調製 [1] Acquisition of DNA of the present invention and preparation of oligonucleotide
シゾサッカロマイセス 'ボンベの pop 1+ [Genes & Development, 11， 1548-1 560 (1997)] と相同性をもつ遺伝子を、 デ一夕べ一スより、 BLAST、 FASTA、 Smit h - Waterman法等を利用したプログラム、 フレームサーチ [イスラエル、 コンビ ュジェン (Compugen) 社] 相同性検索ソフトウエアを利用して検索することが できる。 Genes having homology to Schizosaccharomyces' bomb pop 1+ [Genes & Development, 11, 1548-1 560 (1997)], BLAST, FASTA, Smith-Waterman method, etc. Program used, frame search [Compugen, Israel] Search using homology search software.
デ一夕ベースとしては、 GenBank、 EMBL、 DDBJ等の塩基デ一夕べ一ス、 GenPept 、 PIR, Swiss- Prot等のアミノ酸配列デ一夕ベース等があげられる。  Examples of the database include bases such as GenBank, EMBL, and DDBJ, and databases based on amino acid sequences such as GenPept, PIR, and Swiss-Prot.
pop 1+と相同性をもつ E ST等、 遺伝子の部分塩基配列から、 以下の方法 により本発明の DN Aを取得することができる。  The DNA of the present invention can be obtained from a partial nucleotide sequence of a gene such as EST having homology to pop 1+ by the following method.
(1) cDNAライブラリーの作製 cDNAライブラリーを作製するために、 適切な細胞または組織より全 RN Aあるいは mRN Aを調製する。 (1) Preparation of cDNA library To make a cDNA library, prepare total RNA or mRNA from appropriate cells or tissues.
全 RNAを調製する方法として、 チォシアン酸グァニジン一トリフルォロ酢 酸セシウム法 [Methods in Enzymology, 154, 3 (1987)] 、 酸性グァニジンチ オシァネ一ト 'フエノ一ル 'クロ口ホルム（AGPC)法 [Analytical Biochemistry, 162, 156 (1987)、 実験医学 ， 1937 (1991)] 等を用いることができる。  Methods for preparing total RNA include the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology, 154, 3 (1987)], the acid guanidine thionate “phenol” and “clonal form (AGPC) method” [Analytical Biochemistry , 162, 156 (1987), Experimental Medicine, 1937 (1991)].
全 RNAからポリ （A) +RNAとして mRNAを調製する方法として、 オリ ゴ （dT) 固定化セルロースカラム法 （モレキュラー クローニング 第 2版） やオリゴ dTラテックスを用いる方法等を用いることができる。  As a method for preparing mRNA as poly (A) + RNA from total RNA, an oligo (dT) -immobilized cellulose column method (molecular cloning second edition), a method using oligo dT latex, or the like can be used.
ファ一スト · トラック · mRNA単離キット [Fast Track mRNA Isolation Ki t;インビトロジェン （ Invitrogen) 社] 、 クイック 'プレップ · mR N A精製 キット [Quick Prep mRNA Purification Kit； フアルマシア (Pharmacia) 社] 等のキットを用いて組織や細胞から直接 mRN Aを調製することもできる。 適切な細胞または組織として、 デ一夕ベースから見出された E S T等が含ま れている cDNAライブラリ一の種類を調べ、 該ライブラリーを構築するため に用いた細胞または組織、 あるいは該組織由来の細胞株等を用いることが好ま しい。  Kits such as Fast Track mRNA Isolation Kit [Fast Track mRNA Isolation Kit; Invitrogen] and Quick Prep mRNA Purification Kit [Quick Prep mRNA Purification Kit; Pharmacia] Can be used to prepare mRNA directly from tissues or cells. As a suitable cell or tissue, a type of cDNA library containing ESTs and the like found from a database is examined, and the cell or tissue used for constructing the library, or a cell derived from the tissue is used. It is preferable to use a cell line or the like.
得られた全 RNAあるいは mRNAを用い、 常法により cDNAライブラリ 一を作製する。  Using the obtained total RNA or mRNA, prepare a cDNA library by a conventional method.
cDNAライブラリ一作製法として、 モレキュラー クローニング 第 2版や カレント プロトコ一ルズ イン モレキュラー バイオロジー、 DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995)等に記載された方法、 あるいは市販のキット、 例えばスーパース クリプト · プラスミ ド · システム ' フォー · cDNA ·シンセシス ' アンド ' プラスミ ド *クローニング [SuperScriptPlasmidSystem for c DNA Synthesi s and Plasmid Cloning；ギブコ BR L (Gibco BRL) 社] やザップ— c DNA .シンセシス 'キット [ZAP- cDNA Synthesis Kit, ストラ夕ジーン社] を 用いる方法等をあげることができる。 As a method for preparing a cDNA library, methods described in Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995), or Commercially available kits, such as SuperScript Plasmid System 'for cDNA Synthesis'and' Plasmid * Cloning [SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning; Gibco BRL (Gibco BRL)] or Zap-c DNA .Synthesis' kit [ZAP-cDNA Synthesis Kit, Straugene Co., Ltd.]
cDNAライブラリーを作成するためのクロ一ニングベクタ一としては、 ェ シエリヒア .コリ (Escherichia coli) K 12株中で自立複製できるものであ れば、 ファージベクター、 プラスミ ドベクタ一等いずれでも使用できる。  A phage vector or a plasmid vector can be used as a closing vector for preparing a cDNA library as long as it can replicate autonomously in Escherichia coli K12 strain.
具体的には、 ZAP Express [ストラタジーン社、 Strategies, 5, 58 (1992)] 、 pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)]、 Lambd a ZAP II (ストラタジーン社） 、 AgtlO、 人 gtll [DNA Cloning, A Practica 1 Approach, 1, 49 (1985)] 、 人 TriplEx (クローンテック社） 、 人 ExCell (フ アルマシア社） 、 pT7T318U (フアルマシア社） 、 pcD2 [Mol. Cell. Biol., 3， 2 80 (1983)]、 pUC18 [Gene, 33, 103 (1985)]、 pAmo [J. Biol. Chem. , 268 , 22782-22787 (1993)、 別名 pAMoPRC3Sc (特開平 05-336963) ] 等をあげること ができる。  Specifically, ZAP Express [Stratagene, Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], Lambd a ZAP II (Stratagene) , AgtlO, person gtll [DNA Cloning, A Practica 1 Approach, 1, 49 (1985)], person TriplEx (Clonetech), person ExCell (Pharmacia), pT7T318U (Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)], pAmo [J. Biol. Chem., 268, 22782-22787 (1993), aka pAMoPRC3Sc (Japanese Unexamined Patent Publication No. 05-336963) )] Etc.
宿主微生物としては、 ェシヱリヒア ·コリに属する微生物であればいずれも 用いることができる。 具体的には、 Escherichia coli XLl-Blue MRF' [ストラ 夕ジーン社、 Strategies,互， 81 (1992)]、 Escherichia coli C600 [Genetics, 39, 440 (1954)]、 Escherichia coli Y1088 [Science, 222, 778 (1983)]、 Escherichia coli Y1Q90 [Science, 222, 778 (1983)]、 Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)]、 Escherichia coli K802 [J. Mol. Biol., 16， 118 (1966)]、 Escherichia coli JM105 [Gene, 38, 275 (1985)]、 Escherichi a coli S0LRTM Strain (ストラ夕ジーン社） 、 Escherichia coli LE392 (モレ キユラ一 クローニング 第 2版） 等を用いることができる。  As the host microorganism, any microorganism belonging to Escherichia coli can be used. Specifically, Escherichia coli XLl-Blue MRF '[Strata Gene, Strategies, Mutual, 81 (1992)], Escherichia coli C600 [Genetics, 39, 440 (1954)], Escherichia coli Y1088 [Science, 222, 778 (1983)], Escherichia coli Y1Q90 [Science, 222, 778 (1983)], Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Escherichia coli K802 [J. Mol. Biol., 16, 118 (1966)], Escherichia coli JM105 [Gene, 38, 275 (1985)], Escherichia coli S0LRTM Strain (Stratagene Inc.), Escherichia coli LE392 (Molecular cloning 2nd edition), etc. Can be.
上記方法により作製した c D N Aライブラリ一に加え、 市販の c D N Aライ ブラリーも利用することができる。  In addition to the cDNA library prepared by the above method, a commercially available cDNA library can also be used.
市販の cDNAライブラリ一として、 クローンテック社、 ライフテックオリ ェンタル社等のヒト、 ゥシ、 マウス、 ラヅト、 ゥサギ等由来の各臓器 cDNA ラィブラリ一をあげることができる。 As a commercially available cDNA library, cDNAs from human organs such as Clonetech, Lifetech Oriental, etc. You can give a library.
(2) 本発明の DN Aの取得  (2) Acquisition of the DNA of the present invention
上記 （1) で作製した cDNAライブラリ一より、 本発明の DNAを有する cDNAクローンを、 ァイソトープあるいは蛍光標識したプロ一ブを用いたコ ロニー ·ハイブリダイゼーシヨン法あるいはプラーク ·ハイブリダイゼーショ ン法 [モレキュラー クローニング 第 2版] 等により選択することができる。 プロ一ブとしては、 一部明らかになつている塩基配列に基いたプライマーを 用いて、 ポリメラ一ゼ'チェイン 'リアクション（PCR) [PCR Protocols, Aca demic Press (1990)] を利用した方法 （PCR法ともいう） で cDNAの一部 を増幅した断片や、 一部明らかになつている塩基配列に基いたオリゴヌクレオ チドを利用することができる。  From the cDNA library prepared in the above (1), a cDNA clone having the DNA of the present invention was cloned by colony hybridization or plaque hybridization using an isotope or a fluorescently labeled probe [Molecular Cloning 2nd edition]. As a probe, using a primer based on the nucleotide sequence that has been partially identified, a method using polymerase 'chain' reaction (PCR) [PCR Protocols, Aca demic Press (1990)] (PCR The method can also be used to amplify a fragment of a part of the cDNA or to use an oligonucleotide based on the nucleotide sequence that is partially identified.
プライマ一として、 全長 cDNAの 5， 端側および 3' 端側の両方の塩基配 列が E S T等により明らかになつている場合には、 その塩基配列に基いて調製 したプライマ一を用いることができる。  If the nucleotide sequence at both the 5, 5 and 3 'end of the full-length cDNA is known by EST, etc., a primer prepared based on the nucleotide sequence can be used as the primer. .
該 c D N Aの両端にァダプ夕一を付加し、 このァダブ夕一の塩基配列と一部 明らかになつている塩基配列に基づいたブラィマーで PCRを行う 5， -RA CE (rapid amplification of cDNA ends)および 3， -RACE [Proc. Natl. Acad. Sci. USA, 85, 8998 (1988)] により、 プライマーに用いた配列よ りも 5' 端側および 3' 端側の cDNA断片を得ることができる。  Add an adapter to both ends of the cDNA and perform PCR with a primer based on the nucleotide sequence that is partially identifiable with the nucleotide sequence of the adapter 5, -RACE (rapid amplification of cDNA ends) And 3, -RACE [Proc. Natl. Acad. Sci. USA, 85, 8998 (1988)] to obtain cDNA fragments 5 'and 3' from the sequence used for the primer. .
得られた c D N A断片をつなぎあわせることにより、 本発明の全長 D N Aを 取得することができる。  By joining the obtained cDNA fragments, the full-length DNA of the present invention can be obtained.
上記の方法により取得された DNAの塩基配列は、 該 DNA断片をそのまま、 または適当な制限酵素等で切断後常法によりベクターに組み込んだ後、 通常用 いられる塩基配列解析方法、 例えばサンガー (Sanger)らのジデォキシ法 [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] あるいはパーキン ·エルマ一社 （Perki nElmer： 373 A · DNAシークェンサ一） 、 フアルマシア社、 ライコア （LI- COR) 社等の塩基配列分析装置を用いて分析することにより決定することができ る。 The nucleotide sequence of the DNA obtained by the above method can be obtained by integrating the DNA fragment as it is, or after digestion with an appropriate restriction enzyme or the like, into a vector by a conventional method, and then using a conventional nucleotide sequence analysis method, for example, Sanger (Sanger Natl. Acad. Sci. USA, 74, 5463 (1977)] or Perkin Elmer (373A, DNA Sequencer), Pharmacia, Lycoa (LI- It can be determined by analysis using a base sequence analyzer such as COR).
上記方法により取得された本発明の DN Aを含むプラスミ ドとして、 例えば、 配列番号 2で表される塩基配列からなる DNAを有するプラスミ ド phMD 6 をあげることができる。  The plasmid containing the DNA of the present invention obtained by the above method includes, for example, a plasmid phMD6 having a DNA consisting of the base sequence represented by SEQ ID NO: 2.
プラスミ ド phMD 6を含有するェシエリヒア 'コリ DH 5ひ/ phMD 6 は、 FERM BP— 66 1 1として、 平成 10年 12月 24日付で工業技術 院生命工学工業技術研究所、 日本国茨城県つくば巿東 1丁目 1番 3号 （郵便番 号 305- 8566) に寄託されている。  Escherichia coli DH5 / phMD6 containing plasmid phMD6 was designated as FERM BP-66 11 on December 24, 1998 by the Institute of Biotechnology, Institute of Biotechnology, Tsukuba, Ibaraki, Japan. Deposited at Higashi 1-3-1, 1-3 (zip code 305-8566).
また、 上記方法で取得した DN Aとストリンジェントな条件下でハイブリダ ィズする DNAを選択することにより、 他の組織あるいは、 他の動物由来、 例 えばヒト由来の目的とする DNAを取得することができる。  In addition, by selecting DNA that hybridizes with the DNA obtained by the above method under stringent conditions, it is possible to obtain the target DNA from other tissues or other animals, for example, human. Can be.
上記方法により得られた塩基配列情報に基づき、 DN A合成機で化学合成す ることにより目的とする DN Aを調製することもできる。 DN A合成機として は、 チォホスファイ ト法を利用した島津製作所社の DN A合成機、 フォスフォ アミダイ ト法を利用したパーキン 'エルマ一社の DN A合成機 mo d e 139 2等をあげることができる。  Based on the nucleotide sequence information obtained by the above method, the desired DNA can also be prepared by chemical synthesis using a DNA synthesizer. Examples of the DNA synthesizer include a DNA synthesizer manufactured by Shimadzu Corporation using the thiophosphite method, and a Perkin 'Elma's DNA synthesizer model 1392 using the phosphoramidite method.
得られた塩基配列が新規な配列かどうかは、 BLAST等の相同性検索プログラム を用いて、 GenBank、 EMBLおよび DDBJなどの塩基配列デ一夕ベースを検索するこ とにより、 デ一夕ベース中の既存の遺伝子の塩基配列と一致すると考えられる ような明らかな相同性を示す塩基配列がないことにより確認できる。  Whether or not the obtained nucleotide sequence is a novel sequence can be determined by searching a nucleotide sequence database such as GenBank, EMBL and DDBJ using a homology search program such as BLAST. This can be confirmed by the absence of a nucleotide sequence showing obvious homology that is considered to match the nucleotide sequence of an existing gene.
上記の方法で得られる新規な D N Aの塩基配列として、 例えば配列番号 2で 示される塩基配列があげられる。  The novel DNA sequence obtained by the above method includes, for example, the nucleotide sequence shown in SEQ ID NO: 2.
(3) 本発明のオリゴヌクレオチドの調製  (3) Preparation of oligonucleotide of the present invention
上述の方法で取得した本発明の DN Aおよび DN A断片を用いて、 常法ある いは上記の DNA合成機により、 本発明の DNAの一部の配列を有するアンチ センス ·オリゴヌクレオチド、 センス ·オリゴヌクレオチド等のオリゴヌクレ ォチドを調製することができる。 Using the DNA and the DNA fragment of the present invention obtained by the above-described method, an antibody having a partial sequence of the DNA of the present invention can be obtained by a conventional method or the above-mentioned DNA synthesizer. Oligonucleotides such as sense oligonucleotides and sense oligonucleotides can be prepared.
該オリゴヌクレオチドとしては、 上記 D N Aの有する塩基配列中の連続した 5〜6 0塩基と同じ配列を有する D N Aまたは該 D N Aと相補的な配列を有す る D N Aをあげることができ、 具体的には、 配列番号 2で表される塩基配列中 の連続した 5〜 6 0塩基と同じ配列を有する D N Aまたは該 D N Aと相補的な 配列を有する D N Aをあげることができる。 センスプライマ一およびアンチセ ンスプライマーとして用いる場合には、 両者の融解温度 （T m) および塩基数 が極端に変わることのない上記記載のォリゴヌクレオチドが好ましい。  Examples of the oligonucleotide include a DNA having the same sequence as the contiguous 5 to 60 bases in the base sequence of the DNA or a DNA having a sequence complementary to the DNA. And a DNA having the same sequence as the consecutive 5 to 60 bases in the base sequence represented by SEQ ID NO: 2 or a DNA having a sequence complementary to the DNA. When used as a sense primer and an antisense primer, the above-mentioned oligonucleotides in which the melting temperature (Tm) and the number of bases of both do not extremely change are preferable.
該オリゴヌクレオチドとして、 例えば、 配列番号 3または 4で表されるオリ ゴヌクレオチドをあげることができる。  Examples of the oligonucleotide include an oligonucleotide represented by SEQ ID NO: 3 or 4.
さらに、 これらオリゴヌクレオチドの誘導体 （以下、 誘導体オリゴヌクレオ チドという） も本発明のオリゴヌクレオチドとして利用することができる。 誘導体オリゴヌクレオチドとしては、 オリゴヌクレオチド中のリン酸ジエス テル結合がホスフォロチォエート結合に変換された誘導体オリゴヌクレオチド、 オリゴヌクレオチド中のリン酸ジエステル結合が N 3， - P 5 ' ホスフォアミ デート結合に変換された誘導体オリゴヌクレオチド、 オリゴヌクレオチド中の リボースとリン酸ジエステル結合がぺプチド核酸結合に変換された誘導体ォリ ゴヌクレオチド、 オリゴヌクレオチド中のゥラシルが C— 5プロビニルゥラシ ルで置換された誘導体ォリゴヌクレオチド、 ォリゴヌクレオチド中のゥラシル が C— 5チアゾ一ルゥラシルで置換された誘導体オリゴヌクレオチド、 オリゴ ヌクレオチド中のシトシンが C— 5プロピニルシトシンで置換された誘導体ォ リゴヌクレオチド、 オリゴヌクレオチド中のシトシンがフエノキサジン修飾シ トシン (phenoxazine-modified cytosine)で置換された誘導体オリゴヌクレオ チド、 オリゴヌクレオチド中のリボースが 2， —0—プロピルリボースで置換 された誘導体オリゴヌクレオチド、 あるいはオリゴヌクレオチド中のリボース が 2 ' —メ トキシェトキシリボースで置換された誘導体ォリゴヌクレオチド等 をあげることができる [細胞工学， ， 1463 (1997)] 。 Furthermore, derivatives of these oligonucleotides (hereinafter, referred to as derivative oligonucleotides) can also be used as the oligonucleotide of the present invention. Derivative oligonucleotides include derivative oligonucleotides in which the phosphate ester bond in the oligonucleotide has been converted to a phosphorothioate bond, and phosphodiester bonds in the oligonucleotide have been converted to N3, -P5 'phosphoramidate bonds. Derived oligonucleotide, Derivative oligonucleotide in which ribose and phosphodiester bond in oligonucleotide is converted to peptide nucleic acid bond, Derivative oligonucleotide in which peracyl in oligonucleotide is substituted with C-5 provinyl peroxyl Derivative oligonucleotides in which peracyl in oligonucleotides is substituted with C-5 thiazylperacyl, derivative oligonucleotides in which cytosines in oligonucleotides are substituted with C-5 propynylcytosine, oligonucleotides Derivative oligonucleotides in which cytosine in the oligonucleotide has been replaced with phenoxazine-modified cytosine, derivative oligonucleotides in which the ribose in the oligonucleotide has been replaced by 2, -0-propylribose, or oligonucleotides in the oligonucleotide Ribose Can be exemplified by derivatives oligonucleotides substituted with 2'-methoxetoxyribose [Cell Engineering,, 1463 (1997)].
