WO2003100064A1 - Novel ubiquitin ligase - Google Patents

Novel ubiquitin ligase Download PDF

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Publication number
WO2003100064A1
WO2003100064A1 PCT/JP2003/006749 JP0306749W WO03100064A1 WO 2003100064 A1 WO2003100064 A1 WO 2003100064A1 JP 0306749 W JP0306749 W JP 0306749W WO 03100064 A1 WO03100064 A1 WO 03100064A1
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sequence
protein
kipl
dna
kpc1
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PCT/JP2003/006749
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French (fr)
Japanese (ja)
Inventor
Keiichi Nakayama
Takumi Kamura
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Kyowa Hakko Kogyo Co., Ltd.
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Priority to AU2003241897A priority Critical patent/AU2003241897A1/en
Priority to JP2004508302A priority patent/JPWO2003100064A1/en
Publication of WO2003100064A1 publication Critical patent/WO2003100064A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a ubiquitin ligase protein having an activity of ubiquitinating p27 Kipl , a complex containing the protein, a DNA encoding the protein, a transformant into which a recombinant DNA containing the DNA has been introduced, and the transformant. And a method for detecting or quantifying mRNA encoding the protein using a polynucleotide or an oligonucleotide derived from the DNA, and a method for suppressing the expression of the protein.
  • the present invention also relates to a method for screening a substance that inhibits ubiquitination of p27 Kipl using the protein, the complex, or the transformant.
  • the present invention relates to an antibody that specifically binds to the protein, a method for immunologically detecting or quantifying the protein using the antibody, a diagnostic agent and a therapeutic agent containing the polynucleotide, the oligonucleotide or the antibody.
  • the eukaryotic cell cycle proceeds by activating a series of cyclin and cyclin-dependent kinase (hereinafter abbreviated as CDK) complexes at the right time and in the right order.
  • CDK cyclin and cyclin-dependent kinase
  • This enzyme activity is controlled by various mechanisms. For example, cyclin protein degradation, phosphorylation and dephosphorylation of CDK, binding and dissociation with a CDK inhibitory protein (hereinafter abbreviated as CKI) are involved in the control mechanism.
  • CKI CDK inhibitory protein
  • Proper control of CDK / cyclin by CKI is essential for normal cell cycle progression, and it has been shown that abnormalities in this control lead to cell carcinogenesis [Genes Dev., 13, 1501 ( 1999); BioEssays, 20, 1020 (1998)].
  • mice homozygously deleted for p27 Kipl are larger than normal mice, and have thymus, testis, ovary, and uterus
  • Adjusting the expression level is considered to be important for elucidation of the cell cycle and the mechanism of canceration, and for the development of cancer diagnostics and therapeutics.
  • p27Kipl expression is regulated not at the transcriptional level, but mainly after transcription, especially by controlling proteolysis.
  • the degradation system by ubiquitin-proteasome is the center [FEBS Lett., 490, 179 (2001); Exp. Cell. Res., 264, 148 (2001); Biochem. Biophys. Res. 282, 853 (2001)].
  • Proteolysis by the ubiquitin-proteasome system is based on the following process.
  • ubiquitin is activated by ubiquitin activating enzyme (E1) and ATP to activate the glycine residue at the carboxy terminus, and thioester bonds to a specific cysteine residue of E1.
  • ubiquitin is transferred to the cysteine residue of ubiquitin-conjugating enzyme (E2).
  • E2 ubiquitin-conjugating enzyme
  • Ubiquitin bound to E2 is isopeptide-linked to lysine residues of the target protein via ubiquitin ligase (E3) that specifically recognizes the target protein (ubiquitination of the target protein).
  • Skp2 is an important molecule for recognition of p27 Kipl .
  • Mice deficient in Skp2 showed growth retardation, growth suppression, chromosome / centrosome abnormalities, and accumulation of p27 Kipl , indicating that Skp2 is involved in p27 Kipl degradation. Suggested [Nat. Cell Biol., 1, 193 (1999); Nat. Cell Biol., 1, 207 (1999); EMBO J., 19, 2069 (2000)].
  • knockout mice lacking the Skp2 gene a decrease in p27 Kipl during the G0-G1 transition period occurred even in the absence of Skp2, which resulted in degradation of p27 Kipl during the GO-G1 transition period.
  • the present invention provides a novel ubiquitin ligase protein having activity to ubiquitinate p27 Kipl , which is different from SCFSkp2 and its constituent components, a ubiquitin ligase complex containing the protein, a DNA encoding the protein, and a set containing the DNA.
  • Transformant into which recombinant DNA has been introduced method for producing the protein using the transformant, detection or quantification of mRNA encoding the protein, using a polynucleotide or oligonucleotide derived from the DNA And a method for suppressing the expression of the protein.
  • the present invention provides a method for screening a substance that inhibits ubiquitination of p27 Kipl using the protein, the complex or the transformant, an antibody that specifically binds to the protein, and a protein using the antibody.
  • An object of the present invention is to provide a method for immunologically detecting or quantifying, a diagnostic agent and a therapeutic agent containing the polynucleotide, the oligonucleotide or the antibody.
  • the present inventors have purified a ubiquitin ligase complex KPC having an activity of ubiquitinating p27 Kipl, and isolated a novel protein KPC1, which is a constituent protein thereof, and a DNA encoding human and mouse KPC1, The nucleotide sequence of the DNA and the amino acid sequence of KPC1 encoded by the DNA were determined. Furthermore, KPC1 alone is a ubiquitin ligase that has the activity to ubiquitinate p27 Kipl ; KPC1 activity requires the C-terminal RING-finger domain; They found that high expression promotes the degradation of p27 Kipl , and completed the present invention.
  • the present invention relates to the following (1) to (53).
  • a protein having an activity of ubiquitinating p27 Kipl which is a constituent component of a complex having an activity of ubiquitinating p27 Kipl and having a molecular weight of 140 kDa.
  • a protein comprising an amino acid sequence represented by SEQ ID NO: 2 or 4 with one or more amino acids added, deleted or substituted, and having an activity of ubiquitinating p27 Kipl .
  • a protein comprising an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2 or 4, and having an activity of ubiquitinating p27 Kipl .
  • a complex having the activity of ubiquitinating p27 Kipl comprising the protein according to any one of claims 1 to 4 and the protein according to any one of the following (a) to ().
  • a DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 or 3 DNA that hybridizes under the conditions of a lingent and encodes a protein having an activity of ubiquitinating p27 Kipl .
  • the transformant according to (10) is cultured in a culture medium, and the protein described in any one of (2) to (4) is produced and accumulated in the culture, and the culture is performed.
  • a recombinant DNA obtained by incorporating the recombinant DNA of (9) and the DNA of any of the following (a) to (e) into a vector is introduced into a host cell. The resulting transformant.
  • the transformant according to (13) or (14) is cultured in a culture medium, and the complex according to (5) is produced and accumulated in the culture, and the complex is produced from the culture.
  • the method for producing a complex according to (5), wherein the method comprises collecting a body.
  • a polynucleotide comprising a sequence complementary to the base sequence of the MA according to any one of (6) to (8).
  • the expression level of the protein according to any one of (2) to () is increased or decreased in mRNA level as compared with a healthy person A method for determining or diagnosing a disease.
  • a polynucleotide comprising a continuous sequence of 20 or more bases in a sequence complementary to the base sequence of the DNA according to any one of (6) to (8)
  • a DM comprising a sequence of 20 to 100 consecutive bases in the nucleotide sequence of the DNA according to any one of (6) to (8) and one of any of (6) to (8) DNA containing a continuous 20 to 100 base sequence in the sequence complementary to the base sequence of the DNA described in
  • Any one of (6) to (8) which is an oligonucleotide comprising a continuous 20 to 100 nucleotides sequence in the nucleotide sequence of 1) according to any one of (6) to (8).
  • (2) to (4) using at least one of oligonucleotides containing a continuous sequence of 20 to 100 bases in a sequence complementary to the base sequence of the DNA according to item 1.
  • a method for determining or diagnosing a disease in which a gene encoding the protein described in any one of the above has a mutation.
  • (22) 20 to consecutive nucleotides in the nucleotide sequence of the DNA according to any one of (6) to (8); an oligonucleotide comprising a sequence of 100 nucleotides and any of (6) to (8) Any one of (2) to (4), comprising at least one of oligonucleotides having a continuous sequence of 20 to 100 nucleotides in a sequence complementary to the nucleotide sequence of DM according to Item 1.
  • RNA selected from the group consisting of the following (a) to (c).
  • RNA a double-stranded RNA consisting of the sequence represented by SEQ ID NO: 36 and a sequence complementary to the sequence and having a sequence obtained by adding 2 to 4 nucleotides to the 3 ′ end of the sequence
  • RNA (a) a double-stranded RNA comprising a sequence corresponding to 19 to 25 contiguous bases in the base sequence of the MA according to any one of (6) to (8);
  • RNA (a) a double-stranded RNA comprising a sequence corresponding to 19 to 25 contiguous bases in the base sequence of the DNA according to any one of (6) to (8);
  • a disease caused by abnormal cell cycle or a disease whose symptom can be alleviated by regulating the cell cycle comprising as an active ingredient at least one selected from the group consisting of the following (a) to (d): Remedy. (a) a double-stranded RNA comprising a sequence corresponding to 19 to 25 contiguous bases in the base sequence of the DNA according to any one of (6) to (8);
  • an oligonucleotide or oligonucleotide derivative comprising a continuous sequence of 20 to 100 bases in a sequence complementary to the base sequence of the DNA according to any one of (6) to (8)
  • a step of bringing the protein according to any one of (1) to (4) or the complex according to (5) into contact with p27 Kipl in the presence and absence of a test sample or comprises comparing the amount of binding between the complex and the step of measuring the amount of binding between p27 Kipl, and the protein or plurality in the presence and absence of test sample polymer and p27 Kipl, p27 Kipl
  • (37) a step of bringing the protein according to any one of (1) to (4) or the complex according to (5) into contact with p27 Kipl in the presence and absence of a test sample; Or a step of measuring the amount of binding between the complex and p27 Kipl , and a step of comparing the amount of binding between the protein or the complex and p27 Kipl in the presence and absence of a test sample. Screening method for substances that suppress the degradation of Kipl .
  • a method for screening a substance that suppresses ubiquitination of p27 Kipl comprising a step of comparing the amount of p27 Kipl .
  • test sample in a system comprising the protein according to any one of (1) to (4) or the complex according to (5), ubiquitin activating enzyme, ubiquitin conjugating enzyme, ubiquitin and p27 Kipl Performing ubiquitination of p27 Kipl in the presence and absence of p27, measuring the amount of ubiquitin incorporated in p27 Kipl, and incorporating ⁇ ⁇ 1 in the presence and absence of the test sample
  • a method for screening for a substance that inhibits degradation of p27 Kipl comprising the step of comparing the amount of ubiquitin .
  • test sample in a system comprising the protein according to any one of (4) to (4) or the complex according to (5), ubiquitin activating enzyme, ubiquitin conjugating enzyme, ubiquitin and p27 Kipl.
  • Ubiquitination of p27 Kipl in the presence and absence of ubiquitin, and measuring the amount of ubiquitin incorporated in p27 Kipl , and ubiquitin incorporated in p ⁇ 1 in the presence and absence of the test sample Including comparing the amount of
  • a disease caused by abnormal cell cycle or a symptom by regulating the cell cycle Screening method of remedy for diseases that can be alleviated.
  • an antibody is an antibody that exhibits an activity of inhibiting the activity of ubiquitinating p27 Kipl of the protein according to any one of (1) to (4) or the complex according to (5), The antibody according to (44).
  • the expression level of the protein according to any one of (1) to (4) is higher than that of a healthy person.
  • a therapeutic agent for a disease caused by abnormal cell cycle or a disease whose condition can be alleviated by regulating the cell cycle comprising the antibody according to (45) or (46) as an active ingredient.
  • KPC1 is a ubiquitin ligase that has activity to ubiquitinate p27 Kipl by itself.
  • Examples of the protein of the present invention include the proteins described in (i) to (4) below. You.
  • a protein comprising an amino acid sequence having at least 60% homology with the amino acid sequence represented by SEQ ID NO: 2 or 4, and having an activity of ubiquitinating p27 Kipl
  • the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is human KPC1
  • the protein consisting of the amino acid sequence represented by SEQ ID NO: 4 is mouse KPC1.
  • deletion or substitution of the above amino acids can be performed by site-directed mutagenesis [Zoller, MJ & Smith, M., Nucleic Acids Res., 10, 6487 (1982); Dalbadie-McFarland, G. et al. Natl. Acad. Sci. USA, 79, 6409 (1982); Wells, JA et al., Gene, 34, 315 (1985); Carter, P. et al., Nucleic Acids Res., 13, 4431. Natl. Acad. Sci. USA, 82, 488 (1985)] to encode a protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 4. This can be done by introducing a site-specific mutation into the gene.
  • the number of amino acids to be deleted, substituted or added is not particularly limited, it is a number that can be deleted, substituted or added by a well-known method such as the above-described site-directed mutagenesis method, and is one to several tens. , Preferably 1 to 25, more preferably 1 to 10; and still more preferably 1 to 5.
  • SEQ ID NO: 2 was also obtained by PCR using a set of PCR primers each having a sequence into which the desired mutation (deletion, substitution, addition) had been introduced at the 5 ′ end thereof [Gene, 77, 51 (1989)].
  • a mutation can be introduced into a DNA encoding a protein consisting of the amino acid sequence represented by 4. That is, first, a sense primer corresponding to the 5 'end of the DNA and an antisense primer corresponding to the sequence immediately before (5' side) the mutation introduction site having a sequence complementary to the mutation sequence at the 5 'end.
  • PCR is performed by converting the DNA into type III to amplify a fragment A (3, in which a mutation has been introduced at the 3rd end) from the 5th end to the mutation introduction site of the DNA.
  • the DNA was transformed with a sense primer corresponding to the sequence immediately after (3 'side) having the mutation sequence at the 5' end and an antisense primer corresponding to the 3 'end of the DNA.
  • PCR is performed to amplify a fragment B from the mutation-introduced site of the MA having a mutation introduced at the 5 ′ end to the 3 ′ end.
  • the sense strand of amplified fragment A and the antisense strand of amplified fragment B have the same mutation-introduced site.
  • the DNA is hybridized, the PCR reaction proceeds as a primer-type, and the DNA into which the mutation has been introduced is amplified.
  • a protein consisting of an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence represented by SEQ ID NO: 2 or 4 and having an activity of ubiquitinating p27 Kipl includes SEQ ID NO: 2 or 4
  • His6 oxahistidine
  • the protein of the present invention in order for the protein of the present invention to have the activity of ubiquitinating p27 Kipl , it is necessary to have the RING finger domain present at positions 1254 to 1291 of the amino acid sequence represented by SEQ ID NO: 2 or 4. Preferably, it has at least 60% or more, usually 80% or more, particularly 95% or more homology with the amino acid sequence represented by SEQ ID NO: 2 or 4.
  • Examples of the complex of the present invention include a complex having the activity of ubiquitinating p27 Kipl, which comprises the protein of the present invention described above and the following proteins (a) to () as constituent components.
  • the protein consisting of the amino acid sequence represented by SEQ ID NO: 6 is human KPG2
  • the protein consisting of the amino acid sequence represented by SEQ ID NO: 8 is mouse KPC2.
  • the addition, deletion and substitution of amino acids can be performed in the same manner as the addition, deletion and substitution of amino acids of the amino acid sequence represented by SEQ ID NO: 2 or 4.
  • the white matter is a protein consisting of an amino acid sequence having 1 to 25 amino acids added to the N-terminal or C-terminal of the amino acid sequence represented by SEQ ID NO: 6 or 8, for example, the N-terminal of the amino acid sequence represented by SEQ ID NO: 6 His6 / herpes simplex virus .epitope (hereinafter abbreviated as His6 / HSV) tag represented by SEQ ID NO: 23 and a C-terminal amino acid sequence represented by SEQ ID NO: 6 Influenza virus, hemagglutinin, epitope (hereinafter abbreviated as HA) represented by SEQ ID NO: 24 Can be. Also, in order for this protein to associate with the protein containing the amino acid sequence represented by SEQ ID NO: 2 or 4, at least 60% or more, usually
  • a DNA having a nucleotide sequence complementary to the nucleotide sequence of the DNA of (1) to (3) can be mentioned.
  • DNA that hybridizes under stringent conditions is, for example, SEQ ID NO: 1.
  • a partial DNA fragment thereof such as DNA having the nucleotide sequence represented as a probe, colony hybridization method, plaque hybridization method or Southern plot hybridization method, etc.
  • a 2-fold concentration SSC solution (the composition of a 1-fold concentration SSC solution consists of 150 t ol / L sodium chloride and 15 t ol / L sodium citrate), fill the solution at 65 ° C. MA that can be identified by washing can be given.
  • Hybridization is described in Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular 'Cloning 3rd Edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987 -2001) (Hereinafter abbreviated as "protocols" in "molecular” biology), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995), etc.
  • a DNA having at least 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 1 or 3, preferably 70% or more, more preferably 80% or more, and still more preferably DNAs having 90% or more, particularly preferably 95% or more, and most preferably 98% or more homology can be mentioned.
  • DNA having a base sequence complementary to the base sequence of the DNA of (1) to (3) specifically, a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 3 is given.
  • the DNAs of (1) to (3) are usually obtained as double-stranded DNAs
  • the DNAs of (1) to (3) are used as antisense strands simultaneously with the DNAs of the sense strands (i) to (3).
  • DNA having a base sequence complementary to the base sequence can also be obtained. After heating the double-stranded DNAs of (1) to (3) at 100 ° C for 5 minutes and rapidly cooling them on ice, the DNAs (sense strands) of (1) to (3) and DNA (antisense strand) having a base sequence complementary to the base sequence can be separated.
  • KPC KPC uses the activity of ubiquitination of p27 Kipl as an index to determine the activity of human warm-blooded animals (for example, guinea pigs, rats, mice, chickens, puppies, bushes, puppies, puppies, sal, etc.). It can be purified from cells, or any tissue in which the cells are present, or cells of the blood cell lineage or cultured cells thereof.
  • human warm-blooded animals for example, guinea pigs, rats, mice, chickens, puppies, bushes, puppies, puppies, sal, etc.
  • a method for measuring the activity of ubiquitinating p27 Kipl the method described in 7. (1) can be used.
  • KPC purification methods include centrifugation, salting out with an aqueous solution of ammonium sulfate, or chromatography using a DEAE-Sepharose column, anion exchange or cation exchange column, gel filtration column, etc., alone or in combination. There is a way to handle it. ,
  • the determination of the partial amino acid sequence of the constituent protein contained in the purified KPC can be performed by the following method. That is, from the purified KPC, the protein of the component contained in the KPC was subjected to sodium dodecyl sulfate sodium polyacrylamide gel electrophoresis (
  • the peptide mixture after protease treatment is analyzed using an LCQ ion trap mass spectrometer, MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometry, an electrospray ionization tandem mass spectrometer, or the like.
  • the partial amino acid sequence can be determined by comparing the mass of the peptide theoretically obtained by protease digestion of the sequence on the amino acid sequence database [Pandey, A. & Mann, M., Nature 405, 837 (2000)].
  • the amino acid sequence data of Genpept, PIR, Swiss-Prot, etc. By searching the base, an amino acid sequence having homology to KPC1 can be searched. Search for nucleotide sequence homologous to the cDNA encoding KPC1 by searching the amino acid sequence translated in each frame from the nucleotide sequence of GenBank, EMBL, DDBJ, etc. can do. If the obtained sequence is an EST (Expressed Sequence Tag), the obtained sequence is used as a query to search for sequences with further homology and EST sequences from the other end derived from the same cDNA clone.
  • EST Expressed Sequence Tag
  • a cDNA clone whose EST sequence has been determined is obtained, and the nucleotide sequence of the entire cDNA can be determined.
  • the obtained cDM nucleotide sequence is translated in each frame, and the amino acid sequence of open-reading frame (0RF) having the same amino acid sequence as the partial amino acid sequence of KPC1 can be used as the amino acid sequence of KPC1.
  • IMAGE consortium cDNA clones IMAGE: 3909169 and IMAGE: 3909203 search Genetics each having the EST sequence of human cDNA (Genbank accession numbers BE885419 and BE885914), respectively. (Available from the company).
  • DNA encoding KPC1 can be isolated as follows. First KPC1 properly selected areas of the region encoding including the cDNA, DNA including 5 'end from 20 to 40 nucleotide sequence of the nucleotide sequence of the selected region in 3 5 end 3 of the base sequence of the selected area DNA containing a sequence complementary to the 20 to 40 bases at the 3 'end is synthesized using a DNA synthesizer. Prepare cDNA from tissues and cells expressing KPC1. The DNA encoding KPC1 can be amplified and isolated by PCR using the prepared cDNA as type II and two types of synthetic DNA as primers. Preparation of cDNA from tissues and cells, and PCIU or molecular cloning, 3rd edition, can be performed according to the method described in the third edition.
  • Examples of the DNA encoding KPC1 obtained as described above include a human KPC1 cDNA having the nucleotide sequence represented by SEQ ID NO: 1 and a mouse KPC1 cDNA having the nucleotide sequence represented by SEQ ID NO: 3. it can. Human KPC1 and mouse KPC1 encoded by these cDNAs have novel amino acid sequences represented by SEQ ID NOs: 2 and 4, respectively.
  • the base sequence of each codon in the region encoding KPC1 Is not limited to the codons used in cDNA, and any codon base sequence encoding the same amino acid can be used.
  • KPC2 was the same protein as known glioblastoma cell differentiation factor-related protein (GBDR1) CGenomics 65, 243 (2000)]. Therefore, DNAs encoding KPC2 include DNA having the nucleotide sequence represented by SEQ ID NO: 5 (cDNA of human GBDM) and DNA having the nucleotide sequence represented by SEQ ID NO: 7 (cDNA of mouse GBDR1). it can. Human KPC2 and mouse KPC2 encoded by these cDNAs have the amino acid sequences represented by SEQ ID NOs: 6 and 8, respectively.
  • any region of the obtained nucleotide sequence is complementary to a DNA containing a sequence of 20 to 40 nucleotides at the 5 'end of the nucleotide sequence of the region at the 3' end and 20 to 40 nucleotides of the 3 'end of the nucleotide sequence of the region.
  • a DNA containing a unique sequence at the 3 ′ end can be amplified and isolated by primer-type PCR using human or mouse genomic DNA as type III. Preparation of genomic DNA and PCR can be performed according to the method described in Molecular Cloning, 3rd edition.
  • a genomic DNA library prepared using chromosomal DNA isolated from mouse or human cells or tissues based on the method described in Molecular Cloning 3rd Edition was obtained in (2).
  • mouse or human KPC1 cMA obtained as a probe and screening by a method such as plaque hybridization mouse or human genomic DNA containing the KPC1 gene can be obtained.
  • the exon / intron structure of the KPC1 gene can be clarified by comparing the nucleotide sequence of the genomic DNA containing the KPC1 gene with the nucleotide sequence of the cDNA.
  • the base sequence of the genomic DNA of the KPC1 gene in a region that regulates transcription, such as promoter can be determined. This sequence is useful for analyzing the regulation mechanism of transcription of the KPC1 gene.
  • KPC1 or KPC2 was prepared using the method described in 2 above, using the method described in, for example, Molecular Cloning, Third Edition, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). Alternatively, it can be produced by expressing DNA encoding KPC2 in a host cell. That is, KPC1 or KPC2 is constructed by constructing a recombinant vector in which DNA encoding KPC1 or KPC2 is inserted immediately downstream of an appropriate expression vector, and introducing the vector into host cells. A transformant to be expressed is obtained. By culturing the transformant, KPC1 or KPC2 is produced and accumulated in the culture. KPC1 or KPC2 can be produced by isolating and purifying KPC1 or KPC2 from the culture. The method for producing KPC1 is described below in (1) to (3). KPC2 can be produced in a similar manner using DNA encoding KPC2.
  • the expression vector contains a promoter that can replicate autonomously in the host cell or can be integrated into the chromosome, and can transcribe mA from DNA encoding KPC1 in the host cell. Things are used.
  • any prokaryote, yeast, animal cell, insect cell, plant cell, or the like can be used as long as it can express the gene of interest.
  • animal and plant individuals can be used.
  • the expression vector is capable of autonomous replication in the host prokaryotic organism, and contains a ribosome binding sequence and a DNA encoding KPC1 downstream of the promoter.
  • a clone that has a cloning site is imported.
  • a sequence expressing a marker gene such as a drug resistance gene should be included for selecting a transformant. Insert the DNA encoding KPC1 into the cloning site downstream of the ribosome binding sequence.
  • An appropriate distance between the ribosome binding sequence and the initiation codon for example, 6 to 18 for an E. coli host vector) (Base).
  • Any promoter can be used as long as it can be expressed in the host cell.
  • examples thereof include promoters derived from Escherichia coli and phage, such as trp promoter, lac promoter, PL promoter, T7 promoter, PR promoter and the like. Promoters designed and modified artificially, such as Promoter overnight, tac promoter, T71ac promoter, let I promoter — Yuichi, etc. in which two trp promoters are connected in series can also be used.
  • the promoters of SP01 and SP02 which are phages of Bacillus subtilis, the promoters of PenP and the like can be mentioned.
  • expression vectors examples include pGEMEX-1 (promega), pQE-30 (Qiagen), PKYP200 CAgric. Biol. Chem., 48, 669 (1984)], pLSAl CAgric. Biol. Chem. , 53, 277 (1989)), pGELl (Pro Natl. Acad. Sci., USA, 82, 4306 (1985)), pTrS30 (preparation of E. coli ⁇ 109 1-30 ( ⁇ 1 ⁇ 8?
  • Examples of the host cell include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., for example, Escherichia coli XLl-Blue, Escherichia coli XL2-Blue, Escherichia E.
  • Escherichia coli DH1 Escherichia coli M.C1000s Escherichia coli KY3276, Escherichia coli W1485 N Escherichia coli JM109, Escherichia coli awake 01, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49 N Escherichia coli BL21ic (BL3ic) s Serratia fonticola.
  • Serratia liquefaciens Serratia marcescens Bacillus subtil is, Bacillus amyloliquefaciens, Brevibacterimii ammoniagenes, Brevibacterium i thigh ariophilum ATCC14068 ⁇ Brevibacterium saccharolyticum ATCC14066, Corynebacterium glutamicmn ATCC13032, Corynebacterium glutajnicum ATCC14067, Corynebacterium glutajnicum ATCC13869, Corynebacterium acetoacido philum ATCC13870, Microbacterium ammoniaphilum ATCC15354, and Pseudo cult as sp. D-0110.
  • Any method for introducing the recombinant vector can be used as long as it is a method for introducing DNA into the host cells described above.
  • an electroporation method [NucIeic Acids Res., 16, 6127 (1988) Acad. Sci. USA, 69, 2110 (1972); Gene, 17, 107 (1982)], a protoplast method (Japanese Patent Application Laid-Open No. 63-248394; Mol. Gen. Genet., 168, HI (1979)].
  • expression vectors include promoters that perform transcription in host yeast, transcription termination sequences and genes that serve as transformation markers in yeast, such as drug resistance genes and TRP1, Those containing sequences that can express amino acid synthesis genes such as HIS3 and LEU2 are used.
  • those capable of autonomously replicating and expressing a drug resistance gene serving as a gene transfer marker in E. coli are also preferable.
  • promoters such as the alcohol dehydrogenase gene ADH1 of Saccharomyces cerevisiae and the galactose metabolism genes GAL1 and GAL10 may be used.
  • Acid phosphatase gene PH05 promoter phosphoglycerate kinase gene PGK promoter
  • glyceraldehyde triphosphate dehydrogenase gene GAP promoter heat shock protein gene promoter, mating factor gene MF
  • yeast strains belonging to the genera Saccharomyces, Schizosaccharomyces, Pichia, and the like yeast strains belonging to the genera Saccharomyces, Schizosaccharomyces, Pichia, and the like.
  • Saccharomyces cerevisiae, Schizosaccharomyces pombe, Picnia pastoris, and the like can be mentioned. .
  • any method for introducing a recombinant vector any method can be used as long as it is a method for introducing DNA into yeast, and examples thereof include an electrification method (Methods EnzymoL, 194, 182 (1991)) and a spheroplast method. [Proc. Natl. Acad. Sci. USA, 81, 4889 (1984)], lithium acetate method [J. Bacteriol., 153, 163 (1983)]
  • the expression vector One that promotes transcription one that contains a sequence of transcription termination and polyadenylation signals of the transcript is used.
  • the vector in order to facilitate the production and maintenance of the vector, it is desirable that the vector be capable of autonomous replication and expression of a drug resistance gene as a gene transfer marker in Escherichia coli. Any promoter can be used as long as it can be transcribed in animal cells, but it can be used for the early promoter of SV40 and the promoter of the IE (immediate early) gene of human site megalovirus.
  • Sequences derived from the LTRs of retroviruses such as Yohjanhansa, Rous sarcoma virus, human T-cell leukemia virus I, and Moroni murine leukemia virus, or meta-oral thionine gene,? -Actin gene, and elongation factor 1.
  • promoters of genes derived from animal cells such as 1 can be mentioned.
  • a promoter artificially combining these promoters such as an SRa promoter combining the SV40 early promoter and the LTR of human T cell leukemia virus I, may be used.
  • a constant KPC1-expressing cell in which DNA encoding KPC1 is integrated into the host chromosome MA is prepared by introducing a KPC1-expression vector containing a sequence capable of expressing a resistance gene to a drug such as G418 or hygromycin into the host cell, Can be selected by culturing in the presence of E. coli.
  • a constant expression vector for KPC1 containing a sequence capable of expressing the dihydrofolate reductase (dhfr) gene was introduced into the host cell, By increasing the concentration of methotrexate, a dhfr inhibitor, in a stepwise manner, it is possible to amplify the copy number of the DNA encoding KPC1 together with the dhfr gene.
  • a host cell for performing gene amplification using the dhfr gene a cell in which the dhfr gene does not function, for example, CHO / dhfr- (ATCC: CRL-9096) is used.
  • Specific expression vectors include, for example, PEGFP-C2 (Clontech), PAGE107 (Japanese Patent Application Laid-Open No. 3-22979; Cytotechnol., 3, 133, (1990)), pAS3-3 (Japanese Patent Application Laid-Open No. 2-227075), pCDM8 (Nature, 329, 840, (1987)), pCMV-Tagl (Stratagene) pcDM3.1 (+) (Invitrogen), PHEP4 (Invitrogen), pMSG (Amersham Biosciences) And pAMo [J. Biol. Chem., 268, 22782 (1993)].
  • Host cells include human cells such as HeLa, Namalwa, 293, African black monkey monkey kidney cells C0S-1 and C0S-7, hamster cells CH0 and MK, Cell lines such as NHI3T3 which is a mouse embryo cell, SP2 / 0 and NS0 which are mouse / myeloma cells, and YB2 / 0 which is a rat myeloma cell can be cited.
  • recombinant vector so long as it is a method for introducing DNA into animal cells either can be used, for example, elect port Poreshiyo down method [Cytotechnol., 3 3 133 (1990 ) ], calcium phosphate method ( Pp. 2-227075), the Lipofection method [Proc. Natl. Acad. Sci. USA, S4, 7413 (1987)], and the like.
  • a viral vector for expression can be used.
  • the expression viral vector lacks at least one of the genes encoding the proteins required for packaging the virus, is capable of producing a recombinant virus in packaging cells, and is encoded by DNA introduced into host cells.
  • Those containing a suitable promoter all at once to express the protein can be used.
  • pMX-purOs MFG Proc. Natl. Acad. Sci. USA, 92, 6733 (1995)
  • LL-CG, CL-CG, CS -CG ⁇ CLG J. Virol., 72, 8150 (1998)
  • pAdexl Nucleic Acids Res., 23, 3816 (1995)
  • Necessary proteins for virus packaging include gag, pol, env, etc. derived from mouse retrovirus in the case of retrovirus vector, and gag, pol, env, vpr derived from HIV virus in the case of lentivirus vector. , Vpu, vif, tat, rev, nef, etc .; E1A, E1B, etc. derived from adenovirus in the case of adenovirus vectors; Rep (p5, pl9, p40), Vp (Cap), etc. in the case of adeno-associated virus Can be given.
  • the promoter described in the expression vector using the above animal cell as a host can be used.
  • the active peptide precursor gene is inserted downstream of the promoter in the viral vector to construct a recombinant viral vector.
  • the constructed recombinant virus vector is introduced into a packaging cell adapted to the virus vector to produce a recombinant virus.
  • any cell can be used as long as it can supply a protein necessary for packaging that encodes the above-mentioned gene deficient in the viral vector.
  • Examples of a method for introducing the viral vector into the packaging cell include a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075) and a lipofection method (Proc. Natl. Acad. Sci. USA, M, 7413 (1987)). Can be.
  • the virus vector By infecting a host cell with the produced recombinant virus, the virus vector can be introduced into the host cell.
  • the host cell include the same host cells as those of the expression vector using the above-described animal cell as a host.
  • baculovirus expression system Baculovirus Expression Vectors: A Laboratory Manual, WH Freeman and Company, New York (1992); Bio / Technology, 6, 47 (1988)] is used. That is, after the DNA encoding KPC1 is introduced into the transfer vector, the vector and baculovirus are simultaneously introduced into insect cells, and KPC1 is placed under the polyhedrin gene promoter, which is a strong promoter. KPC1 can be expressed by producing, by homologous recombination, a recombinant baculovirus into which the coding DNA has been inserted, and then infecting insect cells again with this recombinant baculovirus.
  • baculovirus Autographa californica nuclear polyhedrosis virus, glyconucleopolyhedrovirus, etc. are used.
  • insect cells Spodoptera frugiperda cells, Sf9 and Sf21 CBaculovirus Expression Vectors: A Laboratory Manual, WH Freeman and Company, New York (1992)], Trichoplusia ni cells, High5 (manufactured by Invitrogen), etc., are used. Can be done. The silkworm larva can also be used as it is.
  • a polyhedrin promoter and a baculovirus-derived sequence for causing homologous recombination, and genetic manipulations such as maintenance and propagation of the gene and integration of foreign genes are performed in Escherichia coli.
  • sequences autonomously replicable in Escherichia coli and drug resistance genes such as pVL1392, pVL1393, pBlueBac4.5 (both from Invitrogen), pBacPAK9 (from Clontech), etc. Is raised. '
  • KPC1 can also be produced using animal individuals. For example, a known method [Am. J. Clin. Nutr., 63, 639S (1996); Am. J. Clin. Nutr., 63, 627S (1996); Bio / Technology, 9, 830 (1991)] KPC1 can be produced in transgenic non-human animals.
  • Any promoter can be used as long as it can be expressed in animals.
  • mammary gland cell-specific promoters such as ⁇ -casein promoter,-casein promoter, and? Lactoglobulin promoter, whey acidic protein promoter, etc. are preferably used.
  • a transformant derived from a microorganism or animal cell having a recombinant vector into which DNA encoding KPC1 has been incorporated is cultured according to a conventional culture method to produce and accumulate KPC1, and to collect KPC1 from the culture.
  • KPC1 can be manufactured.
  • RPMI 1640 medium As a medium for culturing a transformant using animal cells as a host, commonly used RPMI 1640 medium [J. Am. Med. Assoc., 199, 519 (1967)], Eagle's MEM (Mimimum Essential Medium) (Science, 122 5 501 (1952)), Dalbecco modified Eagle medium CVirology, 8, 396 (1959)), 199 medium CProc. Soc. Exp. Biol. Med., 73, 1 (1950)] or a culture medium obtained by adding fetal serum to the culture medium, etc.
  • an antibiotic such as penicillin-streptomycin may be added to the culture medium. It is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C, and the presence of 5% C02.
  • Tffi-FH medium manufactured by Phanningen
  • Sf-900 II SFM medium manufactured by Invitrogen
  • ExCell400 ExCell405
  • Grace's insect medium preferably pH 6-7, culture temperature 25-30 ° C, and culture time is usually 1-5 days. If necessary, an antibiotic such as genomycin may be added to the medium during the culture.
  • KPC1 can be produced by rearing and producing and accumulating KPC1 according to a usual method, and collecting KPC1 from the animal individual. That is, in the case of an animal individual, for example, a non-human transgenic animal having MA encoding KPC1 is bred, and KPC1 is produced and accumulated in the animal. By collecting KPC1 from inside, KPC1 can be manufactured. Examples of the place of production and accumulation in the animal include milk, eggs and the like of the animal.
  • a culture medium for culturing a transformant obtained by using a prokaryotic organism such as Escherichia coli or a eukaryotic microorganism such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which 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 C.
  • the carbon source may be any one that can be assimilated by the organism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Alcohols such as acid, ethanol, and propanol can be used.
  • nitrogen source examples include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium or inorganic salts of organic acids, other nitrogen-containing compounds, peptone, meat extract, yeast extract, and corn starch. 1. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.
  • dipotassium hydrogen phosphate potassium dihydrogen 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 aeration and stirring culture.
  • the culture temperature is preferably 15 to 40 ° C, and the culture period is usually 16 to 96 hours.
  • the culture maintain the pH at 3.0 to 9.0.
  • the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, aqueous ammonia, or the like. If necessary, an antibiotic such as ampicillin-tetracycline may be added to the medium during the culture period.
  • an inducer may be added to the medium as needed during the culturing.
  • the inducer include isopropyl galactoside which induces the lac promoter, and indole acrylic acid which induces the trp promoter.
  • KPC1 In order to isolate and purify KPC1 accumulated in the culture of the above transformant, the following ordinary protein isolation and purification methods may be used.
  • KPC1 When KPC1 is secreted extracellularly, KPC1 accumulates in the medium. Therefore, after the culture is completed, only the cell-free medium is recovered by a method such as centrifugation.
  • a normal protein isolation and purification method from the medium that is, a solvent extraction method, a salting-out method using ammonium sulfate, and a desalting method
  • Precipitation method with organic solvent DEAE Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), anion exchange chromatography method using resin such as Mono Q ⁇ (Amersham Biosciences), SP Cation exchange chromatography using a resin such as Sepharose (Amersham's Biosciences), hydrophobic chromatography using a resin such as butyl sepharose and phenylsepharose, molecular sieve
  • a purified sample can be obtained by using a gel filtration method, an affinity chromatography method, a chromatofocusing method, or an electrophoresis method such as isoelectric focusing alone or in combination.
  • KPC1 When KPC1 accumulates in the cells of the transformant, the cells of the transformant are collected from the culture after completion of the culture by centrifugation or the like, and suspended in a buffer, and then disrupted by an ultrasonic crusher. The cells are disrupted by a French press or the like to obtain a cell-free extract.
  • a purified sample is obtained from the supernatant obtained by centrifuging the cell-free extract in the same manner as in the above-mentioned purification and isolation from the medium. be able to.
  • KPC1 When KPC1 is present in the form of an insoluble substance in the cells, the cell-free extract is centrifuged, and the insoluble KPC1 is recovered as a precipitate fraction.
  • the lysate is diluted to the extent that the protein denaturing agent does not denature the protein, or the protein denaturing agent is not contained or the protein denaturing agent is not contained.
  • KPC1 is restored to a normal three-dimensional structure, and a purified sample can be obtained by the same isolation and purification method as described above.
  • Structural analysis of the purified KPCl can be performed by a method commonly used in protein chemistry, for example, the method described in "History of Protein Structure for Gene Cloning” (Hisashi Hirano, Tokyo Chemical Dojin, 1993). It is.
  • a transformant expressing both KPC1 and KPC2 is prepared, and the transformant is cultured according to the method described in (2), whereby a complex containing KPC1 and KPC2 as a component in the culture is obtained. (Hereinafter, also referred to as a KPC1-KPC2 complex), and the complex is produced by isolating and purifying the complex from the culture according to the method described in (3). be able to.
  • a transformant expressing both KPC1 and KPC2 can be prepared by introducing both the KPC1 expression vector and the KPC2 expression vector prepared by the method described in (1) into a host. Alternatively, an expression unit consisting of a promoter and DNA encoding KPC2 connected downstream of the promoter is inserted into the KPC1 expression vector to produce KPC1 and KPC2 expression vectors, which are then introduced into host cells. Can also be produced.
  • the KPC1-KPC2 complex can also be produced by mixing KPC1 and KPC2 produced according to the methods described in (1) to (3) and associating them in vitro.
  • a purified preparation of the full-length or partial fragment of KPC1 obtained by the method described in 3 above, or a peptide consisting of a partial amino acid sequence of KPC1 is used as an antigen, and administered to animals to give KPC1 and KPC1.
  • a polyclonal antibody that specifically binds can be produced.
  • a peptide is used as the antigen, it is desirable that the peptide be covalently bound to a carrier protein such as keyhole's phosphate, hemocyanin, or bovine tilogin purine.
  • Peptides used as antigens can be prepared by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method), tBOC method (t-butyloxycarbonyl method), or Applied Biosystems, Advanced ChemTech, Torizu Corporation, etc. Chemical synthesis can be carried out using a peptide synthesizer.
  • Non-human mammals such as rabbits, goats, rats, mice, and hams that are 3-20 weeks old can be used as the animals to be administered.
  • the antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. 3-7 after each dose On the day, blood is collected to prepare serum, and the reaction of the serum with the antigen used for immunization is determined by enzyme-linked immunosorbent assay (ELISA) [Enzyme-linked immunosorbent assay (3rd ed.); A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)].
  • ELISA enzyme-linked immunosorbent assay
  • the dose of the antigen is preferably 50 to 200 mg per administration per animal.
  • the following method can be given as a specific example of ELISA.
  • KPC1 or peptide used as an antigen is coated on an appropriate plate, the serum is reacted, and an antibody to the immunoglobulin of the animal to which the antigen has been administered is labeled with an enzyme such as horseradish peroxidase. Is reacted.
  • the reaction is performed by adding a substrate that develops color with the labeling enzyme, and the amount of color development is measured with a spectrophotometer to determine the serum antibody titer.
  • a polyclonal antibody can be obtained by obtaining whole serum from an animal whose serum has 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 Press, (1988)] or DEAE-Sepharose column, Chromatography using ion-exchange columns, protein A or G columns, gel filtration columns, etc., alone or in combination.
  • the spleen is removed 3 to 7 days after the last administration of the antigenic substance to the mouse or rat showing the antibody titer.
  • the spleen is minced in MEM, crushed with forceps, centrifuged at 200 rpm for 5 minutes, and the supernatant is discarded.
  • the spleen cells in the resulting precipitate fraction are treated with Tris-chlorinated ammonium buffer (pH7.65) for 1 to 2 minutes to remove red blood cells, washed three times with MEM, and the resulting splenocytes are used as antibody-producing cells. Used.
  • myeloma cells cell lines obtained from mice or rats are used.
  • 8-azaguanine-resistant mice derived from BALB / c
  • SP2 / 0-Agl4 Nature, 276, 269 (1978)
  • P3-X63-Ag8653 J. Immunol., 123, 1548 (1979)
  • P3-X63-Ag8 Nature, 256, 495 ( 1975)] etc.
  • These cell lines can be used in an 8-azaguanine medium [RPMI 1640 medium: 1.5 l / L glutamine, 50 / mol / L 2 -mercaptoethanol, 10 ⁇ g / mL Medium containing 10% fetal serum and 10% fetal serum (hereinafter referred to as “normal medium”), followed by 15 zg / mL 8-azaguanine. Culture and use 2 ⁇ 10 7 or more of the cells for fusion.
  • RPMI 1640 medium 1.5 l / L glutamine, 50 / mol / L 2 -mercaptoethanol, 10 ⁇ g / mL Medium containing 10% fetal serum and 10% fetal serum (hereinafter referred to as “normal medium”), followed by 15 zg / mL 8-azaguanine.
  • the cell group of the obtained precipitate fraction was thoroughly disintegrated, and a solution obtained by mixing 2 mL of polyethylene glycol-1000 2 g MEM and 0.7 mL of dimethyl sulfoxide per antibody-producing cell at 37 ° C.
  • KPC1 or peptide used as an antigen is coated on an appropriate plate, and the culture supernatant of the hybridoma is reacted, and further labeled with an enzyme such as horseradish peroxidase.
  • an enzyme such as horseradish peroxidase.
  • React with the recognized anti-mouse immunoglobulin antibody anti-rat immunoglobulin antibody if the antibody-producing cells are derived from rat.
  • the reaction is performed by adding a substrate that develops color using the labeling enzyme, and the amount of color developed is measured with a spectrophotometer to detect antibodies that specifically bind to KPC1 in the culture supernatant.
  • hybridoma repeat cloning twice by limiting dilution [First, use HT medium (medium in which aminopterin is removed from HAT medium), and second, use normal medium]. Similarly, antibodies that bind to KPC1 in the culture supernatant of hybridomas are detected, and hybridomas that exhibit high and stable antibody production are selected as hybridoma strains that produce monoclonal antibodies that specifically bind to KPC1. I do.
  • the subclass of the antibody 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 from the absorbance in 280 dishes.
  • An antibody that specifically binds to KPC1 can be obtained by the methods described in (1) and (2) above. Further, in place of KPC1, a purified preparation of the full-length or partial fragment of KPC2, or a peptide consisting of a partial amino acid sequence of KPC2 is used as an antigen, according to the method described in (1) and (2) above. An antibody that specifically binds to KPC2 can be obtained.
  • KPC1-KPC2 is prepared according to the methods described in (1) and (2) above.
  • An antibody that specifically binds to the complex can be obtained.
  • An antibody that specifically binds to KPC1 or KPC2 is also used as an antibody that specifically binds to the KPC1-KPC2 complex.
  • Antibodies that inhibit the activity of ubiquitinating p27 Kipl possessed by the KPC1 or KPC1-KPC2 complex can be used in a system for measuring the activity of ubiquitinating p27 Kipl described in 7. (1).
  • An antibody that specifically binds to the KPC1 or KPC1-KPC2 complex obtained by the method described in (2) is added as a test sample, and the activity of the KPC1 or KPC1-KPC2 complex to ubiquitinate p27 Kipl is measured.
  • it can be obtained by selecting an antibody in which the activity of the KPC1 or KPC1-KPC2 complex has been reduced as compared with the case where it is not added.
  • An antibody that specifically binds to the KPC1 or KPC1-KPC2 complex obtained by the method described in (3) is added as a test sample, and the amount of p27 Kipl bound to the KPC1 or KPC1-KPC2 complex is measured. It can be obtained by selecting an antibody having a reduced amount of binding as compared with the case where it is not added.
  • a human genomic DNA sequence on the Go sequence database usually contains information about the chromosomal location of that sequence. For example, 2. For the sequence of human genomic DNA containing exon of the human KPC1 genomic gene obtained in (2) (Genbank accession number: NT-022439), it is reported that it is located on human chromosome 3p24.3. It is listed above. Therefore, it can be said that the human KPC1 gene is located on human chromosome 3p24.3.
  • LOH loss of heterozygosity: a chromosomal deletion found in one of two genes
  • KPC1 may be involved in the development of cancers with L0H in this region.
  • polynucleotides derived from MA encoding KPC1 may be used.
  • an antibody that specifically binds to KPC1 such a cancer can be diagnosed or treated based on the method described in 9., 11., or 13.
  • Non-human transgenic animals into which DNA encoding KPC1 has been introduced are described in Proc. Natl. Acad. Sci. USA, 77, 7380 (1980); Nature, 344, 541 (1990); Nature, 315, 680 (1985); Immunol. Immunopathol., 17, 303 (1987)], directly into a fertilized egg of a non-human mammal, in the cells of the animal.
  • a gene construct in which DNA encoding KPC1 is linked downstream of a promoter capable of expressing KPC1 can be prepared.
  • a non-human mammalian embryonic stem cell can be obtained by a known method [Nature, 292, 154 (1981); Proc. Natl.
  • a promoter that can be used for the expression vector in animal cells described in 3. (1) can be used in the same manner.
  • transgenic animals that express KPC1 at high levels systemically or tissue-specifically can be obtained.
  • the activity of KPC1 promotes the ubiquitination and degradation of p27 Kipl in cells, thereby causing cell cycle abnormalities and causing diseases caused by abnormal cell cycles, such as cancer. It is considered to be a disease model such as.
  • the drug can be evaluated by administering a drug such as an anticancer drug to the transgenic animal and observing the disease symptoms and pathology of the transgenic animal.
  • a targeting vector containing an inactive KPC1 gene in which all or part of the exon portion of the genomic DNA has been deleted is prepared. Using a known method (Nature, 326, 295 (1987), Cell, 51, 503 (1987)), the evening-targeting vector was introduced into embryonic stem cells, and the KPC1 gene on the chromosome and the introduced inactive KPC1 gene were homologous. Recombinant embryonic stem cells can be produced [Nature, 350 3 243 (1991)].
  • a germ-line chimeric individual can be produced using the collective chimera method or the injection chimera method.
  • the evening targeting vector can be prepared according to the method described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993) and the like. The evening targeting vector can be used in either a replacement type or an insertion type.
  • Methods for efficiently selecting homologous recombinants include, for example, positive selection, promoter selection, negative selection, poly A selection described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993). The following method can be used. Methods for selecting the target homologous recombinant from the selected cell lines include Southern hybridization (Molecular I. Cloning 3rd Edition) for genomic DNA, PCR, and the like.
  • KPC1 or KPC1-KPC2 complex as follows.
  • the ability of the body to ubiquitinate p27 Kipl can be measured o
  • ubiquitin is added to a mixture of the purified KPC1 or KPC1-KPC2 complex obtained by the method described in the above item 3 and ⁇ 27 ⁇ 1 , El and ⁇ 2 in a test tube, followed by the reaction.
  • the ubiquitinated p27 Kipl is isolated by SDS-PAGE or the like, and the amount of ubiquitin incorporated into the p27 Kipl is measured.
  • the amount of ubiquitin incorporated into p27 Kipl can be determined by using ubiquitin added during the reaction, labeled with fluorescence, biotin, or radioisotope, and measuring a signal based on the label, or by using an anti-ubiquitin antibody.
  • ubiquitin can be measured by detecting ubiquitin [Nature, 373, 81 (1995); FEBS Lett., 377, 193 (1995); Science, 269, 682 (1995)] o After SDS-PAGE, An immunoblot analysis using an antibody that recognizes p27 Kipl (an antibody that specifically binds to p27 Kipl or an antibody that specifically binds to a tag added to p27 Kipl ) was performed, and the increase in the molecular weight of the target protein was used as an index. The amount of ubiquitin can also be measured [J. Biol. Chem., 276, 48937 (2001)].
  • a transformant expressing the KPC1 or KPC1-KPC2 complex is prepared according to the method described in 3. (1), 3. (4), and the transformant or the cell extract or the like is used for the transformant.
  • the system for stopping the reaction of the ubiquitin-proteasome system until the ubiquitination is replaced with the above-mentioned invitro-retrofitting method. It can also be used instead of the component system for measurement.
  • an animal cell or the like expressing p2 Kipl , El and E2 which can use KPC1 or KPC1-KPC2 complex endogenously, such as an animal cell, is used.
  • ubiquitin incorporated into p27 Kipl can be measured by the same method as in the above-mentioned in vitro reconstitution system.
  • ⁇ 27 ⁇ Amino acid labeled with a radioactive isotope to the medium during the culturing of the transformant, after labeling the p27 Kipl, measuring the radioactivity subjected to SDS-PAGE, or recognize p27 Kipl It can be measured by performing an immunoblot analysis using an antibody to be tested.
  • ubiquitin and proteasome were obtained by removing the p27 Kipl from the above-mentioned transformed product expressing the KPC1 or KPC1-KPC2 complex, such as a cell extract, or the in vitro reconstitution system described in (1). Add ⁇ 27 ⁇ 1 to the added system and allow it to react.Collect a part over time, measure the content of p27 Kipl , and treat the host that does not express KPC1 or KPC1-KPC2 complex, or KPC1 or KPC1 -To determine the degradation of p27 Kipl depending on KPC1 or KPC1-KPC2 complex by comparing with the content of p27 Kipl using the above in vitro reconstitution system without KPC2 complex. Can be. The measurement of the content of p27 Kipl can be performed in the same manner as described above.
  • a substance that inhibits ubiquitination of p27 Kipl can be obtained by the screening methods described in (1) and (2) below.
  • an in vitro reconstitution system containing KPC1 or KPC1-KPC2 complex, p27 Kipl , El, E2 and ubiquitin Or a trait that expresses KPC1 or KPC1-KPC2 complex
  • a test sample is added to a system obtained by adding ubiquitin and a proteinase inhibitor to a transformant or a processed product such as a cell extract of the transformant, or not added
  • the ubiquitination reaction of p27 Kipl is performed.
  • the amount of ubiquitin incorporated into p27 Kipl is measured and compared.
  • test sample is selected as a substance that inhibits ubiquitination of p27 Kipl .
  • Test samples include synthetic compounds, naturally occurring proteins, artificially synthesized proteins, antibodies, peptides, carbohydrates, lipids, modified forms and derivatives thereof, and mammals (eg, mouse, rat, guinea pig). Urine, body fluid, tissue extract, cell culture supernatant, and cell extract of hamsters, hamsters, bush, olive, olive, poma, dog, cat, monkey, human, etc., as well as non-peptide compounds, Examples include fermentation products, extracts of plants and other organisms, and the like.
  • test sample is not a single substance but a mixture of many substances such as tissue extracts, cell culture supernatants, and fermentation products
  • selected test sample is further purified, and the By screening, a substance that inhibits the activity of ubiquitinating p27 Kipl can be isolated and identified.
  • the amount of binding between KPC1 or KPC1-KPC2 complex and ⁇ 27 ⁇ 1 is measured.
  • the test sample is the same as in (1). If the test sample is a mixture, the test sample can be further purified and rescreened to isolate and identify substances that inhibit the binding of KPC1 or KPC1-KPC2 complex to p27 Kipl. .
  • KPC1 obtained by the method described in 3. (3), or purified by the method described in 1. or 3. (4)
  • This can be achieved by mixing the KPC1-KPC2 complex and p27 Kipl in vitro.
  • using a host endogenously expressing p27 Kipl Prepare a transformant that expresses the KPC1 or KPCl-KPC2 complex obtained by the method described in 3., or 3.Create a p27 Kipl expression vector according to the method described in (2), and prepare a KPC1 or KPCl-KPC2 complex.
  • the cells By producing a transformant co-transfected with an expression vector for the body, the cells can be brought into contact in a cell.
  • the test sample is added by adding the test sample to the above-mentioned in vitro mixture or a processed product such as a cell extract of the transformant, or by culturing the transformant in a medium containing the test sample. Can be done.
  • immunoprecipitation was performed using an anti-p27 Kipl antibody, and the KPC1 or KPC1-KPC2 complex contained in the immunoprecipitate was specifically bound to KPC1 prepared by the method described in 4.
  • the measurement can be carried out by detecting by immunoblot analysis using an antibody to be used.
  • immunoprecipitation is performed using an antibody that specifically binds to KPC1
  • p27 Kipl contained in the immunoprecipitate is detected by Immunob port analysis using an anti-p27 Kipl antibody. be able to.
  • Antibodies specifically binding to anti-p27 Kipl antibody and KPC1 may be antibodies that specifically bind to p27 Kipl or KPC1-added antibodies.
  • the substance that inhibits the binding between KPC1 or KPC1-KPC2 complex and p27 Kipl obtained by the above method can inhibit ubiquitination of p27 Kipl by KPC1 or KPC1-KPC2 complex.
  • the substance that inhibits p27 Kipl ubiquitination selected by the screening method (1) or (2) can suppress the degradation of p27 Kipl in cells. Furthermore, such substances can suppress the progression of the cell cycle by suppressing the degradation of p27 Kipl , so that diseases caused by abnormal cell cycles and the symptoms can be reduced by regulating the cell cycle. It can be used as a therapeutic agent for various diseases. Diseases caused by abnormal cell cycles and diseases whose symptoms can be reduced by regulating the cell cycle include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, percutaneous transvascular coronary angioplasty. Subsequent vascular restenosis, pulmonary fibrosis, glomerulonephritis, autoimmune disease, etc. can be mentioned.
  • the above-mentioned substance that inhibits ubiquitination of p27 Kipl is particularly effective for treating cancer.
  • the substance that inhibits ubiquitination of p27 Kipl include KPC1 in which the RING finger domain has been deleted, for example, a protein consisting of the amino acid sequence at positions 1 to 1253 of SEQ ID NO: 2.
  • a polynucleotide containing a continuous sequence of 20 or more bases complementary to the base sequence of the DNA encoding KPC1, or (b) a polynucleotide complementary to the base sequence of the DNA encoding KPC1 KPC1-encoding mRNA can be detected or quantified using DNA containing a continuous 20-100 base sequence and DNA containing a continuous 20-100 base sequence of the nucleotide sequence of MA encoding KPC1.
  • the polynucleotide may be DNA or RNA.
  • a DNA containing a sequence of 20 or more consecutive nucleotides complementary to the nucleotide sequence of the DNA encoding KPC1 is a double-stranded DNA encoding KPC1 or a portion containing a sequence of 20 or more consecutive nucleotides thereof. Obtained as fragment antisense strand DNA. Heating the double-stranded DNA at 100 ° C for 5 minutes and then rapidly cooling it on ice can separate the sense and antisense strands.
  • a promoter sequence such as T7 promoter and SP6 promoter is ligated to the 3 'end of DNA encoding KPC1 or a partial fragment containing a continuous sequence of 20 bases or more, and an in polymerase chain reaction using RNA polymerase is performed.
  • a partial fragment containing a sequence of 20 or more consecutive bases of the DNA encoding KPC1 is prepared by cleaving the DNA encoding KPC1 with an appropriate restriction enzyme, or by converting the DNA encoding KPC1 into a ⁇ type.
  • DNA that contains the sequence at the 5 'end 20 to 40 bases at the 3' end of the desired fragment, 3 at the 3 'end complementary to the sequence at the 20 to 40 bases, and DNA containing the DNA at the end as the primer be able to.
  • Primers can be synthesized on a DNA synthesizer.
  • DNA containing a continuous 20 to 100 base sequence with a sequence complementary to the base sequence of the DNA encoding KPC1, 20 to 100 continuous base sequences of the DNA having the DNA encoding KPC1; DNA containing the sequence can be prepared using an MA synthesizer.
  • Methods for detecting the expression level of mRNA encoding KPC1 include, for example, (1) Northern hybridization, (2) Dot blot hybridization, (3) In situ hybridization, (4) RT-PCR, (5) ) Differential hybridization, (6) DNA chip, (7) ribonuclease-protected assay, etc.
  • Samples to be subjected to the above method include cells collected from a living body or various tissues. Biological samples, primary cultured cells prepared from biological samples, various cultured cell lines, mRNAs obtained from the transformants described in 3. (1), or all MA are used. Hereinafter, the mRNA and total RNA are referred to as sample-derived RNA. Preparation of specimen-derived A can be performed by the method described in Molecular-Cloning Third Edition. In the situ hybridization of (3), tissue sections and cells are used instead of specimen-derived A.
  • RNA derived from a sample is separated by gel electrophoresis, transcribed onto a support such as Nylon Fil, and 20 or more consecutive nucleotides complementary to the nucleotide sequence of the DNA encoding KPC1
  • a support such as Nylon Fil
  • mAA encoding KPC1 can be detected as a band.
  • the hybridization and the washing step are desirably performed under stringent conditions.
  • the labeled probe may be, for example, a nick 'translation, a random' priming, or a method such as 5, phosphorylation at the terminal, or the like, in which the radioactive isotope, biotin, digoxygenin, fluorescent group, Can be prepared by incorporation into a polynucleotide. Since the amount of labeled probe reflects the amount of mRNA encoding KPC1, the amount of mRNA bound to KPC1 can be quantified by quantifying the amount of bound labeled probe. Electrophoresis, transfer of membranes, preparation of probes, hybridization, and detection of mRNA can be performed by the methods described in Molecular 'Clothing Third Edition.
  • MA dot-plot hybridization is performed by spot-fixing RNA extracted from tissues or cells onto a membrane in a dot-like manner, hybridizing with a labeled polynucleotide as a probe, and performing hybridization specifically with the probe. This is a method for detecting mRNA that hybridizes. As the probe, the same probe as that of Northern hybridization can be used. Preparation of RNA, spotting of RNA, hybridization, and detection of mRNA can be performed by the methods described in Molecular 'Cloning, Third Edition.
  • In situ hybridization is performed by using a paraffin or cryo-isotope section of tissue obtained from a living body or immobilized cells as a sample, performing labeled probe, hybridization, and washing steps, and then performing microscopic observation. This is a method for examining the distribution and localization of mA in tissues and cells [Methods in Enzymology, 254, 419 (1995)]. As the probe, those similar to those in Northern hybridization can be used. Hybridization and washing steps should be performed under stringent conditions to prevent false positives
  • cDNA synthesized from an RNA derived from a sample using an oligo dT primer or a random primer and a reverse transcriptase (hereinafter referred to as a cDNA derived from the sample) is used. Used for measurement.
  • the sample-derived RNA is niRNA
  • any of the above primers can be used.
  • the sample-derived RNA is total RNA, it is necessary to use an oligo dT primer.
  • cDNA can be synthesized by the method described in Molecular 'Cloning Third Edition.
  • RT-PCI or cDNA derived from the sample was converted to type II, and PCR was performed using KPC1-specific primers designed from the nucleotide sequence of the cDNA encoding KPC1, to amplify a fragment of the cDNA encoding KPC1 to obtain mRNA.
  • the specific primers one, select the appropriate region excluding the poly A chain of cDNA encoding KPC1, its 5 5 ends 20 of the nucleotide sequence of regions; 100 DNA and 3 comprising the nucleotide sequence ' A set of DNAs having a sequence complementary to the terminal 20 to 100 bases can be used.
  • the primer sequence should be designed based on conditions such as no binding between primers or within the primer, specific binding to the target cDNA at the annealing temperature, and removal from the target cMA under denaturing conditions. Is preferred.
  • mRNA encoding a protein such as actin-glyceraldehyde-13-phosphate dehydrogenase (hereinafter abbreviated as G3PDH) that rarely changes in expression level depending on cell types or culture conditions.
  • G3PDH actin-glyceraldehyde-13-phosphate dehydrogenase
  • KPC1 actin-glyceraldehyde-13-phosphate dehydrogenase
  • This number can be determined by performing PCR in which the number of reaction cycles is increased stepwise, collecting DNA fragments to be amplified in each PCR, and quantifying by gel electrophoresis.
  • PCR can be performed by the method described in Molecular Cloning, Third Edition.
  • cDNA derived from the sample matches the nucleotide sequence of the DNA encoding KPC1.
  • MAb encoding KPC1 by performing hybridization and washing on a filter or a base such as slide glass or silicon on which a polynucleotide containing a complementary sequence of consecutive 20 or more bases has been immobilized. Can be detected. Methods based on such a principle include a differential high prescription (Trends Genet., 7, 314 (1991)) and a DNA chip (Genome Res., 6, 639 (1996)).
  • Both methods can immobilize internal controls such as actin and G3PDH on a filter or a substrate to accurately detect the difference in the amount of mRNA encoding KPC1 between the control sample and the target sample. it can.
  • labeled cDNA was synthesized using different labeled dNTPs (a mixture of dATP, dGTP, dCTP, and dTTP) based on RNA from the control sample and the target sample. By simultaneously hybridizing two labeled cDNA probes, accurate quantification of mRNA encoding KPC1 can be performed.
  • a promoter sequence such as T7 promoter, SP6 promoter, etc. is linked to the 3 and 3 ends of the DNA encoding KPC1, and labeled NTP (ATP, GTP, CTP, UTP). Mixture) and an in vitro transcription system using RNA polymerase to synthesize labeled antisense RNA.
  • the labeled antisense RNA is combined with sample-derived A to form an A-RNA hybrid, and then digested with ribonuclease that degrades only single-stranded RNA.
  • the digest is subjected to gel electrophoresis, and an RNA fragment protected from digestion by forming a UNA-RNA hybrid is detected or quantified as mRNA encoding KPC1.
  • Samples to be used for the determination or diagnosis of disease include biological samples such as tissues and blood obtained from patients or mRNA obtained from primary culture cell samples obtained from cells obtained from the biological samples and cultured in an appropriate medium in a test tube. Total RNA is used. In addition, a tissue section obtained from a biological sample can also be used.
  • Diseases in which the expression level of KPC1 is increased at the mRNA level include diseases caused by abnormal cell cycles.
  • Diseases caused by cell cycle abnormalities include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, pulmonary fibrosis, glomerulonephritis, autoimmune diseases, and the like. Effective for determining or diagnosing cancer.
  • the method of quantifying mRNA encoding KPC1 can be used for predicting 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.
  • the drug can be evaluated by comparing the expression level of the mRNA depending on the presence or absence of the drug.
  • the clearest test to assess for the presence of a disease-causing mutation in the KPG1-encoding locus is to directly compare genes from a control population with genes from diseased patients. is there.
  • a biological sample or a sample derived from primary cultured cells established from the biological sample is collected from a disease patient and a healthy person, and MA or RNA is extracted from the biological sample and the sample derived from the primary cultured cell.