[2] 本発明の蛋白質の調製  [2] Preparation of the protein of the present invention
( 1 ) 形質転換体の作製  (1) Preparation of transformant
上記 [1] に記載の方法により取得した本発明の DNAを宿主細胞中で発現 させ、 本発明の蛋白質を製造するためには、 モレキュラー クローニング 第 2 版、 カレント プロトコル イン モレキユラ バイオロジー等に記載された方法 を用いることができる。  Expression of the DNA of the present invention obtained by the method described in the above [1] in a host cell to produce the protein of the present invention is described in Molecular Cloning, Second Edition, Current Protocol in Molecular Biology, etc. Can be used.
即ち、 本発明の DNAを適当な発現べクタ一のプロモ一夕一下流に挿入した 組換えベクターを造成し、 該ベクターを宿主細胞に導入することにより、 本発 明の蛋白質を発現する形質転換体を取得し、 該形質転換体を培養することによ り、 本発明の蛋白質を製造することができる。  That is, by constructing a recombinant vector in which the DNA of the present invention is inserted into a suitable expression vector at one time downstream of a promoter, and introducing the vector into a host cell, transformation for expressing the protein of the present invention is performed. By obtaining a transformant and culturing the transformant, the protein of the present invention can be produced.
宿主細胞としては、 細菌、 酵母、 動物細胞、 昆虫細胞、 植物細胞等、 目的と する遺伝子を発現できるものであればいずれも用いることができる。  As the host cell, any cell that can express the target gene, such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.
発現べクタ一としては、 上記宿主細胞において自立複製可能なレゝしは染色体 中への組込が可能で、 本発明の D N Aを転写できる位置にプロモー夕一を含有 しているものが用いられる。  As an expression vector, a vector capable of autonomously replicating in the above-described host cell, which can be integrated into a chromosome, and which contains a promoter at a position where the DNA of the present invention can be transcribed is used. .
細菌等の原核生物を宿主細胞として用いる場合、 本発明の蛋白質をコードす る D N Aを含有してなる組換えべクタ一は原核生物中で自立複製可能であると 同時に、 プロモーター、 リボソーム結合配列、 本発明の DN Aおよび転写終結 配列から構成される組換えべクタ一であることが好ましい。 プロモー夕一を制 御する遺伝子が含まれていてもよい。  When a prokaryote such as a bacterium is used as a host cell, the recombinant vector containing the DNA encoding the protein of the present invention is capable of autonomous replication in the prokaryote and has a promoter, a ribosome binding sequence, It is preferably a recombinant vector comprising the DNA of the present invention and a transcription termination sequence. The gene controlling the promotion may be included.
発現べクタ一としては、 例えば、 pBTrp2、 pBTac l、 pBTa c 2 (いずれもベーリンガ一マンハイム社）、 PKK233- 2 (フアルマシア社）、 pSE280 Examples of expression vectors include, for example, pBTrp2, pBTac1, pBTac2 (both Boehringer Mannheim), PKK233-2 (Pharmacia), pSE280
(インビトロジェン社） 、 pGEMEX- 1 [プロメガ (Promega)社] 、 pQE- 8 (キアゲ ン （QIAGEN)社） 、 pKYPIO (特開昭 58- 110600)、 pKYP200 [Agric. Biol. Chem., 48, 669 (1984)]、 pLSAl [Agric. Biol. Chem. , 53, 277 (1989)]、 pGELl [ Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)]、 pBluescript II SK (-) (ス トラ夕ジーン社） 、 pTrs32 (FERM BP— 5408) 、 pGHA2 (FERM BP— 400) 、 pGKA2 (FERM B-6798) , pT e rm2 (特閧平 3— 22979、 US468619 K US4939094、 US 5160735) ^ pGEX (フアルマシア社） 、 pE T— 3 (ノバジェ ン社） 、 pSupex、 pUB 110, pTP5、 pC 194、 pTrxFu s (Invitrogen社） 、 MAL- c 2 (New England Biolabs社） 等をあげるこ とができる。 (Invitrogen), pGEMEX-1 [Promega], pQE-8 (QIAGEN), pKYPIO (JP-A-58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277 (1989)], pGELl [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (- ) (Strata Gene Co., Ltd.), pTrs32 (FERM BP-5408), pGHA2 (FERM BP-400), pGKA2 (FERM B-6798), pT e rm2 (Tokumeihei 3—22979, US468619K US4939094, US5160735) ) ^ pGEX (Pharmacia), pET-3 (Novagen), pSupex, pUB110, pTP5, pC194, pTrxFus (Invitrogen), MAL-c2 (New England Biolabs), etc. Can be.
プロモ一夕一としては、 宿主細胞中で発現できるものであれば、 いずれのも のを用いてもよい。 例えば、 卫プロモ一夕一 （P^£)、 プロモー夕一 （P lac)、 PLプロモ一夕一、 ΡΒプロモ一夕一、 Τ7プロモー夕一等の、 ェシエリヒ ァ ·コリゃファージ等に由来するプロモ一夕一、 SP01プロモー夕一、 SP 02プロモ一夕一、 p e ηΡプロモー夕一等をあげることができる。 また、 Ρ trpを 2つ直列させたプロモ一夕一 （P^ x2)、 ；^プロモ一夕ー、 lacT7プ ロモ—夕—、 let Iプロモーターのように人為的に設計改変されたプロモー夕一 等も用いることができる。 Any promoter may be used as long as it can be expressed in a host cell. For example, - defense Promo Isseki one (P ^ £), promoter evening one (P lac), P L promoter Isseki one, Ρ Β promo Isseki one, Τ7 promoter evening one, etc., to Eshierihi § coli Ya phage, etc. The origins of Promo One Night, SP01 Promo One Night, SP 02 Promo One Night, pe ηΡ Promo Night, etc. can be listed. In addition, two sets of promoters, 直列 trp, are connected in series (P ^ x2); ^ promoters, lacT7 promoters, and promoters designed artificially like let I promoter. Etc. can also be used.
リボソーム結合配列としては、 シャイン—ダルガノ （ Shine-Dalgarno)配列 と開始コドンとの間を適当な距離 （例えば 6〜18塩基） に調節したプラスミ ドを用いることが好ましい。  As the ribosome binding sequence, it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
本発明の組換えベクターにおいては、 本発明の DN Aの発現には転写終結配 列は必ずしも必要ではないが、 構造遺伝子の直下に転写終結配列を配置するこ とが好ましい。  In the recombinant vector of the present invention, a transcription termination sequence is not necessarily required for expression of the DNA of the present invention, but it is preferable to arrange a transcription termination sequence immediately below a structural gene.
宿主細胞としては、 ェシエリヒア属、 セラチア属、 バチルス属、 ブレビパク テリゥム属、 コリネバクテリウム属、 ミクロバクテリウム属、 シユードモナス 属等に属する微生物、 例えば、 Escherichia coli XLl-Blueヽ Escherichia coli XL2 - Blue、 Escherichia coli DH1、 Escherichia coli MC1000、 Escherichia coli KY3276, Escherichia coli W1485^ Escherichia coli JM109, Escherichia coli HB101、 Escherichia coli No.49、 Escherichia coli W3110、 Escherichia coli NY49、 Serratia ficaria Serrat ia font i col a Serratia; liquefaciens、 Serrat ia marcescens Bacillus subtil is、 Bacillus amyloliquefaciensx Brevibacte rium ammmoniageiies、 Brevibacterium i腿 ariopniium ATCC14068、 Brevibacteri um saccharolyticum ATCC14066、 Corynebacterium glutamicum ATCC13032、 Cory nebacterium glutamicum ATCC14067、 Corynebacterium glutamicum ATCC13869、 Corynebacterium acetoacidophilum ATCC13870、 Microbacterium ammoniaphilumHost cells include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., such as Escherichia coli XLl-Blue Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485 ^ Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Serratia font Serratia; liquefaciens, Serrat ia marcescens Bacillus subtil is, Bacillus amyloliquefaciens x Brevibacte rium ammmoniageiies, Brevibacterium i thigh ariopniium ATCC14068, Brevibacteri um saccharolyticum ATCC14066, Corynebacterium glutamicum ATCC13032, Cory nebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum
ATCC 15354, Pseudomonas sp. D- 0110等をあげることができる。 ATCC 15354, Pseudomonas sp. D-0110 and the like.
組換えベクターの導入方法としては、 上記宿主細胞へ D N Aを導入する方法 であればいずれも用いることができ、 例えば、 カルシウムイオンを用いる方法 As a method for introducing a recombinant vector, any method for introducing DNA into the above host cells can be used, for example, a method using calcium ions
[Proc. Natl . Acad. Sci . USA, 69, 2110 ( 1972)] 、 プロトプラスト法 （特開 昭 63- 2483942) 、 エレクトロポレーシヨン法 [Gene, 17, 107 ( 1982)、 Molecular & General Genetics, 168, 111 ( 1979)] 等をあげることができる。 USA, 69, 2110 (1972)], the protoplast method (JP-A-63-2483942), the electroporation method [Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].
酵母を宿主細胞として用いる場合には、 発現べクタ一として、 例えば、 YEpl3 If yeast is used as the host cell, use YEpl3 as an expression vector.
(ATCC37115) 、 YEp24 (ATCC37051) 、 YCp50 (ATCC37419) 、 pHS19 pHS15等を 用いることができる。 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19 and pHS15 can be used.
プロモ一夕一としては、 酵母中で発現できるものであればいずれのものを用 いてもよく、 例えば、 ΙΉ05プロモー夕一、 PGKプロモ一夕一、 GAPプロモ一夕一、 ADHプロモー夕一、 gal 1プロモーター、 gal 10プロモーター、 ヒートショック 蛋白質プロモ一夕一、 MF a l プロモー夕一、 CUP 1プロモ一夕一等をあげること ができる。  Any promoter can be used as long as it can be expressed in yeast. For example, ΙΉ05 Promo One, PGK Promo One, GAP Promo One, ADH Promo One, gal 1 promoter, gal 10 promoter, heat shock protein promoter, MF al promoter, CUP 1 promoter, etc.
宿主細胞としては、 サヅカロマイセス属、 シゾサヅカロマイセス属、 クルイ ベロミセス属、 トリコスポロン属、 シヮニォミセス属、 ピチア属等に属する酵 母、 例えは、 Saccharomyces cerevisiaeヽ Schizosaccharomyces pombe、 Kluyveromyces lactis、 Trichosporon pullulans、 Schwanniomyces alluvius、 Pichia pastor i s等をあげることができる。 Examples of host cells include yeasts belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schizinomyces, Pichia, etc., such as Saccharomyces cerevisiae Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastor is and the like.
組換えベクターの導入方法としては、 酵母に DN Aを導入する方法であれば いずれも用いることができ、例えば、エレクトロポレ一シヨン法 [Methods inEnz ymology, 194, 182 (1990)] 、 スフエロプラスト法 [Proc. Natl. Acad. Sci. U SA, 81, 4889 (1984)] 、 酢酸リチウム法 [Journal of Bacteriology, 153, 163 (1983)] 等をあげることができる。  As a method for introducing a recombinant vector, any method for introducing DNA into yeast can be used. For example, electroporation [Methods in Enzymology, 194, 182 (1990)], spheroplast Natl. Acad. Sci. USA, 81, 4889 (1984)], lithium acetate method [Journal of Bacteriology, 153, 163 (1983)], and the like.
動物細胞を宿主として用いる場合には、 発現べクタ一として、 例えば、 pc DNA I/Amp (インビトロジェン社）、 p cDNAI、 p CDM8 [Nature, 329, 840 (1987)] 、 pAGE 107 [特開平 3- 22979、 Cytotechnology, 3, 133 (1990)] 、 pREP4 (インビトロジェン社） 、 pAGE 103 [Journal of Biochemistry, 101, 1307 (1987)] 、 pAMo、 pAMoA、 pAS 3 - 3 ( 特開平 2-227075) 等が用いられる。  When an animal cell is used as a host, examples of expression vectors include pc DNA I / Amp (Invitrogen), pcDNAI, pCDM8 [Nature, 329, 840 (1987)], pAGE107 [ -22979, Cytotechnology, 3, 133 (1990)], pREP4 (Invitrogen), pAGE 103 [Journal of Biochemistry, 101, 1307 (1987)], pAMo, pAMoA, pAS3-3 (Japanese Patent Laid-Open No. 2-227075), etc. Is used.
プロモ一夕一としては、 動物細胞中で発現できるものであればいずれも用い ることができ、 例えば、 サイ トメガロウィルス （CMV) の IE (i麗 ediate early ) 遺伝子のプロモ一夕一、 SV40の初期プロモー夕一、 メタ口チォネインのプロ モーター、 レトロウイルスのプロモ一夕一、 ヒートショックプロモ一夕一、 S Rひプロモ一夕一等をあげることができる。 また、 ヒト CMVの IE遺伝子のェンハ ンサーをプロモーターと共に用いてもよい。  Any promoter can be used as long as it can be expressed in animal cells. The early promoter of the first, the promoter of the meta mouth chainonein, the promoter of the retrovirus, the one of the heat shock promoter, the one of the SR hypromo, etc. Further, the enhancer of the IE gene of human CMV may be used together with the promoter.
宿主細胞としては、 マウス · ミエ口一マ細胞、 ラヅト ' ミエローマ細胞、 マ ウス 'ハイブリ ドーマ細胞、ヒ卜の細胞であるナマルバ（Namalwa)細胞、 Namalwa KJM- 1細胞、 ヒト胎児腎臓細胞またはヒト白血病細胞、 アフリカミ ドリザル腎 臓細胞、 チャイニーズ 'ハムスターの細胞である CH0細胞または HBT5637 (特開 昭 63- 299) 等をあげることができる。  Host cells include mouse myeloma cells, rat 'myeloma cells', mouse' hybridoma cells, human namalwa cells, Namalwa KJM-1 cells, human fetal kidney cells or human leukemia Cell, African green monkey kidney cell, Chinese hamster cell CH0 cell or HBT5637 (JP-A-63-299).
マウス ' ミエローマ細胞としては、 SP2/0、 NS0等、 ラット ' ミエ口一マ細胞 としては YB2/0等、 ヒト胎児腎臓細胞としては HEK293(ATCC: CRL-1573), 293等、 ヒト白血病細胞としては、 BALL- 1等、 アフリカミ ドリザル腎臓細胞としては COS- 1、 COS- 7等をあげることができる。 Mouse '' myeloma cells include SP2 / 0, NS0, etc., Rat '' myeoma cells include YB2 / 0, etc., and human fetal kidney cells HEK293 (ATCC: CRL-1573), 293, etc. Human leukemia cells include BALL-1 and the like, and African green monkey kidney cells include COS-1 and COS-7.
組換えベクターの導入方法としては、 動物細胞に D N Aを導入する方法であ ればいずれも用いることができ、 例えば、 エレクトロボレ一シヨン法 [ Cytotechnology, 3, 133 ( 1990)] 、 リン酸カルシウム法 （特開平 2-227075)、 リポフエクシヨン法 [Proc. Natl . Acad. Sci . USA, 84, 7413 ( 1987)]、 Virology, 52, 456 ( 1973)に記載の方法等をあげることができる。  Any method for introducing a recombinant vector can be used so long as it is a method for introducing DNA into animal cells. Examples of the method include the electroporation method [Cytotechnology, 3, 133 (1990)] and the calcium phosphate method. Kaihei 2-227075), the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], and the methods described in Virology, 52, 456 (1973).
昆虫細胞を宿主細胞として用いる場合には、 例えばバキュロウィルス 'イク スプレッシヨン 'ベクターズ ァ 'ラボラトリー'マニュアル [Baculovirus Expre ssion Vectors, A Laboratory Manual, W. H. Freeman ana Company, New York ( 1992)] 、 モレキュラー ·バイオロジー ァ ·ラボラトリ一 ·マニュアル （Molec ular Biology, A Laboratory Manual) ヽ カレン卜 - フ。口 卜コ——クレス、 ·イン ·モ レキユラ一 'バイオロジー 、 Bio/Technology, 6, 47 ( 1988)等に記載された方 法によって、 蛋白質を発現することができる。  When insect cells are used as host cells, for example, Baculovirus 'Expression' Vectors 'Laboratory' Manual [Baculovirus Expression Vectors, A Laboratory Manual, WH Freemanana Company, New York (1992)], Molecular Bio Logistic Biology, A Laboratory Manual ヽ Current-f. Proteins can be expressed by the method described in, for example, "Kores, Cress, in Molecular Biology", Biology, Bio / Technology, 6, 47 (1988).
即ち、 組換え遺伝子導入ベクターおよびバキュロウィルスを昆虫細胞に共導 入して昆虫細胞培養上清中に組換えゥィルスを得た後、 さらに組換えゥィルス を昆虫細胞に感染させ、 蛋白質を発現させることができる。  That is, the recombinant gene transfer vector and baculovirus are co-transfected into insect cells to obtain recombinant virus in the culture supernatant of insect cells, and then the recombinant virus is infected with insect cells to express the protein. Can be.
該方法において用いられる遺伝子導入べクタ一としては、 例えば、 pVL1392、 pVL1393、 pBlueBacI I I (ともにインビトロジェン社） 等をあげることができる。 バキュロウィルスとしては、 例えば、 夜盗蛾科昆虫に感染するウィルスであ るァゥトグラファ ·力リフォルニ力 ·ヌクレア一 ·ポリへドロシス ·ウィルス (Autographa californica nuclear polyhedrosis virus) 等を用レヽることがで きる。  Examples of the gene transfer vector used in the method include pVL1392, pVL1393, and pBlueBacII (all from Invitrogen) and the like. As the baculovirus, for example, a virus that infects night moth insects, such as Atographa, Ariformi, Nuclea, Polyhedrosis, and the like (Autographa californica nuclear polyhedrosis virus) can be used.
宿主細胞としては、 Spodoptera frugiperdaの卵巣細胞、 Trichoplusia の 卵巣細胞、 力ィコ卵巣由来の培養細胞等を用いることができる。  As the host cells, ovary cells of Spodoptera frugiperda, ovary cells of Trichoplusia, cultured cells derived from phytococcus ovary and the like can be used.
Spodoptera frugiperdaの卵巣細胞としては Sf9、 Sf21 (バキュロウィルス ' イクスプレツシヨン 'ベクタ一ズ ァ 'ラボラトリ一 'マニュアル）等、 Trichoplu ^ の卵巣細胞としては High 5、 BTI-TN-5B1-4 (インビトロジヱン社） 等、 カイコ卵巣由来の培養細胞としては Bombyx mori N4等をあげることができる。 組換えゥィルスを調製するための、 昆虫細胞への上記組換え遺伝子導入べク 夕一と上記バキュロウィルスの共導入方法としては、 例えば、 リン酸カルシゥ ム法 （特開平 2-227075) 、 リポフエクシヨン法 [Proc. Natl . Acad. Sci . USA, 84, 7413 ( 1987)] 等をあげることができる。 The ovarian cells of Spodoptera frugiperda include Sf9 and Sf21 (baculovirus '' High ovarian cells of Trichoplu ^, BTI-TN-5B1-4 (Invitrogen), etc. Bombyx mori as cultured cells derived from silkworm ovary N4 etc. can be given. Examples of a method for co-transferring the above-described recombinant gene into insect cells and the above baculovirus into an insect cell for preparing a recombinant virus include a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a lipofection method, and the like. [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
植物細胞を宿主として用いる場合には、 発現べクタ一として、 例えば、 T i プラスミ ド、 タバコモザイクウィルスベクタ一等を用いることができる。  When a plant cell is used as a host, for example, Ti plasmid, tobacco mosaic virus vector, or the like can be used as an expression vector.
プロモ一夕一としては、 植物細胞中で発現できるものであればいずれのもの を用いてもよく、 例えば、 カリフラワーモザイクウィルス （CaMV) の 35Sプロモ 一夕一、 ィネアクチン 1プロモー夕一等をあげることができる。  Any promoter can be used as long as it can be expressed in plant cells. For example, cauliflower mosaic virus (CaMV) 35S promoter, inineactin 1 promoter, etc. Can be.
宿主細胞としては、 タバコ、 ジャガイモ、 トマト、 ニンジン、 ダイズ、 アブ ラナ、 アルフアルファ、 イネ、 コムギ、 ォォムギ等の植物細胞等をあげること ができる。  Examples of the host cell include plant cells of tobacco, potato, tomato, carrot, soybean, abrana, alfa alfa, rice, wheat, wheat, and the like.