  • DNA encoding KPC1 that has been amplified by PCR using the DNA or a cDNA synthesized from the DNA or the RNA into a type I primer and designed based on the nucleotide sequence of the KPC1 gene (these MAs are referred to as , Which is referred to as sample-derived DNA) can be used as sample DNA.
  • a heteroduplex formed by hybridizing a DNA strand having a wild-type allele and a DNA strand having a mutant allele is used.
  • a detection method can be used.
  • Methods for detecting heteroduplexes include (1) heteroduplex detection method by polyacrylamide gel electrophoresis [Trends Genet., 7, 5 (1991)], and (2) —conformation conformation. Single strand conformation polymorphism analysis (SSCP analysis) [Genomics, 16, 325 (1993)], (3) Chemical cleavage of mismatches (CCM Method, chemical cleavage of mismatches) Human Molecular Genetics, BIOS Scientific Publishers Limited (1996)], (4) Enzymatic cleavage method of mismatch [Nat. Genet., 9, 103 (1995)], (5) Denaturing gel electrophoresis Method (denaturing gradient gel electrophoresis DGGE method) [Mutat. Res., 288, 103 (1993)].
  • Heteroduplex detection by polyacrylamide gel electrophoresis is based on the use of a DNA derived from a sample as type II, and a PCIU using a primer designed based on the nucleotide sequence of the KPC1 gene. Amplify and perform polyacrylamide gel electrophoresis. When a heteroduplex is formed due to a mutation in the KPC1 gene, the mobility is lower than that of a homoduplex having no mutation, and these can be detected as extra bands. Separation is better with special gels such as Hydrolink and MDE. Searching for fragments smaller than 200 bp can detect insertions, deletions, and most single base substitutions. Heteroduplex analysis is preferably performed on a single gel combined with the single-stranded conformation polymorphism analysis described below.
  • the DNA derived from the sample was type- ⁇ , and the gene fragment was amplified as a fragment smaller than 200 bp by PCR using a primer designed based on the base sequence of the KPC1 gene. Electrophoresis in When PCR is performed, the KPC1 gene fragment can be detected as a band by labeling the primer with a radioisotope or a fluorescent dye, or by staining an unlabeled amplified product with silver. If a control sample is also run to clarify the difference from the wild-type pattern, the mutated fragment can be detected from the difference in mobility.
  • the DNA derived from the specimen is type III, and the gene fragment amplified from the PCIU using primers designed based on the nucleotide sequence of the KPC1 gene is incorporated into the DNA encoding KPC1 with a radioisotope or fluorescent dye.
  • a radioisotope or fluorescent dye By hybridizing with the labeled DNA and treating with osmium tetroxide, one strand of the DNA at the mismatched site can be cleaved to detect the mutation.
  • the CCM method is one of the most sensitive detection methods and can be applied to kilobase-length samples.
  • the enzymatic cleavage method of Misumi is a method of enzymatically cleaving mismatches by combining ribonuclease A with an enzyme such as T4 endonuclease VII, which is involved in the repair of mischid in cells, instead of osmium tetroxide. It is.
  • sample-derived DNA is converted into type II, and the gene fragment amplified with primers designed based on the base sequence of the KPC1 gene is electrophoresed on a gel with a concentration gradient or temperature gradient of a chemical denaturant. .
  • the gene fragment moves in the gel to a position where it is denatured into a single strand, and does not move after denaturation. Since the mobility in the gel differs depending on whether the gene fragment has a mutation or not, the presence of the mutation can be detected.
  • a poly (G: C) terminal should be attached to each primer.
  • PTT method protein truncation test
  • Genomics 20, 1 (1994)
  • the PTT method can specifically detect splice site mutations and nonsense mutations.
  • the PPC method uses the T7 promoter sequence as a sequence 20 to 40 bases from the 5 'end of the KPC1 coding region of the KPC1 cDNA.
  • the nucleotide sequence of the sample-derived DNA and the sample-derived cDNA it is possible to use a primer designed based on the nucleotide sequence of the DNA of the present invention. By analyzing the determined base sequence, it can be determined whether or not there is a causative mutation in the sample-derived DNA or the sample-derived cDNA.
  • Mutations other than the coding region of the KPC1 gene can be detected by examining non-coding regions, such as introns and regulatory sequences near or in the gene. Diseases caused by mutations in noncoding regions can be confirmed by detecting abnormally sized or abnormally produced mRNA in diseased patients when compared to control samples according to the method described above. it can.
  • DNA encoding KPC1 was used as a probe for hybridization by the method described in 2.
  • (2). Can be cloned. In non-coding areas Mutations can be searched for according to any of the methods described above.
  • Oligonucleotides used in the method for determining or diagnosing a disease having a mutation in the KPC1 gene include oligonucleotides containing a continuous 20 to 100 nucleotide sequence of the nucleotide sequence of DNA encoding KPC1, and KPC1 Oligonucleotides containing a sequence of 20 to 100 bases consecutive to a sequence complementary to the base sequence of DNA can be given. Oligonucleotides are preferably oligo DNAs. Oligonucleotides can be synthesized by a DNA synthesizer.
  • Diseases having a mutation in the KPC1 gene can be determined or diagnosed by detecting a mutation in the gene in any of human tissues. For example, if a germline mutation is present, individuals who inherit the mutation may be more likely to develop the disease. The mutation can be detected by testing DNA from any tissue of the individual's body. For example, a disease can be determined or diagnosed by collecting blood, extracting DM from cells of the blood, and testing gene mutation using this MA. In addition, prenatal diagnosis can be performed by using fetal cells, placental cells, or amniotic cells to test gene mutations.
  • the type of the disease can be determined or diagnosed and used for selecting a drug to be administered.
  • the obtained tissue is treated with trypsin or the like, and the obtained cells are cultured in an appropriate medium.
  • Chromosomal DNA and RNA can be extracted from the cultured cells.
  • CDNA can be synthesized from RNA.
  • DNA or cDNA obtained from a human specimen by any of the above methods for the purpose of determination or diagnosis is referred to as diagnostic specimen-derived DNA.
  • a continuous nucleotide sequence containing 20 to 100 nucleotides in the base sequence of the DNA encoding KPC1 and a sequence complementary to the nucleotide sequence in the DNA encoding KPC1 are used.
  • oligonucleotides containing a 20 to 100 base sequence To determine or diagnose a disease using at least one of the oligonucleotides containing a 20 to 100 base sequence, (1) detection of a restriction enzyme site, (2) allele-specific oligonucleotide Method using nucleotide probe (AS0: allele specific oligonucleotide hybridization), (3) PCR using allele-specific oligonucleotide (ARMS: amplification refractory mutation system), (4) Oligonucleotide ligation (5) PCR-PHFA (PCR-preferential homoduplex iormation assay), (6) Method using oligo DNA array [Protein nucleic acid enzyme, i ⁇ , 2004 (1998)] Can be.
  • AS0 allele specific oligonucleotide hybridization
  • PCR-specific oligonucleotide amplification refractory mutation system
  • Oligonucleotide ligation (5) PCR-PHFA (PCR
  • the DNA derived from the diagnostic sample is amplified with primers designed based on the sequence of KPC1 cDNA, digested with the restriction enzyme, and the resulting restriction enzyme digested. Mutation can be easily detected by comparing the DNA fragment with that of a normal person. However, since single base changes rarely occur, for the purpose of determination or diagnosis, an Oligo DNA probe is designed by combining the sequence information of KPC1 cDNA and the information of a mutation identified separately. Mutations are detected by reverse dot blot method, in which an Oligo DNA probe is bound to the primer and hybridized.
  • an oligo DNA designed based on the mutation identified as the sequence contained in human KPC1 cDNA was bound to the primer, and a primer designed using the sequence contained in KPC1 cDNA from the DNA from the diagnostic sample was used. It is preferable to use a reverse dot blot in which hybridization is performed using a probe prepared by PCR using labeled dNTP.
  • the DNA chip method which consists of directly synthesizing the sequence of KPC1 cDNA and the oligo DNA designed based on the mutation on a substrate such as slide glass or silicon to create a high-density array, is based on a small amount of diagnostic sample. It is a mutation detection method suitable for large-scale diagnostic purposes because various mutations can be detected more easily in DNA or cDNA derived from diagnostic samples. Base mutations can also be detected with the following oligonucleotide 'ligation' Atsushi (0LA).
  • the oligo DNA consisting of the sequence having the mutation site at the 3 'end with the mutation site in between and the oligo DNA consisting of the sequence adjacent to the 3' side of the mutation site is 2 This is made.
  • the diagnostic sample-derived DNA and the oligo DNA are hybridized. After hybridization, ligate the two oligos with DNA ligase. If the sequence corresponding to the mutation site in the DNA derived from the diagnostic sample matches the sequence of the oligo DNA, the two oligo DNAs are linked, but if they are different, they are not linked.
  • OLA is a mutation detection method that is suitable for efficiently judging or diagnosing many samples in a short period of time because electrophoresis and centrifugation are not required.
  • a small amount of a mutant gene can be quantitatively and easily detected by the following PCR-PHFA method [Br. J. Haematol., 95, 198 (1996)].
  • the PCR-PHFA method combines PCR, hybridization in a liquid phase showing extremely high specificity, and ED-PCR (enzymatic detection of PCR product), which detects PCR products in the same manner as ELISA. It is a thing.
  • ED-PCR enzyme detection of PCR product
  • an oligo MA having a sequence of 20 to 100 bases at the 5 'side of the mutation site in the nucleotide sequence of the DNA encoding KPC1 at the 5' end and 3 'more than the mutation site at the mutation site
  • An oligo DNA containing a sequence complementary to a continuous 20 to 100 base sequence existing on the 5 'side at the 5' end can be used.
  • a primer set having the same sequence without a label and a non-labeled amplified product obtained by amplifying a DNA derived from a diagnostic specimen or a cDNA derived from a diagnostic specimen into a template are mixed in a large excess of 20 to 100 times.
  • the mixture After heat denaturation, the mixture is cooled with a gentle temperature gradient of 1 ° C for 5 to 10 minutes to form a complete complementary strand preferentially.
  • the labeled DNA thus re-formed is captured and adsorbed on streptavidin-immobilized gel via biotin, and bound with an enzyme-labeled anti-DNP antibody via DNP, and detected by a color reaction with an enzyme. If the sample does not contain a gene with the same sequence as the labeled DNA, the original double-stranded labeled DNA is preferentially reformed and shows color. to this On the other hand, when genes having the same sequence are present, the color development is remarkably reduced because the replacement of the complementary strand occurs randomly and the amount of the labeled DNA to be regenerated is reduced. This enables the detection and quantification of known mutation and polymorphism genes.
  • the disease having a mutation in the KPC1 gene includes a disease caused by an abnormality in the cell cycle.
  • Diseases caused by cell cycle abnormalities include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, pulmonary fibrosis, glomerulonephritis, autoimmune diseases, and the like. Is particularly effective for determining or diagnosing cancer.
  • siENA short interfering UNA
  • shRNAi short hairpin RNA
  • the sequence to be selected is located in a region at least 50 bases downstream from the start codon in the translation region and has a GC content of 30 to 70%, preferably around 50%, and matches the nucleotide sequence of another gene. Instead, a sequence specific to KPC1 cDNA is more preferred.
  • sequence X A sequence of 19 to 25 bases excluding AA at the end of the selected sequence or a sequence of 19 bases excluding AA and TT (where T in the sequence is U for RNA, hereinafter referred to as sequence X) , and two to four nucleotide sequence complementary thereto that of 35 ends with the sequence X, 2 pieces of that preferably have a sequence obtained by adding two dT (T of Dokishi body) or ⁇ Make RNA.
  • sequence X include the sequence shown in SEQ ID NO: 36, which corresponds to the 1852th to 1882th nucleotides of the human KPC1 cDNA and mouse KPC1 cDNA.
  • An siRNA can be prepared by chaining the two prepared DNAs.
  • Annealing is performed by dissolving the two RNAs in an appropriate buffer (for example, lOminol / L Tris-HC 50 l / L NaCl, lmmol / L EDTA, pH 7.5), and then heat This can be done by heating at 90-95 ° C for 1-5 minutes in a Thermacycler and then cooling to 25 ° C over 45-60 minutes.
  • the iRNA that suppresses the expression of KPC1 includes a double-stranded RNA comprising a sequence represented by SEQ ID NO: 36 and a sequence complementary to the sequence and having 2 to 4 nucleotides added to the 3 ′ end. And double-stranded RNAs consisting of the sequences represented by SEQ ID NOs: 34 and 35, respectively. These siRNAs can suppress the expression of human KPC1 and mouse KPC1.
  • the above-mentioned sequence X and a sequence complementary to the sequence X are connected by a suitable spacer sequence consisting of 3 to 15 bases, and 3, 2 to 4 nucleotides at the terminal, preferably An RNA having a sequence to which two dTs or ⁇ are added is prepared.
  • the 5 'end of the spacer sequence is preferably dTdT or UU.
  • Either of the sequence X or the position of the sequence complementary to the sequence X may be located first.
  • An example of shRNA that suppresses the expression of KPC1 is an RNA consisting of the sequence represented by SEQ ID NO: 38.
  • the shRNA can suppress the expression of human KPC1 and mouse KPC1.
  • shRNA is cleaved in the cell and converted to siRNA.
  • the two RNAs and shRNAs used for the above siRNA can be chemically synthesized using a DNA synthesizer.
  • a silencer siRNA production kit or the like a double-stranded DNA having the sequence of the T7 promoter sequence and the RNA to be produced is produced, and a T7 polymerase having the MA as a type II is used. It can also be prepared using an in vitro transcription system.
  • the siRNA can be expressed in cells by introducing the KPC1 siRNA expression vector prepared as follows into cultured cells or cells in a living body.
  • the KPC1 siRNA expression vector contains a U6 promoter or a HI promoter, etc.
  • a sirRNA expression vector containing an RNA polymerase III promoter, a primer consisting of 3 to 15 bases starting from the above sequences X and TT It can be prepared by inserting a DNA containing a sequence, a sequence complementary to sequence X, and a sequence consisting of 4 to 6 Ts, which is equivalent to RNA polymerase 111 min.
  • shRNA containing sequence X and a complementary sequence is synthesized by the RNA polymerase III reaction from the U6 promoter, and this sMNA is cleaved in the cell and converted to siRNA.
  • siRNA expression vector examples include pSilencer 1.0-U6 (manufactured by Ambion), pSilencer 3.0 (manufactured by Ambion), pSUPER (manufactured by OligoEngine) and the like.
  • a vector for siRNA expression using a retrovirus vector or a lentivirus vector [Science, 296, 550 (2002); Proc. Natl. Acad. Sci USA, 100, 1844 (2003); Nat. Genet., 33. 401 (2003)].
  • the KPC1 siRNA expression vector was prepared by inserting the U6 promoter and the DNA linked to the sequence shown in SEQ ID NO: 37 downstream of the U6 promoter between Not I / Sal I sites of the retroviral vector pMX-puro II.
  • Retrovirus vectors for expressing KPC1 siRNA By introducing this vector into human or mouse cells, siRNA having the sequences represented by SEQ ID NOs: 34 and 35 is expressed, and the expression of human KPC1 or mouse KPC1 can be
  • oligonucleotide containing a sequence of 20 to 100 nucleotides complementary to the complementary sequence (antisense sequence) or a derivative of the oligonucleotide to the cell or in vivo
  • translation can be suppressed, and as a result, expression of KPC1 can be suppressed.
  • a nucleotide sequence complementary to 20 to 100 nucleotides including the start codon of the region encoding KPC1 is preferable.
  • Oligonucleotide derivatives that are not subject to degradation by deoxyliponuclease or ribonuclease are preferred.
  • oligonucleotide derivative examples include an oligonucleotide derivative in which a phosphoric ester bond in an oligonucleotide is converted into a phosphorothioate bond, and a phosphoric diester bond in an oligonucleotide in which an N3, -P5 'phosphoramide bond is formed.
  • Oligonucleotide derivative Oligonucleotide derivative in which ribose and phosphodiester bond in Oligonucleotide are converted to Peptide Nucleic Acid bond, Peracyl in Oligonucleotide is replaced by C-5 propynyl Peracyl Oligonucleotide derivatives, Oligonucleotide derivatives in which peracyl in the oligonucleotide is substituted with C-5 thiazole peracyl, Cytosine in the oligonucleotide is substituted with C-5 propynylcytosine, Oligonu Oligonucleotide derivatives in which cytosine in the nucleotide is substituted with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2, -0-propylribose, or Oligonucleotide derivatives in which the ribo
  • oligonucleotides or oligonucleotide derivatives can be synthesized using an MA synthesizer.
  • siRNAs shRNAs, antisense oligonucleotides, and derivatives of the oligonucleotides include oligofectamine reagent (manufactured by Invitrogen), lipofectamine (Lipofectamine) 2000 (manufactured by Invitrogen), and transmessenger transfection. (TransMessenger Transfection) It can be introduced into cells using a reagent for ribosome transfection such as a reagent (manufactured by Qiagen). Further, the KPC1 siRNA expression vector can be introduced into cells in the same manner as the method for introducing the expression vector into animal cells described in 2.
  • the recombinant virus prepared using the vector can be administered and the cells can be infected to introduce the virus.
  • the siRNA, shRNA, antisense oligonucleotide, derivative of the oligonucleotide, or KPC1 siRNA expression vector can be administered as it is or as a liposome preparation by intravenous injection or the like. it can.
  • KPC1 ubiquitinates p27 Kipl in cells and promotes its degradation, it suppresses p27 Kipl degradation by suppressing KPC1 expression by the methods described in (1) and (2). be able to. Furthermore, since the progression of the cell cycle can be suppressed by suppressing the degradation of p27 Kipl, the method is used for diseases caused by abnormal cell cycles and diseases in which the symptoms can be alleviated by regulating the cell cycle. Can be used for treatment. Diseases caused by abnormal cell cycle and diseases whose symptoms can be reduced by regulating the cell cycle include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, and percutaneous transvascular coronary angioplasty. Vascular restenosis, pulmonary fibrosis, glomerulonephritis, autoimmune diseases, etc., and the method is particularly effective for treating cancer. 13. Detection and quantification of KPC1 using an antibody that specifically binds to KPC1
  • KPC1 or a cell or tissue containing KPC1 can be immunologically detected and quantified.
  • the measurement sample cell or tissue extract, culture supernatant, body fluids such as blood, urine, saliva, etc., or paraffin section or cryo-section section of the tissue are used.
  • RIA radioimmunoassay
  • immunostaining immunostaining
  • immunofluorescent staining immunoblotting
  • dot plotting immunoprecipitation
  • sandwich ELISA monoclonal antibody experiment manual (Kodansha) Scientific) (1987), Lectures on Sequential Chemistry, 5, Immunobiochemical Research (Tokyo Kagaku Doujinshi) (1986)].
  • MA refers to the reaction of an antibody that specifically binds to KPC1 with a measurement sample, and then reacts with an antibody that is labeled with a radioisotope and binds to the antibody.Then, the antigen-antibody complex is separated. This is a method of measuring radioactivity at scintillation counters and detecting and quantifying KPC1 in measurement samples.
  • the antibody that binds to the antibody include an antibody that binds to IgG of an immunized animal at the time of producing an antibody that specifically binds to KPC1, for example, a rat antibody that is prepared by immunizing a rat with an antibody that specifically binds to KPC1. If present, anti-rat IgG antibodies can be mentioned.
  • the immunostaining method involves reacting an antibody that specifically binds to KPC1 with a measurement sample such as a tissue section or cell, and then reacting with an antibody that binds to an enzyme such as peroxidase or an antibody that binds to the antibody labeled with biotin. After that, a color reaction according to the labeling substance is performed, and KPC1 in the measurement sample is detected by microscopic observation.
  • the immunofluorescent staining method involves reacting an antibody that specifically binds to KPC1 with a measurement sample such as a tissue section or a cell, and then reacts with fluorescein isothiocyanate (FITC), Alexa 546, and tetramethylrhodamine isothiocyanate.
  • FITC fluorescein isothiocyanate
  • Alexa 546 Alexa 546
  • tetramethylrhodamine isothiocyanate tetramethylrhodamine isothiocyanate
  • the immunoblotting (Western plot) method is a method in which a sample to be measured is fractionated by SDS-PAGE (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, ⁇ 988), and then blotted on a PVDF membrane or nitrocellulose membrane. And the membrane With an antibody that specifically binds to KPCl, and an enzyme such as peroxidase.
  • the dot plot method is a method in which a measurement sample is printed in a dot form on a nitrocellulose membrane, an antibody that specifically binds to KPC1 is reacted with the membrane, and enzymes such as peroxidase, biotin, radioisotope, etc. After reacting the antibody that binds to the antibody labeled with, the KPC1 in the measurement sample is detected and quantified by a method according to the labeling substance.
  • the immunoprecipitation method involves reacting a measurement sample with an antibody that specifically binds to KPC1, then adding a carrier that specifically binds to immunoglobulin such as protein A-cepharose, and reacting it. Is a method for isolating a carrier bound to an antigen-antibody complex. The antigen-antibody complex is eluted from the carrier, and KPC1 in the measurement sample is detected in the same manner as in the immunoblot method.
  • a sandwich ELISA is a method in which a measurement sample is reacted with a plate on which an antibody that specifically binds to KPC1 is adsorbed, and then an antibody that specifically binds to KPC1 having a different epitope from the above antibody is reacted. After reacting an antibody labeled with an enzyme such as peroxidase that binds to the enzyme, a color reaction is performed according to the enzyme to detect and quantify KPC1 in the measurement sample.
  • an enzyme such as peroxidase that binds to the enzyme
  • a change in the expression level of KPC1 can be detected to determine or diagnose a disease in which the expression level of KPC1 decreases or increases.
  • Diseases in which the expression level of KPC1 is decreased include diseases caused by abnormal cell cycles, such as cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, pulmonary fibrosis, glomerulonephritis, and autoimmune diseases. Etc., and is particularly effective for cancer.
  • Methods for quantifying and diagnosing or diagnosing the expression level of KPC1 using an antibody include the methods for detecting and quantifying KPC1 described in 13.
  • a biological sample itself such as tissue, blood, serum, urine, stool, saliva, or a cell or cell extract obtained from the biological sample obtained from a patient with a disease is used.
  • a biological sample itself such as tissue, blood, serum, urine, stool, saliva, or a cell or cell extract obtained from the biological sample obtained from a patient with a disease is used.
  • Can be It can also be obtained from biological samples A paraffin or cryostat section of the tissue can also be used.
  • a disease associated with a change in the expression level of KPC1 can be determined or diagnosed as follows. First, the expression levels of KPC1 in multiple samples of patients and healthy subjects are measured and compared by the detection methods described above, and the range of the expression levels of KPC1 in patients and healthy subjects is determined. Judgment or diagnosis is made by comparing the expression level of KPC1 in the subject's sample with the expression level of a healthy person and the expression level of a patient, and examining which expression level falls within the range.
  • antibodies that specifically bind to KPC1 antibodies that inhibit the binding between p27 Kipl and KPC1, or antibodies that inhibit KPC1's activity to ubiquitinate p27 Kipl, are ubiquitination of intracellular KPC1 And its decomposition can be suppressed. Furthermore, the antibody can suppress the progression of the cell cycle by suppressing the degradation of p27 Kipl , so that diseases caused by abnormalities in the cell cycle and diseases in which the symptoms can be alleviated by regulating the cell cycle.
  • Can be used for the treatment of Diseases caused by abnormal cell cycles and diseases whose symptoms can be reduced by regulating the cell cycle include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, percutaneous transvascular coronary angioplasty.
  • the latter can be exemplified by vascular restenosis, pulmonary fibrosis, glomerulonephritis, autoimmune disease and the like, and the antibody is particularly effective for treating cancer.
  • the oligonucleotide or oligonucleotide derivative that suppresses the expression of KPC1 described in 12., the antibody that specifically binds to KPC1 described in 14., which is an antibody that inhibits the binding of p27 Kipl to KPC1, or the KPC1 is An antibody that has the activity of inhibiting the activity of ubiquitinating p27 Kipl can be administered alone as a therapeutic agent, but is usually combined with one or more pharmacologically acceptable carriers. And provided as a pharmaceutical preparation produced by any method well-known in the field of pharmacology.
  • oral administration or parenteral administration such as buccal, respiratory, rectal, subcutaneous, intramuscular and intravenous, Desirably, intravenous administration can be mentioned.
  • Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, Suppositories, injections, ointments, tapes and the like.
  • Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.
  • Liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, soybean oil, P- It can be manufactured using preservatives such as 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 and sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl alcohol It can be produced using additives such as a binder such as hydroxypropylcellulose and gelatin, a surfactant such as a fatty acid ester, and a plasticizer such as glycerin.
  • Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
  • the injection is prepared using a carrier comprising a salt solution, a glucose solution or a mixture of both.
  • Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids.
  • Sprays are prepared using a carrier which does not irritate the oral and respiratory mucosa of the recipient and which disperses the active ingredient as fine particles to facilitate absorption.
  • the carrier include lactose and glycerin.
  • Formulations such as aerosols and dry powders can be made depending on the properties of the DNA or oligonucleotide and the carrier used. Also, in these parenteral preparations, the components exemplified as additives in the oral preparation can be added.
  • 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 20 mg / kg per adult per day.
  • reagents required for quantifying mfiNA or KPC1 encoding KPC1 or detecting mutations in the KPC1 gene such as buffers, salts, reaction enzymes, Binds to the antibody of the invention It may contain a labeled antibody, a color former for detection, and the like.
  • Fig. 1 shows a protocol for purifying KPC from a heron reticulocyte extract.
  • Fig. 2 The upper part of Fig. 2 shows the measurement of the activity of each fraction obtained by the Superose 6 gel filtration column to ubiquitinate ⁇ 27 ⁇ 1 by the in vitro reconstitution system.
  • P27 , p27- (GST-Ub) p27- (GST-Ub) 2, p27- (GST-Ub) 3, p27- (GST-Ub) n on the left , p27 Kipl and GST-Ub are 1 P27 Kipl with two GST-Ub added, p27 Kipl with two GST-Ub added, p27 Kip with three GST-Ub added p27 Kipl with three or more GST-Ub indicates the position on SDS-PAGE The same applies to drawings).
  • the molecular weight marker indicates the position of the fraction eluted by gel filtration.
  • the lower panel shows the results of the same fraction stained with Komasi after SDS-PAGE.
  • the position of the molecular weight marker is shown on the left, and the positions of KPC1 and KPC2 are shown on the right.
  • Fig. 3 Like Fig. 2, Fig. 3 shows the measurement of the activity of each fraction obtained with the mini-Q column to ubiquitinate p27 Kipl by the in vitro reconstitution system. The result of fractionation of the fractions after SDS-PAGE and coomassie staining is shown. The position of the molecular weight marker is shown on the left, and the positions of KPC1 and KPC2 are shown on the right.
  • FIG. 4 shows the results of SDS-PAGE and Komasi-stained KPC purified from a perch reticulocyte extract.
  • the position of the molecular weight marker is shown on the left, the positions of KPC1 and KPC2 are shown on the right, and the amino acid sequence of the partial peptide obtained therefrom is shown in one-letter code.
  • FIG. 5 Fig. 5a shows the structure of KPC1 and KPC2.
  • SPRAY stands for SPRAY domain
  • RING stands for RING finger domain
  • UBU stands for ubiquitin-like domain
  • UBA stands for ubiquitin-associated domain.
  • FIG. 5b shows the amino acid sequence of human KPC1. The part corresponding to the sequence obtained from peptide analysis of the purified sample is underlined, the SPRY domain and the RING finger domain are shown in black and white inverted and boxed, respectively, and the RING finger domain is shown. * Is added to the sequence predicted to be the zinc binding site in the protein.
  • Fig. 6 shows the results of SDS-PAGE of the purified recombinant KPC1-KPC2 complex expressed and expressed in insect cells, followed by coomassie staining. The position of the molecular weight marker is shown on the left, and the positions of the His6 / FLAG-added KPC1 and His6 / HSV-added KPC2 are shown on the right.
  • Fig. 7 shows the results of immunoprecipitation analysis of intracellular association of KPC1 and KPC1 (AR) expressed in insect cells with KPC2.
  • WT indicates wild-type KPC1, ⁇ indicates KPC1 (. ⁇ , + indicates KPC2, and one indicates no expression.
  • Each of the immunoprecipitates by (binding to KPC1 and KPC1 ( ⁇ )) shows the immunoblot analysis using an anti-FLAG antibody, and the lower panel using an anti-HSV antibody (binding to KPC2).
  • Fig. 8 shows the results of immunoprecipitation analysis of the binding of the KPC1-KPC2 complex expressed in insect cells to p27 Kipl at the in vivo opening. + Indicates addition, and 1 indicates no addition.
  • FIG. 9 shows the results of measuring the activity of the KPC1-KPC2 complex expressed in insect cells to ubiquitinate p27 Kipl and phosphorylated Sicl (Sicl-P). + Indicates addition of each component of the in vitro reconstitution system, KPC1-KPC2 complex, and SCF ede4 (denoted as SCF Cdc4 in the figure), and one indicates no addition.
  • the left shows the case where p27 Kipl was used as the substrate, and the right shows the case where Sicl-P was added as the substrate and the immunoblotting analysis was performed using anti-p27 Kipl antibody and anti-HPC4 antibody (binding to Sicl-P).
  • Sicl-P ⁇ Sicl-P- (GST-Ub) l s Sic P- (GST-Ub) n, Sicl-P and GST-Ub with one Sicl-P and GST-Ub added The positions on the SDS-PAGE of two or more Sicl-Ps are shown.
  • FIG. 10 shows the results of measuring the activity of ubiquitinating p27 Kipl of KPC1, PCl (AR), KPC1-KPC2 complex, and KPCl (AR) -KPC2 complex expressed in insect cells.
  • WT indicates wild-type KPC1
  • ⁇ indicates KPC1 (/ ⁇ R)
  • + indicates KPC2, and-indicates no.
  • + indicates that the reaction was performed in the in vitro reconstitution system, and-indicates that the reaction was not performed without adding the components of the in vitro reconstitution system.
  • FIG. 11 shows the results when various proteins shown above each lane were used as E2.
  • FIG. 10 shows the results of measuring the activity of the KPC1-KPC2 complex to ubiquitinate p27 Kipl .
  • (-) Shows the results when E2 was not added.
  • FIG. 12 shows the results of measuring the activity of the KPC1-KPC2 complex to ubiquitinate p27 Kipl and ⁇ 27 ⁇ 1 phosphorylation site variants (S10A, T187A, S10E, T187E).
  • + indicates that the reaction of the in vitro reconstitution system was performed, and-indicates that the reaction was not performed without adding the components of the in vitro reconstitution system.
  • FIG. 13 shows the results of analyzing the degradation of p27 Kipl in cells overexpressing the KPC1-KPC2 complex and the KPC1 ( ⁇ ) -KPC2 complex.
  • Fig. 13a shows the case where the nuclear export of the protein was inhibited in the absence of leptomycin B
  • Fig. 13b shows the case where the nuclear export of the protein was inhibited in the presence of leptomycin B.
  • the left panel shows the results of SDS-PAGE analysis
  • the upper panel shows the anti-p27 Kipl antibody
  • the lower panel shows the immunoblot using the anti- GSK -3 ⁇ antibody.
  • On the right is a graph showing the decomposition of the amount of p27 Kipl over time.
  • KPC1-KPC2 The results of the KPC2 complex, the results of the cells in which the KPCl (AIl) -KPC2 complex was expressed, and the results of the cells in which the control vector alone was introduced are shown.
  • KPCl (WT) -KPC2 indicates KPC1-KPC2 complex
  • KPC1 (AR) -KPC2 indicates cells expressing KPC1 (AR) -KPC2 complex
  • control indicates cells into which only one control vector was introduced.
  • FIG. 14 shows the results of analyzing the suppression of KPC1 expression by RNAi and the degradation of p27 Kipl .
  • the upper part shows the analysis of KPC1 and KPC2 expression in cells into which the KPC1 siRNA expression vector has been introduced. Left shows anti-KPC1 antibody and right shows immunoblotting with anti-KPC2 antibody. .
  • the lower part shows an analysis of the time-dependent degradation of p27 Kipl in cells into which the KPC1 siRNA expression vector has been introduced. The upper two rows show the anti-p27 Kipl antibody, and the lower two rows show the immunoblots using the anti- GSK - 35 antibody. Indicates analysis in cells into which a control ECTP siRNA expression vector was introduced.
  • FIG. 15 shows the results of a time-course analysis of the amounts of various proteins in cells in which KPC1 expression was suppressed by RNAi. Analysis of protein mass at each time on lane by immunoblot using anti-p27 Kipl antibody, anti- cyclin A antibody, anti-GSK-3? Antibody, anti-KPC1 antibody, and anti-KPC2 antibody Is shown.
  • KPC1 is KPC1
  • the cells transfected with the siMA expression vector and EOTP show the analysis in the cells transfected with the control EGFP siRNA expression vector.
  • FIG. 16 is a graph showing cell proliferation of cells in which KPC1 expression was suppressed by RNAi, wherein the horizontal axis indicates time (h) and the vertical axis indicates the number of cells.
  • Garden shows the number of cells transfected with the KPC1 siRNA expression vector
  • Hata shows the number of cells transfected with the control EGFP siRNA expression vector.
  • a new ubiquitin ligase complex was separated and purified from heron reticulocyte extract using the activity of ubiquitination of p27 Kipl as an index.
  • the yeast (Saccharomyces cerevisiae) Ubal which is the E1 molecule of the ubiquitination reaction, is a N-terminal, C-terminal, Myc-tag, and His6-tag-fused protein
  • the E2 molecule, human UbcH5A is an N-terminal, C-terminal As a protein fused with His6 tag and FLAG tag, and mouse ubiquitin as glutathione S-transferase (GST) fusion protein (hereinafter abbreviated as GST-Ub), both E.
  • P27 Kipl which serves as a substrate for the reaction, was expressed in the above Escherichia coli using mouse p27 Kipl as a GST fusion protein, purified with glutathione beads (Amersham, manufactured by Biosciences), and further purified with PreScission protease (Amersham).
  • the GST portion was cleaved and removed with Biosciences, Inc., and a sample purified using a mini Q column (Amersham's Biosciences) was used.
  • reaction was performed using a 10 ⁇ L reaction buffer (40 nnnol / L Hepes-NaOH containing 50 ng of Ubal, 100 ng of UbcH5A, 3 ⁇ g of GST-Ub, and 50 ng of p27 Kipl ). pH 7.9), 60 thigh ol / L potassium acetate, 0.5 thigh ol / L EGTA, thigh ol / dithiothreitol (hereinafter abbreviated as DTT), 5 brain ol / L magnesium chloride, 10% ( ⁇ / ⁇ ) glycerol, 1.5 employment ol / L ATP) for 30 minutes at 26 ° C.
  • a heron reticulocyte extract (approximately 20 g of protein) prepared by the method of Hershko et al. [J. Biol. Chem., 258, 8206 (1983)] was used in buffer A [50 t / l Tris-HCl ( 7.4), 0.1 t ol / L DTT, 10% (v / v) glycerol] and mixed with 700 mL of DE52 resin for 45 minutes. After packing into a 10 cm diameter column, the column was washed with buffer A, and stepwise eluted with buffer A containing 100 and 300 ol / L potassium chloride, and 140 mL each was collected.
  • the activity of each fraction to ubiquitinate p27 Kipl was measured, and the active fractions were collected, concentrated by adding 60% ammonium sulfate, and the resulting precipitate was collected using 150 fiber ol.
  • the suspension was suspended in 10 mL of buffer A containing 1 / L potassium chloride. After dialysis against buffer A to remove residual ammonium sulfate, the supernatant collected by centrifugation (12,000 xg, 15 minutes) was added to buffer A containing 150 t ol / L potassium chloride.
  • the column was passed through a Superdex 200 gel filtration column (manufactured by Amersham Biosciences) at a rate of 10 mL / h.
  • Active fractions (equivalent to the elution area containing 20-50 t ol / L potassium phosphate) This was collected and dialyzed against buffer A containing 40 ⁇ l / L potassium chloride. The supernatant obtained by centrifugation at 60,000 xg for 20 minutes is collected, and a mono-Q column (Amersham 'Bioscience') pretreated with buffer A containing 40 ol / L potassium chloride Passed through the tower. Continuous elution was performed with 30 mL of buffer A containing 40-350 t / l of potassium chloride, and fractions of 1 mL each were collected.
  • Fig. 1 shows the purified protocol
  • Fig. 2 shows a Superose 6 gel filtration column
  • Fig. 3 shows the protein analysis by SDS-PAGE of each fraction obtained using the Mini Q column. The result of measurement of the activity of ubiquitinating ⁇ 27 ⁇ ⁇ 1 is shown. 140 kDa and 50 kDa bands were identified as proteins that corresponded to the activity, which were designated as KPC1 and KPC2.
  • Fractions containing KPC1 and KPC2 were separated by 10% SDS-PAGE, visualized by coomassie staining, and the bands were cut out (Fig. 4). After each reduction, S-carboxamidomethylation and trypsin digestion in the gel, the peptides are separated and recovered on nRPC C2 / C18 column (Amersham Biosciences), and subjected to Edman degradation. Amino acid sequence analysis was performed. The amino acid sequences of 11 peptides obtained from the band of KPC1 are shown in SEQ ID NOS: 9 to 19, and the amino acid sequences of two peptides obtained from the band of KPC2 are shown in SEQ ID NOs: 20 and 21. .
  • Example 1 A salt capable of encoding the partial amino acid sequence of KPC1 and KPC2 obtained in (3) The base sequence was searched from the base sequence database GenBank. As a result, the nucleotide sequence considered to be KPC1 cDNA was Genpunk Accession No. BE885419 and
  • SEQ ID NO: 1 shows the nucleotide sequence of the KPC1 coding region of the human KPC1 cDNA contained in both cDNA clones
  • SEQ ID NO: 2 shows the nucleotide sequence of SEQ ID NO:
  • the amino acid sequence of human KPC1 encoded by the nucleotide sequence represented by 1 is shown. Both the nucleotide sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2, the nucleotide sequence database and the amino acid sequence database had no identical sequences, and were novel sequences. It was confirmed that the amino acid sequence of human KPC1 contained the partial amino acid sequence of KPC1 obtained in Example 1 (3) (FIG. 5).
  • the nucleotide sequence at the 5 'end and 3' end of the region predicted to encode KPC1 in the mouse KPC1 cDNA was determined. I found it. Based on these sequences, DNA (KPC1-M1, KPC1-M2) consisting of the nucleotide sequence represented by SEQ ID NO: 25 or 26 was used as a primer, and mouse T cell cDNA library (Clontech) was used as type II. The cDNA fragment encoding mouse KPC1 was amplified and isolated by PCR.
  • SEQ ID NO: 3 shows the nucleotide sequence of the region encoding KPC1 of mouse KPC1 cDNA
  • SEQ ID NO: 4 shows the amino acid sequence of mouse KPC1 encoded by the nucleotide sequence represented by SEQ ID NO: 3.
  • SEQ ID NO: 3 shows the nucleotide sequence of the region encoding KPC1 of mouse KPC1 cDNA
  • SEQ ID NO: 4 shows the amino acid sequence of mouse KPC1 encoded by the nucleotide sequence represented by SEQ ID NO: 3.
  • Both the nucleotide sequence represented by SEQ ID NO: 3 and the amino acid sequence represented by SEQ ID NO: 4 were novel sequences, with no identical sequences in the nucleotide sequence database and the amino acid sequence database.
  • KPC1 is composed of 1314 amino acids in both human and mouse, and the SPRY domain is in the 132-253 position, and the RING-type ubiquitin ligase such as the SCF complex, APC / C complex and VHL complex is in the 1254-1291 position near the C-terminal There was a common RING finger domain (Figure 5a).
  • human glioblastoma cell differentiation factor-related protein (GBDR1) It was estimated to be the ortholog of the protein [Li, C et al., Genomics 65, 243 (2000)]. Therefore, the cDNA encoding human KPC2 is identical to the human GBDR1 cDNA and consists of the respective nucleotide sequences represented by SEQ ID NOS: 27 and 28 based on the nucleotide sequence of human GBDR1 cDNA (GenBank Accession No. AF 176796).
  • the DNA (KPC2-Hls KPC2-H2) was used as a primer, and a human liver cDNA library (Clontech) was used as a type II PCI.
  • the 0RF region was obtained as a cDNA fragment encoding human KPC2. .
  • the DNA fragment obtained by this PCR encodes human KPC2 with an HSV tag derived from primer KPC2-HI added to the N-terminus, for use in the expression in the insect cells shown in Example 3. 5 1 site at 3 end, 1 site at 3 'end.
  • SEQ ID NO: 8 shows the amino acid sequence of mouse KPC2 encoded by the nucleotide sequence represented by SEQ ID NO: 7 showed that. KPC2 had a ubiquitin-like domain on the N-terminal side and two ubiquitin-associated domains (Fig. 5a).
  • KPC1 and KPC2 encoded by the cDNA obtained in Example 2 constitute a ubiquitin ligase complex using p27 Kipl as a substrate
  • recombinant KPC1 and KPC2 were prepared using the cDNA.
  • the activity of the KPC1-KPC2 complex to ubiquitinate p27 Kipl was measured.
  • human KP02 cDNA fragment amplified in Example 2 since the encoding human KPC2 added with HSV tag at the N-terminal, with Xhol with the KPC1 amplified fragment 5, the I, 3 3 ends at the end After cleavage, similarly, a His6 / HSV tag consisting of the amino acid sequence represented by SEQ ID NO: 23 is added to the N-terminus by inserting between the MI / il sites of pBacPA into which the region encoding the His6 tag described above has been inserted. A transfer vector into which DNA encoding human KPC2 was inserted was prepared.
  • FIG. 6 shows the results of separating the purified KPC1-KPC2 complex by SDS-PAGE and staining with Kumashi.
  • KPC1 is immunoprecipitated from the cell extract of Sf21 cells expressing KPC1 and KPC2 using antibodies against FLAG and protein A-Sepharose beads (manufactured by Sigma). Was detected with an antibody against the HSV tag. As a result, KPC2 was detected in the immunoprecipitate against KPC1, and it was revealed that both proteins were bound intracellularly (Fig. 7).
  • the recombinant p27 Kipl expressed in Escherichia coli obtained in Example 1 and the recombinant KPC1-KPC2 complex prepared in (1) were mixed. After immunoprecipitation with an antibody against p27 Kipi, KPC1 and KPC2 were detected using an antibody against FLAG and an antibody against HSV tag, respectively. As a result, it was confirmed that the immunoprecipitate contained the KPC1-KPC2 complex (FIG. 8). This result indicates that the KPC1-KPC2 complex and p27 Kipl are molecules that bind to each other.
  • KPCl (AR) mutant KPC1 lacking the RING finger domain
  • DNA (KPCl-H-ls KPC1-H-3) consisting of the nucleotide sequence represented by SEQ ID NO: 29 or 31 was used as a primer, and KPCl (AR) was obtained by PCR using human KPC1 cDNA clone IMAGE: 3909169 as type III. (1 ⁇ of SEQ ID NO: 2; consisting of the amino acid sequence at position 1253)
  • a DNA fragment encoding a protein having a FLAG tag added to the N-terminus was amplified.
  • KPC1 (AR) was immunoprecipitated with an antibody against a FLAG tag in the cell extract of Sf21 cells expressing the KPC1 (AI-KPC2 complex, and the presence or absence of co-precipitated KPC2 was detected.
  • KPC2 was detected in the immunoprecipitate against KPC1 (mR), indicating that the RING finger domain was not involved in binding to KPC2 (FIG. 7).
  • KPCl (AR) -KPC2 complex p27 Kipl was subjected to polyubiquitination by the method described in (4). As shown in Fig. 10, the ubiquitination observed in the wild-type KPC1-KPC2 complex was not detected in the KPC1 ( ⁇ -KPC2 complex, and the RING finger domain of KPC1 may be essential in the polyubiquitination reaction. confirmed.
  • p27 Kipl degradation regulating intracellular localization of p27 Kipl is phosphorylation at amino acid position two places of p27 Kipl (10 serine, 187th threonine; it it Ser- 10, abbreviated as Thr- 187) is important Is reported.
  • Thr- 187 a mutant p27 Kipl in which the amino acid at the phosphorylation site was substituted was prepared, and polyubiquitination by the KPC-KPC2 complex was performed. was analyzed.
  • the mutant p27 Kipl was obtained by substituting Ser-10 and Thr-187 with alanine (referred to as S10A and T187A, respectively) and substituting with glutamic acid (respectively referred to as S10E and T187E) in Example 1 ( It was prepared by the same method as the preparation of recombinant p27 Kipl described in 1). As a result of performing a polyubiquitination reaction using these mutant p27 Kipls as substrates, it was confirmed that all mutants were ubiquitinated similarly to wild-type p27 Kipl (Fig. 12).
  • PCR was performed by using DNA (KPC2-H-3, KPC2-H-4) consisting of the nucleotide sequences represented by SEQ ID NOs: 32 and 33 as a primer, and transforming the human KPC2 cDNA fragment amplified in Example 2 into a ⁇ type.
  • NIH3T3 cells ATCC No. CRL-1658
  • NIH3T3 cells ATCC No. CRL-1658
  • the cells were cultured in KPC1 and KPC2 cells by immunofluorescence staining using anti-FLAG antibody (Sigma) and anti-HA antibody (Sanyu Cruise) [J. Biol. Chem., 276, 33111 (2001)].
  • Example 5 Effect of overexpression of the KPC2 complex on degradation of p27 Kipl KPCl with the His6 / FLAG fragment added to the transfer protein for expression of KPC1 prepared in Example 2 (1) Insert the IMHI-2 ⁇ 1 fragment containing MA encoding AR) into the expression retrovirus vector pMX-puro between the iHI / Sml sites to create a retrovirus vector for KPCl (AR) expression did. In the same manner as in Example 4, NIH3T3 cells overexpressing KPCl (AR) and KPC2 were produced.
  • NIH3T3 cells highly expressing KPC1 and KPC2 prepared in Example 4 and NIH3T3 cells infected with a recombinant retrovirus prepared from pMX-puro vector for control, intracellularly
  • the function of KPC for the degradation of p27 Kipl was investigated.
  • the cells synchronized with the cell cycle GO by contact inhibition were replated at a cell density of about 40% to enable entry into the cell cycle G1, and the amount of p27 Kipl thereafter was analyzed over time using an immo plot (No. 13 Figure a).
  • Example 6 Antibody that specifically binds to KPC1 and antibody that specifically binds to KPC2 Mouse KPC1 partial fragment of KPC1 containing 300 amino acids from the N-terminus, mouse KPC2 each as an antigen, immunizing the egret, By collecting the antiserum, a polyclonal antibody that specifically binds to KPC1 and a polyclonal antibody that specifically binds to KPC2 were obtained. As shown in Example 7 and FIG. 14, it was possible to detect KPC1 and KPC2 endogenously expressed in NIH3T3 cells, respectively, by the immunoblotting using these antibodies.
  • the puromycin resistance gene was inserted between the SildIII / ⁇ I sites of the retrovirus vector for expression pffl Exp. HematoL 24, 324 (1996)] to prepare the vector pMX-puro II. Between the Notl / Sall sites of pMX-puro II, a DNA having the sequence represented by SEQ ID NO: 37 connected to mouse U6 promoter and downstream thereof was inserted to prepare a retrovirus vector for expressing KPC1 siRNA. Cells into which the vector has been introduced express siA targeting the 1852th to 1872nd sequence of the mouse KPC1 gene (SEQ ID NO: 3).
  • a DNA having the mouse U6 promoter and the sequence shown in SEQ ID NO: 39 connected downstream thereof was inserted between the Notl / Sall sites of pMX-puro II, and EGFP, an improved green fluorescent protein, was inserted. Gene, 173, 33 (1996)).
  • Cells into which the vector has been introduced express siA targeting the 126th to 146th sequence of the coding region of the EGFP gene.
  • recombinant ⁇ retroviruses were prepared in the same manner as in Example 4, and the resulting recombinant viruses were respectively infected into NIH3T3 cells, and 10 / g / mL puromycin was prepared. By selecting in a medium containing E.
  • KPC1 expression was specifically suppressed in NIH3T3 cells expressing KPC1 siRNA, compared to NIH3T3 cells expressing control EGFP siRNA.
  • the expression of KPC2 was similar in both NIH3T3 cells, and was not suppressed (Fig. 14).
  • NIH3T3 cells After synchronizing these NIH3T3 cells with the cell cycle GO by contact inhibition, they were re-plated at a cell density of about 40% to enter the cell cycle G1, and the amount of p27 Kipl in subsequent cells was analyzed over time by immunoblot. (Fig. 14). As a control, the amount of GSK-3 protein was also analyzed. As a result, NIH3T3 cells expressing control EGFP siMA show degradation of p27 Kipl over time, whereas NIH3T3 cells expressing mouse KPC1 siRNA inhibit degradation of p27 Kipl . Was confirmed. Degradation of the control GSK-3? Was not particularly observed in either cell.
  • NIH3T3 cells were cultured in a medium containing 0.1% serum, and p27 Kipl , cyclin A, KPC1, KPC2, and GSK-3? Protein content and cell number were analyzed over time (Fig. 15). And Figure 16). Protein content was measured by immunoblot.
  • NIH3T3 cells expressing control EGFP siRNA compared to NIH3T3 cells expressing control EGFP siRNA, NIH3T3 cells expressing KPC1 siRNA suppress degradation of p27 Kipl , resulting in more accumulation of p27 Kipl over time, It was confirmed that p27 Kipl promoted the degradation of cyclin A, whose activation and degradation were inhibited.
  • novel Yubikichinriga has an activity of Yubikichin the p27 Kipl - DNA encoding peptidase protein, Yubikichin ligase complex having an activity of Yubikichin the p27 Kipl containing protein, the protein, the set comprising the DNA
  • a recombinant DNA a transformant transformed with the recombinant DNA, and an antibody recognizing the protein.
  • the present invention relates to the treatment of cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, vascular restenosis after percutaneous transluminal coronary angioplasty, pulmonary fibrosis, glomerulonephritis, autoimmune diseases, etc. It is useful for diseases caused by abnormal cell cycle, diseases whose symptoms can be alleviated by regulating the cell cycle, and particularly for diagnosis and treatment of cancer.
  • SEQ ID NO: 1 Inventor: Keiichi Nakayama; Takumi Kamura
  • SEQ ID NO: 34 KPCl siRNA sense strand
  • SEQ ID NO: 35 KPCl siMA antisense strand
  • SEQ ID NO: 37 DM inserted into KPCl siRNA expression vector
  • SEQ ID NO: 39 DNA inserted into EGFP s iRNA expression vector

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Abstract

It is intended to provide a novel protein having an activity of ubiquitinating p27Kip1; a DNA encoding this protein; a process for producing the protein; a method of screening a substance inhibiting ubiquitination of p27Kipl using the protein; an antibody specifically binding to the protein; and a diagnostic and a remedy for a disease caused by abnormal cell cycle such as cancer containing a polynucleotide or an oligonucleotide originating in the above DNA or the above antibody.

Description

明 細 書 新規ュビキチンリガ一ゼ 技術分野  Description New ubiquitin ligase Technical field
本発明は p27Kiplをュビキチン化する活性を有するュビキチンリガ一ゼ蛋白質、 該蛋白質を含む複合体、 該蛋白質をコードする DNA、 該 DNAを含む組換え体 DNAを 導入した形質転換体、 該形質転換体を用いた該蛋白質の製造方法、 該 DNAに由来 するポリヌクレオチドまたはオリゴヌクレオチドを用いた、 該蛋白質をコードす る mRNAの検出または定量する方法および該蛋白質の発現を抑制する方法に関する 。 また本発明は、 該蛋白質、 該複合体または該形質転換体を用いた、 p27Kiplのュ ビキチン化を阻害する物質のスクリーニング方法に関する。 さらに、 該蛋白質と 特異的に結合する抗体、 該抗体を用いて該蛋白質を免疫学的に検出または定量す る方法、 該ポリヌクレオチド、 オリゴヌクレオチドまたは該抗体を含む診断薬、 治療薬に関する。 背景技術 The present invention relates to a ubiquitin ligase protein having an activity of ubiquitinating p27 Kipl , a complex containing the protein, a DNA encoding the protein, a transformant into which a recombinant DNA containing the DNA has been introduced, and the transformant. And a method for detecting or quantifying mRNA encoding the protein using a polynucleotide or an oligonucleotide derived from the DNA, and a method for suppressing the expression of the protein. The present invention also relates to a method for screening a substance that inhibits ubiquitination of p27 Kipl using the protein, the complex, or the transformant. Furthermore, the present invention relates to an antibody that specifically binds to the protein, a method for immunologically detecting or quantifying the protein using the antibody, a diagnostic agent and a therapeutic agent containing the polynucleotide, the oligonucleotide or the antibody. Background art
真核細胞の細胞周期は、 一連のサイクリンとサイクリン依存性キナーゼ （以下 、 CDKと略す） の複合体が適切な時期に適切な順序で活性化されることにより進 行する。 この酵素活性は種々の機構により制御されている。 例えば、 サイクリン 蛋白質の分解、 CDKのリン酸化、 脱リン酸化、 CDK阻害蛋白質 （以下、 CKIと略す ) との結合と解離、 などがその制御機構に関与している。 CKIによる CDK/サイク リンの適切な制御は正常な細胞周期の進行に必須であり、 この制御の異常は細胞 の癌化につながることが明らかになってきている 〔Genes Dev. , 13, 1501 (1999) ; BioEssays, 20, 1020 (1998)〕 。  The eukaryotic cell cycle proceeds by activating a series of cyclin and cyclin-dependent kinase (hereinafter abbreviated as CDK) complexes at the right time and in the right order. This enzyme activity is controlled by various mechanisms. For example, cyclin protein degradation, phosphorylation and dephosphorylation of CDK, binding and dissociation with a CDK inhibitory protein (hereinafter abbreviated as CKI) are involved in the control mechanism. Proper control of CDK / cyclin by CKI is essential for normal cell cycle progression, and it has been shown that abnormalities in this control lead to cell carcinogenesis [Genes Dev., 13, 1501 ( 1999); BioEssays, 20, 1020 (1998)].
なかでも細胞周期の G1期から S期への進行の制御が、 細胞癌化の抑制に重要で あることが明らかになつてきており、 その過程で最も重要な役割を担っているの が CKIの一つである p27Kiplの量の制御である。 〔FEBS Lett. , 490. 179 (2001) ; Exp. Cell. Res. , 64, 148 (2001 ) ; Biochem. Biophys. Res. Co腿 un.， 282, 853 (2001)〕。 GO期から Gl期、 そして S期の移行には、 G1サイクリンと CDKの複合 体の活性化が重要であり、 p27Kiplはこの活性化を阻害する活性を有する。 正常細 胞では、 GO期においては p27Kiplの発現量は高く、 増殖刺激に伴い、 G1後期から S 期にかけて減少する (Nature, 372, 570 ( 1994) ; Genes Dev. , 9, 1831 (1995)In particular, it has become clear that controlling the progression of the cell cycle from the G1 phase to the S phase is important for suppressing cell carcinogenesis, and CKI plays the most important role in this process. One is the control of the amount of p27 Kipl . [FEBS Lett., 490.179 (2001); Exp. Cell. Res., 64, 148 (2001); Biochem. Biophys. Res. Co., 282, 853 (2001)]. In the transition from GO phase to Gl phase and S phase, G1 cyclin and CDK complex Activation of the body is important, and p27 Kipl has the activity of inhibiting this activation. In normal cells, the expression level of p27 Kipl is high in the GO phase and decreases from late G1 to S phase with growth stimulation (Nature, 372, 570 (1994); Genes Dev., 9, 1831 (1995)
〕 。 p27Kiplの強制発現で細胞周期は G1期に停止し、 また p27Kiplのアンチセンスォ リゴヌクレオチドによる発現抑制は、 S期細胞の割合を増加させる 〔Cell, 78,]. cell cycle forced expression of p27 Kipl stops in phase G1, also expressed suppression by p27 Kipl of Anchisensuo Rigo nucleotides increases the percentage of S phase cells [Cell, 78,
59 ( 1994) ; Cell, 78, 67 (1994) ; Science, 272, 877 ( 1996)〕 。 さらに p27Kipl をホモに欠失したマウスは、 通常のマウスより大きく、 胸腺、 精巣、 卵巣、 子宮59 (1994); Cell, 78, 67 (1994); Science, 272, 877 (1996)]. Furthermore, mice homozygously deleted for p27 Kipl are larger than normal mice, and have thymus, testis, ovary, and uterus
、 下垂体、 副腎、 前立腺など多くの組織で過剰な細胞増殖による過形成が認めら れ、 さらに放射線や化学物質による腫瘍形成の頻度が高いことが判明した 〔In many tissues, such as the pituitary gland, adrenal gland, and prostate, hyperplasia due to excessive cell proliferation was observed, and the frequency of tumor formation due to radiation and chemicals was also high [
Cell, 85, 707 (1996) ; Cell, 85, 733 (1996) ; Cell, 855 721 (1996) ;Cell, 85, 707 (1996) ; Cell, 85, 733 (1996); Cell, 85 5 721 (1996);
Nature, 396, 177 (1998)〕 。 また、 ヒト腫瘍においても、 p27Kiplの発現量と癌 の悪性度には相関関係が認められ、 乳癌、 大腸癌をはじめ、 リンパ腫、 胃癌、 喉 頭癌、 甲状腺癌、 前立腺癌、 神経據腫、 肺癌、 卵巣腫瘍など、 多くの癌種で悪性 度の高さと p27Kiplの発現量の間に負の相関関係があることが報告されている 〔Nature, 396, 177 (1998)]. In human tumors, there is also a correlation between the expression level of p27 Kipl and the malignancy of cancer, including breast cancer, colorectal cancer, lymphoma, stomach cancer, laryngeal cancer, thyroid cancer, prostate cancer, neural tumor, It has been reported that many cancer types, such as lung cancer and ovarian tumor, have a negative correlation between high malignancy and p27 Kipl expression [
Nat. Med.， 3, III ( 1997) ; Nat. Med. , 3, 227 (1997) ; Nat. Med. , 3, 231Nat. Med., 3, III (1997); Nat. Med., 3, 227 (1997); Nat. Med., 3, 231.
( 1997) ; Nat. Med. , 3, 593 (1997)〕 。 従って、 p27Kiplの発現制御機構を理解し(1997); Nat. Med., 3, 593 (1997)]. Therefore, understanding the expression control mechanism of p27 Kipl
、 発現量の調整を行うことは、 細胞周期、 癌化機構の解明、 癌診断薬、 治療薬の 開発に重要であると考えられる。 Adjusting the expression level is considered to be important for elucidation of the cell cycle and the mechanism of canceration, and for the development of cancer diagnostics and therapeutics.
p27Kiplの発現制御は転写レベルではなく、 主として転写後、 特に蛋白質分解制 御によって行われている。 特にュビキチン-プロテアゾームによる分解系がその 中心である 〔FEBS Lett. , 490, 179 (2001 ) ; Exp. Cell. Res. , 264, 148 (2001 ) ; Biochem. Biophys. Res. Co醒 un.， 282, 853 (2001 )〕 。 p27Kipl expression is regulated not at the transcriptional level, but mainly after transcription, especially by controlling proteolysis. In particular, the degradation system by ubiquitin-proteasome is the center [FEBS Lett., 490, 179 (2001); Exp. Cell. Res., 264, 148 (2001); Biochem. Biophys. Res. 282, 853 (2001)].
ュビキチン-プロテアソ一ム系による蛋白質分解は以下のプロセスによる。 ま ず、 ュビキチンはュビキチン活性化酵素 （E1) と ATPによりカルボキシ末端のグ リシン残基が活性化され、 E1の特定のシスティン残基にチォエステル結合する。 次にュビキチンはュビキチン結合酵素 （E2) のシスティン残基に転移される。 E2 に結合したュビキチンは、 標的蛋白質を特異的に認識するュビキチンリガ一ゼ （ E3) を介して、 標的蛋白質のリジン残基にイソペプチド結合される （標的蛋白質 のュビキチン化） 。 結合したュビキチンの 48番目のリジン残基にさらにュビキチ ン化反応が繰り返されることによって、 基質蛋白質にュビキチンが鎖状に付加さ れる （ポリュビキチン化） 。 最後に、 ポリュビキチン鎖が分解シグナルとして、 巨大な蛋白質分解酵素複合体であるプロテアゾームに認識され、 標的蛋白質が分 解される。 この一連の過程からなるュビキチン-プロテアソ一ム系において最も 重要なのが、 基質特異性を担うとされる E3である 〔Nature, 3733 81 ( 1995)〕。 p27Kiplをュビキチン化する E3としては、 Skp2、 Skpl、 Cullおよび Rbxlを構成成 分として含む蛋白質複合体である SCFSkp2が知られている。 なかでも Skp2が p27Kiplの認識に重要な分子である。 Skp2をホモで欠失したマウスは成長遅延、 増 殖抑制、 染色体 ·中心体異常が見られ、 p27Kiplの蓄積が見られたことからも、 Skp2が p27Kiplの分解に関与していることが示唆される 〔Nat. Cell Biol . , 1, 193 (1999) ; Nat. Cell Biol. , 1, 207 (1999) ; EMBO J. , 19, 2069 (2000)〕。 しかし、 Skp2遺伝子を欠失したノックアウトマウスにおいて、 G0-G1移行期にお ける p27Kiplの減少は Skp2が存在しなくても起こることが判明し、 GO- G1移行期に おける p27Kiplの分解には SCFSkp2に依存しない他の未知の経路が存在することが 示唆された 〔J. Biol . Chem. , 276, 48937 (2001 )〕 。 この G0-G1移行期における SCFSkp2に依存しない p27Kiplの分解機構を解明し、 それを司る E3を同定すること は、 細胞周期、 癌化機構の解明、 癌の診断薬および治療薬の開発に極めて重要で あると考えられる。 発明の開示 Proteolysis by the ubiquitin-proteasome system is based on the following process. First, ubiquitin is activated by ubiquitin activating enzyme (E1) and ATP to activate the glycine residue at the carboxy terminus, and thioester bonds to a specific cysteine residue of E1. Next, ubiquitin is transferred to the cysteine residue of ubiquitin-conjugating enzyme (E2). Ubiquitin bound to E2 is isopeptide-linked to lysine residues of the target protein via ubiquitin ligase (E3) that specifically recognizes the target protein (ubiquitination of the target protein). By repeating the ubiquitination reaction on the lysine residue at position 48 of the bound ubiquitin, ubiquitin is added in a chain to the substrate protein. (Polyubiquitination). Finally, the polyubiquitin chain is recognized as a degradation signal by the proteosome, a giant proteolytic enzyme complex, and the target protein is degraded. Yubikichin comprising the series of steps - the most important in the proteasome one beam system is a E3 that is responsible for the substrate specificity [Nature, 373 3 81 (1995)]. As E3 that ubiquitinates p27 Kipl , SCFSkp2, a protein complex containing Skp2, Skpl, Cull, and Rbxl as constituent components, is known. Among them, Skp2 is an important molecule for recognition of p27 Kipl . Mice deficient in Skp2 showed growth retardation, growth suppression, chromosome / centrosome abnormalities, and accumulation of p27 Kipl , indicating that Skp2 is involved in p27 Kipl degradation. Suggested [Nat. Cell Biol., 1, 193 (1999); Nat. Cell Biol., 1, 207 (1999); EMBO J., 19, 2069 (2000)]. However, it was found that in knockout mice lacking the Skp2 gene, a decrease in p27 Kipl during the G0-G1 transition period occurred even in the absence of Skp2, which resulted in degradation of p27 Kipl during the GO-G1 transition period. Suggested that there is another unknown pathway independent of SCFSkp2 [J. Biol. Chem., 276, 48937 (2001)]. Elucidation of the SCFSkp2-independent degradation mechanism of p27 Kipl during the G0-G1 transition phase, and identification of E3, which controls it, is extremely important for elucidation of the cell cycle, canceration mechanism, and development of cancer diagnostics and therapeutics. Deemed important. Disclosure of the invention
本発明は SCFSkp2およびその構成成分とは異なる、 p27Kiplをュビキチン化する 活性を有する新規なュビキチンリガーゼ蛋白質、 該蛋白質を含むュビキチンリガ ーゼ複合体、 該蛋白質をコードする DNA、 該 DNAを含む組換え体 DNAを導入した形 質転換体、 該形質転換体を用いた該蛋白質の製造方法、 該 DNAに由来するポリヌ クレオチドまたはォリゴヌクレオチドを用いた、 該蛋白質をコードする mRNAの検 出または定量する方法および該蛋白質の発現を抑制する方法を提供することを目 的とする。 また本発明は、 該蛋白質、 該複合体または該形質転換体を用いた、 p27Kiplのュビキチン化を阻害する物質のスクリーニング方法、 該蛋白質と特異的 に結合する抗体、 該抗体を用いて該蛋白質を免疫学的に検出または定量する方法 、 該ポリヌクレオチド、 オリゴヌクレオチドまたは該抗体を含む診断薬、 治療薬 を提供することを目的とする。 本発明者は、 p27Kiplをュビキチン化する活性を有するュビキチンリガーゼ複合 体 KPCを精製し、 その構成蛋白質である新規な蛋白質 KPC1、 およびヒトおよびマ ゥスの KPC1をコードする DNAを単離し、 該 DNAの塩基配列および該 DNAがコードす る KPC1のァミノ酸配列を明らかにした。 さらに KPC1が単独で p27Kiplをュビキチン 化する活性を有するュビキチンリガーゼであること、 KPC1の活性には C末の RING フィンガー （RING-finger) ドメインが必要なこと、 KPC1を含む複合体を細胞に 高発現させることにより、 p27Kiplの分解が促進されることを見出し、 本発明を完 成し/こ。 The present invention provides a novel ubiquitin ligase protein having activity to ubiquitinate p27 Kipl , which is different from SCFSkp2 and its constituent components, a ubiquitin ligase complex containing the protein, a DNA encoding the protein, and a set containing the DNA. Transformant into which recombinant DNA has been introduced, method for producing the protein using the transformant, detection or quantification of mRNA encoding the protein, using a polynucleotide or oligonucleotide derived from the DNA And a method for suppressing the expression of the protein. Also, the present invention provides a method for screening a substance that inhibits ubiquitination of p27 Kipl using the protein, the complex or the transformant, an antibody that specifically binds to the protein, and a protein using the antibody. An object of the present invention is to provide a method for immunologically detecting or quantifying, a diagnostic agent and a therapeutic agent containing the polynucleotide, the oligonucleotide or the antibody. The present inventors have purified a ubiquitin ligase complex KPC having an activity of ubiquitinating p27 Kipl, and isolated a novel protein KPC1, which is a constituent protein thereof, and a DNA encoding human and mouse KPC1, The nucleotide sequence of the DNA and the amino acid sequence of KPC1 encoded by the DNA were determined. Furthermore, KPC1 alone is a ubiquitin ligase that has the activity to ubiquitinate p27 Kipl ; KPC1 activity requires the C-terminal RING-finger domain; They found that high expression promotes the degradation of p27 Kipl , and completed the present invention.