組換えべクタ一の導入方法としては、 植物細胞に D N Aを導入する方法であ ればいずれも用いることができ、 例えば、 ァグロパクテリゥム (Agrobacterium ) (特開昭 59-140885、 特閧昭 60- 70080、 W094/00977) 、 エレクトロポレーショ ン法 （特閧昭 60- 251887) 、 パ一ティクルガン （遺伝子銃） を用いる方法 （特許 第 2606856、 特許第 2517813) 等をあげることができる。  As a method for introducing the recombinant vector, any method for introducing DNA into plant cells can be used. For example, Agrobacterium (Japanese Patent Application Laid-Open No. 59-140885, 60-70080, W094 / 00977), an electroporation method (Japanese Patent Application 60-251887), a method using a particle gun (gene gun) (Patent No. 2606856, Patent No. 2517813), and the like.
遺伝子の発現方法としては、 直接発現以外に、 モレキュラー 'クローニング 第 2版に記載されている方法等に準じて、 分泌生産、 融合タンパク質発現等を 行うことができる。  As a method for expressing the gene, secretory production, fusion protein expression and the like can be performed according to the method described in Molecular 'Cloning 2nd edition, etc., in addition to direct expression.
酵母、 動物細胞、 昆虫細胞または植物細胞により発現させた場合には、 糖あ るいは糖鎖が付加された蛋白質を得ることができる。  When expressed by yeast, animal cells, insect cells or plant cells, a sugar or sugar chain-added protein can be obtained.
以上のようにして得られる形質転換体を培地に培養し、 培養物中に本発明の 蛋白質を生成蓄積させ、 該培養物から採取することにより、 本発明の蛋白質を 製造することができる。 本発明の形質転換体を培地に培養する方法は、 宿主の 培養に用レ、られる通常の方法に従って行うことができる。 The transformant obtained as described above is cultured in a medium, and the culture of the present invention is added to the culture. The protein of the present invention can be produced by producing and accumulating the protein and collecting the protein from the culture. The method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
また、 患者の生体内から採取した細胞に、 適切な本発明の蛋白質を発現させ るための発現べクタ一を導入した後、 細胞を生体内に戻すことにより、 本発明 の蛋白質を患者の生体内で発現させることもできる。  In addition, after introducing an appropriate expression vector for expressing the protein of the present invention into cells collected from a patient's living body, the protein of the present invention is returned to the living body by introducing the protein of the present invention into the patient. It can also be expressed in the body.
( 2 ) 形質転換体の培養  (2) Culture of transformants
本発明の形質転換体を培地に培養する方法は、 宿主の培養に用いられる通常 の方法に従って行うことができる。  The method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
ェシヱリヒア ·コリ等の原核生物あるいは酵母等の真核生物を宿主として得 られた形質転換体を培養する培地としては、 該生物が資化し得る炭素源、 窒素 源、 無機塩類等を含有し、 形質転換体の培養を効率的に行える培地であれば天 然培地、 合成培地のいずれを用いてもよい。  A culture medium for culturing a transformant obtained using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like that can be used by the organism. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.
炭素源としては、 該生物が資化し得るものであればよく、 グルコース、 フラ クト一ス、 スクロース、 これらを含有する糖蜜、 デンプンあるいはデンプン加 水分解物等の炭水化物、 酢酸、 プロピオン酸等の有機酸、 エタノール、 プロパ ノール等のアルコ一ル類等を用 、ることができる。  The carbon source may be any one that can be assimilated by the organism, such as glucose, fructose, sucrose, molasses containing them, carbohydrates such as starch or hydrolyzed starch, and organic acids such as acetic acid and propionic acid. Alcohols such as acid, ethanol, and propanol can be used.
窒素源としては、 アンモニア、 塩化アンモニゥム、 硫酸アンモニゥム、 酢酸 アンモニゥム、 リン酸アンモニゥム等の無機酸もしくは有機酸のアンモニゥム 塩、 その他の含窒素化合物、 並びに、 ペプトン、 肉エキス、 酵母エキス、 コ一 ンスチープリカ一、 カゼイン加水分解物、 大豆粕および大豆粕加水分解物、 各 種発酵菌体、 およびその消化物等を用 、ることができる。  Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other inorganic or organic acid ammonium salts, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, and copper extract. In addition, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.
無機物としては、 リン酸第一カリウム、 リン酸第二カリウム、 リン酸マグネ シゥム、 硫酸マグネシウム、 塩化ナトリウム、 硫酸第一鉄、 硫酸マンガン、 硫 酸銅、 炭酸カルシウム等を用いることができる。  As the inorganic substance, potassium potassium phosphate, potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
培養は、 通常振盪培養または深部通気攪拌培養等の好気的条件下で行う。 培 養温度は 1 5〜4 0 °Cがよく、 培養時間は、 通常 1 6時間〜 7日間である。 培 養中 p Hは 3 . 0〜9 . 0に保持する。 p Hの調整は、 無機または有機の酸、 アルカリ溶液、 尿素、 炭酸カルシウム、 アンモニア等を用いて行う。 The cultivation is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. Culture The fermentation temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days. During the cultivation, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
また、 培養中必要に応じて、 アンピシリンやテトラサイクリン等の抗生物質 を培地に添加してもよい。  If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.
プロモーターとして誘導性のプロモーターを用いた組換えベクターで形質転 換した微生物を培養するときには、 必要に応じてインデューサーを培地に添加 してもよい。 例えば、 1^プロモー夕一を用いた発現べクタ一で形質転換した微 生物を培養するときにはイソプロピル一 5— D—チォガラクトビラノシド等を、 ^2プロモー夕一を用いた発現ベクターで形質転換した微生物を培養するとき にはィンドールァクリル酸等を培地に添加してもよい。  When culturing a microorganism transformed with a recombinant vector using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using a 1 ^ promoter, isopropyl-15-D-thiogalactoviranoside or the like is transformed with an expression vector using a ^ 2 promoter. When culturing the transformed microorganism, indoleacrylic acid or the like may be added to the medium.
動物細胞を宿主として得られた形質転換体を培養する培地としては、 一般に 使用されている RPMI 1640培地 [The Journal of the American Medical Associatio n， 199, 519 ( 1967)]、 Eagleの MEM培地 [Science, 122, 501 ( 1952)] 、 ダル べッコ改変醒培地 [Virology, 8, 396 ( 1959)]、 199培地 [Proceeding of the Society for the Biological Medicine, 73, 1 ( 1950)] またはこれら培地に牛 胎児血清等を添加した培地等を用いることができる。  As culture media for culturing transformants obtained using animal cells as hosts, commonly used RPMI 1640 media [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM media [Science , 122, 501 (1952)], Dar-Becco's modified medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] or A medium containing bovine fetal serum or the like can be used.
培養は、 通常 p H 6〜8、 3 0〜4 0 ° 5 % C 02存在下等の条件下で 1〜 7日間行う。 Culture is carried out usually p H 6~8, 3 0~4 0 ° 5% C 0 2 under the conditions such as the presence 1-7 days.
また、 培養中必要に応じて、 カナマイシン、 ペニシリン、 ストレプトマイシ ン等の抗生物質を培地に添加してもよい。  If necessary, antibiotics such as kanamycin, penicillin, and streptomycin may be added to the medium during the culture.
昆虫細胞を宿主として得られた形質転換体を培養する培地としては、 一般に 使用されている TNM-FH培地 [ファーミンジェン （Pharmingen) 社] 、 Sf- 900 I I SFM培地 （ライフ 'テクノロジ一ズ社） 、 ExCell400、 ExCell405 [いずれも JRH ノ ィォサイエンシーズ (JRH Biosciences)社]、 Grace' s Insect Medium [Nature 195， 788 ( 1962)] 等を用いることができる。 培養は、 通常 p H 6〜7、 2 5〜3 0 °C等の条件下で 1 ~ 5日間行う。 As culture media for transformants obtained using insect cells as hosts, commonly used TNM-FH media [Pharmingen], Sf-900 II SFM media (Life's Technologies, Inc.) ), ExCell400, ExCell405 [JRH Biosciences, Inc.], Grace's Insect Medium [Nature 195, 788 (1962)] and the like can be used. The cultivation is usually performed under conditions of pH 6 to 7, 25 to 30 ° C, etc. for 1 to 5 days.
また、 培養中必要に応じて、 ゲン夕マイシン等の抗生物質を培地に添加して もよい。  If necessary, an antibiotic such as genyumycin may be added to the medium during the culture.
植物細胞を宿主として得られた形質転換体は、 細胞として、 または植物の細 胞ゃ器官に分化させて培養することができる。 該形質転換体を培養する培地と しては、 一般に使用されているムラシゲ 'アンド 'スク一グ (MS)培地、 ホワイ ト（White)培地、 またはこれら培地にオーキシン、 サイ トカイニン等、 植物ホル モンを添加した培地等を用いることができる。  A transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a cell organ of a plant. As a medium for culturing the transformant, commonly used Murashige 'and' squeeg (MS) medium, white (White) medium, or plant medium such as auxin, cytokinin, etc. And the like can be used.
培養は、 通常 p H 5〜9、 2 0〜4 0 °Cの条件下で 3〜6 0日間行う。  The cultivation is usually performed at pH 5 to 9, 20 to 40 ° C for 3 to 60 days.
また、 培養中必要に応じて、 カナマイシン、 ハイグロマイシン等の抗生物質 を培地に添加してもよい。  If necessary, antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.
上記のとおり、 本発明の蛋白質をコードする D N Aを組み込んだ組換え体べ クタ一を保有する原核生物、 酵母、 動物細胞、 昆虫細胞または植物細胞由来の 形質転換体を、 通常の培養方法に従って培養し、 該蛋白質を生成蓄積させ、 該 培養物より該蛋白質を採取することにより、 該蛋白質を製造することができる。 遺伝子の発現方法としては、 直接発現以外に、 モレキュラー 'クローニング 第 2版に記載されている方法等に準じて、 分泌生産、 融合蛋白質発現等を行う ことができる。  As described above, a transformant derived from a prokaryote, yeast, animal cell, insect cell or plant cell having a recombinant vector into which the DNA encoding the protein of the present invention has been incorporated is cultured according to a conventional culture method. The protein can be produced by accumulating and producing the protein, and collecting the protein from the culture. As a method for expressing a gene, in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular 'Cloning 2nd edition.
本発明の蛋白質の生産方法としては、 宿主細胞内に生産させる方法、 宿主細 胞外に分泌させる方法、 あるいは宿主細胞外膜上に生産させる方法があり、 使 用する宿主細胞や、 生産させる蛋白質の構造を変えることにより、 該方法を選 択することができる。  The method for producing the protein of the present invention includes a method of producing the protein in a host cell, a method of secreting the protein out of the host cell, and a method of producing the protein on the outer membrane of the host cell. The method can be selected by changing the structure.
本発明の蛋白質が宿主細胞内あるいは宿主細胞外膜上に生産される場合、 ポ ールソンらの方法 [J. Biol . Chem. , 264, 17619 ( 1989)]、 ロウらの方法 [Proc. Natl. Acad. Sci. , USA, 86, 8227 ( 1989)、 Genes Develop. , 4, 1288 (1990) ] 、 または特開平 5- 336963、 特閧平 6- 823021等に記載の方法を準用することに P9 When the protein of the present invention is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)] and the method of Lowe et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)] or JP-A-5-336963, JP-A-6-823021, etc. P9
より、 該蛋白質を宿主細胞外に積極的に分泌させることができる。 Thus, the protein can be actively secreted out of the host cell.
すなわち、 遺伝子組換えの手法を用いて、 本発明の蛋白質の活性部位を含む 蛋白質の手前にシグナルべプチドを付加した形で発現させることにより、 本発 明の蛋白質を宿主細胞外に積極的に分泌させることができる。  That is, the protein of the present invention is expressed in a form in which a signal peptide is added in front of the protein containing the active site of the protein of the present invention using a gene recombination technique, so that the protein of the present invention can be positively extracellularly outside the host cells. It can be secreted.
また、 特開平 2-227075に記載されている方法に準じて、 ジヒドロ葉酸還元酵 素遺伝子等を用いた遺伝子増幅系を利用して生産量を上昇させることもできる。 さらに、 遺伝子導入した動物または植物の細胞を再分化させることにより、 遺伝子が導入された動物個体 （トランスジエニック非ヒト動物） または植物個 体 （トランスジエニック植物） を造成し、 これらの個体を用いて本発明の蛋白 質を製造することもできる。  Further, according to the method described in Japanese Patent Application Laid-Open No. 2-227075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like. Furthermore, the transgenic animal or plant cells are redifferentiated to create transgenic non-human animals or transgenic plants (transgenic plants) into which the gene has been introduced. Can be used to produce the protein of the present invention.
形質転換体が動物個体または植物個体の場合は、 通常の方法に従って、 飼育 または栽培し、 該蛋白質を生成蓄積させ、 該動物個体または植物個体より該蛋 白質を採取することにより、 該蛋白質を製造することができる。  When the transformant is an animal or plant individual, the protein is produced by breeding or cultivating according to a usual method to produce and accumulate the protein, and collecting the protein from the animal or plant individual. can do.
動物個体を用いて本発明の蛋白質を製造する方法としては、 例えば公知の方 法 [American Journal of Clinical Nutrition, 63， 639S 996)、 American Jour nal of Clinical Nutrition, 63, 627S ( 1996 )、 Bio/Technology, 9, 830 ( 1991 )] に準じて遺伝子を導入して造成した動物中に本発明の蛋白質を生産する方法 があげられる。  Methods for producing the protein of the present invention using animal individuals include, for example, known methods (American Journal of Clinical Nutrition, 63, 639S996), American Journal of Clinical Nutrition, 63, 627S (1996), Bio / Technology, 9, 830 (1991)], and a method for producing the protein of the present invention in an animal constructed by introducing a gene.
動物個体の場合は、 例えば、 本発明の蛋白質をコードする D N Aを導入した トランスジエニック非ヒト動物を飼育し、 該蛋白質を該動物中に生成 ·蓄積さ せ、 該動物中より該蛋白質を採取することにより、 該蛋白質を製造することが できる。 該動物中の生成 ·蓄積場所としては、 例えば、 該動物のミルク （特開 昭 63- 309192) 、 卵等をあげることができる。 この際に用いられるプロモ一夕一 としては、 動物で発現できるものであればいずれも用いることができるが、 例 えば、 乳腺細胞特異的なプロモーターであるひカゼインプロモー夕一、 カゼ インプロモーター、 ラクトグロブリンプロモ一夕一、 ホェ一酸性プロテイン プロモー夕一等が好適に用いられる。 In the case of an animal individual, for example, a transgenic non-human animal into which DNA encoding the protein of the present invention has been introduced is bred, the protein is produced and accumulated in the animal, and the protein is collected from the animal. By doing so, the protein can be produced. Examples of the place of production and accumulation in the animal include milk of the animal (JP-A-63-309192), eggs, and the like. As the promoter used at this time, any promoter that can be expressed in animals can be used. For example, the promoter, casein promoter, casein promoter, and lactate, which are breast cell-specific promoters, can be used. Globulin Promo overnight, whey mono-protein Promoter or the like is preferably used.
植物個体を用いて本発明の蛋白質を製造する方法としては、 例えば本発明の 蛋白質をコードする D NAを導入したトランスジェニヅク植物を公知の方法 [ 組織培養， 20 ( 1994)、 組織培養， 21 ( 1995 )、 Trends in Biotechnology, 15, 45 ( 1997)] に準じて栽培し、 該蛋白質を該植物中に生成 ·蓄積させ、 該植物中よ り該蛋白質を採取することにより、 該蛋白質を生産する方法があげられる。 ( 3 ) 発現させた蛋白質の単離精製  Examples of a method for producing the protein of the present invention using plant individuals include, for example, transgenic plants into which DNA encoding the protein of the present invention has been introduced [Tissue Culture, 20 (1994), 21 (1995), Trends in Biotechnology, 15, 45 (1997)], producing and accumulating the protein in the plant, and collecting the protein from the plant to produce the protein. There is a way to do it. (3) Isolation and purification of expressed protein
本発明の形質転換体により製造された蛋白質を単離精製するためには、 通常 の酵素の単離、 精製法を用いればよい。 また、 該蛋白質自体に対する抗体を用 いるァフィ二ティ一クロマトグラフィーによって効率的に精製することができ る。  In order to isolate and purify the protein produced by the transformant of the present invention, a conventional enzyme isolation and purification method may be used. Further, the protein can be efficiently purified by affinity chromatography using an antibody against the protein itself.
本発明の形質転換体により製造された蛋白質は、 例えば本発明の蛋白質が、 細胞内に溶解状態で発現した場合には、 培養等の終了後、 細胞を遠心分離によ り回収し、 水系緩衝液にけん濁後、 超音波破砕機、 フレンチプレス、 マントン ガウリンホモゲナイザー、 ダイノミル等により細胞を破砕し、 無細胞抽出液を 得る。 該無細胞抽出液を遠心分離することにより得られる上清から、 通常の酵 素の単離精製法、 即ち、 溶媒抽出法、 硫安等による塩析法、 脱塩法、 有機溶媒 による沈殿法、 ジェチルアミノエチル （DEAE) —セファロ一ス、 DIAION HPA- 75 (三菱化成社） 等レジンを用いた陰イオン交換クロマトグラフィー法、 S- Sepharose FF (Pharmacia社） 等のレジンを用いた陽イオン交換クロマトグラフ ィ一法、 プチルセファロース、 フエ二ルセファロ一ス等のレジンを用いた疎水 性クロマトグラフィー法、 分子篩を用いたゲルろ過法、 ァフィ二ティークロマ トグラフィ一法、 クロマトフォ一カシング法、 等電点電気泳動等の電気泳動法 等の手法を単独あるいは組み合わせて用い、 精製標品を得ることができる。 また、 該蛋白質が細胞内に不溶体を形成して発現した場合は、 同様に細胞を 回収後破砕し、 遠心分離を行うことにより、 沈殿画分として蛋白質の不溶体を /JP 7335 For example, when the protein of the present invention is expressed in a dissolved state in cells, the cells produced by the transformant of the present invention are collected by centrifugation after completion of culturing, etc. After suspending the liquid, the cells are crushed by an ultrasonic crusher, French press, Menton-Gaurin homogenizer, Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal enzyme isolation and purification method, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Getylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (Mitsubishi Kasei) Anion exchange chromatography using resin, cation exchange using S-Sepharose FF (Pharmacia) etc. Chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenylsepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectrics A purified sample can be obtained using an electrophoresis method such as point electrophoresis alone or in combination. When the protein is expressed by forming an insoluble form in the cells, the cells are similarly collected, crushed, and centrifuged to remove the protein insoluble form as a precipitate fraction. / JP 7335
回収する。 回収した蛋白質の不溶体を蛋白質変性剤等で可溶化する。 該可溶化 液を希釈または透析することにより、 該蛋白質を正常な立体構造に戻した後、 上記と同様の単離精製法により該蛋白質の精製標品を得ることができる。 to recover. The insoluble form of the recovered protein is solubilized with a protein denaturant or the like. After diluting or dialyzing the solubilized solution to return the protein to a normal three-dimensional structure, a purified sample of the protein can be obtained by the same isolation and purification method as described above.
本発明の蛋白質あるいはその糖修飾体等の誘導体が細胞外に分泌された場合 には、 培養上清に該蛋白質あるいはその糖鎖付加体等の誘導体を回収すること ができる。  When the protein of the present invention or its derivative such as a modified sugar is secreted extracellularly, the protein or its derivative such as a sugar chain adduct can be recovered in the culture supernatant.
即ち、 該培養物を上記と同様の遠心分離等の手法により処理することにより 可溶性画分を取得し、 該可溶性画分から、 上記と同様の単離精製法を用いるこ とにより、 精製標品を得ることができる。  That is, a purified fraction is obtained by treating the culture by a method such as centrifugation as described above to obtain a soluble fraction, and by using the same isolation and purification method as described above from the soluble fraction. Obtainable.