すなわち、 本発明は以下の（1 )〜（53)に関する。  That is, the present invention relates to the following (1) to (53).
( 1 ) p27Kiplをュビキチン化する活性を有する複合体の構成成分であり、 分子量が 140kDaである、 p27Kiplをュビキチン化する活性を有する蛋白質。 (1) A protein having an activity of ubiquitinating p27 Kipl , which is a constituent component of a complex having an activity of ubiquitinating p27 Kipl and having a molecular weight of 140 kDa.
( 2 )配列番号 2または 4で表されるアミノ酸配列を含む蛋白質。  (2) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 4;
(3 )配列番号 2または 4で表されるアミノ酸配列において 1つ以上のアミノ酸が付 加、 欠失あるいは置換したアミノ酸配列からなり、 かつ p27Kiplをュビキチン化す る活性を有する蛋白質。 (3) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 or 4 with one or more amino acids added, deleted or substituted, and having an activity of ubiquitinating p27 Kipl .
(4)配列番号 2または 4で表されるアミノ酸配列と 60%以上の相同性を有するアミ ノ酸配列からなり、 かつ p27Kiplをュビキチン化する活性を有する蛋白質。 (4) A protein comprising an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2 or 4, and having an activity of ubiquitinating p27 Kipl .
(5 )請求項 1〜4のいずれか 1項に記載の蛋白質および以下の（a)〜（ のいずれか に記載の蛋白質を構成成分として含む、 p27Kiplをュビキチン化する活性を有する 複合体。 (5) A complex having the activity of ubiquitinating p27 Kipl , comprising the protein according to any one of claims 1 to 4 and the protein according to any one of the following (a) to ().
(a)配列番号 6または 8で表されるァミノ酸配列を含む蛋白質  (a) a protein containing an amino acid sequence represented by SEQ ID NO: 6 or 8
(b)配列番号 6または 8で表されるアミノ酸配列において 1つ以上のアミノ酸が付 加、 欠失あるいは置換したアミノ酸配列からなり、 かつ配列番号 2または 4で表さ れるアミノ酸配列を含む蛋白質と会合する蛋白質  (b) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8 with one or more amino acids added, deleted or substituted, and comprising the amino acid sequence represented by SEQ ID NO: 2 or 4; Associated proteins
( c )配列番号 6または 8で表されるアミノ酸配列と 60%以上の相同性を有するアミ ノ酸配列からなり、 かつ配列番号 2または 4で表されるァミノ酸配列を含む蛋白質 と会合する蛋白質  (c) a protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 6 or 8, and being associated with the protein containing the amino acid sequence represented by SEQ ID NO: 2 or 4
(6 )請求項 2〜4のいずれか 1項に記載の蛋白質をコードする DNA。  (6) A DNA encoding the protein according to any one of claims 2 to 4.
(7)配列番号 1または 3で表される塩基配列を含む DNA。  (7) DNA containing the base sequence represented by SEQ ID NO: 1 or 3.
(8)配列番号 1または 3で表される塩基配列と相補的な塩基配列からなる 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 p27Kiplをュビキチン化する活性 を有する蛋白質をコ一ドする DNA。 (8) A DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 or 3 DNA that hybridizes under the conditions of a lingent and encodes a protein having an activity of ubiquitinating p27 Kipl .
(9) (6 )〜（8)のいずれか 1項に記載の DNAをべクタ一に組み込んで得られる組換え 体 DNA0 (9) (6) to recombinant DNA 0 obtained by incorporating the DNA of base Kuta one to any one of (8)
(10) ( 9)に記載の組換え体 DNAを宿主細胞に導入して得られる形質転換体。  (10) A transformant obtained by introducing the recombinant DNA according to (9) into a host cell.
(11 ) ( 10)に記載の形質転換体を培養液中で培養し、 （2)〜(4)のいずれか 1項に記 載の蛋白質を該培養物中に生成、 蓄積させ、 該培養物中より該蛋白質を採取する ことを特徴とする（2)〜(4)のいずれか 1項に記載の蛋白質の製造法。  (11) The transformant according to (10) is cultured in a culture medium, and the protein described in any one of (2) to (4) is produced and accumulated in the culture, and the culture is performed. The method for producing a protein according to any one of (2) to (4), wherein the protein is collected from a product.
(12) (6)〜（8)のいずれか 1項に記載の DNAおよび以下の（a)〜（e)のいずれかに記 載の DNAをべクタ一に組み込んで得られる組換え体 DNA。  (12) A recombinant DNA obtained by incorporating the DNA described in any one of (6) to (8) and the DNA described in any of the following (a) to (e) into a vector: .
(a)配列番号 6または 8で表されるァミノ酸配列を含む蛋白質をコードする MA (a) MA encoding a protein containing an amino acid sequence represented by SEQ ID NO: 6 or 8
(b)配列番号 6または 8で表されるアミノ酸配列において 1つ以上のアミノ酸が付 加、 欠失あるいは置換したアミノ酸配列からなり、 かつ配列番号 2または 4で表さ れるアミノ酸配列を含む蛋白質と会合する蛋白質をコードする DNA (b) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8 with one or more amino acids added, deleted or substituted, and comprising the amino acid sequence represented by SEQ ID NO: 2 or 4; DNA encoding the associated protein
(c)配列番号 6または 8で表されるアミノ酸配列と 60%以上の相同性を有するアミ ノ酸配列からなり、 かつ配列番号 2または 4で表されるァミノ酸配列を含む蛋白質 と会合する蛋白質をコードする MA  (c) a protein consisting of an amino acid sequence having at least 60% homology with the amino acid sequence represented by SEQ ID NO: 6 or 8, and being associated with the protein containing the amino acid sequence represented by SEQ ID NO: 2 or 4 Code MA
( d)配列番号 5または 7で表される塩基配列を含む DNA  (d) DNA containing the nucleotide sequence represented by SEQ ID NO: 5 or 7
(e)配列番号 5または 7で表される塩基配列と相補的な塩基配列からなる DNAとスト リンジェントな条件下でハイブリダイズし、 かつ配列番号 2または 4で表されるァ ミノ酸配列を含む蛋白質と会合する蛋白質をコードする DNA  (e) hybridizing with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 5 or 7 under stringent conditions, and encoding the amino acid sequence represented by SEQ ID NO: 2 or 4 DNA encoding a protein that associates with the containing protein
(13) ( 12)に記載の組換え体 DNAを宿主細胞に導入して得られる形質転換体。  (13) A transformant obtained by introducing the recombinant DNA according to (12) into a host cell.
( 14) ( 9)に記載の組換え体 DNAおよび以下の（a)〜（ e)のいずれかに記載の DNAをべ クタ一に組み込んで得られる組換え体 DNAを宿主細胞に導入して得られる形質転 換体。  (14) A recombinant DNA obtained by incorporating the recombinant DNA of (9) and the DNA of any of the following (a) to (e) into a vector is introduced into a host cell. The resulting transformant.
(a)配列番号 6または 8で表されるアミノ酸配列を含む蛋白質をコードする DNA (A) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8
(b)配列番号 6または 8で表されるァミノ酸配列において 1つ以上のァミノ酸が付 加、 欠失あるいは置換したアミノ酸配列からなり、 かつ配列番号 2または 4で表さ れるアミノ酸配列を含む蛋白質と会合する蛋白質をコードする MA (b) an amino acid sequence represented by SEQ ID NO: 6 or 8 to which one or more amino acids have been added, deleted or substituted, and comprising the amino acid sequence represented by SEQ ID NO: 2 or 4 MAs encoding proteins that associate with proteins
(c)配列番号 6または 8で表されるアミノ酸配列と 60%以上の相同性を有するアミ ノ酸配列からなり、 かつ配列番号 2または 4で表されるアミノ酸配列を含む蛋白質 と会合する蛋白質をコ一ドする MA (c) an amino acid having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 6 or 8; MA that encodes a protein consisting of the amino acid sequence and associated with the protein containing the amino acid sequence represented by SEQ ID NO: 2 or 4.
(d)配列番号 5または 7で表される塩基配列を含む DNA  (d) DNA containing the nucleotide sequence represented by SEQ ID NO: 5 or 7
( e )配列番号 5または 7で表される塩基配列と相補的な塩基配列からなる DNAとスト リンジヱントな条件下でハイプリダイズし、 かつ配列番号 2または 4で表されるァ ミノ酸配列を含む蛋白質と会合する蛋白質をコードする DNA  (e) It hybridizes with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 5 or 7 under stringent conditions, and contains the amino acid sequence represented by SEQ ID NO: 2 or 4. DNA encoding a protein that associates with the protein
( 15 ) ( 13)または（14)に記載の形質転換体を培養液中で培養し、 （5 )に記載の複合 体を該培養物中に生成、 蓄積させ、 該培養物中より該複合体を採取することを特 徴とする（5 )に記載の複合体の製造法。  (15) The transformant according to (13) or (14) is cultured in a culture medium, and the complex according to (5) is produced and accumulated in the culture, and the complex is produced from the culture. The method for producing a complex according to (5), wherein the method comprises collecting a body.
( 16 ) (6 )〜（8 )のいずれかの 1項に記載の MAが有する塩基配列と相補的な配列か らなるポリヌクレオチド。  (16) A polynucleotide comprising a sequence complementary to the base sequence of the MA according to any one of (6) to (8).
( 17)以下の（a)または (b)を用いて、 （2 )〜(4)のいずれか 1項に記載の蛋白質をコ 一ドする mRNAの検出または定量を行う方法。  (17) A method for detecting or quantifying mRNA encoding the protein according to any one of (2) to (4) using the following (a) or (b).
(a) (6 )~(8 )のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中の 連続する 20塩基以上の配列を含むポリヌクレオチド  (a) a polynucleotide comprising a sequence of 20 or more consecutive nucleotides in a sequence complementary to the nucleotide sequence of the DNA according to any one of (6) to (8);
(b) ( 6 )ん（8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 20〜； 100 塩基の配列を含む MAおよび ( 6 )〜（ 8 )のいずれかの 1項に記載の DNAが有する塩基 配列と相補的な配列中の連続する 20〜100塩基の配列を含む DNA  (b) 20 to 20 consecutive nucleotides in the nucleotide sequence of the DNA according to any one of (6) to (8); MA containing a sequence of 100 nucleotides and one of any of (6) to (8) DNA containing a continuous 20-100 base sequence in the sequence complementary to the base sequence of the DNA described in section
( 18)以下の（a)または (b)を用いて、 （2 )〜( のいずれか 1項に記載の蛋白質の発 現量が健常人と比較して mRNAレベルで増加または減少している疾患の判定または 診断を行う方法。  (18) Using the following (a) or (b), the expression level of the protein according to any one of (2) to () is increased or decreased in mRNA level as compared with a healthy person A method for determining or diagnosing a disease.
(a) ( 6 )〜（8)のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中の 連続する 20塩基以上の配列を含むポリヌクレオチド  (a) a polynucleotide comprising a continuous sequence of 20 or more nucleotides in a sequence complementary to the nucleotide sequence of the DNA according to any one of (6) to (8)
(b) ( 6 ) ~(8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 20〜； 100 塩基の配列を含む DNAおよび ( 6 )〜（ 8 )のいずれかの 1項に記載の DNAが有する塩基 配列と相補的な配列中の連続する 20〜100塩基の配列を含む DNA  (b) continuous 20 to in the base sequence of the DNA according to any one of (6) to (8); DNA containing a sequence of 100 bases and any one of (6) to (8) DNA containing a continuous 20-100 base sequence in the sequence complementary to the base sequence of the DNA described in section
( 19 )以下の（a)または (b)を含む、 （2)〜(4)のいずれか 1項に記載の蛋白質の発現 量が健常人と比較して ΛΝΑレベルで増加または減少している疾患の診断薬。  (19) The expression level of the protein according to any one of (2) to (4) is increased or decreased at a ΛΝΑ level compared to a healthy person, including the following (a) or (b): Diagnostics for disease.
(a) (6 )〜（8)のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中の 連続する 20塩基以上の配列を含むポリヌクレオチド (b) (6)〜（8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 20〜100 塩基の配列を含む DMおよび (6 )〜（8)のいずれかの 1項に記載の DNAが有する塩基 配列と相補的な配列中の連続する 20〜100塩基の配列を含む DNA (a) a polynucleotide comprising a continuous sequence of 20 or more bases in a sequence complementary to the base sequence of the DNA according to any one of (6) to (8) (b) a DM comprising a sequence of 20 to 100 consecutive bases in the nucleotide sequence of the DNA according to any one of (6) to (8) and one of any of (6) to (8) DNA containing a continuous 20 to 100 base sequence in the sequence complementary to the base sequence of the DNA described in
(20 ) (6)〜（8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 20〜； 100 塩基の配列を含むォリゴヌクレオチドおよび (6)〜（8)のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中の連続する 20〜100塩基の配列を含むオリ ゴヌクレオチドのうちの少なくとも 1つを用いて、 （2 )~(4)のいずれか 1項に記 載の蛋白質をコ一ドする遺伝子の変異を検出する方法。  (20) continuous 20 to in the base sequence of the DNA according to any one of (6) to (8); an oligonucleotide having a sequence of 100 bases and any of (6) to (8) Any one of (2) to (4), using at least one of oligonucleotides containing a continuous sequence of 20 to 100 bases in the sequence complementary to the base sequence of the DNA according to item 1 Or a method for detecting a mutation in a gene encoding the protein described in item 1.
(21 ) (6)〜（8)のぃずれか1項に記載の1) が有する塩基配列中の連続する20〜100 塩基の配列を含むォリゴヌクレオチドおよび (6 )〜（ 8 )のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中の連続する 20〜100塩基の配列を含むオリ ゴヌクレオチドのうちの少なくとも 1つを用いて、 （2)~(4)のいずれか 1項に記 載の蛋白質をコードする遺伝子が変異を有している疾患を判定または診断する方 法。  (21) Any one of (6) to (8), which is an oligonucleotide comprising a continuous 20 to 100 nucleotides sequence in the nucleotide sequence of 1) according to any one of (6) to (8). (2) to (4), using at least one of oligonucleotides containing a continuous sequence of 20 to 100 bases in a sequence complementary to the base sequence of the DNA according to item 1. A method for determining or diagnosing a disease in which a gene encoding the protein described in any one of the above has a mutation.
(22) ( 6)〜（8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 20〜； 100 塩基の配列を含むオリゴヌクレオチドおよび (6 )〜（8)のいずれかの 1項に記載の DMが有する塩基配列と相補的な配列中の連続する 20〜100塩基の配列を含むオリ ゴヌクレオチドのうちの少なくとも 1つを含む、 （2)〜(4)のいずれか 1項に記載 の蛋白質をコードする遺伝子が変異を有している疾患の診断薬。  (22) 20 to consecutive nucleotides in the nucleotide sequence of the DNA according to any one of (6) to (8); an oligonucleotide comprising a sequence of 100 nucleotides and any of (6) to (8) Any one of (2) to (4), comprising at least one of oligonucleotides having a continuous sequence of 20 to 100 nucleotides in a sequence complementary to the nucleotide sequence of DM according to Item 1. A diagnostic agent for a disease in which a gene encoding the protein according to Item has a mutation.
(23 ) 以下の（a)〜（c)からなる群から選ばれる RNA。  (23) An RNA selected from the group consisting of the following (a) to (c).
(a)配列番号 36で表される配列および該配列と相補的な配列の 3'端にそれそれ 2〜 4個のヌクレオチドを付加した配列からなる 2本鎖 RNA  (a) a double-stranded RNA consisting of the sequence represented by SEQ ID NO: 36 and a sequence complementary to the sequence and having a sequence obtained by adding 2 to 4 nucleotides to the 3 ′ end of the sequence
(b)配列番号 34および 35で表される配列からなる 2本鎖 RNA  (b) a double-stranded RNA consisting of the sequences represented by SEQ ID NOs: 34 and 35
( c )配列番号 38で表される配列からなる A  (c) A consisting of the sequence represented by SEQ ID NO: 38
(24)以下の（a)〜（d)からなる群から選ばれる少なくとも 1つを用いて、 （2)〜（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。  (24) A method for suppressing the expression of the protein according to any one of (2) to (4), using at least one selected from the group consisting of the following (a) to (d).
(a) (6 )〜(8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 19〜25塩 基に相当する配列を含む二本鎖 RNA  (a) a double-stranded RNA containing a sequence corresponding to 19 to 25 contiguous bases in the base sequence of the DNA according to any one of (6) to (8);
(b) (23 )に記載の RNA  (b) RNA according to (23)
( c ) (6 )〜（8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 19〜25塩 基に相当する配列および該配列と相補的な配列を含みヘアピン構造を形成する(c) continuous 19 to 25 salts in the base sequence of the DNA according to any one of (6) to (8); Contains a sequence corresponding to the group and a sequence complementary to the sequence to form a hairpin structure
RNA RNA
(d) (6 )〜（8 )のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中の 連続する 20〜； 100塩基の配列を含むォリゴヌクレオチドまたはォリゴヌクレオチ ド誘導体  (d) 20 to continuous nucleotides in the sequence complementary to the nucleotide sequence of the DNA according to any one of (6) to (8); an oligonucleotide or oligonucleotide derivative comprising a sequence of 100 nucleotides
(25 )以下の（a)〜（d)からなる群から選ばれる少なくとも 1つを用いて、 細胞内の p27mPlのュビキチン化を阻害する方法。 (25) below (a) using ~ at least one selected from the group consisting of (d), a method of inhibiting Yubikichin of p27 m Pl intracellular.
(a) (6 )〜（8 )のいずれか 1項に記載の MAが有する塩基配列中の連続する 19〜25塩 基に相当する配列を含む二本鎖 RNA  (a) a double-stranded RNA comprising a sequence corresponding to 19 to 25 contiguous bases in the base sequence of the MA according to any one of (6) to (8);
(b) (23)に記載の RNA  (b) RNA according to (23)
(c ) (6 )〜（8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 19〜25塩 基に相当する配列おょぴ該配列と相補的な配列を含みヘアピン構造を形成する RNA  (c) a hairpin structure containing a sequence corresponding to a continuous 19 to 25 bases in the base sequence of the DNA according to any one of (6) to (8), or a sequence complementary to the sequence; RNA forming
(d) (6 )〜（8 )のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中の 連続する 20〜； 100塩基の配列を含むォリゴヌクレオチドまたはォリゴヌクレオチ ド誘導体  (d) an oligonucleotide or oligonucleotide derivative containing a sequence of 20 to 100 consecutive nucleotides in the sequence complementary to the nucleotide sequence of the DNA according to any one of (6) to (8);
(26 )以下の（a)〜（d)からなる群から選ばれる少なくとも 1つを用いて、 細胞内の p27Kiplの分解を抑制する方法。 (26) A method for suppressing the degradation of p27 Kipl in cells, using at least one selected from the group consisting of the following (a) to (d).
(a) (6 )〜（8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 19〜25塩 基に相当する配列を含む二本鎖 RNA  (a) a double-stranded RNA comprising a sequence corresponding to 19 to 25 contiguous bases in the base sequence of the DNA according to any one of (6) to (8);
(b) (23)に記載の RNA  (b) RNA according to (23)
(c) (6 )〜（8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 19〜25塩 基に相当する配列および該配列と相補的な配列を含みヘアピン構造を形成する RNA  (c) forming a hairpin structure comprising a sequence corresponding to 19 to 25 contiguous bases in the base sequence of the DNA according to any one of (6) to (8) and a sequence complementary to the sequence; RNA
(d) (6 )〜（8 )のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中の 連続する 20〜： 100塩基の配列を含むォリゴヌクレオチドまたはォリゴヌクレオチ ド誘導体  (d) 20 to: continuous nucleotides complementary to the nucleotide sequence of the DNA according to any one of (6) to (8): Oligonucleotide or oligonucleotide derivative containing a 100-nucleotide sequence
(27)以下の（a)〜（d)からなる群から選ばれる少なくとも 1つを有効成分として含 有する、 細胞周期の異常を原因とする疾患または細胞周期を調節することにより 症状を軽減できる疾患の治療薬。 (a) (6 )〜（8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 19〜25塩 基に相当する配列を含む二本鎖 RNA (27) A disease caused by abnormal cell cycle or a disease whose symptom can be alleviated by regulating the cell cycle, comprising as an active ingredient at least one selected from the group consisting of the following (a) to (d): Remedy. (a) a double-stranded RNA comprising a sequence corresponding to 19 to 25 contiguous bases in the base sequence of the DNA according to any one of (6) to (8);
(b) (23)に記載の RNA  (b) RNA according to (23)
(c) (6 )~ (8)のいずれか 1項に記載の DNAが有する塩基配列中の連続する 19〜25塩 基に相当する配列および該配列と相補的な配列を含みヘアピン構造を形成する RNA  (c) forming a hairpin structure containing a sequence corresponding to 19 to 25 contiguous bases in the base sequence of the DNA according to any one of (6) to (8) and a sequence complementary to the sequence. RNA
(d) (6 )〜（8)のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中の 連続する 20~100塩基の配列を含むォリゴヌクレオチドまたはォリゴヌクレオチ ド誘導体  (d) an oligonucleotide or oligonucleotide derivative comprising a continuous sequence of 20 to 100 bases in a sequence complementary to the base sequence of the DNA according to any one of (6) to (8)
(28)疾患が癌である（26 )に記載の治療薬。  (28) The therapeutic agent according to (26), wherein the disease is cancer.
(29 ) (6 )〜（8)のいずれかの 1項に記載の DNAを導入することにより作製した非ヒ トトランスジエニック動物。  (29) A non-human transgenic animal produced by introducing the DNA according to any one of (6) to (8).
(30 ) (29)に記載の非ヒトトランスジヱニック動物を細胞周期の異常を原因とする 疾患のモデル動物として用いる方法。  (30) A method using the non-human transgenic animal according to (29) as a model animal for a disease caused by abnormal cell cycle.
(31 )疾患が癌である、 請求項 30に記載の方法。  (31) The method according to claim 30, wherein the disease is cancer.
(32 ) (29)に記載の非ヒトトランスジヱニック動物を用いて薬剤を評価する方法。 (32) A method for evaluating a drug using the non-human transgenic animal according to (29).
(33 )薬剤が抗癌剤である、 （32 )に記載の方法。 (33) The method according to (32), wherein the drug is an anticancer drug.
(34) (2)〜(4)のいずれか 1項に記載の蛋白質をコードする遺伝子が欠損した非ヒ ト動物。  (34) A non-human animal lacking a gene encoding the protein according to any one of (2) to (4).
(35 )被験試料の存在下および非存在下で、 （1 )〜(4)のいずれか 1項に記載 ©蛋白 質または（ 5 )に記載の複合体と p27Kiplとを接触させる工程、 該蛋白質または該複 合体と p27Kiplとの結合量を測定する工程、 および被験試料の存在下および非存在 下での該蛋白質または該複合体と p27Kiplとの結合量を比較する工程を含む、 p27Kiplとひ）〜 (4)のいずれか 1項に記載の蛋白質との結合を阻害する物質のスク リーニング方法。 (35) a step of contacting the protein according to any one of (1) to (4) with the protein or the complex according to (5) and p27 Kipl in the presence and absence of a test sample; measuring the amount of binding between the protein or conjugate and p27 Kipl, and the step of comparing the amount of binding between the presence and in the absence protein or conjugate and p27 Kipl the test sample, p27 The method for screening a substance that inhibits binding to a protein according to any one of (1) to (4).
(36 )被験試料の存在下および非存在下で、 （1 )~ (4)のいずれか 1項に記載の蛋白 質または（ 5 )に記載の複合体と p27Kiplを接触させる工程、 該蛋白質または該複合 体と p27Kiplとの結合量を測定する工程、 および被験試料の存在下および非存在下 での該蛋白質または該複合体と p27Kiplとの結合量を比較する工程を含む、 p27Kipl のュビキチン化を抑制する物質のスクリーニング方法。 (37)被験試料の存在下および非存在下で、 （1 )〜(4)のいずれか 1項に記載の蛋白 質または（ 5 )に記載の複合体と p27Kiplを接触させる工程、 該蛋白質または該複合 体と p27Kiplとの結合量を測定する工程、 および被験試料の存在下および非存在下 での該蛋白質または該複合体と p27Kiplとの結合量を比較する工程を含む、， 27Kipl の分解を抑制する物質のスクリーニング方法。 (36) a step of bringing the protein according to any one of (1) to (4) or the complex according to (5) into contact with p27 Kipl in the presence and absence of a test sample, or comprises comparing the amount of binding between the complex and the step of measuring the amount of binding between p27 Kipl, and the protein or plurality in the presence and absence of test sample polymer and p27 Kipl, p27 Kipl A method for screening for a substance that suppresses ubiquitination of a substance. (37) a step of bringing the protein according to any one of (1) to (4) or the complex according to (5) into contact with p27 Kipl in the presence and absence of a test sample; Or a step of measuring the amount of binding between the complex and p27 Kipl , and a step of comparing the amount of binding between the protein or the complex and p27 Kipl in the presence and absence of a test sample. Screening method for substances that suppress the degradation of Kipl .
(38)被験試料の存在下および非存在下で、 ）〜 (4)のいずれか 1項に記載の蛋白 質または ( 5 )に記載の複合体と p27Kiplを接触させる工程、 該蛋白質または該複合 体と p27Kiplとの結合量を測定する工程、 および被験試料の存在下および非存在下 での該蛋白質または該複合体と p27Kiplとの結合量を比較する工程を含む、 細胞周 期の異常を原因とする疾患または細胞周期を調節することにより症状を軽減でき る疾患の治療薬のスクリ一ニング方法。 (38) a step of bringing the protein according to any one of) to (4) or the complex according to (5) into contact with p27 Kipl in the presence and absence of a test sample; Measuring the amount of binding between the complex and p27 Kipl , and comparing the amount of binding between the protein or the complex and p27 Kipl in the presence and absence of the test sample. A method for screening for a therapeutic agent for a disease caused by an abnormality or a disease whose symptoms can be alleviated by regulating the cell cycle.
(39 )疾患が癌である、 （38)に記載の方法。  (39) The method according to (38), wherein the disease is cancer.
(40) ( 1)〜(4)のいずれか 1項に記載の蛋白質または（5 )に記載の複合体、 ュビキ チン活性化酵素、 ュビキチン結合酵素、 ュビキチンおよび p27Kiplを含む系におい て被験試料の存在下および非存在下で p27Kiplのュビキチン化を行う工程、 p27Kipl にとりこまれたュビキチンの量を測定する工程、 および被験試料の存在下および 非存在下での p27Kiplにとりこまれたュビキチンの量を比較する工程を含む、 p27Kiplのュビキチン化を抑制する物質のスクリ一二ング方法。 (40) A test sample in a system containing the protein according to any one of (1) to (4) or the complex according to (5), ubiquitin activating enzyme, ubiquitin conjugating enzyme, ubiquitin and p27 Kipl step of performing a presence and absence p27 Kipl Yubikichin of the step of measuring the amount of Yubikichin taken into p27 Kipl, and was taken in p27 Kipl in the presence and absence of test sample Yubikichin A method for screening a substance that suppresses ubiquitination of p27 Kipl , comprising a step of comparing the amount of p27 Kipl .
(41 ) ( 1)〜(4)のいずれか 1項に記載の蛋白質または（5 )に記載の複合体、 ュビキ チン活性化酵素、 ュビキチン結合酵素、 ュビキチンおよび p27Kiplを含む系におい て被験試料の存在下および非存在下で p27Kiplのュビキチン化を行う工程、 および p27Kiplにとりこまれたュビキチンの量を測定する工程、 および被験試料の存在下 および非存在下での ρΖγ^1にとりこまれたュビキチンの量を比較する工程を含む 、 p27Kiplの分解を抑制する物質のスクリーニング方法。 (41) A test sample in a system comprising the protein according to any one of (1) to (4) or the complex according to (5), ubiquitin activating enzyme, ubiquitin conjugating enzyme, ubiquitin and p27 Kipl Performing ubiquitination of p27 Kipl in the presence and absence of p27, measuring the amount of ubiquitin incorporated in p27 Kipl, and incorporating ρΖγ ^ 1 in the presence and absence of the test sample A method for screening for a substance that inhibits degradation of p27 Kipl , comprising the step of comparing the amount of ubiquitin .
(42) ）〜 (4)のいずれか 1項に記載の蛋白質または（5 )に記載の複合体、 ュビキ チン活性化酵素、 ュビキチン結合酵素、 ュビキチンおよび p27Kiplを含む系におい て被験試料の存在下および非存在下で p27Kiplのュビキチン化を行う工程、 および p27Kiplにとりこまれたュビキチンの量を測定する工程、 および被験試料の存在下 および非存在下での p ^1にとりこまれたュビキチンの量を比較する工程を含む(42)) The presence of the test sample in a system comprising the protein according to any one of (4) to (4) or the complex according to (5), ubiquitin activating enzyme, ubiquitin conjugating enzyme, ubiquitin and p27 Kipl. Ubiquitination of p27 Kipl in the presence and absence of ubiquitin, and measuring the amount of ubiquitin incorporated in p27 Kipl , and ubiquitin incorporated in p ^ 1 in the presence and absence of the test sample Including comparing the amount of
、 細胞周期の異常を原因とする疾患または細胞周期を調節することにより症状を 軽減できる疾患の治療薬のスクリ一ニング方法。 A disease caused by abnormal cell cycle or a symptom by regulating the cell cycle. Screening method of remedy for diseases that can be alleviated.
(43)疾患が癌である、 （38)に記載の方法。  (43) The method according to (38), wherein the disease is cancer.
(44) ( 1)〜(4)のいずれかの 1項に記載の蛋白質、 または（5)に記載の複合体と特 異的に結合する抗体。  (44) An antibody that specifically binds to the protein of any one of (1) to (4) or the complex of (5).
(45)抗体が、 p27Kiplと（1 )〜(4)のいずれか 1項に記載の蛋白質または（5 )に記載 の複合体との結合を阻害する抗体である、 （44)に記載の抗体。 (45) The antibody according to (44), wherein the antibody inhibits binding between p27 Kipl and the protein according to any one of (1) to (4) or the complex according to (5). antibody.
(46 )抗体が、 （1)〜(4)のいずれか 1項に記載の蛋白質または (5 )に記載の複合体 が有する p27Kiplをュビキチン化する活性を阻害する作用を示す抗体である、 （44) に記載の抗体。 (46) an antibody is an antibody that exhibits an activity of inhibiting the activity of ubiquitinating p27 Kipl of the protein according to any one of (1) to (4) or the complex according to (5), The antibody according to (44).
(47) (44)〜(46)のいずれか 1項に記載の抗体を用いる、 （1 )〜(4)のいずれか 1項 に記載の蛋白質または (5 )に記載の複合体を免疫学的に検出又は定量する方法。 (47) immunologically using the protein according to any one of (1) to (4) or the complex according to (5), using the antibody according to any one of (44) to (46); Method for quantitative detection or quantification.
(48) (44)〜（46)のいずれか 1項に記載の抗体を用いて、 （1 )〜（4)のいずれか 1項 に記載の蛋白質の発現量が、 健常人と比較して蛋白質レベルで増加または減少し ている疾患を判定または診断する方法。 (48) Using the antibody according to any one of (44) to (46), the expression level of the protein according to any one of (1) to (4) is higher than that of a healthy person. A method for determining or diagnosing a disease in which the protein level is increased or decreased.
(49) (44)〜（46 )のいずれか 1項に記載の抗体を含有する、 （1 )〜（4)のいずれか 1 項に記載の蛋白質の発現量が、 健常人と比較して蛋白質レベルで増加または減少 している疾患の診断薬。  (49) The expression level of the protein according to any one of (1) to (4), containing the antibody according to any one of (44) to (46), Diagnostics for diseases with increased or decreased protein levels.
(50) (45)または（46)に記載の抗体を用いて、 p27Kiplのュビキチン化を阻害する方 法。 (50) A method for inhibiting ubiquitination of p27 Kipl using the antibody according to (45) or (46).
(51) (45)または (46)に記載の抗体を用いて、 p27Kiplの分解を抑制する方法。 (51) A method for suppressing the degradation of p27 Kipl using the antibody according to (45) or (46).
(52) (45)または (46)に記載の抗体を有効成分として含有する細胞周期の異常を原 因とする疾患または細胞周期を調節することにより症状を軽減できる疾患の治療 剤。  (52) A therapeutic agent for a disease caused by abnormal cell cycle or a disease whose condition can be alleviated by regulating the cell cycle, comprising the antibody according to (45) or (46) as an active ingredient.
(53)疾患が癌である、 （52)に記載の治療剤。  (53) The therapeutic agent according to (52), wherein the disease is cancer.
本明細書においては、 本発明において同定された p27Kiplをュビキチン化する活 性を有する新規のュビキチンリガ一ゼ複合体を KPC(Kipl ubiquitylation- promoting complex) N KPCの構成成分である 2種の蛋白質を分子量の大きい順に KPC1および KPC2と称する。 KPC1は単独で p27Kiplをュビキチン化する活性を有する ュビキチンリガーゼである。 In the present specification, the two proteins novel Yubikichinriga Ichize complex which is a component of the KPC (Kipl ubiquitylation- promoting complex) N KPC having activity to Yubikichin the p27 Kipl identified herein They are called KPC1 and KPC2 in order of decreasing molecular weight. KPC1 is a ubiquitin ligase that has activity to ubiquitinate p27 Kipl by itself.
本発明の蛋白質としては、 以下のひ）〜 (4)に記載の蛋白質をあげることができ る。 Examples of the protein of the present invention include the proteins described in (i) to (4) below. You.
(1) p27Kiplをュビキチン化する活性を有する複合体の構成成分であり、 分子量が 140kDaである、 p27Kiplをュビキチン化する活性を有する蛋白質 (1) a component of a complex having an activity of Yubikichin the p27 Kipl, molecular weight of 140 kDa, protein having an activity of Yubikichin the p27 Kipl
(2)配列番号 2または 4で表されるアミノ酸配列を含む蛋白質  (2) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 4
(3)配列番号 2または 4で表されるアミノ酸配列において 1つ以上のアミノ酸が付 加、 欠失あるいは置換したアミノ酸配列からなり、 かつ p27Kiplをュビキチン化す る活性を有する蛋白質 (3) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4 with one or more amino acids added, deleted or substituted, and having an activity of ubiquitinating p27 Kipl
(4)配列番号 2または 4で表されるアミノ酸配列と 60%以上の相同性を有するアミ ノ酸配列からなり、 かつ p27Kiplをュビキチン化する活性を有する蛋白質 (4) a protein comprising an amino acid sequence having at least 60% homology with the amino acid sequence represented by SEQ ID NO: 2 or 4, and having an activity of ubiquitinating p27 Kipl
配列番号 2で表されるアミノ酸配列からなる蛋白質はヒト KPC1、 配列番号 4で 表されるアミノ酸配列からなる蛋白質はマウス KPC1である。  The protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is human KPC1, and the protein consisting of the amino acid sequence represented by SEQ ID NO: 4 is mouse KPC1.
上記のアミノ酸の付加、 欠失、 あるいは置換は、 部位特異的変異導入法 〔 Zoller, M. J. & Smith, M.， Nucleic Acids Res. , 10, 6487 (1982) ; Dalbadie-McFarland, G. et al.， Proc. Natl. Acad. Sci. USA, 79, 6409 (1982) ; Wells, J. A. et al.， Gene, 34, 315 (1985) ; Carter, P. et al .， Nucleic Acids Res. , 13, 4431 ( 1985) ; Kunkel, T. A. , Proc. Natl . Acad. Sci. USA, 82, 488 ( 1985)〕 を用いて、 配列番号 2または 4で表されるアミノ酸配 列からなる蛋白質をコ一ドする DNAに部位特異的変異を導入することにより行う ことができる。  The addition, deletion or substitution of the above amino acids can be performed by site-directed mutagenesis [Zoller, MJ & Smith, M., Nucleic Acids Res., 10, 6487 (1982); Dalbadie-McFarland, G. et al. Natl. Acad. Sci. USA, 79, 6409 (1982); Wells, JA et al., Gene, 34, 315 (1985); Carter, P. et al., Nucleic Acids Res., 13, 4431. Natl. Acad. Sci. USA, 82, 488 (1985)] to encode a protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 4. This can be done by introducing a site-specific mutation into the gene.
欠失、 置換もしくは付加されるアミノ酸の数は特に限定されないが、 上記の部 位特異的変異法等の周知の方法により欠失、 置換もしくは付加できる程度の数で あり、 1個から数十個、 好ましくは 1 ~25個、 より好ましくは 1〜； 10個、 さらに 好ましくは 1〜 5個である。  Although the number of amino acids to be deleted, substituted or added is not particularly limited, it is a number that can be deleted, substituted or added by a well-known method such as the above-described site-directed mutagenesis method, and is one to several tens. , Preferably 1 to 25, more preferably 1 to 10; and still more preferably 1 to 5.
目的の変異 （欠失、 置換、 付加） を導入した配列をそれそれの 5'端に持つ 1組 の PCRプライマ一を用いた PCR 〔Gene， 77, 51 (1989)〕 によっても、 配列番号 2ま たは 4で表されるアミノ酸配列からなる蛋白質をコードする DNAに変異を導入する ことができる。 すなわち、 まず該 DNAの 5，端に対応するセンスプライマ一と、 5， 端に変異の配列と相補的な配列を有する、 変異導入部位の直前 （5'側） の配列に 対応するアンチセンスプライマ一で該 DNAを錶型にして PCRを行い、 該 DNAの 5，端 から変異導入部位までの断片 A (3，端に変異が導入されている） を増幅する。 次 いで、 5'端に変異の配列を有する、 変異導入部位の直後 （3'側） の配列に対応す るセンスブラィマ一と、 該 DNAの 3，端に対応するアンチセンスプライマーで該 DNA を铸型にして PCRを行い、 5'端に変異が導入された該 MAの変異導入部位から 3'端 までの断片 Bを増幅する。 これらの増幅断片同士精製後、 混合して錶型ゃプライ マ一を加えずに PCRを行うと、 増幅断片 Aのセンス鎖と増幅断片 Bのアンチセン ス鎖は変異導入部位が共通しているのでハイブリダィズし、 プライマー兼錶型と して PCRの反応が進行し、 変異が導入された該 DNAが増幅する。 SEQ ID NO: 2 was also obtained by PCR using a set of PCR primers each having a sequence into which the desired mutation (deletion, substitution, addition) had been introduced at the 5 ′ end thereof [Gene, 77, 51 (1989)]. Alternatively, a mutation can be introduced into a DNA encoding a protein consisting of the amino acid sequence represented by 4. That is, first, a sense primer corresponding to the 5 'end of the DNA and an antisense primer corresponding to the sequence immediately before (5' side) the mutation introduction site having a sequence complementary to the mutation sequence at the 5 'end. First, PCR is performed by converting the DNA into type III to amplify a fragment A (3, in which a mutation has been introduced at the 3rd end) from the 5th end to the mutation introduction site of the DNA. Next Then, the DNA was transformed with a sense primer corresponding to the sequence immediately after (3 'side) having the mutation sequence at the 5' end and an antisense primer corresponding to the 3 'end of the DNA. Then, PCR is performed to amplify a fragment B from the mutation-introduced site of the MA having a mutation introduced at the 5 ′ end to the 3 ′ end. When these amplified fragments are purified from each other and mixed and subjected to PCR without adding type I primer, the sense strand of amplified fragment A and the antisense strand of amplified fragment B have the same mutation-introduced site. The DNA is hybridized, the PCR reaction proceeds as a primer-type, and the DNA into which the mutation has been introduced is amplified.
配列番号 2または 4で表されるアミノ酸配列において 1つ以上のアミノ酸が付加 、 欠失または置換したアミノ酸配列からなり、 かつ p27Kiplをュビキチン化する活 性を有する蛋白質としては、 配列番号 2または 4で表されるァミノ酸配列の N末に 1〜 25個のァミノ酸を付加したァミノ酸配列からなる蛋白質、 例えば配列番号 2 で表されるアミノ酸配列の N末に配列番号 22で表されるへキサヒスチジン （以下 His6と略す） /FLAGタグを付加したアミノ酸配列からなる蛋白質をあげることが できる。 A protein consisting of an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence represented by SEQ ID NO: 2 or 4 and having an activity of ubiquitinating p27 Kipl includes SEQ ID NO: 2 or 4 A protein comprising an amino acid sequence having 1 to 25 amino acids added to the N-terminal of the amino acid sequence represented by, for example, a protein represented by SEQ ID NO: 22 at the N-terminal of the amino acid sequence represented by SEQ ID NO: 2 A protein consisting of an amino acid sequence to which oxahistidine (hereinafter abbreviated as His6) / FLAG tag is added can be mentioned.
また、 本発明の蛋白質が p27Kiplをュビキチン化する活性を有するためには、 配 列番号 2または 4で表されるァミノ酸配列のうち 1254〜； 1291番目に存在する RINGフ インガ一ドメインを有し、 かつ配列番号 2または 4で表されるアミノ酸配列と、 少 なくとも 60%以上、 通常は 80%以上、 特に 95%以上の相同性を有していることが 好ましい。 In addition, in order for the protein of the present invention to have the activity of ubiquitinating p27 Kipl , it is necessary to have the RING finger domain present at positions 1254 to 1291 of the amino acid sequence represented by SEQ ID NO: 2 or 4. Preferably, it has at least 60% or more, usually 80% or more, particularly 95% or more homology with the amino acid sequence represented by SEQ ID NO: 2 or 4.
本明細書に記載される相同性の数値は、 特に明示した場合を除き、 BLAST CJ. Mol . Biol. , 215, 403-410 ( 1990)〕 、 FASTA (Methods. Enzymol . , 1835 63-98 ( 1990)〕等の当業者に公知の相同性検索プログラムを用いて算出される数値であ つてよいが、 好ましくは BLASTにおいてデフォルト （初期設定） のパラメ一夕を 用いて算出される数値あるいは、 FASTAにおいてデフォルト （初期設定） のパラ メ一夕を用いて算出される数値である。 Numerical homology as described herein is unless otherwise stated, BLAST CJ. Mol. Biol. , 215, 403-410 (1990) ], FASTA (Methods. Enzymol., 183 5 63-98 (1990)], or a numerical value calculated using a homology search program known to those skilled in the art, but is preferably a numerical value calculated using a default (initial setting) parameter in BLAST, or This is a value calculated using the default (initial setting) parameters in FASTA.
本発明の複合体としては、 上記の本発明の蛋白質および以下の（a)〜（ に記載 の蛋白質を構成成分として含む、 p27Kiplをュビキチン化する活性を有する複合体 があげられる。 Examples of the complex of the present invention include a complex having the activity of ubiquitinating p27 Kipl, which comprises the protein of the present invention described above and the following proteins (a) to () as constituent components.
(a)配列番号 6または 8で表されるァミノ酸配列を含む蛋白質  (a) a protein containing an amino acid sequence represented by SEQ ID NO: 6 or 8
(b)配列番号 6または 8で表されるアミノ酸配列において 1つ以上のアミノ酸が付 加、 欠失あるいは置換したアミノ酸配列からなり、 かつ配列番号 2または 4で表 されるアミノ酸配列を含む蛋白質と会合する蛋白質 (b) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 6 or 8; A protein consisting of an added, deleted or substituted amino acid sequence and associated with the protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 4
(c)配列番号 6または 8で表されるアミノ酸配列と 60%以上の相同性を有するアミ ノ酸配列からなり、 かつ配列番号 2または 4で表されるァミノ酸配列を含む蛋白質 と会合する蛋白質  (c) a protein consisting of an amino acid sequence having at least 60% homology with the amino acid sequence represented by SEQ ID NO: 6 or 8, and being associated with the protein containing the amino acid sequence represented by SEQ ID NO: 2 or 4
配列番号 6で表されるアミノ酸配列からなる蛋白質はヒト KPG2、 配列番号 8で表 されるアミノ酸配列からなる蛋白質はマウス KPC2である。 アミノ酸の付加、 欠失 、 置換は上記の配列番号 2または 4で表されるアミノ酸配列のアミノ酸の付加、 欠 失、 置換と同様に行なうことができる。 配列番号 6または 8で表されるアミノ酸 配列において 1つ以上のアミノ酸が付加、 欠失または置換したアミノ酸配列から なり、 かつ配列番号 2または 4で表されるァミノ酸配列を含む蛋白質と会合する蛋 白質としては、 配列番号 6または 8で表されるアミノ酸配列の N末または C末に 1〜 25個のアミノ酸を付加したアミノ酸配列からなる蛋白質、 例えば配列番号 6で表 されるアミノ酸配列の N末に配列番号 23で表される His6/ヘルぺス ·シンプレック ス ·ウィルス .ェピトープ （以下 His6/HSVと略す） タグを付加したアミノ酸配列 からなる蛋白質、 配列番号 6で表されるアミノ酸配列の C末に配列番号 24で表され るィンフルェンザ ·ウィルス ·へマグルチニン ·ェピト一プ (以下 HAと略す) 夕 グを付加したアミノ酸配列からなる蛋白質をあげることができる。 また、 この蛋 白質が配列番号 2または 4で表されるァミノ酸配列を含む蛋白質と会合するために は、 配列番号 6または 8で表されるアミノ酸配列と、 少なくとも 60%以上、 通常は 80%以上、 特に 95%以上の相同性を有していることが好ましい。  The protein consisting of the amino acid sequence represented by SEQ ID NO: 6 is human KPG2, and the protein consisting of the amino acid sequence represented by SEQ ID NO: 8 is mouse KPC2. The addition, deletion and substitution of amino acids can be performed in the same manner as the addition, deletion and substitution of amino acids of the amino acid sequence represented by SEQ ID NO: 2 or 4. A protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8 with one or more amino acids added, deleted or substituted, and associated with the protein containing the amino acid sequence represented by SEQ ID NO: 2 or 4 The white matter is a protein consisting of an amino acid sequence having 1 to 25 amino acids added to the N-terminal or C-terminal of the amino acid sequence represented by SEQ ID NO: 6 or 8, for example, the N-terminal of the amino acid sequence represented by SEQ ID NO: 6 His6 / herpes simplex virus .epitope (hereinafter abbreviated as His6 / HSV) tag represented by SEQ ID NO: 23 and a C-terminal amino acid sequence represented by SEQ ID NO: 6 Influenza virus, hemagglutinin, epitope (hereinafter abbreviated as HA) represented by SEQ ID NO: 24 Can be. Also, in order for this protein to associate with the protein containing the amino acid sequence represented by SEQ ID NO: 2 or 4, at least 60% or more, usually 80% It is preferable that the homology is at least 95% or more.
本発明の DNAとしては、  As the DNA of the present invention,
( 1 )本発明の蛋白質をコ一ドする DNA、  (1) DNA encoding the protein of the present invention,
(2)配列番号 1または 3で表される塩基配列を含む DNA、  (2) a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 or 3,
(3)配列番号 1または 3で表される塩基配列と相補的な塩基配列からなる DNAとスト . リンジェントな条件下でハイブリダイズし、 かつ p27Kiplをュビキチン化する活性 を有する蛋白質をコードする DNA、 および (3) Encodes a protein that hybridizes with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 or 3 under stringent conditions and has an activity to ubiquitinate p27 Kipl DNA, and
( 4 ) ( 1 ) ~ ( 3 )の DNAの塩基配列と相補的な塩基配列からなる DNAをあげることがで ぎる。  (4) A DNA having a nucleotide sequence complementary to the nucleotide sequence of the DNA of (1) to (3) can be mentioned.
ストリンジェントな条件下でハイプリダイズする DNAとは、 例えば配列番号 1で 表される塩基配列を有する DNAなどの本発明の MAまたはその一部の DNA断片をプ ローブとして、 コロニー 'ハイブリダィゼーシヨン法、 プラーク 'ハイブリダィ ゼーション法ぁるいはサザンプロットハイブリダイゼーション法等を用いること により得られる MA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〜； L Omol/Lの塩化ナトリウム存在下、 65 °Cでハイブリダィゼーシヨンを行った後、 0.；！〜 2倍濃度の SSC溶液 （ 1倍濃度の SSC溶液の組成は、 150腿 ol/L塩化ナトリゥム、 15腿 ol/Lクェン酸ナトリウムより なる） を用い、 65°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 MAを あげることができる。 ハイブリダィゼ一シヨンは、 Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press (2001) (以下、 モレキュラー ' クローニング第 3版と略す） 、 Current Protocols in Molecular Biology, John Wiley & Sons (1987 -2001) (以下、 力 レント 'プロトコ一ルズ 'イン 'モレキュラー 'バイオロジーと略す） 、 DNA Cloning 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。 ハイブ リダイズ可能な DNAとして具体的には、 配列番号 1または 3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 70%以上、 より好ましく は 80%以上、 さらに好ましくは 90%以上、 特に好ましくは 95%以上、 最も好まし くは 98%以上の相同性を有する DNAをあげることができる。 DNA that hybridizes under stringent conditions is, for example, SEQ ID NO: 1. Using the MA of the present invention or a partial DNA fragment thereof such as DNA having the nucleotide sequence represented as a probe, colony hybridization method, plaque hybridization method or Southern plot hybridization method, etc. Refers to MA obtained by using the filter, specifically, using a filter on which DNA derived from colonies or plaques is immobilized, from 0.7 to; hybridized at 65 ° C in the presence of L Omol / L sodium chloride. After performing the demonstration, 0.;! Using a 2-fold concentration SSC solution (the composition of a 1-fold concentration SSC solution consists of 150 t ol / L sodium chloride and 15 t ol / L sodium citrate), fill the solution at 65 ° C. MA that can be identified by washing can be given. Hybridization is described in Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular 'Cloning 3rd Edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987 -2001) (Hereinafter abbreviated as "protocols" in "molecular" biology), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995), etc. It can be carried out. Specifically, as a hybridizable DNA, a DNA having at least 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 1 or 3, preferably 70% or more, more preferably 80% or more, and still more preferably DNAs having 90% or more, particularly preferably 95% or more, and most preferably 98% or more homology can be mentioned.
(1)〜(3)の DNAの塩基配列と相補的な塩基配列からなる DNAとしては、 具体的に は配列番号 1または 3で表わされる塩基配列と相補的な塩基配列からなる DNAをあ げることができる。 （1 )〜（3)の DNAは、 通常 2本鎖 DNAとして得られるので、 セン ス鎖であるひ）〜（3)の DNAと同時にアンチセンス鎖として（1)〜（3)の DMの塩基配 列と相補的な塩基配列を有する DNAも得ることができる。 （1)〜（3)の 2本鎖 DNAを 100°Cで 5分間熱した後、 氷上で急速に冷却することにより、 （1)〜(3)の DNA (セ ンス鎖） と該 MAの塩基配列と相補的な塩基配列を有する DNA (アンチセンス鎖） を分離することができる。  As a DNA having a base sequence complementary to the base sequence of the DNA of (1) to (3), specifically, a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 3 is given. Can be Since the DNAs of (1) to (3) are usually obtained as double-stranded DNAs, the DNAs of (1) to (3) are used as antisense strands simultaneously with the DNAs of the sense strands (i) to (3). DNA having a base sequence complementary to the base sequence can also be obtained. After heating the double-stranded DNAs of (1) to (3) at 100 ° C for 5 minutes and rapidly cooling them on ice, the DNAs (sense strands) of (1) to (3) and DNA (antisense strand) having a base sequence complementary to the base sequence can be separated.
以下、 本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail.
1. KPC1および KPC2の精製と構造決定 1. Purification and structure determination of KPC1 and KPC2
(l)KPCの精製 KPCは、 p27Kiplをュビキチン化する活性を指標にして、 ヒトゃ温血動物 （例え ば、 モルモット、 ラット、 マウス、 ニヮトリ、 ゥサギ、 ブ夕、 ヒ'ヅジ、 ゥシ、 サ ルなど） の細胞、 もしくはそれらの細胞が存在するあらゆる組織、 または血球系 の細胞もしくはその培養細胞から、 精製することができる。 p27Kiplをュビキチン 化する活性の測定法としては、 7. (1)に記載した方法を用いることができる。 (l) Purification of KPC KPC uses the activity of ubiquitination of p27 Kipl as an index to determine the activity of human warm-blooded animals (for example, guinea pigs, rats, mice, chickens, puppies, bushes, puppies, puppies, sal, etc.). It can be purified from cells, or any tissue in which the cells are present, or cells of the blood cell lineage or cultured cells thereof. As a method for measuring the activity of ubiquitinating p27 Kipl , the method described in 7. (1) can be used.
KPCの精製法としては、 遠心分 、 硫酸アンモニゥム水溶液による塩析、 また は DEAE-セファロ一スカラム、 陰イオン交換または陽イオン交換カラム、 ゲル濾 過カラムなどを用いるクロマトグラフィーなどを、 単独または組み合わせて処理 する方法が上げられる。 ，  KPC purification methods include centrifugation, salting out with an aqueous solution of ammonium sulfate, or chromatography using a DEAE-Sepharose column, anion exchange or cation exchange column, gel filtration column, etc., alone or in combination. There is a way to handle it. ,
(2)KPC1および KPC2の部分アミノ酸配列の決定 (2) Determination of partial amino acid sequences of KPC1 and KPC2
精製された KPCに含まれる構成成分の蛋白質の部分アミノ酸配列の決定は、 以 下の方法で行うことができる。 すなわち、 精製した KPCから、 KPCに含まれる構成 成分の蛋白質をドデシル硫酸ナトリゥムーポリァクリルアミドゲル電気泳動 （ The determination of the partial amino acid sequence of the constituent protein contained in the purified KPC can be performed by the following method. That is, from the purified KPC, the protein of the component contained in the KPC was subjected to sodium dodecyl sulfate sodium polyacrylamide gel electrophoresis (
SDS-PAGE) などで分離し、 クマシ一染色などで可視化する。 各バンドを切り出し 、 必要に応じてゲル中での還元、 S-カルボキシアミドメチル化、 トリプシン等の 適当なプロテア一ゼによる切断を行なう。 適切なカラム、 例えば/ RPC C2/C18力 ラム （アマシャム .バイオサイェンシズ社製） 等を用いて、 その中に含まれるぺ プチドを分離回収し、 Procise 494 Protein Sequencer (アプライド ·バイオシ ステムズ社製） 等のぺプチドシークェンサ一を用いた自動ェドマン分解法により 、 部分アミノ酸配列を決定することができる。 あるいは、 プロテア一ゼ処理後の ぺプチド混合物を LCQイオントラップ質量分析計、 MALDI-TOF (matrix assisted laser desorption ionization -time of flight ) 質量分析言十、 エレクトロスプレ —イオン化タンデム質量分析計等により解析し、 アミノ酸配列デ一夕ベース上の 配列のプロテア一ゼ消化により理論上得られるペプチドの質量と比較することに より、 部分アミノ酸配列を決定することができる 〔Pandey， A. & Mann, M.， Nature 405, 837 (2000)〕。 Separate by SDS-PAGE) and visualize by Komashi-stain. Each band is cut out and, if necessary, subjected to reduction in a gel, S-carboxyamidomethylation, and cleavage with an appropriate protease such as trypsin. Using an appropriate column, for example, / RPC C2 / C18 column (Amersham Biosciences), the peptide contained therein is separated and recovered, and the Procise 494 Protein Sequencer (Applied Biosystems) ) The partial amino acid sequence can be determined by an automatic Edman degradation method using a peptide sequencer or the like. Alternatively, the peptide mixture after protease treatment is analyzed using an LCQ ion trap mass spectrometer, MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometry, an electrospray ionization tandem mass spectrometer, or the like. The partial amino acid sequence can be determined by comparing the mass of the peptide theoretically obtained by protease digestion of the sequence on the amino acid sequence database [Pandey, A. & Mann, M., Nature 405, 837 (2000)].
. KPC1および KPC2をコードする MAの調製  Preparation of MA encoding KPC1 and KPC2
( 1 )KPC1および KPC2をコードする DNAの調製 (1) Preparation of DNA encoding KPC1 and KPC2
得られた KPC1の部分アミノ酸配列をクエリーに用いて、 BLAST、 FASTA等の相同 性検索プログラムを用いて、 Genpept、 PIR、 Swiss- Prot等のアミノ酸配列デ一夕 ベースを検索することにより、 KPC1と相同性をもつアミノ酸配列を検索すること ができる。 ジヱンバンク （GenBank) 、 EMBL、 DDBJ等の塩基配列デ一夕べ一スを 各フレームで翻訳したアミノ酸配列に対して検索をかけることにより、 KPC1をコ ―ドする cDNAと相同性のある塩基配列を検索することができる。 得られた配列が EST (Expressed Sequence Tag ) の場合は、 得られた配列をクエリーにしてさら に相同性を有する配列、 同一の cDNAクローンに由来するもう一方の端からの EST の配列を検索することにより、 さらに KPC1をコードする cDNAと推定される配列を 検索することができる。 ESTの配列を決定した cDNAクローンを入手し、 その cDNA 全体の塩基配列を明らかにすることができる。 得られた cDMの塩基配列を各フレ ームで翻訳し、 KPC1の部分アミノ酸配列と同一のアミノ酸配列を有するオープン - リーディング . フレーム （0RF) のアミノ酸配列を KPC1のアミノ酸配列とする ことができる。 Using the obtained partial amino acid sequence of KPC1 as a query, and using a homology search program such as BLAST or FASTA, the amino acid sequence data of Genpept, PIR, Swiss-Prot, etc. By searching the base, an amino acid sequence having homology to KPC1 can be searched. Search for nucleotide sequence homologous to the cDNA encoding KPC1 by searching the amino acid sequence translated in each frame from the nucleotide sequence of GenBank, EMBL, DDBJ, etc. can do. If the obtained sequence is an EST (Expressed Sequence Tag), the obtained sequence is used as a query to search for sequences with further homology and EST sequences from the other end derived from the same cDNA clone. As a result, it is possible to further search for a putative cDNA encoding KPC1. A cDNA clone whose EST sequence has been determined is obtained, and the nucleotide sequence of the entire cDNA can be determined. The obtained cDM nucleotide sequence is translated in each frame, and the amino acid sequence of open-reading frame (0RF) having the same amino acid sequence as the partial amino acid sequence of KPC1 can be used as the amino acid sequence of KPC1.
このようにして得られるヒト KPC1をコ一ドする cDNAとしては、 ヒト cDNAの EST の配列 （ジェンバンク登録番号 BE885419および BE885914) をそれそれ有する IMAGEコンソーシアム cDNAクローン IMAGE :3909169および IMAGE :3909203 ( Research Genetics社より入手可能） があげられる。  As the cDNA encoding human KPC1 thus obtained, IMAGE consortium cDNA clones IMAGE: 3909169 and IMAGE: 3909203 (Research Genetics) each having the EST sequence of human cDNA (Genbank accession numbers BE885419 and BE885914), respectively. (Available from the company).
あるいは、 上記で得られた cDNAの塩基配列を元にして、 以下のようにしても KPC1をコードする DNAを単離することができる。 まず KPC1をコードする領域を含 む cDNAの領域を適当に選択し、 選択した領域の塩基配列の 5'端 20〜40塩基の配列 を 35端に含む DNA、 選択した領域の塩基配列の 3'端 20〜40塩基と相補的な配列を 3'端に含む DNAをそれそれ DNA合成機で合成する。 KPC1が発現している組織や細胞 から cDNAを調製する。 調製した cDNAを鎵型とし、 2種類の合成 DNAをプライマーと して用いた PCRにより KPC1をコードする DNAを増幅し単離することができる。 組織 や細胞からの cDNAの調製、 および PCIUまモレキュラー ·クローニング第 3版に記 載の方法に従って行うことができる。 Alternatively, based on the nucleotide sequence of the cDNA obtained above, DNA encoding KPC1 can be isolated as follows. First KPC1 properly selected areas of the region encoding including the cDNA, DNA including 5 'end from 20 to 40 nucleotide sequence of the nucleotide sequence of the selected region in 3 5 end 3 of the base sequence of the selected area DNA containing a sequence complementary to the 20 to 40 bases at the 3 'end is synthesized using a DNA synthesizer. Prepare cDNA from tissues and cells expressing KPC1. The DNA encoding KPC1 can be amplified and isolated by PCR using the prepared cDNA as type II and two types of synthetic DNA as primers. Preparation of cDNA from tissues and cells, and PCIU or molecular cloning, 3rd edition, can be performed according to the method described in the third edition.
以上のようにして得られる KPC1をコードする DNAとして、 配列番号 1で表される 塩基配列を有するヒト KPC1の cDNA、 配列番号 3で表される塩基配列を有するマウ ス KPC1の cDNAをあげることができる。 これらの cDNAがコードするヒト KPC1、 マウ ス KPC1はそれそれ、 配列番号 2および 4で表される新規なアミノ酸配列を有する。 KPC1をコードする DNAにおいては、 KPC1をコードする領域の各コドンの塩基配列 は cDNAで用いられているコドンに限られるものではなく、 同じアミノ酸をコード するあらゆるコドンの塩基配列を用いることができる。 Examples of the DNA encoding KPC1 obtained as described above include a human KPC1 cDNA having the nucleotide sequence represented by SEQ ID NO: 1 and a mouse KPC1 cDNA having the nucleotide sequence represented by SEQ ID NO: 3. it can. Human KPC1 and mouse KPC1 encoded by these cDNAs have novel amino acid sequences represented by SEQ ID NOs: 2 and 4, respectively. In the DNA encoding KPC1, the base sequence of each codon in the region encoding KPC1 Is not limited to the codons used in cDNA, and any codon base sequence encoding the same amino acid can be used.
KPC2は、 公知の glioblastoma cell differentiation factor -related protein (GBDR1 ) CGenomics 65, 243 (2000)〕 と同じ蛋白質であった。 したがって KPC2を コードする DNAとしては、 配列番号 5で表される塩基配列を有する DNA (ヒト GBDMの cDNA) 、 配列番号 7で表される塩基配列を有する DNA (マウス GBDR1の cDNA) をあげることができる。 これらの cDNAがコードするヒト KPC2、 マウス KPC2 はそれそれ、 配列番号 6および 8で表されるアミノ酸配列を有する。  KPC2 was the same protein as known glioblastoma cell differentiation factor-related protein (GBDR1) CGenomics 65, 243 (2000)]. Therefore, DNAs encoding KPC2 include DNA having the nucleotide sequence represented by SEQ ID NO: 5 (cDNA of human GBDM) and DNA having the nucleotide sequence represented by SEQ ID NO: 7 (cDNA of mouse GBDR1). it can. Human KPC2 and mouse KPC2 encoded by these cDNAs have the amino acid sequences represented by SEQ ID NOs: 6 and 8, respectively.