また、 本発明の蛋白質を他のタンパク質との融合タンパク質として生産し、 融合したタンパク質に親和性をもつ物質を用いたァフィ二ティークロマトグラ フィーを利用して精製することもできる。 例えば、 ロウらの方法 [Proc. Natl . Acad. Sci . USA, 86, 8227 ( 1989)、 Genes Develop. , 4, 1288 ( 1990)] 、 特開 平 5- 336963、 特閧平 6- 823021に記載の方法に準じて、 本発明の蛋白質をプロテ ィン Aとの融合タンパク質として生産し、 ィムノグロプリン Gを用いるァフィ 二ティ一クロマトグラフィーにより精製することができる。 また、 本発明の蛋 白質を F 1 a gぺプチドとの融合タンパク質として生産し、 抗 F 1 a g抗体を 用いるァフィ二ティ一クロマトグラフィーにより精製することができる [Proc. Natl . Acad. Sci. USA, 86, 8227 ( 1989)、 Genes Develop. , 4, 1288 ( 1990)] 。 さらに、 該蛋白質自身に対する抗体を用いたァフィ二ティーク口マトグラフ ィ一で精製することもできる。  Alternatively, the protein of the present invention can be produced as a fusion protein with another protein, and purified using affinity chromatography using a substance having an affinity for the fused protein. For example, the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], JP-A-5-336963, JP-A-6-823021. According to the method described, the protein of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using imnoglobulin G. In addition, the protein of the present invention can be produced as a fusion protein with an F1ag peptide and purified by affinity chromatography using an anti-F1ag antibody [Proc. Natl. Acad. Sci. USA , 86, 8227 (1989), Genes Develop., 4, 1288 (1990)]. Further, the protein can be purified by affinity chromatography using an antibody against the protein itself.
このようにして取得される蛋白質として、 例えば、 配列番号 1に示されるァ ミノ酸配列を有する蛋白質をあげることができる。  Examples of the protein obtained in this manner include a protein having the amino acid sequence shown in SEQ ID NO: 1.
また、 本発明の蛋白質は、 F m o c法 （フルォレニルメチルォキシカルボ二 ル法） 、 t B o c法 （t _ブチルォキシカルボニル法） 等の化学合成法によつ ても製造することができる。 また、 ァドバンスト ·ケムテック （Advanced ChemTech) 社、 パーキン ·エル マ一社、 フアルマシア社、 プロテイン 'テクノロジ f ンストウルメント （The protein of the present invention can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t_butyloxycarbonyl method). be able to. Also, Advanced ChemTech, Perkin Elma, Pharmacia, and Protein Technology
Protein Technology Instrument) 社ヽ シンセセノレ ·ベガ (Synthecell-Vega) 社、 パーセプティブ （PerSeptive) 社、 島津製作所等のペプチド合成機を利用 し化学合成することもできる。 Protein Technology Instrument Company Chemical synthesis can also be performed using a peptide synthesizer such as Synthecell-Vega, PerSeptive, or Shimadzu Corporation.
精製した本発明の蛋白質の構造解析は、 蛋白質化学で通常用いられる方法、 例えば遺伝子クローニングのためのタンパク質構造解析 （平野久著、 東京化学 同人発行、 1 9 9 3年） に記載の方法により行うことができる。  Structural analysis of the purified protein of the present invention is performed by a method generally used in protein chemistry, for example, a method described in Protein Structural Analysis for Gene Cloning (Hisashi Hirano, published by Tokyo Kagaku Dojin, 1993). be able to.
[ 3 ] 本発明の蛋白質を認識する抗体の調製 [3] Preparation of antibody recognizing the protein of the present invention
本発明の蛋白質または該蛋白質の部分断片ポリべプチドの精製標品、 あるい は本発明の蛋白質の一部のアミノ酸配列を有するぺプチドを抗原として用いる ことにより、 ポリクローナル抗体、 モノクローナル抗体等、 本発明の蛋白質を 認識する抗体を作製することができる。  By using a purified sample of the protein of the present invention or a polypeptide fragment of the protein fragment, or a peptide having a partial amino acid sequence of the protein of the present invention as an antigen, polyclonal antibodies, monoclonal antibodies, etc. An antibody that recognizes the protein of the present invention can be prepared.
( 1 ) ポリクローナル抗体の調製  (1) Preparation of polyclonal antibody
上記 [ 2 ] に記載の方法により取得した本発明の蛋白質の全長または部分断 片精製標品を抗原として用い、 動物に投与することによりポリクロ一ナル抗体 を作製することができる。  A polyclonal antibody can be prepared by using a purified full-length or partial fragment of the protein of the present invention obtained by the method described in the above [2] as an antigen and administering it to an animal.
投与する動物として、 ゥサギ、 ャギ、 3 ~ 2 0週令のラット、 マウス、 ハム スター等を用いることができる。  As animals to be administered, egrets, goats, rats, mice, hamsters, etc., which are 3 to 20 weeks old can be used.
該抗原の投与量は動物 1匹当たり 5 0〜1 0 0 gが好ましい。  The dose of the antigen is preferably 50 to 100 g per animal.
ペプチドを用いる場合は、 ペプチドをスカシガイへモシァニン （keyhole limp et haemocyanin) ゃ牛チログロブリン等のキャリア蛋白に共有結合させたもの を抗原とするのが望ましい。 抗原とするペプチドは、 ペプチド合成機で合成す ることができる。  When a peptide is used, it is preferable to use, as the antigen, a peptide covalently bonded to a carrier protein such as keyhole limp et haemocyanin or bovine thyroglobulin. The peptide serving as the antigen can be synthesized by a peptide synthesizer.
該抗原の投与は、 1 回目の投与の後 1〜2週間おきに 3 ~ 1 0回行う。 各投 与後、 3〜7日目に眼底静脈叢より採血し、 該血清が免疫に用いた抗原と反応 5 The administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the serum reacts with the antigen used for immunization. Five
することを酵素免疫測定法 [酵素免疫測定法 （ELISA法） ：医学書院刊 1976年、 Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory Press ( 1988)Enzyme-linked immunosorbent assay [ELISA method: published by Medical Shoin 1976, Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)]
] 等で確認する。 ] And confirm.
免疫に用いた抗原に対し、 該血清が充分な抗体価を示した非ヒトほ乳動物よ り血清を取得し、 該血清を分離、 精製することによりポリクロ一ナル抗体を取 得することができる。  A polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
分離、 精製する方法としては、 遠心分離、 4 0〜 5 0 %飽和硫酸アンモニゥ ムによる塩析、 力プリル酸沈殿 [Antibodies,A Laboratory Manual, Cold Spring Harbor Laboratory, ( 1988)] 、 または D E A E—セファロ一スカラム、 陰ィォ ン交換カラム、 プロティン Aまたは G—力ラムあるいはゲル濾過カラム等を用 いるクロマトグラフィー等を、 単独または組み合わせて処理する方法があげら れる。  Methods for separation and purification include centrifugation, salting out with 40-50% saturated ammonium sulfate, and prillic acid precipitation [Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)], or DEAE-cephalo. A single column, an anion exchange column, a chromatography using a protein A or G-column or a gel filtration column, etc., may be used alone or in combination.
( 2 ) モノクローナル抗体の調製  (2) Preparation of monoclonal antibody
(2-1 )抗体産生細胞の調製 (2-1) Preparation of antibody-producing cells
上記（1 )において、 免疫に用いた抗原に対し、 その血清が十分な抗体価を示し たラットを抗体産生細胞の供給源として供する。  In the above (1), a rat whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody-producing cells.
該抗体価を示したラッ卜に抗原物質を最終投与した後 3〜 7日目に、 脾臓を 摘出する。  Three to seven days after the final administration of the antigenic substance to the rat showing the antibody titer, the spleen is removed.
該脾臓を M E M培地 （日水製薬社） 中で細断し、 ピンセットでほぐし、 1， 2 0 0 r p mで 5分間遠心分離した後、 上清を捨てる。  The spleen is shredded in MEM medium (Nissui Pharmaceutical), loosened with tweezers, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
得られた沈殿画分の脾細胞をトリス—塩化アンモニゥム緩衝液 （P H 7 . 6 5 ) で 1〜2分間処理し赤血球を除去した後、 M E M培地で 3回洗浄し、 得ら れた脾細胞を抗体産生細胞として用いる。  The spleen cells in the precipitate fraction thus obtained are treated with Tris-ammonium chloride buffer (PH7.65) for 1 to 2 minutes to remove red blood cells, and then washed three times with MEM medium. Is used as an antibody-producing cell.
(2- 2)骨髄腫細胞の調製 (2-2) Preparation of myeloma cells
骨髄腫細胞としては、 マウスまたはラットから取得した株化細胞を使用する。 例えば、 8-ァザグァニン耐性マウス （BALB/c由来）骨髄腫細胞株 P3- X63Ag8- U1(P 3-Ul) [Curr. Topics Microbiol. Immunol., 81, 1 (1978)、 Eur. J. Immunol., 6, 511 (1976)]、 SP2/0-Agl4(SP-2) [Nature, 276, 269 (1978)]、 P3-X63- Ag8653(653) [J. Immunol. , 123, 1548 (1979)]、 P3-X63-Ag8(X63) [Nature, 256 , 495 (1975)]等を用いることができる。 これらの細胞株は、 8—ァザグァニ ン培地 [RPMI— 1640培地にグルタミン （1.5mM)、 2—メルカプトェ 夕ノール （5 X 10"5M)、 ジェン夕マイシン （ 10〃g/ml) および牛胎児 血清 （FCS) (CSL社、 10%) を加えた培地 （以下、 正常培地という） に、 さらに 8—ァザグァニン （15〃g/ml) を加えた培地] で継代するが、 細胞融合の 3〜4日前に正常培地で培養し、 融合には該細胞を 2 X 107個以上 用いる。 As myeloma cells, cell lines obtained from mice or rats are used. For example, 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line P3-X63Ag8-U1 (P 3-Ul) [Curr. Topics Microbiol. Immunol., 81, 1 (1978), Eur. J. Immunol., 6, 511 (1976)], SP2 / 0-Agl4 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunol., 123, 1548 (1979)], P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)], etc. Can be. These cell lines consisted of 8-azaguanine medium [RPMI-1640 medium containing glutamine (1.5 mM), 2-mercaptoethanol (5 × 10 " 5 M), genyumycin (10 μg / ml) and fetal calf A medium containing serum (FCS) (CSL, 10%) (hereinafter referred to as a normal medium) and a medium supplemented with 8-azaguanine (15 µg / ml) is used. Culture in a normal medium 4 days before, and use 2 × 10 7 or more of the cells for fusion.
(2 - 3)ハイプリ ドーマの作製  (2-3) Preparation of Hypri-Doma
(2 - 1)で取得した抗体産生細胞と（2- 2)で取得した骨髄腫細胞を MEM培地ま たは PBS (リン酸ニナトリウム 1. 83g、 リン酸一カリウム 0. 21g、 食塩 7. 65g、 蒸留水 1リツトル、 pH7. 2) でよく洗浄し、 細胞数が、 抗体産生細胞：骨髄腫細胞 =5〜： L 0 ： 1になるよう混合し、 1, 200 rp mで 5分間遠心分離した後、 上清を捨てる。  The antibody-producing cells obtained in (2-1) and the myeloma cells obtained in (2-2) were added to a MEM medium or PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, salt 7. Wash well with 65 g, 1 liter of distilled water, pH 7.2), mix the cells so that the number of antibody-producing cells: myeloma cells = 5 ~: L 0: 1 and centrifuge at 1,200 rpm for 5 minutes After separation, discard the supernatant.
得られた沈澱画分の細胞群をよくほぐし、 該細胞群に、 攪拌しながら、 37 °Cで、 108抗体産生細胞あたり、 ポリエチレングライコール— 1000 (PE G— 1000) 2 g MEM 2 m 1およびジメチルスルホキシド （DM SO ) 0. 7mlを混合した溶液を 0. 2〜 lml添加し、 さらに 1〜 2分間毎に M E M培地 1〜 2 m 1を数回添加する。 Thoroughly loosened The resulting precipitated fraction of cell groups, the said group of cells, with stirring, at 37 ° C, 10 8 antibody-producing cells per polyethylene of glycol - 1000 (PE G- 1000) 2 g MEM 2 m Add 0.2 to 1 ml of a solution obtained by mixing 0.7 ml of 1 and dimethyl sulfoxide (DMSO), and then add 1 to 2 ml of MEM medium several times every 1 to 2 minutes.
添加後、 MEM培地を加えて全量が 50mlになるように調製する。  After addition, add MEM medium to adjust the total volume to 50 ml.
該調製液を 900 r p mで 5分間遠心分離後、 上清を捨てる。  After centrifuging the preparation at 900 rpm for 5 minutes, discard the supernatant.
得られた沈殿画分の細胞を、 ゆるやかにほぐした後、 メスピペットによる吸 込み、 吹出しでゆるやかに HAT培地 [正常培地にヒポキサンチン （10—4M) 、 チミジン （ 1. 5 X 10"5M) およびアミノブテリン （4 X 10— 7M) を加え た培地] 1 0 0 m l中に懸濁する。 After the precipitated fraction of the cells obtained was gently loosened, intake included by a measuring pipette, gently HAT medium [normal medium hypoxanthine in balloon (10- 4 M), thymidine (1. 5 X 10 "5 M) and aminopterin a (4 X 10- 7 M) was added Medium] in 100 ml.
該懸濁液を 9 6穴培養用プレートに 1 0 0〃 1 /穴ずつ分注し、 5 % C 02 インキュベータ一中、 3 7 °Cで？〜 1 4日間培養する。 The suspension was dispensed into a 96-well culture plate at 100 ° C./well, and the suspension was placed at 37 ° C. in a 5% CO 2 incubator. Incubate for ~ 14 days.
培養後、 培養上清の一部をとりアンチボディィズ [Antibodies- A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapterl4 ( 1988)] 等に述べ られている酵素免疫測定法により、 本発明の蛋白質に特異的に反応するハイブ リ ドーマを選択する。  After culturing, a part of the culture supernatant is removed to obtain the protein of the present invention by the enzyme immunoassay described in Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapter 14 (1988). Select hybridomas that react specifically.
酵素免疫測定法の具体的例として、 以下の方法をあげることができる。  The following method can be given as a specific example of the enzyme immunoassay.
免疫の際、 抗原に用いた本発明の蛋白質の全長または部分断片精製標品を適 当なプレートにコートし、 ハイプリ ドーマ培養上清もしくは後述の（2- 4)で得ら れる精製抗体を第一抗体として反応させ、 さらに第二抗体としてピオチン、 酵 素、 化学発光物質あるいは放射線化合物等で標識した抗ラッ トイムノグロプリ ン抗体を反応させた後に標識物質に応じた反応を行ない、 本発明の蛋白質に特 異的に反応するものを本発明の蛋白質に対するモノクローナル抗体を生産する ハイプリ ドーマとして選択する。  At the time of immunization, a purified sample of the full-length or partial fragment of the protein of the present invention used as the antigen was coated on an appropriate plate, and the hybridoma culture supernatant or the purified antibody obtained in (2-4) described later was purified. After reacting as an antibody, and further reacting an anti-rat immunoglobulin antibody labeled with a biotin, an enzyme, a chemiluminescent substance, a radioactive compound, or the like as a second antibody, a reaction corresponding to the labeling substance is carried out. Those that react specifically are selected as hybridomas that produce monoclonal antibodies against the protein of the present invention.
該ハイプリ ドーマを用いて、 限界希釈法によりクローニングを 2回繰り返し [ 1回目は、 H T培地 （H A T培地からアミノプテリンを除いた培地） 、 2回 目は、 正常培地を使用する] 、 安定して強い抗体価の認められたものを本発明 の蛋白質に対するモノクローナル抗体を生産するハイプリ ドーマ株として選択 する。  Using the hybridoma, cloning was repeated twice by the limiting dilution method [the first time using HT medium (medium from which aminopterin was removed from HAT medium), and the second time using normal medium]. Those with a strong antibody titer are selected as hybridoma strains producing a monoclonal antibody against the protein of the present invention.
(2-4)モノクローナル抗体の調製  (2-4) Preparation of monoclonal antibody
プリスタン処理 [ 2 , 6， 1 0， 1 4—テトラメチルペン夕デカン （Pristane ) 0 . 5 m lを腹腔内投与し、 2週間飼育する] した 8〜1 0週令のマウスま たはヌードマウスに、 （2- 3 )で取得した本発明の蛋白質に対するモノクローナル 抗体を生産するハイプリ ドーマ細胞 5〜2 0 X 1 06細胞/匹を腹腔内に注射す る。 1 0〜2 1日間でハイプリ ドーマは腹水癌化する。 0 P T/JP9 / 7335 8- to 10-week-old mice or nude mice treated with pristane [0.5 ml of 2,6,10,14-tetramethyl-pen-decane (Pristane) administered intraperitoneally and bred for 2 weeks] Next, 5 to 20 × 10 6 hybridoma cells producing a monoclonal antibody against the protein of the present invention obtained in (2-3) are intraperitoneally injected. In 10 to 21 days, the hybridoma becomes ascites cancer. 0 PT / JP9 / 7335
該腹水癌化したマウスから腹水を採取し、 3, 000 rpmで 5分間遠心分 離して固形分を除去する。 Ascites is collected from the mouse with ascites tumor and centrifuged at 3,000 rpm for 5 minutes to remove solids.
得られた上清より、 ポリクローナルで用いた方法と同様の方法でモノクロ一 ナル抗体を精製、 取得することができる。  From the obtained supernatant, a monoclonal antibody can be purified and obtained in the same manner as in the polyclonal method.
抗体のサブクラスの決定は、 マウスモノクローナル抗体タイピングキットま たはラットモノクローナル抗体タイピングキットを用いて行う。 蛋白質量は、 ローリ一法あるいは 280 nmでの吸光度より算出する。  The antibody subclass is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The protein content is calculated by the Lowry method or the absorbance at 280 nm.
[ 4 ] 本発明の蛋白質の存在下でのュビキチンリガーゼ活性および蛋白質分解 促進活性の測定  [4] Measurement of ubiquitin ligase activity and proteolysis-promoting activity in the presence of the protein of the present invention
ュビキチンリガ一ゼ活性は、 本発明の蛋白質の存在下で、 標的蛋白質と蛍光 標識、 ピオチン標識、 アイソトープ標識等をしたュビキチン蛋白質とを細胞内 または試験管内で反応させ、 標的蛋白質に取り込まれたュビキチン蛋白質の量 を測定することにより求めることができる。 また、 取り込まれたュビキチン蛋 白質を抗ュビキチン抗体を用いて検出することによつても求めることができる [Science, 373(5), 81-83 (1995)、 FEBS Letters, 377, 193-196 (1995)、 Science, 269, 682-685 (1995)] 。  The ubiquitin ligase activity is determined by reacting a target protein with a ubiquitin protein labeled with a fluorescent label, a biotin label, or an isotope in the presence of the protein of the present invention in a cell or in a test tube, and the ubiquitin protein incorporated into the target protein. Can be determined by measuring the amount of It can also be determined by detecting the incorporated ubiquitin protein using an anti-ubiquitin antibody [Science, 373 (5), 81-83 (1995), FEBS Letters, 377, 193-196 ( 1995), Science, 269, 682-685 (1995)].
ュビキチンリガーゼ活性を測定する場合、 必要に応じて E 3蛋白質複合体の 他の構成蛋白質、 E l、 E 2、 ュビキチン等、 標的蛋白質のュビキチン化に必 要な他の成分を添加し、 これを用いてもよい [Proc. Natl. Acad. Sci. USA, 92, 2563-2567 (1995)、 Science, 269, 682-685 (1995)、 Cell, 91, 221-230 (1997) When measuring ubiquitin ligase activity, other components necessary for ubiquitination of the target protein, such as other constituent proteins of the E3 protein complex, El, E2, and ubiquitin, are added as necessary. Natl. Acad. Sci. USA, 92, 2563-2567 (1995), Science, 269, 682-685 (1995), Cell, 91, 221-230 (1997).
] o ] o
蛋白質分解促進活性は、 本発明の蛋白質の存在下で、 蛍光標識、 ピオチン標 識、 あるいはァイソト一プ標識した標的蛋白質を細胞内または試験管内で反応 させ、 該標的蛋白質の変動量を測定することにより求めることができる。 また、 標的蛋白質の変動量は、 標的蛋白質の特異抗体を用いて検出することもできる。 標的蛋白質として融合蛋白質を用いた場合には、 融合した蛋白質に対する特異 抗体を用いて検出することもできる。 また、 標的蛋白質に取り込まれたュビキ チンを抗ュビキチン抗体により免疫学的に検出することによつても求めること ができる [Genes & Development, 12, 2587-2597 ( 1998)、 Molecular Cell, 1, 565-574 ( 1998)、 Genes & Development, 11, 3046-3060 ( 1998)、 Genes & Development, U, 1548-1560 ( 1997)] 。 The proteolysis-promoting activity is determined by reacting a fluorescent-labeled, biotin-labeled, or isotopically-labeled target protein in a cell or a test tube in the presence of the protein of the present invention, and measuring the amount of change in the target protein. Can be obtained by The amount of change in the target protein can also be detected using a specific antibody for the target protein. When a fusion protein is used as the target protein, specificity for the fused protein Detection can also be performed using an antibody. It can also be determined by immunologically detecting ubiquitin incorporated into the target protein using an anti-ubiquitin antibody [Genes & Development, 12, 2587-2597 (1998), Molecular Cell, 1, 565]. -574 (1998), Genes & Development, 11, 3046-3060 (1998), Genes & Development, U, 1548-1560 (1997)].