(2)KPC1遺伝子を含むゲノム DNAの取得 (2) Acquisition of genomic DNA containing KPC1 gene
( 1 )で得られた KPC1 cDNAの塩基配列をクエリ一にして、 ジェンバンク等の塩基 配列データベースから、 BLASTや FASTA等の相同性検索プログラムを用いて、 KPC1 cDNAの一部の領域と連続して一致する配列を有するヒトまたはマウスのゲノム DNAを検索することにより、 KPC1遺伝子を含むゲノム DNAの塩基配列の情報を得る ことができる。 このような塩基配列として、 ジェンバンク登録番号： NT— 022439 をあげることができる。 得られた塩基配列の任意の領域は、 その領域の塩基配列 の 5'端 20〜40塩基の配列を 3'端に含む DNAおよびその領域の塩基配列の 3'端 20〜 40塩基と相補的な配列を 3'端に含む DNAをプライマ一、 ヒトまたはマウスゲノム DNAを錶型とした PCRにより増幅し単離することができる。 ゲノム DNAの調製、 お よび PCRはモレキュラー ·クロ一ニング第 3版に記載の方法に従って行うことが できる。  Using the base sequence of the KPC1 cDNA obtained in (1) as a query, and using a homology search program such as BLAST or FASTA from a base sequence database such as GenBank to connect to a partial region of the KPC1 cDNA By searching for human or mouse genomic DNA having a matching sequence, information on the nucleotide sequence of the genomic DNA containing the KPC1 gene can be obtained. An example of such a base sequence is Genbank accession number: NT-022439. Any region of the obtained nucleotide sequence is complementary to a DNA containing a sequence of 20 to 40 nucleotides at the 5 'end of the nucleotide sequence of the region at the 3' end and 20 to 40 nucleotides of the 3 'end of the nucleotide sequence of the region. A DNA containing a unique sequence at the 3 ′ end can be amplified and isolated by primer-type PCR using human or mouse genomic DNA as type III. Preparation of genomic DNA and PCR can be performed according to the method described in Molecular Cloning, 3rd edition.
また、 モレキュラー 'クロ一ニング第 3版に記載の方法基づいて、 マウスある いはヒトの細胞や組織から単離した染色体 DNAを用いて作製したゲノム DNAライブ ラリーに対して、 （2)で得られたマウスあるいはヒトの KPC1 cMAをプローブにし て、 プラーク ·ハイブリダィゼーシヨン等の方法でスクリーニングすることによ り、 KPC1遺伝子を含むマウスあるいはヒトのゲノム DNAを得ることができる。  In addition, a genomic DNA library prepared using chromosomal DNA isolated from mouse or human cells or tissues based on the method described in Molecular Cloning 3rd Edition was obtained in (2). By using the mouse or human KPC1 cMA obtained as a probe and screening by a method such as plaque hybridization, mouse or human genomic DNA containing the KPC1 gene can be obtained.
KPC1遺伝子を含むゲノム DNAの塩基配列と cDNAの塩基配列を比較することによ り、 KPC1遺伝子のェキソン/イントロン構造を明らかにすることができる。 また 、 特に cDNAの 5'側の部分をプローブにすることにより、 KPC1遺伝子の、 プロモー 夕一など転写を制御する領域のゲノム DNAの塩基配列を明らかにすることができ る。 この配列は KPC1遺伝子の転写の制御機構を解析するのに役立つ。 また、 相同 性組換えの手法 Nature, 326, 295 (1987) ; Cell, 51, 503 (1987)〕 により、 染色体上の KPCl遺伝子を不活化または任意の配列と置換したクローンを作製する ことができる。 The exon / intron structure of the KPC1 gene can be clarified by comparing the nucleotide sequence of the genomic DNA containing the KPC1 gene with the nucleotide sequence of the cDNA. In particular, by using the 5 ′ portion of the cDNA as a probe, the base sequence of the genomic DNA of the KPC1 gene in a region that regulates transcription, such as promoter, can be determined. This sequence is useful for analyzing the regulation mechanism of transcription of the KPC1 gene. Also, homology According to the method of sexual recombination, Nature, 326, 295 (1987); Cell, 51, 503 (1987)], a clone in which the KPCl gene on the chromosome is inactivated or replaced with an arbitrary sequence can be produced.
3. KPCls KPC2、 ならびに KPClおよび KPC2を構成成分として含む複合体の製造法 3. Production method of KPCls KPC2 and composites containing KPCl and KPC2 as constituents
KPC1または KPC2は、 モレキユラ一 ' クローニング第 3版、 DNA Cloning 1 : Core Techniques, A Practical Approach, Second Edition, Oxford University Press ( 1995)等に記載された方法を用いて、 上記 2.で調製した KPC1または KPC2を コ一ドする DNAを宿主細胞中で発現させることにより製造することができる。 すなわち、 KPC1または KPC2をコードする DNAを適当な発現べクタ一のプロモー 夕一下流に挿入した組換え体べクタ一を造成し、 該ベクターを宿主細胞に導入す ることにより、 KPC1または KPC2を発現する形質転換体を取得する。 該形質転換体 を培養することにより、 培養物中に KPC1または KPC2が生成蓄積する。 該培養物か ら KPC1または KPC2を単離精製することにより、 KPC1または KPC2を製造することが できる。 以下（1)〜（3)に KPC1の製造方法について記載するが、 KPC2についても、 KPC2をコードする DNAを用いて同様にして製造することができる。 KPC1 or KPC2 was prepared using the method described in 2 above, using the method described in, for example, Molecular Cloning, Third Edition, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). Alternatively, it can be produced by expressing DNA encoding KPC2 in a host cell. That is, KPC1 or KPC2 is constructed by constructing a recombinant vector in which DNA encoding KPC1 or KPC2 is inserted immediately downstream of an appropriate expression vector, and introducing the vector into host cells. A transformant to be expressed is obtained. By culturing the transformant, KPC1 or KPC2 is produced and accumulated in the culture. KPC1 or KPC2 can be produced by isolating and purifying KPC1 or KPC2 from the culture. The method for producing KPC1 is described below in (1) to (3). KPC2 can be produced in a similar manner using DNA encoding KPC2.
( 1)形質転換体の作製 (1) Preparation of transformant
発現べクタ一としては、 宿主細胞において自律複製可能ないしは染色体中への 組込が可能で、 宿主細胞中で KPC1をコ一ドする DNAから m Aを転写できるプロモ —夕一を含有しているものが用いられる。  The expression vector contains a promoter that can replicate autonomously in the host cell or can be integrated into the chromosome, and can transcribe mA from DNA encoding KPC1 in the host cell. Things are used.
宿主細胞としては、 原核生物、 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。 また、 動 物個体や植物個体を用いることができる。  As the host cell, any prokaryote, yeast, animal cell, insect cell, plant cell, or the like can be used as long as it can express the gene of interest. In addition, animal and plant individuals can be used.
細菌等の原核生物を宿主細胞として用いる場合は、 発現べクタ一は宿主原核生 物中で自律複製可能であり、 プロモ一夕一およびその下流にリボゾーム結合配列 および KPC1をコ一ドする DNAを揷入するクローニングサイトを有するものを用い る。 必ずしも必要ではないが、 該クローニングサイトの直後に転写終結配列を配 置する方が好ましい。 また、 形質転換体の選択のため、 薬剤耐性遺伝子等のマー カーとなる遺伝子を発現する配列を含むようにする。 リボソーム結合配列の下流 のクローニングサイトに KPC1をコードする DNAを挿入する。 リボソーム結合配列 と開始コドンとの間は適当な距離 （例えば、 大腸菌宿主のベクターの場合 6〜18 塩基） に調節されていることが好ましい。 When a prokaryote such as a bacterium is used as a host cell, the expression vector is capable of autonomous replication in the host prokaryotic organism, and contains a ribosome binding sequence and a DNA encoding KPC1 downstream of the promoter. Use a clone that has a cloning site to be imported. Although not necessary, it is preferable to arrange a transcription termination sequence immediately after the cloning site. In addition, a sequence expressing a marker gene such as a drug resistance gene should be included for selecting a transformant. Insert the DNA encoding KPC1 into the cloning site downstream of the ribosome binding sequence. An appropriate distance between the ribosome binding sequence and the initiation codon (for example, 6 to 18 for an E. coli host vector) (Base).
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かなるもので もよい。 例えば大腸菌を宿主とした場合は、 trpプロモーター、 lacプロモー夕一 、 PLプロモー夕一、 T7プロモー夕一、 PRプロモー夕一等の、 大腸菌やファージ等 に由来するプロモーター等をあげることができる。 また trpプロモーターを 2つ 直列させたプロモ一夕一、 tacプロモータ一、 T71acプロモー夕一、 let Iプロモ —夕一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。 枯草菌を宿主とした場合は、 枯草菌のファージである SP01や SP02のプロモー夕一 、 PenP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 the host cell. For example, when Escherichia coli is used as a host, examples thereof include promoters derived from Escherichia coli and phage, such as trp promoter, lac promoter, PL promoter, T7 promoter, PR promoter and the like. Promoters designed and modified artificially, such as Promoter overnight, tac promoter, T71ac promoter, let I promoter — Yuichi, etc. in which two trp promoters are connected in series can also be used. When Bacillus subtilis is used as a host, the promoters of SP01 and SP02 which are phages of Bacillus subtilis, the promoters of PenP and the like can be mentioned.
発現べクタ一としては、 例えば、 pGEMEX- 1 (プロメガ社製） 、 pQE- 30 (キアゲ ン社製） 、 PKYP200 CAgric. Biol. Chem. , 48, 669 ( 1984)〕 、 pLSAl CAgric. Biol . Chem. , 53, 277 ( 1989)〕 、 pGELl 〔Pro Natl . Acad. Sci. , USA, 82, 4306 (1985)〕 、 pTrS30 〔大腸菌^109 1½30(^1^ 8? -5407)ょり調製〕 、 pGEX- 5X-3 (アマシャム 'バイオサイェンシズ社製） 、 PET14 (ノバジヱン社製） 、 pPROTet.E (クロンテック社製） pRSET A (インビトロジヱン社製） 等を例示する ことができる。  Examples of expression vectors include pGEMEX-1 (promega), pQE-30 (Qiagen), PKYP200 CAgric. Biol. Chem., 48, 669 (1984)], pLSAl CAgric. Biol. Chem. , 53, 277 (1989)), pGELl (Pro Natl. Acad. Sci., USA, 82, 4306 (1985)), pTrS30 (preparation of E. coli ^ 109 1-30 (^ 1 ^ 8? -5407)), pGEX-5X-3 (manufactured by Amersham Biosciences), PET14 (manufactured by Novagene), pPROTet.E (manufactured by Clontech), pRSETA (manufactured by Invitrogen) and the like can be exemplified.
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、 ブレビバクテ リウム属、 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、 Escherichia coli XL2- Blue、 Escherichia coli DH1、 Escherichia coli M.C1000ヽ Escherichia coli KY3276、 Escherichia coli W1485N Escherichia coli JM109、 Escherichia coli 醒01、 Escherichia coli No.49、 Escherichia coli W3110、 Escherichia coli NY49 N Escherichia coli BL21 (DE3) pLysSヽ Serratia ficaria s Serratia fonticola. Serratia liquefaciens、 Serratia marcescens Bacillus subtil is 、 Bacillus amyloliquefaciens 、 Brevibacterimii ammoniagenes 、 Brevibacterium i腿 ariophilum ATCC14068ヽ Brevibacterium saccharolyticum ATCC14066 、 Corynebacterium glutamicmn ATCC13032 、 Corynebacterium glutajnicum ATCC14067 、 Corynebacterium glutajnicum ATCC13869 、 Corynebacterium acetoacidophilum ATCC13870、 Microbacterium ammoniaphilum ATCC15354、 Pseudo讓 as sp. D- 0110等をあげることができる。 組換えべク夕一の導入方法としては、 上記宿主細胞へ DNA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、 エレクトロポレーシヨン法 〔NucIeic Acids Res. , 16, 6127 ( 1988)〕 、 カルシウムイオンを用いる方法 〔Proc. Natl . Acad. Sci. USA, 69, 2110 (1972) ; Gene, 17, 107 (1982)〕 、 プロトプラスト 法 〔特開昭 63- 248394; Mol. Gen. Genet. , 168, HI ( 1979)〕 等をあげることが できる。 Examples of the host cell include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., for example, Escherichia coli XLl-Blue, Escherichia coli XL2-Blue, Escherichia E. coli DH1, Escherichia coli M.C1000s Escherichia coli KY3276, Escherichia coli W1485 N Escherichia coli JM109, Escherichia coli awake 01, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49 N Escherichia coli BL21ic (BL3ic) s Serratia fonticola. Serratia liquefaciens, Serratia marcescens Bacillus subtil is, Bacillus amyloliquefaciens, Brevibacterimii ammoniagenes, Brevibacterium i thigh ariophilum ATCC14068ヽBrevibacterium saccharolyticum ATCC14066, Corynebacterium glutamicmn ATCC13032, Corynebacterium glutajnicum ATCC14067, Corynebacterium glutajnicum ATCC13869, Corynebacterium acetoacido philum ATCC13870, Microbacterium ammoniaphilum ATCC15354, and Pseudo cult as sp. D-0110. Any method for introducing the recombinant vector can be used as long as it is a method for introducing DNA into the host cells described above. For example, an electroporation method [NucIeic Acids Res., 16, 6127 (1988) Acad. Sci. USA, 69, 2110 (1972); Gene, 17, 107 (1982)], a protoplast method (Japanese Patent Application Laid-Open No. 63-248394; Mol. Gen. Genet., 168, HI (1979)].
酵母を宿主細胞として用いる場合の発現べクタ一としては、 宿主酵母で転写を 行なうプロモ一夕一、 転写の終止配列および酵母での形質転換マーカーとなる遺 伝子、 たとえば薬剤耐性遺伝子や TRP1、 HIS3、 LEU2等のアミノ酸合成系の遺伝子 を発現できる配列を含有しているものが用いられる。 また、 発現ベクターの作製 や維持を容易にするため、 大腸菌内でも自律複製と遺伝子導入マーカーとなる薬 剤耐性遺伝子を発現できるものが好ましい。  When yeast is used as a host cell, expression vectors include promoters that perform transcription in host yeast, transcription termination sequences and genes that serve as transformation markers in yeast, such as drug resistance genes and TRP1, Those containing sequences that can express amino acid synthesis genes such as HIS3 and LEU2 are used. In addition, in order to facilitate the production and maintenance of the expression vector, those capable of autonomously replicating and expressing a drug resistance gene serving as a gene transfer marker in E. coli are also preferable.
プロモー夕一としては、 酵母中で転写を行なえるものであればいずれのものを 用いてもよく、 例えば Saccharomyces cerevisiaeのアルコ一ルデヒドロゲナ一ゼ 遺伝子 ADH1、 ガラクトース代謝系遺伝子 GAL1や GAL10等のプロモー夕一、 酸性フ ォスファタ一ゼ遺伝子 PH05プロモーター、 フォスフォグリセレートキナーゼ遺伝 子 PGKプロモーター、 グリセルアルデヒド 3リン酸デヒドロゲナーゼ遺伝子 GAPプ ロモ—夕—、 ヒートショック蛋白質遺伝子プロモーター、 ひ接合因子遺伝子 MFひ Any promoter may be used as long as it can perform transcription in yeast.For example, promoters such as the alcohol dehydrogenase gene ADH1 of Saccharomyces cerevisiae and the galactose metabolism genes GAL1 and GAL10 may be used. , Acid phosphatase gene PH05 promoter, phosphoglycerate kinase gene PGK promoter, glyceraldehyde triphosphate dehydrogenase gene GAP promoter, heat shock protein gene promoter, mating factor gene MF
1プロモーター、 銅メタ口チォネイン遺伝子 CUP1プロモーター、 Pichia pastoris のアルコールォキシダーゼ遺伝子 A0X1のプロモーター等が用いられる。 1 promoter, copper metamouth thionein gene CUP1 promoter, Pichia pastoris alcoholoxidase gene A0X1 promoter and the like are used.
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、 ピヒア属 等に属する酵母菌株をあげることができ、 具体的には、 Saccharomyces cerevisiae、 Schizosaccharomyces pombe、 Picnia pastoris等をめげることがで きる。  Examples of the host cell include yeast strains belonging to the genera Saccharomyces, Schizosaccharomyces, Pichia, and the like.Specifically, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Picnia pastoris, and the like can be mentioned. .
組換えベクターの導入方法としては、 酵母に DNA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 〔Methods EnzymoL , 194, 182 ( 1991 )〕 、 スフエロプラスト法 〔Proc. Natl. Acad. Sci . USA, 81, 4889 ( 1984)〕 、 酢酸リチウム法 〔J. Bacteriol. , 153, 163 (1983) As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into yeast, and examples thereof include an electrification method (Methods EnzymoL, 194, 182 (1991)) and a spheroplast method. [Proc. Natl. Acad. Sci. USA, 81, 4889 (1984)], lithium acetate method [J. Bacteriol., 153, 163 (1983)]
〕等をあげることができる。 ] Etc. can be given.
動物細胞を宿主として用いる場合の発現ベクターとしては、 宿主動物細胞で転 写を行なうプロモー夕一、 転写の終止と転写物のポリアデニル化のシグナルの配 列を含有しているものが用いられる。 またベクターの作製や維持を容易にするた め、 大腸菌内でも自律複製と遺伝子導入マーカーとなる薬剤耐性遺伝子を発現で きるものが望ましい。 プロモー夕一としては、 動物細胞中で転写を行なえるもの であればいずれも用いることができるが、 SV40の初期プロモ一夕一、 ヒトサイ ト メガロウィルスの IE ( immediate early) 遺伝子のプロモー夕一およぴェンハン サ一、 ラウス肉腫ウィルス、 ヒト T細胞白血病ウィルス I、 モロニ一マウス白血 病ウィルス等のレトロウィルスの LTR等のウィルス由来の配列、 あるいはメタ口 チォネイン遺伝子や ?—ァクチン遺伝子、 伸長因子一 1などの動物細胞由来の遺 伝子のプロモー夕一等をあげることができる。 また SV40の初期プロモーターとヒ ト T細胞白血病ウィルス Iの LTRを組み合わせた SRaプロモーター等これらのプ 口モー夕—を人為的に組み合わせたプロモーターも用いられる。 When an animal cell is used as a host, the expression vector One that promotes transcription, one that contains a sequence of transcription termination and polyadenylation signals of the transcript is used. In addition, in order to facilitate the production and maintenance of the vector, it is desirable that the vector be capable of autonomous replication and expression of a drug resistance gene as a gene transfer marker in Escherichia coli. Any promoter can be used as long as it can be transcribed in animal cells, but it can be used for the early promoter of SV40 and the promoter of the IE (immediate early) gene of human site megalovirus. Sequences derived from the LTRs of retroviruses such as Yohjanhansa, Rous sarcoma virus, human T-cell leukemia virus I, and Moroni murine leukemia virus, or meta-oral thionine gene,? -Actin gene, and elongation factor 1. Examples of promoters of genes derived from animal cells such as 1 can be mentioned. Also, a promoter artificially combining these promoters, such as an SRa promoter combining the SV40 early promoter and the LTR of human T cell leukemia virus I, may be used.
宿主染色体 MAに KPC1をコ一ドする DNAが組み込まれた恒常的な KPC1発現細胞は 、 G418、 ハイグロマイシン等の薬剤に対する耐性遺伝子を発現できる配列を含む KPC1発現ベクターを宿主細胞に導入し、 薬剤の存在下で培養することにより選択 することができる。 また、 宿主細胞中での KPC1の生産量を上昇させるために、 ジ ヒドロ葉酸レダク夕ーゼ （dhfr) 遺伝子を発現できるような配列を含む KPC1の恒 常的発現ベクターを宿主細胞に導入し、 dhfr阻害剤であるメトトレキセート （ methotrexate ) の濃度を段階的に上げながら培養することにより、 dhfr造伝子と ともに KPC1をコードする DNAのコピー数を増幅させることもできる。 この dhfr遺 伝子を用いた遺伝子増幅を行なう場合の宿主細胞としては、 dhfr遺伝子が機能し ていない細胞、 例えば CHO/dhfr- (ATCC :CRL- 9096) などを用いる。  A constant KPC1-expressing cell in which DNA encoding KPC1 is integrated into the host chromosome MA is prepared by introducing a KPC1-expression vector containing a sequence capable of expressing a resistance gene to a drug such as G418 or hygromycin into the host cell, Can be selected by culturing in the presence of E. coli. To increase the production of KPC1 in the host cell, a constant expression vector for KPC1 containing a sequence capable of expressing the dihydrofolate reductase (dhfr) gene was introduced into the host cell, By increasing the concentration of methotrexate, a dhfr inhibitor, in a stepwise manner, it is possible to amplify the copy number of the DNA encoding KPC1 together with the dhfr gene. As a host cell for performing gene amplification using the dhfr gene, a cell in which the dhfr gene does not function, for example, CHO / dhfr- (ATCC: CRL-9096) is used.
具体的な発現べクタ一として、 例えば、 PEGFP-C2 (クロンテヅク社） 、 PAGE107 〔特開平 3- 22979; Cytotechnol . , 3, 133, (1990)〕、 pAS3-3 (特開平 2 - 227075)、 pCDM8 (Nature, 329, 840， （1987)〕、 pCMV-Tagl (ストラタジーン社 製） pcDM3.1(+) (インビトロジヱン社） 、 PHEP4 (インビトロジェン社製） 、 . pMSG (アマシャム 'バイオサイェンシズ社製） 、 pAMo 〔J. Biol. Chem. , 268, 22782 (1993)〕 等があげられる。  Specific expression vectors include, for example, PEGFP-C2 (Clontech), PAGE107 (Japanese Patent Application Laid-Open No. 3-22979; Cytotechnol., 3, 133, (1990)), pAS3-3 (Japanese Patent Application Laid-Open No. 2-227075), pCDM8 (Nature, 329, 840, (1987)), pCMV-Tagl (Stratagene) pcDM3.1 (+) (Invitrogen), PHEP4 (Invitrogen), pMSG (Amersham Biosciences) And pAMo [J. Biol. Chem., 268, 22782 (1993)].
宿主細胞としては、 ヒト細胞である HeLa、 ナマルバ （Namalwa)、 293、 ァフリ カミドリザル腎臓細胞である C0S-1や C0S-7、 ハムスターの細胞である CH0や MK、 マウス胎児細胞である NHI3T3、 マウス · ミエ口一マ細胞である SP2/0や NS0、 ラヅ ト · ミエローマ細胞である YB2/0等の細胞株をあげることができる。 Host cells include human cells such as HeLa, Namalwa, 293, African black monkey monkey kidney cells C0S-1 and C0S-7, hamster cells CH0 and MK, Cell lines such as NHI3T3 which is a mouse embryo cell, SP2 / 0 and NS0 which are mouse / myeloma cells, and YB2 / 0 which is a rat myeloma cell can be cited.
組換えベクターの導入方法としては、 動物細胞に DNA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 口ポレーシヨ ン法 〔 Cytotechnol. , 33 133 (1990)〕 、 リン酸カルシウム法 （特閧平 2-227075 ) 、 リ ポフエクシヨン法 〔Proc. Natl. Acad. Sci. USA , S4, 7413 (1987)〕 等をあげ ることができる。 Introduction of the recombinant vector so long as it is a method for introducing DNA into animal cells either can be used, for example, elect port Poreshiyo down method [Cytotechnol., 3 3 133 (1990 ) ], calcium phosphate method ( Pp. 2-227075), the Lipofection method [Proc. Natl. Acad. Sci. USA, S4, 7413 (1987)], and the like.
あるいは、 発現用ウィルスベクターを利用することもできる。 発現用ウィルス ベクターとしては、 該ウィルスのパッケージングに必要なタンパク質をコードす る遺伝子の少なくとも 1つが欠損しており、 パッケージング細胞において組換え ウィルスが生産でき、 宿主細胞で導入する DNAがコードする蛋白質を発現させる ために好適なプロモ一夕一を含有しているものを用いることができる。 例えば、 pMX-purOs MFG 〔Proc. Natl. Acad. Sci. USA, 92, 6733 (1995)〕 、 pBabePuro CNucleic Acids Res. , 18, 3587-3596 (1990)〕 、 LL-CG, CL-CG、 CS-CGヽ CLG 〔 J. Virol. , 72, 8150 (1998)〕 、 および pAdexl (Nucleic Acids Res. , 23, 3816 (1995)〕 等をあげることができる。  Alternatively, a viral vector for expression can be used. The expression viral vector lacks at least one of the genes encoding the proteins required for packaging the virus, is capable of producing a recombinant virus in packaging cells, and is encoded by DNA introduced into host cells. Those containing a suitable promoter all at once to express the protein can be used. For example, pMX-purOs MFG [Proc. Natl. Acad. Sci. USA, 92, 6733 (1995)], pBabePuro CNucleic Acids Res., 18, 3587-3596 (1990)], LL-CG, CL-CG, CS -CG ヽ CLG [J. Virol., 72, 8150 (1998)], and pAdexl (Nucleic Acids Res., 23, 3816 (1995)).
ウィルスのパッケージングに必要なタンパク質として、 レトロウィルスベクタ —の場合にはマウスレトロウイルス由来の gag、 pol、 env等、 レンチウィルスべ クタ一の場合には HIVウィルス由来の gag、 pol、 env、 vpr、 vpu、 vif、 tat、 rev 、 nef等、 アデノウイルスベクターの場合にはアデノウイルス由来の E1A、 E1B等 、 アデノ随伴ウィルスの場合は Rep(p5,pl9,p40)、 Vp(Cap)等の蛋白質をあげるこ とができる。  Necessary proteins for virus packaging include gag, pol, env, etc. derived from mouse retrovirus in the case of retrovirus vector, and gag, pol, env, vpr derived from HIV virus in the case of lentivirus vector. , Vpu, vif, tat, rev, nef, etc .; E1A, E1B, etc. derived from adenovirus in the case of adenovirus vectors; Rep (p5, pl9, p40), Vp (Cap), etc. in the case of adeno-associated virus Can be given.
プロモータ一としては、 上記の動物細胞を宿主とする発現ベクターに記載のプ 口モーターを使用することができる。  As the promoter, the promoter described in the expression vector using the above animal cell as a host can be used.
活性ペプチド前駆体遺伝子をウィルスベクター内のプロモーターの下流に揷入 し、 組換えウィルスベクターを造成する。 造成した該組換えウィルスぺク夕一を 、 該ウィルスベクターに適合したパッケージング細胞に導入し、 組換えウィルス を生産する。  The active peptide precursor gene is inserted downstream of the promoter in the viral vector to construct a recombinant viral vector. The constructed recombinant virus vector is introduced into a packaging cell adapted to the virus vector to produce a recombinant virus.
パッケージング細胞としては、 該ウィルスベクタ一が欠損する上記遺伝子のコ ードするパッケージングに必要なタンパク質を補給できる細胞であればいかなる ものも用いることができ、 該遺伝子を発現させたヒト腎臓由来の HEK293細胞やマ ウス線維芽細胞 NIH3T3を、 レトロウイルスベクタ一の場合は Plat E細胞 〔Gene Ther. , 7, 1063 (2000)〕 を用いることができる。 As the packaging cell, any cell can be used as long as it can supply a protein necessary for packaging that encodes the above-mentioned gene deficient in the viral vector. HEK293 cells and mouse fibroblast NIH3T3 derived from human kidney expressing the gene, and Plat E cells in the case of a retrovirus vector [Gene Ther., 7, 1063 (2000)]. Can be used.
上記パッケージング細胞への上記ウィルスベクターの導入法として、 例えば、 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, M, 7413 (1987)〕 等をあげることができる。  Examples of a method for introducing the viral vector into the packaging cell include a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075) and a lipofection method (Proc. Natl. Acad. Sci. USA, M, 7413 (1987)). Can be.
生産された組換えウィルスを宿主細胞に感染させることにより、 宿主細胞にゥ ィルスべクタ一を導入することができる。 宿主細胞としては、 上記の動物細胞を 宿主とする発現べクタ一の宿主細胞と同様のものをあげることができる。  By infecting a host cell with the produced recombinant virus, the virus vector can be introduced into the host cell. Examples of the host cell include the same host cells as those of the expression vector using the above-described animal cell as a host.
昆虫細胞を宿主細胞として用いる場合は、 バキュロウィルス発現系 〔 Baculovirus Expres sion Vectors : A Laboratory Manual, W.H. Freeman and Company, New York ( 1992) ; Bio/Technology, 6, 47 (1988)〕 が用いられる。 即 ち、 トランスファ一ベクターに KPC1をコードする DNAを揷入した後、 該ベクター とバキュロウィルスを昆虫細胞に同時に導入し、 強力なプロモ一夕一であるポリ へドリン遺伝子プロモ一夕一下に KPC1をコ一ドする DNAが揷入された組換えバキ ュロウィルスを相同組換えによつて作製した後、 この組換えバキュロウィルスを 再度昆虫細胞に感染させることにより、 KPC1を発現することができる。  When an insect cell is used as a host cell, a baculovirus expression system [Baculovirus Expression Vectors: A Laboratory Manual, WH Freeman and Company, New York (1992); Bio / Technology, 6, 47 (1988)] is used. That is, after the DNA encoding KPC1 is introduced into the transfer vector, the vector and baculovirus are simultaneously introduced into insect cells, and KPC1 is placed under the polyhedrin gene promoter, which is a strong promoter. KPC1 can be expressed by producing, by homologous recombination, a recombinant baculovirus into which the coding DNA has been inserted, and then infecting insect cells again with this recombinant baculovirus.
バキュロウィルスとしては Autographa californica 核多角体病ウィルス、 力 ィコ核多角体病ウィルス等が用いられる。 昆虫細胞としては Spodoptera frugiperdaの細胞である Sf 9および Sf21 CBaculovirus Expression Vectors: A Laboratory Manual, W. H. Freeman and Company, New York (1992) 〕 、 Trichoplusia niの細胞である High5 (インビトロジェン社製） 等を用いることが できる。 また、 カイコ幼虫体をそのまま用いることもできる。 トランスファ一ベ クタ一には、 ポリヘドリンプロモーターおよび相同組換えを起こさせるためのバ キュロウィルス由来の配列、 ぺク夕一の維持 ·増殖や外来遺伝子の組み込み等の 遺伝子操作を大腸菌内で行なうための配列 （大腸菌での自律複製可能な配列およ び薬剤耐性遺伝子） が含まれており、 具体的には pVL1392、 pVL1393、 pBlueBac4.5 (ともにインビトロジヱン社製） 、 pBacPAK9 (クロンテヅク社製） 等があげられる。 '  As the baculovirus, Autographa californica nuclear polyhedrosis virus, glyconucleopolyhedrovirus, etc. are used. As insect cells, Spodoptera frugiperda cells, Sf9 and Sf21 CBaculovirus Expression Vectors: A Laboratory Manual, WH Freeman and Company, New York (1992)], Trichoplusia ni cells, High5 (manufactured by Invitrogen), etc., are used. Can be done. The silkworm larva can also be used as it is. In the transfer vector, a polyhedrin promoter and a baculovirus-derived sequence for causing homologous recombination, and genetic manipulations such as maintenance and propagation of the gene and integration of foreign genes are performed in Escherichia coli. (E.g., sequences autonomously replicable in Escherichia coli and drug resistance genes), such as pVL1392, pVL1393, pBlueBac4.5 (both from Invitrogen), pBacPAK9 (from Clontech), etc. Is raised. '
動物個体を用いて KPC1を生産することもできる。 例えば、 公知の方法 〔Am. J. Clin. Nutr. , 63, 639S (1996) ; Am. J. Clin. Nutr. , 63, 627S (1996); Bio/Technology, 9, 830 (1991)〕 に準じて、 KPC1をコードする DNAを導入した非 ヒトトランスジエニック動物中に KPC1を生産することができる。 KPC1 can also be produced using animal individuals. For example, a known method [Am. J. Clin. Nutr., 63, 639S (1996); Am. J. Clin. Nutr., 63, 627S (1996); Bio / Technology, 9, 830 (1991)] KPC1 can be produced in transgenic non-human animals.
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一夕一である αカゼインプロモー夕 ―、 ^カゼインプロモ一夕一、 ?ラクトグロブリンプロモー夕一、 ホエー酸性プ ロティンプロモ一夕一等が好適に用いられる。  Any promoter can be used as long as it can be expressed in animals.For example, mammary gland cell-specific promoters such as α-casein promoter,-casein promoter, and? Lactoglobulin promoter, whey acidic protein promoter, etc. are preferably used.
(2)形質転換体の培養 (2) Culture of transformants
KPC1をコードする DNAを組み込んだ組換え体べクタ一を保有する微生物、 動物 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、 KPC1を生成蓄積させ 、 該培養物より KPC1を採取することにより、 KPC1を製造することができる。  A transformant derived from a microorganism or animal cell having a recombinant vector into which DNA encoding KPC1 has been incorporated is cultured according to a conventional culture method to produce and accumulate KPC1, and to collect KPC1 from the culture. Thus, KPC1 can be manufactured.