[ 5 ] 本発明の蛋白質の阻害物質または活性化物質の探索 ·同定および医薬ま たは診断薬としての利用  [5] Search and identification of inhibitors or activators of the protein of the present invention and their use as pharmaceuticals or diagnostics
本発明の蛋白質、 あるいは後述 [ 7 ] の方法で本発明の蛋白質を発現してい ることの確認された組織、 細胞等を用い、 これに被験試料を添加し、 [ 4 ] 記 載の方法によりュビキチンリガーゼ活性または蛋白質分解促進活性を測定する。 被験試料との接触の有無における、 本発明の蛋白質のュビキチンリガーゼ活 性あるいは蛋白質分解促進活性を比較することにより、 被験試料の中からュビ キチンリガーゼ活性または蛋白質分解促進活性を変動させる化合物をスクリ一 ニングすることができる。  A test sample is added to the protein of the present invention or a tissue, cell, or the like in which the expression of the protein of the present invention has been confirmed by the method of [7] described below, and a test sample is added thereto. Ubiquitin ligase activity or proteolysis promoting activity is measured. By comparing the ubiquitin ligase activity or the proteolysis-promoting activity of the protein of the present invention in the presence or absence of contact with the test sample, a compound that varies the ubiquitin ligase activity or the proteolysis-promoting activity from the test sample is determined. Can be screened.
以下、 該活性を変動させる化合物のうち、 該活性を増強する化合物をァゴニ スト、 該活性を阻害する化合物をアン夕ゴニストともいう。  Hereinafter, among the compounds that change the activity, a compound that enhances the activity is also referred to as an agonist, and a compound that inhibits the activity is also referred to as an antagonist.
また、 ュビキチンリガーゼ活性または蛋白質分解促進活性を変動させる化合 物は、 以下の方法によりスクリ一ニングすることができる。  In addition, a compound that changes ubiquitin ligase activity or proteolysis-promoting activity can be screened by the following method.
本発明の蛋白質を発現している細胞、 組織、 これらから調製した細胞膜、 精 製した本発明の蛋白質あるいは該蛋白質の部分断片と標識化合物とを接触させ、 本発明の蛋白質と標識化合物との結合の有無を確認する。  Cells and tissues expressing the protein of the present invention, cell membranes prepared therefrom, purified protein of the present invention or a partial fragment of the protein are contacted with a labeling compound, and the protein of the present invention binds to the labeling compound. Check for
結合が認められた標識化合物を用い、 上記と同様の条件下で被験試料を添加 し、 上記と同様に標識化合物の結合量を測定する。  A test sample is added under the same conditions as above using a labeled compound that has been observed to bind, and the amount of binding of the labeled compound is measured in the same manner as above.
被験試料の添加の有無における、 標識化合物の結合量を比較することにより、 被験試料の中からァゴニストまたはアン夕ゴニストをスクリーニングすること ができる。 被験試料としては、 合成化合物、 天然に存在する蛋白質、 人工的に合成され た蛋白質、 ペプチド、 糖質、 脂質、 これらの修飾体、 誘導体を、 また哺乳動物（ 例えばマウス、 ラット、 モルモット、 ハムスター、 ブ夕、 ヒッジ、 ゥシ、 ゥマ、 ィヌ、 ネコ、 サル、 ヒト等)の尿、 体液、 組織抽出物、 細胞培養上清、 細胞抽出 物を、 さらに、 非ペプチド性化合物、 発酵生産物、 植物その他の生物の抽出物 等をあげることができる。 By comparing the amount of the labeled compound bound before and after the addition of the test sample, it is possible to screen the test sample for an agonist or an engonist. Test samples include synthetic compounds, naturally occurring proteins, artificially synthesized proteins, peptides, carbohydrates, lipids, modified forms and derivatives thereof, and mammals (eg, mice, rats, guinea pigs, hamsters, Urine, body fluids, tissue extracts, cell culture supernatants, cell extracts, and other non-peptide compounds, fermentation products And extracts of plants and other organisms.
上記の方法により取得される、 本発明の蛋白質のァゴニストまたはアン夕ゴ 二ストは、 予防薬または治療薬 （併せて医薬ともいう） として単独で用いるこ とが可能ではあるが、 通常は薬理学的に許容される一つあるいはそれ以上の担 体と一緒に混合し、 製剤学の技術分野においてよく知られる任意の方法により 製造した医薬製剤として用いることが望ましい。  The agonist or angonist of the protein of the present invention obtained by the above method can be used alone as a prophylactic or therapeutic agent (also referred to as a drug), but usually it is used in pharmacology. It is admixed with one or more commercially acceptable carriers and used as a pharmaceutical preparation produced by any method well known in the pharmaceutical art.
該治療薬の投与方法としては、 治療に際し最も効果的な方法を使用すること が望ましく、 経口投与または、 口腔内、 気道内、 直腸内、 皮下、 筋肉内および 静脈内等の非経口投与による方法を用いることができる。  The method of administering the therapeutic agent is desirably the most effective method for the treatment, and is orally or parenterally, such as oral, respiratory, rectal, subcutaneous, intramuscular, and intravenous. Can be used.
該治療薬の剤形としては、 軟膏剤、 噴霧剤、 カプセル剤、 錠剤、 顆粒剤、 シ ロップ剤、 乳剤、 座剤、 注射剤、 テープ剤等をあげることができる。  Examples of the dosage form of the therapeutic agent include ointments, sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, tapes and the like.
経口投与に適当な製剤としては、 乳剤、 シロップ剤、 カプセル剤、 錠剤、 散 剤、 顆粒剤等をあげることができる。  Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
乳剤およびシロップ剤のような液体調製物は、 水、 ショ糖、 ソルビトール、 果糖等の糖類、 ポリエチレングリコ一ル、 プロピレングリコ一ル等のグリコ一 ル類、 ごま油、 オリープ油、 大豆油等の油類、 P—ヒドロキシ安息香酸エステ ル類等の防腐剤、 ストロベリーフレーバー、 ペパーミント等のフレーバ一類等 を添加剤として用いて製造することができる。  Liquid preparations such as emulsions and syrups are prepared from water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, and oils such as sesame oil, olive oil and soybean oil. And preservatives such as P-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint as additives.
カプセル剤、 錠剤、 散剤、 顆粒剤等は、 乳糖、 ブドウ糖、 ショ糖、 マンニト —ル等の賦形剤、 デンプン、 アルギン酸ナトリウム等の崩壊剤、 ステアリン酸 マグネシウム、 タルク等の滑沢剤、 ポリビニルアルコール、 ヒドロキシプロビ ルセルロース、 ゼラチン等の結合剤、 脂肪酸エステル等の界面活性剤、 グリセ リン等の可塑剤等を添加剤として用い製造することができる。 Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl alcohol , Hydroxyprobi It can be produced using additives such as binders such as cellulose and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin as additives.
非経口投与に適当な製剤としては、 注射剤、 座剤、 噴霧剤等があげられる。 注射剤は、 例えば、 塩溶液、 ブドウ糖溶液、 あるいは両者の混合物からなる 担体等を用いて調製することができる。  Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. The injection can be prepared, for example, using a carrier comprising a salt solution, a glucose solution, or a mixture of both.
座剤は、 例えば、 カカオ脂、 水素化脂肪またはカルボン酸等の担体を用いて 調製することができる。  Suppositories can be prepared using a carrier such as cocoa butter, hydrogenated fat or carboxylic acid.
噴霧剤は、 上記で取得されたァゴニストまたはアン夕ゴニストをそのまま噴 霧剤として用いることが可能であるが、 受容者の口腔および気道粘膜を刺激せ ず、 かつ該化合物を微細な粒子として分散させ吸収を容易にさせる担体等を用 いて調製した噴霧剤が好ましい。  As the propellant, the agonist or angonist obtained above can be directly used as a propellant, but it does not irritate the oral cavity and airway mucosa of the recipient and disperses the compound as fine particles. Sprays prepared using a carrier or the like that facilitates absorption are preferred.
担体として、 具体的には乳糖、 グリセリン等を例示することができる。  Specific examples of the carrier include lactose and glycerin.
上記で取得されたァゴニストまたはアン夕ゴニスト、 および担体の性質によ り、 エアロゾル、 ドライパウダー等の製剤を調製することが可能である。  Formulations such as aerosols and dry powders can be prepared depending on the properties of the agonist or angonist obtained above and the carrier.
これらの非経口剤においても、 経口剤で添加剤として例示した成分を添加す ることができる。  In these parenteral preparations, the components exemplified as additives for oral preparations can be added.
投与量または投与回数は、 目的とする治療効果、 投与方法、 治療期間、 年齢、 体重等により異なるが、 通常成人 1日当たり 1 0〃g/k g〜8 m g/k gで ある。  The dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 mg / kg to 8 mg / kg per day for an adult.
[ 6 ]本発明の蛋白質の発現を調節する化合物 （以下、 発現調節化合物と略す） の探索および同定  [6] Search and identification of compounds that regulate the expression of the protein of the present invention (hereinafter abbreviated as expression regulating compounds)
( 1 )本発明の抗体を用いた発現調節化合物の探索および同定  (1) Search and identification of expression regulating compounds using the antibody of the present invention
本発明の蛋白質を発現する細胞を被験試料と接触させた後、 本発明の抗体を 用いることにより、 その細胞中、 細胞培養上清中に存在する発現調節化合物を 探索、 同定することができる。  After contacting a cell expressing the protein of the present invention with a test sample and using the antibody of the present invention, an expression regulating compound present in the cell and in a cell culture supernatant can be searched and identified.
細胞としては、 本発明の蛋白質を発現している細胞、 細胞株、 組織ならいか なるものでも用いることができる。 The cell may be a cell, cell line or tissue expressing the protein of the present invention. Can be used.
また、 下記 [7] に記載した抗体により免疫学的に検出する方法を用い、 該 蛋白質の発現が認められた細胞、 細胞株あるレ、は組織を用いることができる。 好適な細胞株として、 例えば、 ヒト類表皮癌由来 A431細胞 (ATCC:CRL-1555)を あげることができる。  In addition, a cell, a cell line, or a tissue in which the expression of the protein has been observed can be used by the method of immunologically detecting with the antibody described in [7] below. Suitable cell lines include, for example, human epidermoid carcinoma-derived A431 cells (ATCC: CRL-1555).
被験試料としては、 上記 [5] の被験試料であげたものを用いることができ る。  As the test sample, those mentioned in the test sample in the above [5] can be used.
本発明の蛋白質を発現する細胞を、 該細胞の増殖することのできる培地に懸 濁し、 被験試料を該培地に添加し、 該細胞と接触させた後、 本発明の抗体を用 い、 該細胞の発現した蛋白質含量を定量する。 定量する方法としては、 例えば 下記の免疫細胞染色を利用した方法をあげることができる。  Cells expressing the protein of the present invention are suspended in a medium capable of growing the cells, a test sample is added to the medium, and the cells are contacted with the cells. Of the expressed protein is quantified. As a method for quantification, for example, a method using the following immune cell staining can be mentioned.
培養付着細胞を P B S緩衝液で洗浄し、 0. 05 % トリプシン、 0. 02 % ED T A (エチレンジァミン 4酢酸）を含む PB S緩衝液 3mlを加え、 余分な 溶液を除いた後、 37°C、 5分間インキュベートすることによりフラスコより 細胞を剥がす。  The adherent cells in the culture were washed with PBS buffer, and 3 ml of PBS buffer containing 0.05% trypsin and 0.02% EDTA (ethylenediamine tetraacetic acid) was added. After removing excess solution, 37 ° C, Remove cells from flask by incubating for 5 minutes.
浮遊細胞については培養細胞をそのまま用いることができる。  Cultured cells can be used as they are for suspension cells.
免疫細胞染色を行う細胞を免疫細胞染色用緩衝液 （1% BSA、 0. 02% EDTA、 0. 05% アジ化ナトリウムを含む PB S)等に懸濁し、 1〜20 X 105個ずつ丸底 96穴プレートに分注する。 Cells immune cell staining buffer to perform immunocytochemistry were suspended in such (1% BSA, 0. 02% EDTA, PB S containing 0.05% sodium azide), 1~20 X 10 5 cells each round Dispense into the bottom 96-well plate.
該プレートに、 本発明のモノクローナル抗体を分注する。  The monoclonal antibody of the present invention is dispensed on the plate.
モノクローナル抗体としては、 [3] (2- 3)で取得した本発明のモノクロ一ナ ル抗体を産生するハイプリ ドーマの培養上清、 [3] (2- 4)で取得した精製モノ クローナル抗体をあげることができる。 さらに、 該モノクローナル抗体を標識 した抗体も用いることができる。  Examples of the monoclonal antibody include the culture supernatant of a hybridoma producing the monoclonal antibody of the present invention obtained in [3] (2-3), and the purified monoclonal antibody obtained in [3] (2-4). I can give it. Furthermore, an antibody labeled with the monoclonal antibody can also be used.
モノクローナル抗体を標識した抗体としては、 例えばピオチン標識した抗体 をあげることができる。 ピオチン標識した抗体は公知の方法 （酵素抗体法：学際企画刊 1985年） で調 製することができる。 An example of an antibody labeled with a monoclonal antibody is a biotin-labeled antibody. A biotin-labeled antibody can be prepared by a known method (enzyme antibody method: interdisciplinary project, 1985).
上記抗体を、 免疫細胞染色用緩衝液あるいは 10%動物血清を含む免疫細胞 染色用緩衝液を用いて 0. 1 ~ 50〃 g/m 1の濃度になるように希釈する。 該希釈抗体を 20〜 500〃 1/穴となるように分注し、 氷冷下で 30分間 放置する。  The above antibody is diluted to a concentration of 0.1 to 50 μg / ml using an immune cell staining buffer or an immune cell staining buffer containing 10% animal serum. The diluted antibody is dispensed at 20-500 抗体 1 / well and left under ice-cooling for 30 minutes.
標識されていない抗体を用いた場合には、 上記プレートに免疫細胞染色用緩 衝液を添加し、 細胞を洗浄後、 FITC (fluorescein isothiocyanate)ある いはフィコエリスリン等の蛍光色素で標識した抗マウスィムノグロブリン抗体 あるいは抗ラットイムノグロプリン抗体を 0. 1〜5 Ο zg/ml程度の濃度 で含む免疫細胞染色用緩衝液を 50〜500 /1/穴ほど分注し、 氷冷下で 3 0分間遮光して放置する。  If an unlabeled antibody is used, add an immunocell staining buffer to the plate, wash the cells, and label the anti-mouse with a fluorescent dye such as FITC (fluorescein isothiocyanate) or phycoerythrin. Dispense 50-500 / 1 / well of immunoglobulin antibody or anti-rat immunoglobulin antibody at a concentration of about 0.1 to 5 μg / ml in an amount of about 50 to 500/1 / well. Leave for 5 minutes in the shade.
ビォチン標識した該モノクロ—ナル抗体を用レ、た場合には、 上記プレートに F I T Cあるいはフィコエリスリン等の蛍光色素で標識したストレプトァビジ ンを 50〜500 1/穴ほど分注し、 氷冷下で 30分間遮光して放置する。 両ケースとも、 放置後、 プレートに免疫細胞染色用緩衝液を添加し、 細胞を 良く洗浄し、 蛍光顕微鏡、 セルソ一夕一等により解析する。  When using the biotin-labeled monoclonal antibody, dispens 50-500 1 / well of streptavidin labeled with a fluorescent dye such as FITC or phycoerythrin on the above plate, and cool on ice. Leave under the light for 30 minutes. In both cases, after allowing to stand, add a buffer for immunocell staining to the plate, wash the cells well, and analyze with a fluorescence microscope, Celso overnight.
被験試料を添加しない系と比較し、 本発明の蛋白質含量を増加あるいは減少 させることのできた被験試料を探索することにより、 発現調節化合物を同定す ることができる。  An expression-regulating compound can be identified by searching for a test sample capable of increasing or decreasing the protein content of the present invention, as compared with a system to which no test sample is added.
(2) 本発明の蛋白質をコードする mRN Aを検出する方法、 および該方法を 用レ >た発現調節化合物の探索および同定  (2) A method for detecting mRNA encoding the protein of the present invention, and a search and identification of an expression regulating compound using the method.
本発明の蛋白質あるいは該蛋白質をコードする mRN Aを発現する細胞を、 該細胞の増殖することのできる培地に懸濁し、 被験試料を該培地に添加し、 該 細胞を接触させた後、 該細胞の発現した該 mRNAの含量を、 通常のノーザン ハイブリダイゼーシヨン法、 ： R N Aのドットブロットハイブリダイゼ一ション 法、 RT— PCR法等を用いて定量する。 Cells expressing the protein of the present invention or mRNA encoding the protein are suspended in a medium capable of growing the cells, a test sample is added to the medium, and the cells are contacted. The content of the expressed mRNA was determined by the usual Northern hybridization method: dot blot hybridization of RNA. Method, RT-PCR, etc.
ハイブリダィゼ一シヨン法等に用いるプローブとしては、 本発明の D N Aお よびオリゴヌクレオチドが、 RT— P CR法等に用いることのできるプライマ 一としては、 本発明のオリゴヌクレオチドが好適に用いられる。  The DNA and the oligonucleotide of the present invention are preferably used as a probe used in the hybridization method and the like, and the oligonucleotide of the present invention is suitably used as a primer that can be used in the RT-PCR method and the like.
被験試料を添加しない系と比較し、 本発明の蛋白質をコ一ドする mRNA含 量を増加あるいは減少させることのできた被験試料を探索することにより、 発 現調節化合物を同定することができる。  An expression-regulating compound can be identified by searching for a test sample capable of increasing or decreasing the content of mRNA encoding the protein of the present invention, as compared to a system to which no test sample is added.
本発明の蛋白質あるいは該蛋白質をコ一ドする mRNAを発現する細胞を被 験試料と接触させた後、 該 mRNA含量を定量することにより、 本発明の蛋白 質をコードする遺伝子の発現を変動させる化合物を探索、 同定することができ る。  After contacting a cell that expresses the protein of the present invention or an mRNA encoding the protein with a test sample, the content of the mRNA is quantified to vary the expression of the gene encoding the protein of the present invention. Compounds can be searched and identified.
本発明の蛋白質あるいは該蛋白質 mRNAを発現する細胞および被験試料と して、 上記 [6] (1) のものを用いることができる。  The above-mentioned [6] (1) can be used as a cell or a test sample which expresses the protein of the present invention or the protein mRNA.
(3) レポーター遺伝子を用いた発現調節化合物の探索および同定  (3) Search and identification of expression regulatory compounds using reporter genes
本発明の蛋白質をコードする遺伝子の転写を制御するプロモー夕一 DNA ( 以下、 単にプロモーター DN Aという） の下流にレポ一夕一遺伝子の連結され た D N Aを含むプラスミ ドで形質転換された形質転換体と被験試料とを接触さ せた後、 レポーター遺伝子によりコードされた蛋白質の発現量を定量すること により転写の効率を変動させる化合物を探索、 同定することができる。  Transformation transformed with a plasmid containing DNA linked to a repo overnight gene downstream of a promoter DNA (hereinafter simply referred to as a promoter DNA) that controls transcription of a gene encoding the protein of the present invention. After contacting the test sample with the body, the expression level of the protein encoded by the reporter gene can be quantified to search for and identify compounds that alter transcription efficiency.
プロモー夕一 DN Aは、 通常、 遺伝子の 5' 上流に含まれることが多い。 本 発明の蛋白質をコードする遺伝子のプロモーター DN Aは、 本発明の DN Aま たはオリゴヌクレオチドを用いて、 例えば Genome Walker kits (Clontech社） 等、 公知の方法により調製することができる。 また、 該領域を適当な制限酵素 を用い、 適切な長さに切断した断片を転写制御領域として用いることができる。 レポ一夕—遺伝子としては、 該遺伝子の翻訳産物が細胞内で安定であり、 該 翻訳産物の存在量が容易に定量できるものであればいかなるものでも用いるこ とができ、 例えば、 クロラムフエニコ一ルァセチルトランスフェラーゼ（CAT )、 ガラクトシダーゼ （ 5— ga 1) 、 ルシフェラ一ゼ （luc) 、 グリー ンフルォレヅセントプロテイン （GFP) 等をあげることができる。 Promoter DNA is usually contained 5 'upstream of the gene. The promoter DNA of the gene encoding the protein of the present invention can be prepared using the DNA or the oligonucleotide of the present invention by a known method such as Genome Walker kits (Clontech). In addition, a fragment obtained by cutting this region to an appropriate length using an appropriate restriction enzyme can be used as a transcription control region. As a repo gene, any gene can be used as long as the translation product of the gene is stable in cells and the amount of the translation product can be easily quantified. Examples thereof include chloramphenicylacetyltransferase (CAT), galactosidase (5-ga1), luciferase (luc), and green fluorescent protein (GFP).
該転写制御領域を含むレポータープラスミ ドを導入する宿主細胞としては、 いかなる細胞も用いることができるが、 好適には、 [6] ( 1) 記載の本発明 の蛋白質あるいは該蛋白質 mR N Aの発現が認められている細胞株を用いるこ とができる。  As the host cell into which the reporter plasmid containing the transcription control region is introduced, any cell can be used. Preferably, the expression of the protein of the present invention or the protein mRNA described in [6] (1) is preferred. Recognized cell lines can be used.
被験試料として、 上記 [5] の被験試料であげたものを用いることができる。 転写制御領域の下流に常法によりレポ一夕一遺伝子を連結し、 作製したブラ スミ ドを用い、 常法により宿主細胞を形質転換する。  As the test sample, those mentioned in the test sample in the above [5] can be used. The repo overnight gene is ligated to the downstream of the transcription control region by an ordinary method, and the prepared cells are used to transform host cells by an ordinary method.
また、 ポジティブセレクション用マーカー （G418耐性遺伝子等） および ネガティブセレクション用マーカ一 （単純へルぺスウィルスのチミジンキナ一 ゼゃジフテリア毒素 Aフラグメント遺伝子等） をつないだジーン夕一ゲティング ベクターを作成し、 本発明の蛋白質をコードする遺伝子の一部をレポ一夕一遺 伝子で置換した細胞株を作成することもできる [Nature, 336, 348 (1988)、 Analytical Biochemistry, 214, 77 (1993)、 Gene Targeting, The Practical Approach Series, IRL Press (1993)] 。  In addition, we created a gene targeting vector by linking a marker for positive selection (such as the G418 resistance gene) and a marker for negative selection (such as the gene for thymidine kinase and zediphteria toxin A fragment of simple virus virus). Cell lines in which a part of the gene encoding the protein of the present invention is substituted with a repo overnight gene can also be prepared [Nature, 336, 348 (1988), Analytical Biochemistry, 214, 77 (1993), Gene Targeting, The Practical Approach Series, IRL Press (1993)].
該形質転換体を、 例えば該細胞の増殖することのできる培地に懸濁し、 被験 試料を該培地に添加し、 該細胞を接触させた後、 該細胞の発現したレポーター 遺伝子にコードされた蛋白質の量を、 該蛋白質に適した方法で検出または定量 する。  The transformant is suspended, for example, in a medium in which the cells can grow, a test sample is added to the medium, and the cells are brought into contact with each other, and then the protein encoded by the reporter gene expressed in the cells is removed. The amount is detected or quantified by a method appropriate for the protein.
検出または定量する方法として、 CATの場合には、 例えば、 モレキュラー クロ一ニング第 2版， 16章， 60頁に記載の方法を、 一 ga lの場合には 、 例えば、 モレキュラー クローニング 第 2版， 16章， 66頁に記載の方法 を、 lucの場合には、 例えば、 実験医学別冊バイオマニュアルシリーズ 4遺 伝子導入と発現'解析法， 81 (1994)に記載の方法を、 GFPの場合には、 例え ば、 Proc. Natl. Acad. Sci. USA, 94, 4653 (1997)記載の方法等をあげること ができる。 As a method for detection or quantification, in the case of CAT, for example, the method described in Molecular Cloning, Second Edition, Chapter 16, page 60, and in the case of one gal, for example, Molecular Cloning, Second Edition, In the case of luc, the method described in Chapter 16, p. 66, for example, the method described in Experimental Medicine Separate Volume Bio Manual Series 4 Gene Transfer and Expression 'Analysis Method, 81 (1994) is used in the case of GFP. Is, for example, For example, the method described in Proc. Natl. Acad. Sci. USA, 94, 4653 (1997) can be mentioned.
被験試料を添加しない系と比較し、 レポーター遺伝子にコードされた蛋白質 含量を増加あるいは減少させることのできた被験試料を探索することにより、 プロモー夕一遺伝子による転写の効率を変動させる化合物を取得することがで きる。  To obtain a compound that changes the efficiency of transcription by the promoter gene by searching for a test sample that can increase or decrease the content of the protein encoded by the reporter gene compared to a system without the test sample. I can do it.
[7]本発明の DNA、 蛋白質、 抗体、 ァゴニスト、 アン夕ゴニストおよび発 現調節化合物の利用  [7] Utilization of DNA, protein, antibody, agonist, angelic gonist and expression regulating compound of the present invention
( 1) 本発明の DN Aは、 本発明の DN Aまたはオリゴヌクレオチドをプロ一 ブとして用いて、 ヒトの組織ゃヒト由来の細胞から [1] (1)と同様にして抽出 した RNAについてノーザンハイプリダイゼ一シヨンを行うことにより、 その 組織や細胞における本発明の蛋白質遺伝子の mRN Aを検出または定量をする ことができる。 各種の組織でその mRN Aの発現量を比較することにより本発 明の蛋白質の組織発現分布を知ることができる。  (1) The DNA of the present invention is obtained by using the DNA or the oligonucleotide of the present invention as a probe and extracting RNA from human tissue ゃ human-derived cells in the same manner as in [1] (1) using Northern. By performing the hybridization, mRNA of the protein gene of the present invention in the tissue or cell can be detected or quantified. By comparing the mRNA expression levels in various tissues, the tissue expression distribution of the protein of the present invention can be known.
(2) 本発明のオリゴヌクレオチドは、 本発明の DN Aの特異的プライマーと して用いて、 ヒトの組織ゃヒト由来の細胞から [1] (1)と同様にして抽出した RNAについて RT-P CR [reverse transcription PCR； PCR Protocols (1990) ] を行うことにより、 本発明の蛋白質をコードする mRN Aの検出や定量を行 うことができる。  (2) The oligonucleotide of the present invention was used as a specific primer for the DNA of the present invention to detect RNA extracted from human tissue ゃ human-derived cells in the same manner as in [1] (1). By performing PCR [reverse transcription PCR; PCR Protocols (1990)], it is possible to detect and quantify mRNA encoding the protein of the present invention.
該 mR N Aを定量する方法は、 本遺伝子が関与する病態の診断や細胞障害性 ヌクレオシド誘導体 （抗腫瘍剤、 抗ウィルス剤） の効果の予測等に用いること ができる。  The method for quantifying the mRNA can be used for diagnosis of pathological conditions involving the present gene, prediction of the effects of cytotoxic nucleoside derivatives (antitumor agents, antiviral agents), and the like.
各種病態モデル動物において、 該 mRNAを定量することにより、 病態にお ける該遺伝子産物の重要性を明らかにすることができる。 また、 薬剤の有無に よる該 mRN Aの発現量を比較することにより薬剤を評価することができる。  By quantifying the mRNA in various disease model animals, the importance of the gene product in the disease condition can be clarified. Further, the drug can be evaluated by comparing the expression amount of the mRNA depending on the presence or absence of the drug.
(3)本発明の DN Aまたはオリゴヌクレオチドは、 これをプローブとして用 いて、 ヒトの組織切片に対して in situハイブリダイゼ一ション [Methods in Enzymology, 254, 419 (1995)] を行うことにより、 組織内での本発明の蛋白質 の発現細胞の特定等のより細かい発現分布を知ることができる。 (3) The DNA or oligonucleotide of the present invention is used as a probe By performing in situ hybridization [Methods in Enzymology, 254, 419 (1995)] on human tissue sections, finer expression distribution such as identification of cells expressing the protein of the present invention in tissues can be achieved. You can know.
これらの方法によって得られる、 本発明の蛋白質がどのような組織や細胞で 発現しているかに関する情報および細胞がどのような刺激を受けたときに発現 量が変化するかに関する情報は、 本発明の蛋白質の生理機能や病態への関与を 解析するために有用である。  Information on the tissue and cells expressing the protein of the present invention and the information on the stimulus to which the cells change their expression level obtained by these methods are described in the present invention. It is useful for analyzing the involvement of proteins in physiological functions and disease states.
(4)本発明の DNAまたはオリゴヌクレオチドをプローブとして用い、 ゲノ ム DNAに対してサザンハイブリダィゼ一シヨン 〔モレキュラー クローニング 第 2版] を行うことにより、 本発明の蛋白質をコードする遺伝子の変異、 欠失、 増幅、 多型を検出することができる。  (4) Mutation of the gene encoding the protein of the present invention by subjecting the genomic DNA to Southern hybridization [molecular cloning second edition] using the DNA or oligonucleotide of the present invention as a probe , Deletion, amplification and polymorphism can be detected.
(5) 本発明のオリゴヌクレオチドは、 本発明の DN Aの特異的プライマーと して用いて、 ヒ卜の組織ゃヒト由来の細胞のゲノム DNAに対して P CR反応 を行うことにより、 本発明の蛋白質をコードする遺伝子の変異、 欠失、 増幅、 多型を検出することができる。 (5) The oligonucleotide of the present invention can be used as a specific primer for the DNA of the present invention to perform PCR reaction on genomic DNA of human tissue / human-derived cells. Mutations, deletions, amplifications, and polymorphisms in the gene encoding the protein can be detected.
変異、 欠失、 増幅、 多型の検出を行うことにより、 該遺伝子の変異、 欠失、 増幅、 多型が原因あるいは危険因子となっている可能性のある、 例えば悪性腫 瘍、 神経変性疾患、 自己免疫疾患、 炎症性疾患等、 蛋白質分解の亢進または低 下により引き起こされる疾患の診断を行うことができる。  Detection of mutations, deletions, amplifications, and polymorphisms may result in mutations, deletions, amplifications, and polymorphisms in the gene, which may be a cause or risk factor, such as malignant tumors and neurodegenerative diseases It is possible to diagnose diseases caused by increased or decreased protein degradation, such as autoimmune diseases and inflammatory diseases.
(6) 本発明のアンチセンス'オリゴヌクレオチド（RNA/DNA)を用い、 本 発明の蛋白質をコードする遺伝子の転写もしくは mRN Aの翻訳を抑制するこ とにより [化学， 46, 681 (1991)、 Bio/Technology, 9, 358 (1992)]、 該遺伝 子が発症に関与している可能性のある、 例えば悪性腫瘍、 神経変性疾患、 自己 免疫疾患、 炎症性疾患等、 蛋白質分解の亢進または低下により引き起こされる 疾患の予防や治療に用いることができる。  (6) By using the antisense 'oligonucleotide (RNA / DNA) of the present invention to suppress the transcription of the gene encoding the protein of the present invention or the translation of mRNA, [Chemistry, 46, 681 (1991), Bio / Technology, 9, 358 (1992)], where the gene may be involved in the pathogenesis, such as malignant tumors, neurodegenerative diseases, autoimmune diseases, inflammatory diseases, etc. It can be used for prevention and treatment of diseases caused by.
上述のアンチセンス'オリゴヌクレオチドは、 本発明の蛋白質をコ一ドする D N Aの塩基配列中の連続した 5〜60塩基と相補的な配列を有するオリゴヌク レオチド、 好ましくは本発明の蛋白質をコードする DNAの翻訳開始領域にあ る 5〜 60塩基と相補的な塩基配列を基にして設計 ·調製し、 生体内に投与す る。 The antisense 'oligonucleotide described above encodes a protein of the present invention. Oligonucleotides having a sequence complementary to consecutive 5 to 60 bases in the base sequence of NA, preferably a base sequence complementary to 5 to 60 bases in the translation initiation region of DNA encoding the protein of the present invention. Based on the design and preparation, it is administered in vivo.
(7) 本発明の DN Aを含有する医薬は、 上記 [5] の本発明の蛋白質のァゴ 二ストまたはアン夕ゴニストの医薬製剤の調製法と同様な方法を用いて調製す ることができ、 調製された該医薬製剤を上記 [5] の場合と同様の方法で投与 することができる。  (7) The medicament containing the DNA of the present invention can be prepared by the same method as the above-mentioned [5] for preparing the pharmaceutical preparation of the agonist or angonist of the protein of the present invention. The prepared pharmaceutical preparation can be administered in the same manner as in the above [5].
本発明の蛋白質を含有する医薬は、 上記 [5] の本発明の蛋白質のァゴニス トまたはアン夕ゴニス卜の医薬製剤の調製法と同様な方法を用いて調製するこ とができ、 調製された該医薬製剤を上記 [5]の場合と同様の方法で投与する ことができる。  The medicine containing the protein of the present invention can be prepared and prepared by using the same method as the above-mentioned [5] for preparing the pharmaceutical preparation of the agonist or angonisto of the protein of the present invention. The pharmaceutical preparation can be administered in the same manner as in the above [5].
本発明の抗体を含有する医薬は、 上記 [5] の本発明の蛋白質のァゴニスト またはアン夕ゴニス卜の医薬製剤の調製法と同様な方法を用いて調製すること ができ、 調製された該医薬製剤を上記 [5] の場合と同様の方法で投与するこ とができる。  The drug containing the antibody of the present invention can be prepared by the same method as the above-mentioned [5], which is the same as the method for preparing the agonist of the protein of the present invention or the pharmaceutical preparation of an gonist, and the prepared drug is used. The preparation can be administered in the same manner as in [5] above.
本発明の DN Aまたはオリゴヌクレオチドは一本鎖または二本鎖としてレト ロウィルス、 アデノウイルス、 アデノ随伴ウィルス等のウィルスベクタ一、 そ の他のベクタ一に組み込んで遺伝子治療用べクタ一とし、 遺伝子治療に用いる ことができる。  The DNA or oligonucleotide of the present invention may be incorporated as a single-stranded or double-stranded DNA vector such as retrovirus, adenovirus, adeno-associated virus, and other vectors into a vector for gene therapy to produce a gene therapy vector. Can be used for treatment.
(8)本発明の蛋白質に対する抗体を用いて、 本発明の蛋白質を免疫学的に検 出または定量することができる。  (8) The protein of the present invention can be immunologically detected or quantified using an antibody against the protein of the present invention.
具体的にはマイクロタイ夕一プレートを用いる EL I SA法、 酵素標識抗体 法や蛍光抗体法による免疫組織染色、 ウエスタンブロット法等を用いた検出法 をあげることができる。  Specific examples include the ELISA method using a micro-tie plate, immunohistochemical staining using an enzyme-labeled antibody method or a fluorescent antibody method, and a detection method using a Western blot method.
具体的には、 液相中で本発明の蛋白質と反応する抗体のうちェピトープが異 なる 2種類のモノクローナル抗体を用いたサンドィツチ EL I SA法、 1251等の 放射性同位体で標識した本発明の蛋白質と本発明の蛋白質を認識する抗体を用 いるラジオイムノアヅセィ法等があげることができる。 Specifically, among the antibodies that react with the protein of the present invention in the liquid phase, the epitope is different. Two monoclonal antibodies Sanditsuchi EL I SA method using, be given such radioimmunoprecipitation Adzu Si method are use antibodies that recognize proteins and protein of the present invention of the present invention labeled with a radioisotope such as 125 1 comprising Can be.
また、 本発明の抗体は、 病理組織切片を用いた免疫組織染色にも利用できる。 本発明の抗体を用い、 健常者および被験者の細胞または組織に存在する本発明 の蛋白質を免疫学的に検出または定量し、 その量を健常者と被験者とで比較し、 発現量が変化しているかどうかを調べることにより、 被験者の例えば悪性腫瘍、 神経変性疾患、 自己免疫疾患、 炎症性疾患等、 蛋白質分解の亢進または低下に より引き起こされる疾患についての病態の診断薬等に用いることができる。 上記のとおり、 本発明の抗体は、 免疫組織染色剤として用いることができる。 図面の簡単な説明  Further, the antibody of the present invention can also be used for immunohistological staining using a pathological tissue section. Using the antibody of the present invention, the protein of the present invention present in cells or tissues of healthy subjects and subjects is immunologically detected or quantified, and the amount is compared between healthy subjects and subjects. By examining whether or not the compound is used, it can be used as a diagnostic agent for a disease state of a subject caused by an increase or decrease in protein degradation such as a malignant tumor, a neurodegenerative disease, an autoimmune disease, an inflammatory disease and the like. As described above, the antibody of the present invention can be used as an immunohistochemical stain. BRIEF DESCRIPTION OF THE FIGURES
第 1図はプラスミ ド phMD 6の構造を簡単に示した図である。 FIG. 1 is a diagram simply showing the structure of plasmid phMD6.
第 2図はプラスミ ド phMD 6にコードされる蛋白質のアミノ酸配列と、 既知 の F— WD蛋白質であるマウス MD 6(mMD 6 )、 ヒト — TRCP (hbTRCP ) 、 サッカロマイセス 'セレピシェ CDC 4 (scCDC4) 、 シゾサッカロマイセ ス .ボンべ pop 1+ (sppopl) のアミノ酸配列を比較した図である。 星印は全 ての配列で一致しているアミノ酸残基を示す （アミノ酸残基は一文字表記で示 す） 。 配列の上に、 Fボックス （F— boxと記載） および WDドメイン （W D - 1〜WD - 7と記載） の位置を示す。 Figure 2 shows the amino acid sequence of the protein encoded by the plasmid phMD6, and the known F-WD proteins mouse MD6 (mMD6), human — TRCP (hbTRCP), Saccharomyces' Celepiche CDC4 (scCDC4), It is a figure which compared the amino acid sequence of Shizosaccharomyces. Bomb pop 1+ (sppopl). An asterisk indicates an amino acid residue that is identical in all sequences (amino acid residues are indicated by one letter). Above the sequence, the positions of the F box (denoted as F-box) and the WD domain (denoted as WD-1 to WD-7) are shown.
第 3図はプラスミ ド phMD 6にコードされる蛋白質のアミノ酸配列と、 既知 の F— WD蛋白質であるマウス MD 6(mMD 6)、 ヒト ^— TRCP (hbTRCP ) 、 サッカロマイセス 'セレピシェ CD C 4 (scCDC4) 、 シゾサッカロマイセ ス 'ボンべ pop 1+ (sppopl) のアミノ酸配列を比較した図 （図 2の続き） で ある。 Figure 3 shows the amino acid sequence of the protein encoded by the plasmid phMD6, and the known F-WD proteins mouse MD6 (mMD6), human ^ TRCP (hbTRCP), and Saccharomyces' Celepiche CDC4 (scCDC4 ) And a comparison of the amino acid sequences of Schizosaccharomyces ′ s bomb pop 1+ (sppopl) (continuation of FIG. 2).
第 4図は、 4種のヒト癌細胞株 （Jurkat, HeLa, A431, Saos-2) 由来の RNAを 鎵型として、 hMD 6に含まれる配列に対応するプライマー （配列番号 5に示 される 5 '端側 D N Aプライマーおよび、 配列番号 4に示される 3 '端側 D N A プライマーを用いて、 RT— PCR実験を行った結果を示す。 FIG. 4 shows primers corresponding to the sequence contained in hMD6 (SEQ ID NO: 5), using RNAs from four human cancer cell lines (Jurkat, HeLa, A431, Saos-2) as type III. FIG. 9 shows the results of RT-PCR experiments performed using the 5′-end DNA primer used and the 3′-end DNA primer shown in SEQ ID NO: 4.
符号の説明  Explanation of reference numerals
kb：キロ塩基対 (kilobase pairs) kb: kilobase pairs
Ap：アンビシリン耐性遺伝子  Ap: Ambicillin resistance gene
ori:プラスミ ド複製起点 ori: Plasmid replication origin
knt:キロヌクレオチド (kilonucleotides) knt: kilonucleotides
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
以下により具体的な実施例をあげて説明するが、 これにより本発明の範囲が 限定されるものではない。  Hereinafter, the present invention will be described with reference to specific examples, but the scope of the present invention is not limited thereto.
実施例 1 ヒト p op 1+関連蛋白質 （hMD 6) cDNAのクローン化 (1) ヒト類表皮癌細胞 A431からのクローン化 Example 1 Cloning of human pop 1 + -related protein (hMD6) cDNA (1) Cloning from human epidermoid carcinoma cell A431
遺伝子操作的手法は特に断らない限り公知のモレキュラー クロ一ニング 第 2版に記載されている方法により行った。  Unless otherwise specified, the genetic engineering method was performed by the method described in the known Molecular Cloning 2nd edition.
シゾサッカロマイセス ·ボンべ pop 1+遺伝子 [Genes & Development, 11， 1548-1560 (1997)] と相同性をもつヒト E S T配列 (Genbank, ACCESSION AA252 600, AA452542) より、 配列番号 3および 5に示される塩基配列からなる 5'端 側 DN Aプライマーを設計し、化学合成した。また、同様に E S T配列（Genbank, ACCESSION AA579079, AA243378) より、 配列番号 4に示される塩基配列からな る 3'端側 DNAプライマ一を設計し、 化学合成した。 DNAの化学合成はアブ ライ ドバイオシステムズ（Applied Biosystems)社の D N A合成機 Model 392を使 用した通常の固相合成法により行った。  SEQ ID NOS: 3 and 5 from human EST sequence (Genbank, ACCESSION AA252 600, AA452542) having homology to Schizosaccharomyces bombi pop 1+ gene [Genes & Development, 11, 1548-1560 (1997)] A 5′-end DNA primer consisting of a base sequence to be used was designed and chemically synthesized. Similarly, based on the EST sequence (Genbank, ACCESSION AA579079, AA243378), a 3′-end DNA primer consisting of the nucleotide sequence shown in SEQ ID NO: 4 was designed and chemically synthesized. Chemical synthesis of DNA was carried out by a conventional solid phase synthesis method using a DNA synthesizer Model 392 manufactured by Applied Biosystems.
ヒト類表皮癌由来細胞株 A431 (ATCC CRL— 1555)より、 Total RNA Separator Kit (Clontech社）を用い、 添付のプロトコ一ルに従って、 全 RNAを調製した。 全 RNA各 5〃gより、 Superscript TM Preamplification System(GIBC0 BRL 社）を用い、 添付のプロトコールに従ってオリゴ （dT) プライマ一より逆転 写反応を行い、 cDNAを調製した。 Total RNA was prepared from human epidermoid carcinoma-derived cell line A431 (ATCC CRL-1555) using a Total RNA Separator Kit (Clontech) according to the attached protocol. From 5 μg of total RNA, SuperscriptTM Preamplification System (GIBC0 BRL Using the oligo (dT) primer according to the attached protocol to prepare cDNA.
21〃1の該反応液を 50倍希釈し、 そのうちの 10 1を鍊型に用いて、 PCR反応を行った。 プライマーとしては、 配列番号 3に示される 5'端側 DN Aプライマー、 および配列番号 4に示される 3，端側 DN Aプライマ一を用いた 錡型 cDNA10〃l、 2種類のプライマ一各々 0. 25〃M、 各成分 40 0〃MのdNTP (dATP、 dGTP、 dCTP、 dTTP)混合液、 2. 5 mM塩酸マグネシゥム、 TaKaRa LA Taq (宝酒造社）2. 5単位および 1 x La Taq 緩衝液 （マグネシウム 'フリー） を含む反応溶液 40〃1を用い、 下記条件下 で PC Rを行った。 即ち、 DNA Thermal Cycler 480 (パーキンエルマ一 ·シ一 タス（Perkin Elmer Cetus)社）を用い、 94°Cで 30秒間、 55°Cで 60秒間、 72°Cで 120秒間の工程を 1サイクルとして 25サイクル行い、 さらに 72 °Cで 7分間加熱した。  The reaction solution (21 液 1) was diluted 50-fold, and a PCR reaction was performed using 101 of the diluted solution. As primers, the 5′-end DNA primer shown in SEQ ID NO: 3 and the 3 ′ end DNA primer shown in SEQ ID NO: 4, 10 μl of 錡 type cDNA, each of the two types of primers were used. 25〃M, 400〃M each component dNTP (dATP, dGTP, dCTP, dTTP) mixture, 2.5 mM magnesium hydrochloride, TaKaRa LA Taq (Takara Shuzo) 2.5 units and 1 x La Taq buffer ( PCR was performed under the following conditions using a reaction solution containing magnesium (free) 40〃1. That is, using a DNA Thermal Cycler 480 (Perkin Elmer Cetus), a process consisting of 94 ° C for 30 seconds, 55 ° C for 60 seconds, and 72 ° C for 120 seconds is defined as one cycle. 25 cycles were performed and heating was further performed at 72 ° C for 7 minutes.
得られた該 PCR溶液を 5〃 1分取し、 ァガロース電気泳動にかけた。 その 結果、 予想される約 1. 5 kbの DNA断片が増幅されていることを確認した。 確認後、 上記 PC R反応液をァガロース電気泳動にかけ、 約 1. 5kbの該 DNA断片を、 Gene Clean III Kit (BI0101社）を用い、 添付のプロトコ一ルに 従って精製した。  The PCR solution thus obtained was collected in a 5 1-minute fraction and subjected to agarose electrophoresis. As a result, it was confirmed that an expected DNA fragment of about 1.5 kb was amplified. After confirmation, the PCR reaction solution was subjected to agarose electrophoresis, and the DNA fragment of about 1.5 kb was purified using a Gene Clean III Kit (BI0101) according to the attached protocol.
上記で回収した末端平滑化された精製 c D N A ( h M D 6 )および pT7B lue T- Vector(Novagen社）を、 TaKaRa DNA Ligation Kit Ver.l (宝酒造社) を用いて、 添付のプロトコールに従ってライゲ一シヨン反応を行った。  Using the TaKaRa DNA Ligation Kit Ver.l (Takara Shuzo), the purified blunt-ended cDNA (hMD6) and pT7Blue T-Vector (Novagen) collected above were collected according to the attached protocol. A Chillon reaction was performed.
該反応により得られた組み換えプラスミ ド DN Aを用いてェシヱリヒア ·コ リ DH5ひ株 （モレキュラー クロ一ニング第 2版、 A.10) を形質転換し、 モレ キユラ一 クローニング第 2版に記載の方法によりプラスミ ド phMD6を得 た。 得られたプラスミ ド p hMD 6を用いて、 クロ一ン化した D N A断片 hMD 6の塩基配列を以下の通り決定した。 p hMD 6を銪型とし、 Big Dye Primer Cyc le Sequencing Kit (Applied Biosystems社) を用い、 添付のプロトコ一ルに従 つて塩基配列決定用の試料を作製し、 ABI PRISifM 377 DNA sequencer (Applied Biosystems社） を用いて、 塩基配列を決定した。 p hMD 6には配列番号 2に 記載された c D NAが含まれ、 1 3 6 2塩基対のオープンリーディングフレー ム （以下、 O R Fと略す） が存在する。 p hMD 6の構造を図 1に示す。 The recombinant plasmid DNA obtained by the reaction was used to transform Escherichia coli DH5 strain (Molecular Cloning, 2nd ed., A.10), and the method described in Molecular cloning 2nd ed. As a result, plasmid phMD6 was obtained. Using the obtained plasmid pMD6, the nucleotide sequence of the cloned DNA fragment hMD6 was determined as follows. Using phMD6 as type I, a sample for nucleotide sequence determination was prepared using Big Dye Primer Cycle Sequencing Kit (Applied Biosystems) according to the attached protocol, and ABI PRISif M 377 DNA sequencer (Applied Biosystems). Was used to determine the nucleotide sequence. PhMD6 contains the cDNA described in SEQ ID NO: 2, and has an open reading frame (hereinafter abbreviated as ORF) of 1362 base pairs. The structure of phMD6 is shown in FIG.
p hMD 6の O R Fには、 配列番号 1に示される 4 5 4アミノ酸残基からな る蛋白質 （以下、 hMD 6と略記する） がコードされていた。  In the ORF of phMD6, a protein consisting of 454 amino acid residues shown in SEQ ID NO: 1 (hereinafter abbreviated as hMD6) was encoded.
前記のように Fボックス蛋白質は、 該蛋白質のァミノ末端近くに Fボックス と呼ばれる 4 0〜5 0残基のアミノ酸からなる領域を有し、 Fボックス蛋白質 のうち F— WD蛋白質は、 該蛋白質のカルボキシ末端近くに WDドメインと呼 ばれる配列を有し、 WDドメインは、 通常はトリブトファンーァスパラギン酸 (WD ) をその最後のアミノ酸残基としてもち、 かつ約 2 0〜4 0残基からな るアミノ酸配列が 4〜 8回反復する配列を有する構造を持つ。  As described above, the F-box protein has a region consisting of amino acids of 40 to 50 residues called an F-box near the amino terminal of the protein. Among the F-box proteins, the F-WD protein is It has a sequence called the WD domain near the carboxy terminus, which usually has tributophan-aspartic acid (WD) as its last amino acid residue and from about 20 to 40 residues. The amino acid sequence has a structure having a sequence repeated 4 to 8 times.
今回、 得られたアミノ酸配列を検討したところ、 上記 F— WD蛋白質の構造 特性を備えていることが確認された。  When the obtained amino acid sequence was examined this time, it was confirmed that the amino acid sequence had the structural characteristics of the F-WD protein.
該ァミノ酸配列を、 解析プログラム [Wisconsin Package ( Genetics Compute r Group，米国） に含まれる Gap] を用いて、 既知の F—WD蛋白質と比較したと ころ、 全長に渡ってマウス MD 6遺伝子 （Genbank Accession X54352) にコ一 ドされる蛋白質 （mMD 6 ) と高い相同性を有しており、 hMD 6と mMD 6 の間で 9 7 %の相同性（Similarity)を示した。 また、 hMD 6遺伝子と mMD 6遺伝子の翻訳領域全長を上記解析プログラムを用いて比較したところ、 塩基 配列においても 9 5 %の相同性を示した。 この極めて高い相同性から、 hMD 6は mMD 6のヒトォ一ソログ （ortholog) であると考えられる。  The amino acid sequence was compared with a known F-WD protein using an analysis program [Gap included in the Wisconsin Package (Genetics Computer Group, USA)]. Accession X54352) has a high homology with the protein (mMD 6), and shows 97% homology (Similarity) between hMD 6 and mMD 6. In addition, when the total length of the translation regions of the hMD6 gene and the mMD6 gene was compared using the above-mentioned analysis program, the nucleotide sequences showed 95% homology. Due to this extremely high homology, hMD6 is considered to be the ortholog of mMD6.
さらに、 F—WD蛋白質のアミノ酸配列中で特によく保存されており、 ュビ JP Furthermore, it is particularly well conserved in the amino acid sequence of F-WD protein. JP
キチンリガ一ゼ E 3の機能発現にも重要であると考えられる、 Fボックス、 W Dドメインについて、 ュビキチンリガ一ゼ活性あるいは蛋白質分解促進活性が 報告されている既知の蛋白質の配列と hMD 6の配列の比較を、 上記解析プロ グラムを用いて行った。 なお、 Fボックスは、 配列番号 6で示される 43残基 からなるアミノ酸配列 （配列番号 1の 59〜101番目のアミノ酸配列） に該 当すると考えられる。 また、 WDドメインは、 配列番号 7で示される 29残基 からなるアミノ酸配列 （WD— 1 ：配列番号 1の 146〜174番目のァミノ 酸配列） 、 配列番号 8で示される 27残基からなるアミノ酸配列 （WD— 2 ： 配列番号 1の 186〜 212番目のァミノ酸配列） 、 配列番号 9で示される 3 1残基からなるアミノ酸配列 （WD - 3 ：配列番号 1の 224〜 254番目の アミノ酸配列） 、 配列番号 10で示される 30残基からなるアミノ酸配列 （W D-4 ：配列番号 1の 266〜 295番目のアミノ酸配列） 、 配列番号 11で 示される 29残基からなるアミノ酸配列 （WD— 5 ：配列番号 1の 307〜3 35番目のアミノ酸配列） 、 配列番号 12で示される 6残基からなるアミノ酸 配列 （WD— 6 ：配列番号 1の 346〜 351番目のァミノ酸配列） 、 配列番 号 13で示される 40残基からなるアミノ酸配列 （WD - 7 ：配列番号 1の 3 63〜402番目のアミノ酸配列） を含む、 配列番号 1の 146〜402番目 のアミノ酸配列に該当すると考えられる。 Comparison of the hMD6 sequence with a known protein sequence that has been reported to exhibit ubiquitin ligase activity or proteolysis-promoting activity in the F box and WD domain, which is also considered to be important for the functional expression of chitin ligase E3. Was performed using the above analysis program. The F box is considered to correspond to the amino acid sequence consisting of 43 residues shown in SEQ ID NO: 6 (amino acid sequence at positions 59 to 101 in SEQ ID NO: 1). The WD domain has an amino acid sequence consisting of 29 residues represented by SEQ ID NO: 7 (WD-1: the amino acid sequence at positions 146 to 174 of SEQ ID NO: 1), an amino acid consisting of 27 residues represented by SEQ ID NO: 8 Sequence (WD-2: amino acid sequence at positions 186 to 212 of SEQ ID NO: 1), an amino acid sequence consisting of 31 residues represented by SEQ ID NO: 9 (WD-3: amino acid sequence at positions 224 to 254 of SEQ ID NO: 1) ), An amino acid sequence consisting of 30 residues represented by SEQ ID NO: 10 (WD-4: amino acid sequence at positions 266 to 295 of SEQ ID NO: 1); an amino acid sequence consisting of 29 residues represented by SEQ ID NO: 11 (WD- 5: Amino acid sequence consisting of 6 residues shown in SEQ ID NO: 12 (amino acid sequence at positions 307 to 335 of SEQ ID NO: 1) (WD-6: amino acid sequence of positions 346 to 351 in SEQ ID NO: 1), SEQ ID NO: No. 13 amino acid sequence consisting of 40 residues (WD-7: sequence Including No. 1 of 3 63 to 402 amino acid sequence) believed to correspond to the 146-402 amino acid sequence of SEQ ID NO: 1.
Fボックスでは、 ヒト5— TRCPに対して 43%、 シゾサッカロマイセス .ボンべ p op 1+に対して 35%、 サヅカロマイセス 'セレピシェ CD C4に 対して 47%、 という高い相同性（Similarity)を示した。 さらに、 WDドメイ ンでも、 ヒト ?一 TRCPに対して 34%、 シゾサッカロマイセス 'ボンべ p op 1+に対して 28%、 サッカロマイセス 'セレピシェ CDC4に対して 33 %、 という高い相同性を示した。 なお、 hMD 6とその他の F—WD蛋白質の 配列を比較したものを図 2および 3に示す。  The F-box shows high homology (43% for human 5-TRCP, 35% for Schizosaccharomyces .bomb 1) and 47% for Saccharomyces' Celepiche CD C4. Was. In addition, the WD domain showed high homology of 34% with human TRCP, 28% with Schizosaccharomyces 'bomb pop 1+, and 33% with Saccharomyces' Celepiche CDC4. . FIGS. 2 and 3 show a comparison of the sequences of hMD6 and other F-WD proteins.
以上の結果から、 hMD 6遺伝子にコードされる OR Fはュビキチンリガ一 ゼ E 3のサブュニヅトである新規 F— WD蛋白質をコードしていると考えられ る。 Based on the above results, the ORF encoded by the hMD6 gene was It is thought to encode a novel F-WD protein that is a submittal of zE3.
実施例 2 RT—P CRによる hMD 6 mRNAの発現解析 Example 2 Analysis of hMD6 mRNA expression by RT-PCR
ヒト T細胞白血病由来細胞株 Jurkat (ATCC TIB- 152)、 ヒト子宮類癌由来細 胞株 HeLa (ATCC CCL- 2)、 ヒト類表皮癌由来細胞株 A431 (ATCC CRL— 1555), ヒ ト骨肉腫由来細胞株 Saos-2 (ATCC HTB-85) より、 Total RNA Separator Kit (Clo ntech社）を用い、 添付のプロトコールに従って、 全 RNAを調製した。 全 RN A各 5 gより、 Superscript TM Preamplification System(GIBC0 BRL社）を 用い、 添付のプロトコールに従ってオリゴ （dT) プライマーより逆転写反 応を行い、 cDNAを調製した。  Human T-cell leukemia-derived cell line Jurkat (ATCC TIB-152), human uterine cancer-derived cell line HeLa (ATCC CCL-2), human epidermoid carcinoma-derived cell line A431 (ATCC CRL-1555), human osteosarcoma Total RNA was prepared from the derived cell line Saos-2 (ATCC HTB-85) using a Total RNA Separator Kit (Clontech) according to the attached protocol. From 5 g of each RNA, reverse transcription reaction was performed with oligo (dT) primer using Superscript ™ Preamplification System (GIBC0 BRL) according to the attached protocol to prepare cDNA.
21〃1の該反応液を 50倍希釈し、 そのうちの 10 1を錡型に用いて、 PCR反応を行った。 プライマーとしては、 配列番号 5に示される 5'端側 DN Aブラィマー、 および配列番号 4に示される 3 '端側 D N Aプライマ一を用いた 。 錡型 cDNA各 10〃 1、 2種類のプライマー各々 0. 5〃M、 各成分 40 0〃Mの dNTP (dATP、 dGTP、 dCTP、 dTTP)混合液、 2. 5 mM塩化マグネシゥム、 TaKaRa LA Taq(宝酒造社）2. 5単位および 1 x La Taq 緩衝液 （マグネシウム 'フリー） を含む反応溶液 20 1を用い、 下記条件下 で PC Rを行った。  The reaction solution (21 液 1) was diluted 50-fold, and a PCR reaction was performed using 101 of the diluted solution. As primers, a 5′-end DNA primer shown in SEQ ID NO: 5 and a 3′-end DNA primer shown in SEQ ID NO: 4 were used. Type II cDNA 10〃1, each of the two primers 0.5〃M each, each component 400〃M dNTP (dATP, dGTP, dCTP, dTTP) mixture, 2.5 mM magnesium chloride, TaKaRa LA Taq ( PCR was performed under the following conditions using a reaction solution 201 containing 2.5 units and 1 × La Taq buffer (magnesium-free).
即ち、 MA Thermal Cycler 480 (パ一キンエルマ一 ·シ一タス（Perkin Elmer Cetus)社）を用い、 94°Cで 30秒間、 55°Cで 60秒間、 72°Cで 90秒間の 工程を 1サイクルとして 25サイクル行い、 さらに 72°Cで 7分間加熱した。 得られた該 PCR溶液より 10〃1を分取し、 ァガロース電気泳動にかけた結 果を図 4に示す。 試した 4種のヒト癌細胞由来 P CR溶液の全てにおいて、 h MD 6をコードする塩基配列から予想される約 1. 5 k bの大きさの DNA断 片が増幅されることを確認した。 以上の結果から、 これら 4種の癌細胞で hM D6をコードする cD N Aが発現していることが示された。 実施例 3 h M D 6遺伝子の染色体マッピング That is, using a MA Thermal Cycler 480 (Perkin Elmer Cetus), one cycle of a process at 94 ° C for 30 seconds, 55 ° C for 60 seconds, and 72 ° C for 90 seconds. 25 cycles, and further heated at 72 ° C for 7 minutes. FIG. 4 shows the results obtained by collecting 10〃1 of the obtained PCR solution and subjecting it to agarose electrophoresis. It was confirmed that in all of the four types of PCR solutions derived from human cancer cells, a DNA fragment having a size of about 1.5 kb predicted from the nucleotide sequence encoding hMD6 was amplified. From the above results, it was shown that cDNA encoding hMD6 was expressed in these four types of cancer cells. Example 3 h Chromosome mapping of MD6 gene
h M D 6遺伝子の一部を含む E S Tである M579079、 AI066748について、 ァ メリカ National Center for Biotechnology Information (NCBI )の UniGeneサ一 バを用いて検索した結果、 Hs. 13755の UniGeneクローンにアセンブリされた。 該 クローンは、 既にヒト染色体上にマップされている S T S (Sequence-tagged site) である SHGC- 15152を含んでいた。  As a result of searching for M579079 and AI066748, which are ESTs containing a part of the hMD6 gene, using the UniGene server of the American National Center for Biotechnology Information (NCBI), they were assembled to the Hs. 13755 UniGene clone. The clone contained SHGC-15152, an STS (Sequence-tagged site) already mapped on the human chromosome.
該 S T Sは、 ヒト 9番染色体上のマーカ一、 D9S258と D9S1821の間に位置して いた。 この位置は、 第 9番染色体長腕の 3 1—3 2領域（9q31- 32)に相当する。 この領域は、 食道癌、 基底細胞癌、 非ホジキン型リンパ腫、 （日本臨床、 ， 98 6 - 991( 1996 )、 Cancer Res. , 56, 1629-1634 ( 1996 )、 Cancer Lett, 79, 67-72 ( 1994)、 Genes Chromosomes Cancer, 12, 32-36 ( 1995) などで欠失が見られる 領域であり、 癌抑制遺伝子の存在が示唆されている。 また、 T細胞白血病にお ける t(7; 9)染色体点座の切断点の近傍でもある。 さらに、 虚血性心疾患の危険 因子である低 H D L (高密度リポ蛋白質） を引き起こすタンジール病 （Tangier disease) の原因遺伝子や家族性自律神経障害の原因遺伝子が、 この領域にマツ プされている [Nature Genetics, 4, 160-164 ( 1993), Nature Genetics, 20, 96-98 ( 1998)] 。 該クローンは、 これらの疾患と関与している可能性がある。  The STS was located between markers D9S258 and D9S1821 on human chromosome 9. This position corresponds to the 31-32 region (9q31-32) of the long arm of chromosome 9. This area includes esophageal cancer, basal cell carcinoma, non-Hodgkin's lymphoma, (Japanese clinical,, 986-991 (1996), Cancer Res., 56, 1629-1634 (1996), Cancer Lett, 79, 67-72 (1994), Genes Chromosomes Cancer, 12, 32-36 (1995), etc., where deletions are seen, suggesting the presence of a tumor suppressor gene, and t (7; 9) It is also near the breakpoint of the chromosomal locus, as well as the genes responsible for Tangier disease (Tangier disease), which causes low HDL (high density lipoprotein), a risk factor for ischemic heart disease, and familial autonomic dysfunction. The causative gene has been mapped to this region [Nature Genetics, 4, 160-164 (1993), Nature Genetics, 20, 96-98 (1998)] The clone is involved in these diseases there is a possibility.
産業上の利用可能性 Industrial applicability
本発明により、 新規 F—WD蛋白質、 該蛋白質をコードする D N A、 該 D N Aを含有する組換えベクター、 および該組換えベクターを保有する形質転換体、 が提供される。 本発明により得られる蛋白質、 該蛋白質をコードする D N A、 該蛋白質を認識する抗体該蛋白質、 該 D N Aの一部配列からなるオリゴヌクレ ォチドは、 神経変性疾患、 悪性腫瘍、 自己免疫疾患、 炎症性疾患、 ウィルス性 疾患等、 蛋白質分解の亢進または低下により引き起こされる疾患の予防、 治療、 診断等に有効に用いられる。  The present invention provides a novel F-WD protein, a DNA encoding the protein, a recombinant vector containing the DNA, and a transformant containing the recombinant vector. A protein obtained by the present invention, a DNA encoding the protein, an antibody recognizing the protein, the protein, and an oligonucleotide comprising a partial sequence of the DNA may be a neurodegenerative disease, a malignant tumor, an autoimmune disease, an inflammatory disease, It is effectively used for the prevention, treatment, diagnosis, etc. of diseases caused by enhanced or reduced protein degradation such as viral diseases.
「配列フリーテキスト」 配列番号 3—人工配列の説明：合成 D N A 配列番号 4—人工配列の説明：合成 D N A 配列番号 5—人工配列の説明：合成 D N A "Sequence free text" SEQ ID NO: 3—Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 4—Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 5—Description of Artificial Sequence: Synthetic DNA

Claims

請求の範囲 The scope of the claims
1. 配列番号 1に示されるアミノ酸配列からなる蛋白質。  1. A protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
2. 配列番号 1に示されるアミノ酸配列において 1以上のァミノ  2. one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1
酸が欠失、 置換または付加されたァミノ酸配列からなる F— WD蛋白質。An F-WD protein comprising an amino acid sequence in which an acid has been deleted, substituted or added.
3. 配列番号 6で示されるアミノ酸配列を有してなる F—WD蛋白質。 3. An F-WD protein having the amino acid sequence represented by SEQ ID NO: 6.
4. 配列番号 7〜13で示されるアミノ酸配列の 1以上の配列を有してなる F— WD蛋白質。  4. An F-WD protein having one or more amino acid sequences represented by SEQ ID NOs: 7 to 13.
5. 請求項 1〜4のいずれか 1項に記載の蛋白質をコ一ドする DN Aまたは 該 DNAと相補的な配列を有する DNA。  5. A DNA encoding the protein according to any one of claims 1 to 4, or a DNA having a sequence complementary to the DNA.
6. 配列番号 2記載の塩基配列を有する D N Aまたは該 D N Aと相補的な配列 を有する DNA。  6. DNA having the nucleotide sequence of SEQ ID NO: 2 or DNA having a sequence complementary to the DNA.
7. 請求項 5または 6記載の DNAとストリンジェン卜な条件下でハイブリダ ィズし、 かつ F— WD蛋白質をコードする DNAまたは該 DNAと相補的な配 列を有する DNA。  7. A DNA that hybridizes with the DNA according to claim 5 or 6 under stringent conditions, and that encodes a F-WD protein or has a sequence complementary to the DNA.
8. 請求項 5〜7のいずれか 1項に記載の DNAを含有する組換えべクタ一。 8. A recombinant vector containing the DNA according to any one of claims 5 to 7.
9. プラスミ ド phMD 6である、 請求項 8記載の組換えぺク夕一。 9. The recombinant protein of claim 8, which is plasmid phMD6.
10. 請求項 8または 9記載の組換えべクタ一を保有する形質転換体。  10. A transformant having the recombinant vector according to claim 8 or 9.
11. 形質転換体が、 微生物、 動物細胞、 植物細胞および昆虫細胞から選ばれ る形質転換体である、 請求項 10記載の形質転換体。  11. The transformant according to claim 10, wherein the transformant is a transformant selected from a microorganism, an animal cell, a plant cell, and an insect cell.
12. 微生物がェシエリヒア (Escherichia)属に属する微生物である、 請求項 11記載の形質転換体。  12. The transformant according to claim 11, wherein the microorganism is a microorganism belonging to the genus Escherichia.
13. ェシエリヒア属に属する微生物がェシエリヒア ' コリ (Escherichia coli ) DH5a/phMD6 (FERM BP— 6611) である、 請求項 12記載の形質転換体。  13. The transformant according to claim 12, wherein the microorganism belonging to the genus Escherichia is Escherichia coli DH5a / phMD6 (FERM BP-6611).
14. 請求項 10〜 13のいずれか 1項に記載の形質転換体を培地に培養し、 培養物中に請求項 1〜4のいずれか 1項に記載の蛋白質を生成蓄積させ、 該培 養物から該蛋白質を採取することを特徴とする、 請求項 1〜4のいずれか 1項 に記載の蛋白質の製造方法。 14. The transformant according to any one of claims 10 to 13 is cultured in a medium, and the protein according to any one of claims 1 to 4 is produced and accumulated in the culture. The method for producing a protein according to any one of claims 1 to 4, wherein the protein is collected from a nutrient.
15. 請求項 5〜 7のいずれか 1項に記載の D N Aの有する塩基配列中の連続 した 5〜6 0塩基と同じ配列を有するオリゴヌクレオチド、 該オリゴヌクレオ チドと相補的な配列を有するオリゴヌクレオチド、 およびこれらオリゴヌクレ ォチドの誘導体ォリゴヌクレオチドから選ばれるォリゴヌクレオチド。  15. An oligonucleotide having the same sequence as 5 to 60 consecutive bases in the base sequence of the DNA according to any one of claims 5 to 7, an oligonucleotide having a sequence complementary to the oligonucleotide. And oligonucleotides selected from oligonucleotide derivatives of these oligonucleotides.
16. 請求項 1〜4のいずれか 1項に記載の蛋白質を認識する抗体。  16. An antibody that recognizes the protein according to any one of claims 1 to 4.
17. 請求項 1 6記載の抗体を用いることを特徴とする、 請求項 1〜4のいず れか 1項に記載の蛋白質の免疫学的検出法。  17. The method for immunologically detecting a protein according to any one of claims 1 to 4, wherein the antibody according to claim 16 is used.
18. 請求項 1 6記載の抗体を用いることを特徴とする、 請求項 1〜4のいず れか 1項に記載の蛋白質の免疫組織染色法。  18. The method for immunohistological staining of a protein according to any one of claims 1 to 4, wherein the antibody according to claim 16 is used.
19. 請求項 1 6記載の抗体を含有する、 免疫組織染色剤。  19. An immunohistochemical staining agent comprising the antibody according to claim 16.
20. 請求項 1〜 4のいずれか 1項に記載の蛋白質と被験試料とを接触させ、 該蛋白質の有するュビキチンリガーゼ活性を変動させることを特徴とする化合 物のスクリーニング方法。  20. A method for screening a compound, which comprises bringing a protein according to any one of claims 1 to 4 into contact with a test sample, and varying the ubiquitin ligase activity of the protein.
21. 請求項 1〜4のいずれか 1項に記載の蛋白質を発現する細胞と被験試料 とを接触させ、 該蛋白質の有するュビキチンリガーゼ活性を変動させることを 特徴とする化合物のスクリーニング方法。  21. A method for screening a compound, which comprises bringing a cell that expresses the protein according to any one of claims 1 to 4 into contact with a test sample, and varying the ubiquitin ligase activity of the protein.
22. 請求項 2 0または 2 1記載の方法により得られる化合物。  22. A compound obtained by the method according to claim 20 or 21.
23. 請求項 1〜 4のいずれか 1項に記載の蛋白質と被験試料とを接触させ、 該蛋白質の有する蛋白質分解促進活性を変動させることを特徴とする化合物の スクリーニング方法。  23. A method for screening a compound, which comprises bringing a protein according to any one of claims 1 to 4 into contact with a test sample to vary the activity of the protein in promoting protein degradation.
24. 請求項 1〜4のいずれか 1項に記載の蛋白質を発現する細胞と被験試料 とを接触させ、 該蛋白質の有する蛋白質分解促進活性を変動させることを特徴 とする化合物のスクリーニング方法。  24. A method for screening a compound, which comprises bringing a cell that expresses the protein according to any one of claims 1 to 4 into contact with a test sample, and fluctuating the protein degradation promoting activity of the protein.
25. 請求項 2 3または 2 4記載の方法により得られる化合物。 25. A compound obtained by the method according to claim 23.
26. 請求項 1〜4のいずれか 1項に記載の蛋白質を発現する細胞と被験試料 とを接触させることを特徴とする、 該蛋白質をコ一ドする遺伝子の発現を変動 させる化合物のスクリーニング方法。 26. A method for screening a compound that alters the expression of a gene encoding the protein, comprising contacting a cell expressing the protein according to any one of claims 1 to 4 with a test sample. .
27. 請求項 2 6記載の方法により得られる化合物。  27. A compound obtained by the method according to claim 26.
28. 請求項 5〜7のいずれか 1項に記載の D N Aを用いることを特徴とする、 請求項 1〜4のいずれか 1項に記載の蛋白質をコードする mR NAを検出する 方法。  28. A method for detecting mRNA encoding the protein according to any one of claims 1 to 4, wherein the DNA according to any one of claims 5 to 7 is used.
29. 請求項 1 5記載のオリゴヌクレオチドを用いることを特徴とする、 請求 項 1〜4のいずれか 1項に記載の蛋白質をコ一ドする mR NAを検出する方法。 29. A method for detecting an mRNA encoding the protein according to any one of claims 1 to 4, wherein the oligonucleotide according to claim 15 is used.
30. 請求項 5〜 7のいずれか 1項に記載の D N Aを用いることを特徴とする、 請求項 1〜 4のいずれか 1項に記載の蛋白質の発現を抑制する方法。 30. A method for suppressing the expression of the protein according to any one of claims 1 to 4, wherein the DNA according to any one of claims 5 to 7 is used.
31. 請求項 1 5記載のオリゴヌクレオチドを用いることを特徴とする、 請求項 1〜 4のいずれか 1項に記載の蛋白質の発現を抑制する方法。  31. A method for suppressing the expression of the protein according to any one of claims 1 to 4, wherein the oligonucleotide according to claim 15 is used.
32. 請求項 5〜7のいずれか 1項に記載の D N Aを用いることを特徴とする、 請求項 1〜4のいずれか 1項に記載の蛋白質をコードする遺伝子のプロモー夕 一 D N Aを取得する方法。  32. Obtaining a promoter DNA of a gene encoding the protein according to any one of claims 1 to 4, wherein the DNA according to any one of claims 5 to 7 is used. Method.
33. 請求項 1 5記載のオリゴヌクレオチドを用いることを特徴とする、 請求 項 1〜4のいずれか 1項に記載の蛋白質をコードする遺伝子のプロモーター D N Aを取得する方法。  33. A method for obtaining a promoter DNA of a gene encoding the protein according to any one of claims 1 to 4, wherein the oligonucleotide according to claim 15 is used.
34. 請求項 1〜4のいずれか 1項に記載の蛋白質をコードする遺伝子の転写 を司るプロモーター D N A。  34. A promoter DNA which controls transcription of a gene encoding the protein according to any one of claims 1 to 4.
35. 請求項 3 4記載のプロモー夕一 D NAおよび該プロモーター D NAの 下流に連結させたレポ一夕一遺伝子を含有するプラスミ ドを保有する形質転換 体と被験試料とを接触させ、 該レポ一夕一遺伝子の翻訳産物含量を測定するこ とを特徴とする、 該プロモータ一による転写の効率を変動させる化合物のスク リ一ニング法。 35. A test sample is contacted with a transformant having a plasmid containing the promoter DNA and the repo overnight gene linked downstream of the promoter DNA according to claim 34, and A method for screening a compound that changes the efficiency of transcription by said promoter, comprising measuring the content of a translation product of the gene overnight.
36. レポ一夕一遺伝子が、 クロラムフエニコ一ル ' ァセチルトランスフェラ —ゼ遺伝子、 一ガラクトシダーゼ遺伝子、 ルシフヱラ一ゼ遺伝子およびグリ —ン ·フルォレツセント ·プロテイン遺伝子から選ばれる遺伝子である、 請求 項 3 5記載のスクリ一二ング方法。 36. The repo overnight gene according to claim 35, wherein the gene is selected from a chloramphenicil 'acetyltransferase gene, a monogalactosidase gene, a luciferase gene and a green fluorescein protein gene. Screening method.
37. 請求項 3 5または 3 6記載の方法により得られる化合物。  37. A compound obtained by the method according to claim 35 or 36.
38. 請求項 1〜4のいずれか 1項に記載の蛋白質を含有する、 蛋白質分解の 亢進または低下により引き起こされる疾患の予防薬。  38. A preventive agent for a disease caused by enhanced or reduced proteolysis, comprising the protein according to any one of claims 1 to 4.
39. 請求項 1〜4のいずれか 1項に記載の蛋白質を含有する、 蛋白質分解の 亢進または低下により引き起こされる疾患の治療薬。  39. A therapeutic agent for a disease caused by enhanced or reduced proteolysis, comprising the protein according to any one of claims 1 to 4.
40. 請求項 1〜4のいずれか 1項に記載の蛋白質を含有する、 蛋白質分解の 亢進または低下により引き起こされる疾患の診断薬。  40. A diagnostic agent for a disease caused by enhanced or reduced proteolysis, comprising the protein according to any one of claims 1 to 4.
41. 請求項 5〜 7のいずれか 1項に記載の D N Aを含有する、 蛋白質分解の 亢進または低下により引き起こされる疾患の予防薬。  41. A preventive agent for a disease caused by enhanced or reduced proteolysis, comprising the DNA of any one of claims 5 to 7.
42. 請求項 5〜7のいずれか 1項に記載の D N Aを含有する、 蛋白質分解の 亢進または低下により引き起こされる疾患の治療薬。  42. A therapeutic agent for a disease caused by enhanced or reduced proteolysis, comprising the DNA according to any one of claims 5 to 7.
43. 請求項 5〜 7のいずれか 1項に記載の D N Aを含有する、 蛋白質分解の 亢進または低下により引き起こされる疾患の診断薬。  43. A diagnostic agent for a disease caused by enhanced or reduced proteolysis, comprising the DNA of any one of claims 5 to 7.
44. 請求項 1 5記載のオリゴヌクレオチドを含有する、 蛋白質分解の亢進ま たは低下により引き起こされる疾患の予防薬。  44. A preventive agent for a disease caused by enhanced or reduced proteolysis, comprising the oligonucleotide according to claim 15.
45. 請求項 1 5記載のオリゴヌクレオチドを含有する、 蛋白質分解の亢進ま たは低下により引き起こされる疾患の治療薬。  45. A therapeutic agent for a disease caused by enhanced or reduced proteolysis, comprising the oligonucleotide according to claim 15.
46. 請求項 1 5記載のオリゴヌクレオチドを含有する、 蛋白質分解の亢進ま たは低下により引き起こされる疾患の診断薬。  46. A diagnostic agent for a disease caused by enhanced or decreased proteolysis, comprising the oligonucleotide according to claim 15.
47. 請求項 1 6記載の抗体を含有する、 蛋白質分解の亢進または低下により 引き起こされる疾患の予防薬。  47. A preventive agent for a disease caused by enhanced or reduced protein degradation, comprising the antibody according to claim 16.
48. 請求項 1 6記載の抗体を含有する、 蛋白質分解の亢進または低下により 引き起こされる疾患の治療薬。 48. The antibody according to claim 16, wherein the protein degradation is enhanced or reduced. Remedies for the disease caused.
49. 請求項 1 6記載の抗体を含有する、 蛋白質分解の亢進または低下により 引き起こされる疾患の診断薬。  49. A diagnostic agent for a disease caused by enhanced or reduced protein degradation, comprising the antibody according to claim 16.
PCT/JP1999/007335 1998-12-28 1999-12-27 Novel protein WO2000040609A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021252A2 (en) * 1994-02-01 1995-08-10 The Board Of Trustees Of The Leland Stanford Junior University Wd-40-derived peptides and uses thereof
WO1999018989A1 (en) * 1997-10-16 1999-04-22 Baylor College Of Medicine F-box proteins and genes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021252A2 (en) * 1994-02-01 1995-08-10 The Board Of Trustees Of The Leland Stanford Junior University Wd-40-derived peptides and uses thereof
WO1999018989A1 (en) * 1997-10-16 1999-04-22 Baylor College Of Medicine F-box proteins and genes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
C. CENCIARELLI ET AL.: "Identification of a family of human F-box proteins", CURRENT BIOLOGY, vol. 9, no. 20, 1999, pages 1177 - 1179, XP002924396 *
JEFFREY T. WINSTON ET AL.: "A family of mammalian F-box proteins", CURRENT BIOLOGY, vol. 9, no. 20, 1999, pages 1180 - 1182, XP002924397 *
K. DUFF J. ET AL.: "Secondary structure analysis identifies a putative mouse protein demonstrating similarity to the repeat units found in CDC4, the G protein beta subunits and related proteins", DNA SEQUENCE, vol. 3, no. 4, 1992, pages 213 - 220, XP002924394 *
KIN-ICHIRO KOMINAMI ET AL.: "Fission yeast WD-repeat protein Pop1 regulates genome ploidy through ubiquitin-proteasome-mediated degradation of the CDK inhibitor Rum1 and the S-phase initiator Cdc18", GENES & DEVELOPMENT, vol. 11, no. 12, 1997, pages 1548 - 1560, XP002924395 *
KIN-ICHIRO KOMINAMI ET AL.: "Two F-box/WD-repeat proteins Pop1 and Pop2 form hetero- and homo-complexes together with cullin-1 in the fission yeast SCF (Skp1-Cullin-1-F-box) ubiquitin ligase", GENES TO CELLS, vol. 3, no. 11, 1998, pages 721 - 735, XP000908732 *
LYAPINA S.A. ET AL.: "Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein", PROC. NATL. ACAD. SCI. USA, vol. 95, no. 13, 1998, pages 7451 - 7456, XP000892178 *

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