動物細胞を宿主とし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 〔J. Am. Med. Assoc.， 199, 519 (1967)〕 、 イーグル （ Eagle) の MEM (Mimimum Essential Medium ) (Science, 1225 501 (1952)〕 、 ダ ルべッコ (Dalbecco) 改変イーグル培地 CVirology, 8, 396 (1959)〕、 199培地 CProc. Soc. Exp. Biol. Med. , 73, 1 (1950)〕 またはこれら培地にゥシ胎児血 清等を添加した培地等を用いることができる。 必要に応じてペニシリンゃストレ プトマイシン等の抗生物質を培地に添加してもよい。 培養は、 通常 pH6〜8、 30〜 40°C、 5%C02存在下等の条件下で 1〜7日間行う。 As a medium for culturing a transformant using animal cells as a host, commonly used RPMI 1640 medium [J. Am. Med. Assoc., 199, 519 (1967)], Eagle's MEM (Mimimum Essential Medium) (Science, 122 5 501 (1952)), Dalbecco modified Eagle medium CVirology, 8, 396 (1959)), 199 medium CProc. Soc. Exp. Biol. Med., 73, 1 (1950)] or a culture medium obtained by adding fetal serum to the culture medium, etc. If necessary, an antibiotic such as penicillin-streptomycin may be added to the culture medium. It is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C, and the presence of 5% C02.
昆虫細胞を宿主細胞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 Tffi- FH培地 〔ファーミンジェン （Phanningen) 社製〕 、 Sf-900 II SFM培地 （インビトロジェン社製） 、 ExCell400、 ExCell405 〔いずれも JRHバ ィォサイエンシズ （Jffi Biosciences ) 社製〕 、 Graceの昆虫培地 〔Nature, 195, 788 (1962)〕等を用いることができる。 培養条件は、 pH 6〜7、 培養温度 25〜30 °Cがよく、 培養時間は、 通常 1〜5日間である。 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。  As a medium for culturing a transformant obtained by using an insect cell as a host cell, generally used Tffi-FH medium (manufactured by Phanningen), Sf-900 II SFM medium (manufactured by Invitrogen) ), ExCell400, ExCell405 (all manufactured by JRH Biosciences), Grace's insect medium [Nature, 195, 788 (1962)] and the like can be used. Culture conditions are preferably pH 6-7, culture temperature 25-30 ° C, and culture time is usually 1-5 days. If necessary, an antibiotic such as genomycin may be added to the medium during the culture.
形質転換体が動物個体の場合は、 通常の方法に従って、 飼育し、 KPC1を生成蓄 積させ、 該動物個体より KPC1を採取することにより、 KPC1を製造することができ る。 すなわち、 動物個体の場合、 例えば、 KPC1をコードする MAを保有する非ヒ トトランスジヱニック動物を飼育し、 KPC1を該動物中に生成 '蓄積させ、 該動物 中より KPC1を採取することにより、 KPC1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、 卵等をあげることができる。 大腸菌等の原核生物あるいは酵母等の真核微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。 When the transformant is an animal individual, KPC1 can be produced by rearing and producing and accumulating KPC1 according to a usual method, and collecting KPC1 from the animal individual. That is, in the case of an animal individual, for example, a non-human transgenic animal having MA encoding KPC1 is bred, and KPC1 is produced and accumulated in the animal. By collecting KPC1 from inside, KPC1 can be manufactured. Examples of the place of production and accumulation in the animal include milk, eggs and the like of the animal. A culture medium for culturing a transformant obtained by using a prokaryotic organism such as Escherichia coli or a eukaryotic microorganism such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which 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 C.
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 などのアルコ一ル類等を用いることができる。  The carbon source may be any one that can be assimilated by the organism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, 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、 酢酸ァ 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、 コーンスチ 一プリカ一、 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いることができる。  Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium or inorganic salts of organic acids, other nitrogen-containing compounds, peptone, meat extract, yeast extract, and corn starch. 1. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.
無機塩としては、 リン酸水素二カリウム、 リン酸二水素カリウム、 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 salt, dipotassium hydrogen phosphate, potassium dihydrogen 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、 振盪培養または通気攪拌培養などの好気的条件下で行う。 培養温 度は 15〜40°Cがよく、 培養期間は、 通常 16〜96時間である。 培養中 pHは 3.0〜9.0 に保持する。 pH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 シゥム、 アンモニア水などを用いて行う。 必要に応じて、 培養期間中にアンピシ リンゃ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。  The cultivation is usually carried out under aerobic conditions such as shaking culture or aeration and stirring culture. The culture temperature is preferably 15 to 40 ° C, and the culture period is usually 16 to 96 hours. During the culture, maintain the pH at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, aqueous ammonia, or the like. If necessary, an antibiotic such as ampicillin-tetracycline may be added to the medium during the culture period.
誘導性のプロモーター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、 培養中に必要に応じてインデューサーを培地に添加してもよい。 イン デューサ一としては、 例えば、 lacプロモーターを誘導するイソプロピルガラク トシド、 trpプロモーターを誘導するィンドールァクリル酸等があげられる。 (3)発現させた蛋白質の単離精製  When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as needed during the culturing. Examples of the inducer include isopropyl galactoside which induces the lac promoter, and indole acrylic acid which induces the trp promoter. (3) Isolation and purification of expressed protein
上記形質転換体の培養物中に蓄積した KPC1を単離精製するには、 以下のような 通常の蛋白質の単離精製法を用いればよい。 KPC1が細胞外に分泌される場合には、 培地中に KPC1が蓄積する。 従って培養終 了後、 遠心分離等の手法により細胞を含まない培地のみを回収する。 該培地から 、 通常の蛋白質の単離精製法、 即ち、 溶媒抽出法、 硫安等による塩析法、 脱塩法In order to isolate and purify KPC1 accumulated in the culture of the above transformant, the following ordinary protein isolation and purification methods may be used. When KPC1 is secreted extracellularly, KPC1 accumulates in the medium. Therefore, after the culture is completed, only the cell-free medium is recovered by a method such as centrifugation. A normal protein isolation and purification method from the medium, that is, a solvent extraction method, a salting-out method using ammonium sulfate, and a desalting method
、 有機溶媒による沈殿法、 DEAEセファロース、 DIAION HPA-75 (三菱化学社製） 、 モノ Q (Mono Qヽ アマシャム ·バイオサイェンシズ社製) 等のレジンを用いた 陰イオン交換クロマトグラフィー法、 SPセファロース （アマシャム 'バイオサイ ェンシズ社製） 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 セファロ一ス、 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 ィ一法、 分子篩を用いたゲルろ過法、 ァフィ二ティ一クロマトグラフィー法、 ク ロマトフオーカシング法、 および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。 Precipitation method with organic solvent, DEAE Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), anion exchange chromatography method using resin such as Mono Q ヽ (Amersham Biosciences), SP Cation exchange chromatography using a resin such as Sepharose (Amersham's Biosciences), hydrophobic chromatography using a resin such as butyl sepharose and phenylsepharose, molecular sieve A purified sample can be obtained by using a gel filtration method, an affinity chromatography method, a chromatofocusing method, or an electrophoresis method such as isoelectric focusing alone or in combination.
KPC1が、 形質転換体の細胞内に蓄積する場合には、 培養終了後の培養物から、 形質転換体の細胞を遠心分離等の手法により回収し、 緩衝液にけん濁後、 超音波 破砕機、 フレンチプレス等により細胞を破碎し、 無細胞抽出液を得る。 KPC1が細 胞内で溶解状態で存在する場合には、 該無細胞抽出液を遠心分離することにより 得られた上清から、 上記の培地からの精製単離と同様にして精製標品を得ること ができる。 また、 KPC1が細胞内に不溶体を形成して存在する場合は、 該無細胞抽 出液を遠心分離後、 沈殿画分として KPC1の不溶体を回収する。 この KPC1の不溶体 を蛋白質変性剤で可溶化した後、 該可溶化液を、 蛋白質変性剤の濃度を蛋白質が 変性しない程度まで希釈するか、 あるいは、 蛋白質変性剤を含まないかまたは蛋 白質変性剤の濃度が蛋白質が変性しない程度まで希薄な溶液に透析し、 KPC1を正 常な立体構造に復元させた後、 上記と同様の単離精製法により精製標品を得るこ とができる。  When KPC1 accumulates in the cells of the transformant, the cells of the transformant are collected from the culture after completion of the culture by centrifugation or the like, and suspended in a buffer, and then disrupted by an ultrasonic crusher. The cells are disrupted by a French press or the like to obtain a cell-free extract. When KPC1 is present in a dissolved state in the cells, a purified sample is obtained from the supernatant obtained by centrifuging the cell-free extract in the same manner as in the above-mentioned purification and isolation from the medium. be able to. When KPC1 is present in the form of an insoluble substance in the cells, the cell-free extract is centrifuged, and the insoluble KPC1 is recovered as a precipitate fraction. After solubilizing the insoluble KPC1 with a protein denaturing agent, the lysate is diluted to the extent that the protein denaturing agent does not denature the protein, or the protein denaturing agent is not contained or the protein denaturing agent is not contained. After dialysis against a solution diluted to such an extent that the protein does not denature the protein, KPC1 is restored to a normal three-dimensional structure, and a purified sample can be obtained by the same isolation and purification method as described above.
また、 公知の方法 〔J. Biomol . NMR, 6, 129 ( 1998) ; Science, 242, 1162 ( 1988) ; J. Biochem. , 110, 166 ( 1991)〕 に準じて、 インビトロ転写 '翻訳系を 用いて KPC1を生産することができる。 すなわち、 KPC1をコードする DNAを SP6、 T7 、 T3等のプロモータ一の下流につなげ、 それぞれのプロモ一夕一特異的な RNAポ リメラ一ゼを反応させることにより大量の KPC1をコードする RNAをインビトロで 合成した後、 無細胞系の翻訳系例えばゥサギ網状赤血球ライセートやコムギ胚芽 抽出液を用いた翻訳系を利用して、 KPC1を生産することができる。 精製した KPCl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 子クロ一ニングのための蛋白質構造解析」 （平野久著、 東京化学同人発行、 1993 年） に記載の方法により実施可能である。 Further, according to a known method [J. Biomol. NMR, 6, 129 (1998); Science, 242, 1162 (1988); J. Biochem., 110, 166 (1991)], an in vitro transcription Can be used to produce KPC1. That is, DNA encoding KPC1 is connected downstream of a promoter such as SP6, T7, T3, etc., and a large amount of RNA encoding KPC1 is converted in vitro by reacting each promoter-specific RNA polymerase. After the synthesis, KPC1 can be produced using a cell-free translation system such as a renal erythrocyte lysate or a translation system using a wheat germ extract. Structural analysis of the purified KPCl can be performed by a method commonly used in protein chemistry, for example, the method described in "History of Protein Structure for Gene Cloning" (Hisashi Hirano, Tokyo Chemical Dojin, 1993). It is.
(4)KPC1および KPC2を構成成分として含む複合体の製造方法 (4) Method for producing a composite containing KPC1 and KPC2 as constituent components
KPC1および KPC2の両者を発現する形質転換体を作製し、 （2 )に記載の方法に準 じて該形質転換体を培養することにより、 培養物中に KPC1および KPC2を構成成分 として含む複合体 （以下、 KPC1-KPC2複合体とよぶこともある） を生成蓄積させ 、 （3 )に記載の方法に準じて該培養物から該複合体を単離精製することにより、 該複合体を製造することができる。 KPC1および KPC2の両者を発現する形質転換体 は、 （1 )に記載した方法で作製した KPC1の発現ベクターと KPC2の発現べクタ一の 両者を宿主に導入することにより作製できる。 あるいは、 KPC1の発現ベクターに さらに、 プロモーターおよびその下流に接続した KPC2をコ一ドする DNAからなる 発現ユニットを揷入して、 KPC1および KPC2の発現ベクターを作製し、 宿主細胞に 導入することによつても作製できる。  A transformant expressing both KPC1 and KPC2 is prepared, and the transformant is cultured according to the method described in (2), whereby a complex containing KPC1 and KPC2 as a component in the culture is obtained. (Hereinafter, also referred to as a KPC1-KPC2 complex), and the complex is produced by isolating and purifying the complex from the culture according to the method described in (3). be able to. A transformant expressing both KPC1 and KPC2 can be prepared by introducing both the KPC1 expression vector and the KPC2 expression vector prepared by the method described in (1) into a host. Alternatively, an expression unit consisting of a promoter and DNA encoding KPC2 connected downstream of the promoter is inserted into the KPC1 expression vector to produce KPC1 and KPC2 expression vectors, which are then introduced into host cells. Can also be produced.
KPC1 - KPC2複合体は、 （ 1 ) ~ ( 3 )に記載の方法に準じて製造した KPC1および KPC2 を混合し、 インビトロで会合させることによつても製造することができる。  The KPC1-KPC2 complex can also be produced by mixing KPC1 and KPC2 produced according to the methods described in (1) to (3) and associating them in vitro.
4. KPC1と特異的に結合する抗体の調製 4. Preparation of antibodies that specifically bind to KPC1
( 1 )ポリクローナル抗体の調製 (1) Preparation of polyclonal antibody
上記 3.に記載の方法により取得した KPC1の全長または部分断片の精製標品、 あ るいは KPC1の一部のアミノ酸配列からなるぺプチドを抗原として用い、 動物に投 与することにより、 KPC1と特異的に結合するポリクロ一ナル抗体を作製すること ができる。 抗原としてペプチドを用いる場合は、 ペプチドをキ一ホール ' リンぺ ヅト ·へモシァニンゃ牛チログ口プリン等のキヤ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、 Fmoc法 （フルォレニルメチ ルォキシカルボニル法） 、 tBOC法 (t-ブチルォキシカルボニル法）等の化学合成法 あるいは、 アプライ ド ·バイオシステムズ社、 Advanced ChemTech社、 鳥津製作 所等のぺプチド合成機を利用して化学合成することができる。  A purified preparation of the full-length or partial fragment of KPC1 obtained by the method described in 3 above, or a peptide consisting of a partial amino acid sequence of KPC1 is used as an antigen, and administered to animals to give KPC1 and KPC1. A polyclonal antibody that specifically binds can be produced. When a peptide is used as the antigen, it is desirable that the peptide be covalently bound to a carrier protein such as keyhole's phosphate, hemocyanin, or bovine tilogin purine. Peptides used as antigens can be prepared by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method), tBOC method (t-butyloxycarbonyl method), or Applied Biosystems, Advanced ChemTech, Torizu Corporation, etc. Chemical synthesis can be carried out using a peptide synthesizer.
投与する動物として、 ゥサギ、 ャギ、 3〜20週令のラット、 マウス、 ハムス夕 —等の非ヒトほ乳動物を用いることができる。  Non-human mammals such as rabbits, goats, rats, mice, and hams that are 3-20 weeks old can be used as the animals to be administered.
抗原の投与は、 1回目の投与の後 1〜2週間おきに 3〜10回行う。 各投与後、 3〜7 日目に採血して血清を調製し、 該血清が免疫に用いた抗原と反応することを酵素 免疫測定法 （ELISA ) 〔酵素免疫測定法 （第 3版） 、 医学書院 （1987) ; Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Pre ss (1988)〕 等で確認する。 抗原の投与量は動物 1匹に投与 1回当たり 50〜200〃gが 好ましい。 The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. 3-7 after each dose On the day, blood is collected to prepare serum, and the reaction of the serum with the antigen used for immunization is determined by enzyme-linked immunosorbent assay (ELISA) [Enzyme-linked immunosorbent assay (3rd ed.); A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)]. The dose of the antigen is preferably 50 to 200 mg per administration per animal.
ELISAの具体的例として、 以下の方法をあげることができる。 免疫の際、 抗原 に用いた KPC1またはペプチドを適当なプレートにコートし、 血清を反応させ、 さ らに抗原を投与した動物のィムノグロブリンに対する抗体をホースラディッシュ •ペルォキシダーゼ等の酵素で標識した抗体を反応させる。標識酵素により発色 する基質を添加して反応を行ない、 発色量を分光光度計により測定し、 血清の抗 体価とする。 、  The following method can be given as a specific example of ELISA. At the time of immunization, KPC1 or peptide used as an antigen is coated on an appropriate plate, the serum is reacted, and an antibody to the immunoglobulin of the animal to which the antigen has been administered is labeled with an enzyme such as horseradish peroxidase. Is reacted. The reaction is performed by adding a substrate that develops color with the labeling enzyme, and the amount of color development is measured with a spectrophotometer to determine the serum antibody titer. ,
免疫に用いた抗原に対し、 血清が充分な抗体価を示した動物より全血清を取得 し、 該血清を分離、 精製することによりポリクローナル抗体を取得することがで きる。 分離、 精製する方法としては、 遠心分離、 40〜50%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 Press, (1988)〕 、 または DEAE—セファロースカラム、 陰ィ オン交換カラム、 プロティン Aまたは Gカ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  A polyclonal antibody can be obtained by obtaining whole serum from an animal whose serum has 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 Press, (1988)] or DEAE-Sepharose column, Chromatography using ion-exchange columns, protein A or G columns, gel filtration columns, etc., alone or in combination.
(2)モノクローナル抗体の調製 (2) Preparation of monoclonal antibody
(a)抗体産生細胞の調製  (a) 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 たマウスまたは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 mouse or rat whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody-producing cells.
該抗体価を示したマウスまたはラット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。 該脾臓を MEM中で細断し、 ピンセットでほく、し、 l，200rpmで 5 分間遠心分離した後、 上清を捨てる。得られた沈殿画分の脾細胞をトリスー塩化 アンモニゥム緩衝液 （pH7.65) で 1〜2分間処理し赤血球を除去した後、 MEM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 is removed 3 to 7 days after the last administration of the antigenic substance to the mouse or rat showing the antibody titer. The spleen is minced in MEM, crushed with forceps, centrifuged at 200 rpm for 5 minutes, and the supernatant is discarded. The spleen cells in the resulting precipitate fraction are treated with Tris-chlorinated ammonium buffer (pH7.65) for 1 to 2 minutes to remove red blood cells, washed three times with MEM, and the resulting splenocytes are used as antibody-producing cells. Used.
(b)骨髄腫細胞の調製  (b) 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- Ul C Curr. Topics Microbiol . Immunol . , 81, 1 ( 1978)、 Eur. J. Immunol . , 6, 511 (1976)〕 、 SP2/0-Agl4 〔 Nature, 276, 269 (1978)〕 、 P3-X63-Ag8653 〔J. Immunol. , 123, 1548 (1979)〕、 P3-X63-Ag8 (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 1.5腿 ol/Lグルタミン、 50 /mol/L 2 —メルカプトエタノール、 10〃g/mLゲン 夕マイシンおよび 10%ゥシ胎児血清を加えた培地 （以下、 正常培地という） に、 さらに 15 zg/mL 8-ァザグァニンを加えた培地〕 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 mice (derived from BALB / c) myeloma cell line P3-X63Ag8-UlC Curr. Topics Microbiol. Immunol., 81, 1 (1978), Eur. J. Immunol., 6, 511 (1976) )), SP2 / 0-Agl4 (Nature, 276, 269 (1978)), P3-X63-Ag8653 (J. Immunol., 123, 1548 (1979)), P3-X63-Ag8 (Nature, 256, 495 ( 1975)] etc. These cell lines can be used in an 8-azaguanine medium [RPMI 1640 medium: 1.5 l / L glutamine, 50 / mol / L 2 -mercaptoethanol, 10 μg / mL Medium containing 10% fetal serum and 10% fetal serum (hereinafter referred to as “normal medium”), followed by 15 zg / mL 8-azaguanine. Culture and use 2 × 10 7 or more of the cells for fusion.
(c)ハイプリ ドーマの作製 (c) Preparation of Hypri-Doma
(a)で取得した抗体産生細胞と（b)で取得した骨髄腫細胞を MEMまたは PBS ( 1.83g/Lリン酸水素ニナトリゥム、 0.21g/Lリン酸ニ水素一力リゥム、 7.65g/L塩 化ナトリウム、 PH7.2) でよく洗浄し、 細胞数が、 抗体産生細胞：骨髄腫細胞 =5 〜10： 1になるよう混合し、 l, 200rpmで 5分間遠心分離した後、 上清を捨てる。 得られた沈澱画分の細胞群をよくほぐし、 攪拌しながら 37°Cで、 抗体産生細 胞あたり、ポリエチレングリコール— 1000 2g MEM 2mLおよびジメチルスルホキ シド 0.7mLを混合した溶液を 0.2〜； ImL添加し、 更に；！〜 2分間毎に MEM l〜2mLを数 回添加する。 添加後、 MEMを加えて全量が 50mLになるように調製する。 該調製液 を 900rpmで 5分間遠心分離後、 上清を捨てる。  The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) were combined with MEM or PBS (1.83 g / L sodium hydrogen phosphate, 0.21 g / L sodium phosphate monophosphate, 7.65 g / L salt). Wash well with sodium chloride, PH7.2), mix so that the number of cells becomes antibody-producing cells: myeloma cells = 5 to 10: 1, centrifuge at 200 rpm for 5 minutes, and discard the supernatant . The cell group of the obtained precipitate fraction was thoroughly disintegrated, and a solution obtained by mixing 2 mL of polyethylene glycol-1000 2 g MEM and 0.7 mL of dimethyl sulfoxide per antibody-producing cell at 37 ° C. with stirring was 0.2 to ImL. Add; Add ~ 2 mL of MEM several times every ~ 2 min. After addition, add MEM to adjust the total volume to 50 mL. 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 0.4麵 ol/Lヒポキサンチン、 15 z mol/Lチミジンおよび 0.4〃mol/Lアミノブテリンを加えた培地〕 lOOmL中に懸濁す る。 該懸濁液を 96ゥエル培養用プレートに 100 Lノウエルずつ分注し、 5% C02 インキュベータ一中、 37°Cで？〜 14日間培養する。 Gently loosen the cells in the obtained precipitate fraction, then slowly suck in and blow out with a female pipette.Hat medium (0.4 l / l hypoxanthine, 15 zmol / l thymidine and 0.4 lmol in normal medium) / L aminobuterin added medium] Suspend in lOOmL. The suspension dispensed at 100 L Noueru the plate for 96 Ueru culturing, in 5% C0 2 incubator primary, at 37 ° C? Incubate for ~ 14 days.
培養後、 培養上清の一部をとり、 ELISAにより、 培養上清中の KPC1に結合する 抗体を検出することにより、 KPC1に特異的に結合するモノクローナル抗体を生産 するハイプリド一マを選択する。  After culturing, a portion of the culture supernatant is taken, and antibodies that bind to KPC1 in the culture supernatant are detected by ELISA, thereby selecting a hybridoma that produces a monoclonal antibody that specifically binds to KPC1.
ELISAの具体的例として、 以下の方法をあげることができる。 免疫の際、 抗原 に用いた KPC1またはペプチドを適当なプレートにコートし、 ハイプリドーマの培 養上清を反応させ、 さらにホースラディッシュ ·ペルォキシダ一ゼ等の酵素で標 識した抗マウスィムノグロブリン抗体 （抗体産生細胞がラット由来の場合は抗ラ ヅトイムノグロブリン抗体） を反応させる。 標識酵素により発色する基質を添加 して反応を行ない、 発色量を分光光度計により測定して、 培養上清中の KPC1に特 異的に結合する抗体を検出する。 The following method can be given as a specific example of ELISA. At the time of immunization, KPC1 or peptide used as an antigen is coated on an appropriate plate, and the culture supernatant of the hybridoma is reacted, and further labeled with an enzyme such as horseradish peroxidase. React with the recognized anti-mouse immunoglobulin antibody (anti-rat immunoglobulin antibody if the antibody-producing cells are derived from rat). The reaction is performed by adding a substrate that develops color using the labeling enzyme, and the amount of color developed is measured with a spectrophotometer to detect antibodies that specifically bind to KPC1 in the culture supernatant.
選択したハイプリ 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、 HT培地 （HAT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〕、 上記と同様にして、 ハイプリドーマの培養上清中の KPC1に結合する抗体を検出し、 安定して高い抗体生産量を示すハイプリ ドーマを KPC1に特異的に結合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 selected hybridoma, repeat cloning twice by limiting dilution [First, use HT medium (medium in which aminopterin is removed from HAT medium), and second, use normal medium]. Similarly, antibodies that bind to KPC1 in the culture supernatant of hybridomas are detected, and hybridomas that exhibit high and stable antibody production are selected as hybridoma strains that produce monoclonal antibodies that specifically bind to KPC1. I do.
( d)モノクロ一ナル抗体の調製  (d) Preparation of monoclonal antibody
プリスタン処理 〔プリスタン （2, 6, 10, 14—テトラメチルペン夕デカン） 0.5Λ を腹腔内投与し、 2週間飼育する〕 した 8~10週令のマウスまたはヌードマウス に、 （c )で取得した KPC1に特異的に結合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 5〜20 x l06細胞/匹を腹腔内に注射する。 10〜21日間でハイプ リドーマは腹水癌化する。 該腹水癌化したマウスから腹水を採取し、 3, 000rpmで 5分間遠心分離して固形分を除去する。 得られた上清より、 ポリクローナル抗体 の精製で用いた方法と同様の方法でモノク口一ナル抗体を精製、 取得することが できる。 Obtained in (c) in 8-10 week old mice or nude mice treated with pristane (0.5% pristane (2,6,10,14-tetramethylpentyldecane) administered intraperitoneally and bred for 2 weeks) high pre dormer cells 5 to 20 x l0 6 cells / mouse to produce a monoclonal antibody that specifically binds to KPC1 was injected intraperitoneally. In 10 to 21 days, the hybridoma becomes ascites cancer. Ascites is collected from the mouse with ascites tumor and centrifuged at 3,000 rpm for 5 minutes to remove solids. From the obtained supernatant, a monoclonal antibody can be purified and obtained by the same method as used in the purification of the polyclonal antibody.
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 リー法あるいは 280皿での吸光度より算出する。  The subclass of the antibody 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 from the absorbance in 280 dishes.
上記 ( 1 )および (2)に記載の方法で KPC1と特異的に結合する抗体を得ることがで きる。 また、 KPC1に代えて KPC2の全長または部分断片の精製標品、 あるいは KPC2 の一部のアミノ酸配列からなるぺプチドを抗原として用い、 上記 (1 )および (2)に 記載の方法に準じて、 KPC2と特異的に結合する抗体を得ることができる。  An antibody that specifically binds to KPC1 can be obtained by the methods described in (1) and (2) above. Further, in place of KPC1, a purified preparation of the full-length or partial fragment of KPC2, or a peptide consisting of a partial amino acid sequence of KPC2 is used as an antigen, according to the method described in (1) and (2) above. An antibody that specifically binds to KPC2 can be obtained.
( 3 )KPC1-KPC2複合体と特異的に結合する抗体 (3) Antibodies that specifically bind to the KPC1-KPC2 complex
1.または 3. (4)に記載の方法で得られる KPC1- KPC2複合体の精製標品を抗原と して用い、 上記 ( 1 )および (2)に記載の方法に準じて、 KPC1- KPC2複合体と特異的 に結合する抗体を得ることができる。 KPC1または KPC2と特異的に結合する抗体も、 KPC1 - KPC2複合体と特異的に結合 する抗体として用いられる。 1. or 3.Using the purified preparation of the KPC1-KPC2 complex obtained by the method described in (4) as an antigen, KPC1-KPC2 is prepared according to the methods described in (1) and (2) above. An antibody that specifically binds to the complex can be obtained. An antibody that specifically binds to KPC1 or KPC2 is also used as an antibody that specifically binds to the KPC1-KPC2 complex.
(4)p27Kiplのュビキチン化を阻害する抗体 (4) Antibodies that inhibit p27 Kipl ubiquitination
p27Kiplのュビキチン化を阻害する抗体としては、 KPC1または KPC1- KPC2複合体 が有する ρΖγ1^1をュビキチン化する活性を阻害する抗体、 および p27Kiplと KPC1ま たは KPC KPC2複合体との結合を阻害する抗体があげられる。 Antibodies that inhibit Yubikichin of p27 Kipl, binding of KPC1 or KPC1- KPC2 antibody that inhibits the activity of Yubikichin the ρΖγ 1 ^ 1 the complex has, and p27 Kipl and KPC1 or the KPC KPC2 complex Antibodies that inhibit the activity.
KPC1または KPC1- KPC2複合体が有する p27Kiplをュビキチン化する活性を阻害す る抗体は、 7. (1)に記載した p27Kiplをュビキチン化する活性を測定する系に、 (1)〜(3)に記載した方法で得られた KPC1または KPC1- KPC2複合体と特異的に結合 する抗体を被験試料として添加して、 KPC1または KPC1- KPC2複合体の p27Kiplをュ ビキチン化する活性を測定し、 添加しない場合と比較して、 KPC1または KPC1- KPC2複合体の活性が低下した抗体を選択することにより、 得ることができる。 p27Kiplと KPC1または KPC1- KPC2複合体との結合を阻害する抗体は、 8. (2)に記 載した p27Kiplと KPC1または KPC1 -KPC2複合体との結合を測定する系に、 U )〜（ 3 ) に記載した方法で得られた KPC1または KPC1-KPC2複合体と特異的に結合する抗体 を被験試料として添加して、 p27Kiplと KPC1または KPC1 - KPC2複合体との結合量を 測定し、 添加しない場合と比較して、 結合量が低下した抗体を選択することによ り、 得ることができる。 Antibodies that inhibit the activity of ubiquitinating p27 Kipl possessed by the KPC1 or KPC1-KPC2 complex can be used in a system for measuring the activity of ubiquitinating p27 Kipl described in 7. (1). An antibody that specifically binds to the KPC1 or KPC1-KPC2 complex obtained by the method described in (2) is added as a test sample, and the activity of the KPC1 or KPC1-KPC2 complex to ubiquitinate p27 Kipl is measured. However, it can be obtained by selecting an antibody in which the activity of the KPC1 or KPC1-KPC2 complex has been reduced as compared with the case where it is not added. Antibodies that inhibit the binding of p27 Kipl and KPC1 or KPC1- KPC2 complexes, to a system for measuring the binding of 8. (2) serial mounting the p27 Kipl to the KPC1 or KPC1 -KPC2 complex, U) ~ An antibody that specifically binds to the KPC1 or KPC1-KPC2 complex obtained by the method described in (3) is added as a test sample, and the amount of p27 Kipl bound to the KPC1 or KPC1-KPC2 complex is measured. It can be obtained by selecting an antibody having a reduced amount of binding as compared with the case where it is not added.
5. KPC1遺伝子の染色体上の位置 5. Location of the KPC1 gene on the chromosome
塩碁配列データペース上のヒトゲノム DNAの配列には通常、 その配列の染色体 の位置についての情報が記載されている。 例えば、 2. (2)で得られたヒト KPC1ゲ ノム遺伝子のェキソンを含むヒトゲノム DNAの配列 （ジヱンバンク登録番号： NT— 022439) についてはヒト染色体 3p24.3に位置することがデ一夕べ一ス上に記 載されている。 したがってヒト KPC1遺伝子はヒト染色体 3p24.3に位置するという ことができる。  A human genomic DNA sequence on the Go sequence database usually contains information about the chromosomal location of that sequence. For example, 2. For the sequence of human genomic DNA containing exon of the human KPC1 genomic gene obtained in (2) (Genbank accession number: NT-022439), it is reported that it is located on human chromosome 3p24.3. It is listed above. Therefore, it can be said that the human KPC1 gene is located on human chromosome 3p24.3.
このようにして得られた KPC1遺伝子の染色体上の位置の情報は、 疾患と KPC1遺 伝子の関連を研究するのに役立つ。 例えば、 癌では癌抑制遺伝子の存在する可能 性の高い染色体上の領域として、 多くの癌について高頻度に LOH ( loss of heterozygosity： 2対の内の一方の遺伝子に見られる染色体欠失） が検出される 領域の特定が進んでいるが、 このような領域と KPC1遺伝子が存在する染色体の位 置が一致すれば、 KPC1がこの領域に L0Hを持つ癌の発症に関与する可能性がある ことになる。 この場合、 このような癌における KPC1遺伝子の変異や KPC1の発現量 を解析することにより、 KPC1と癌との関連が明らかになれば、 KPC1をコードする MAに由来するポリヌクレオチドゃォリゴヌクレオチド、 KPC1と特異的に結合す る抗体を用いて、 9.、 11.または 13.に記載する方法に基づいて、 このような癌の 診断や治療を行なうことができる。 Information on the chromosomal location of the KPC1 gene obtained in this way is useful for studying the association between the disease and the KPC1 gene. For example, in cancer, LOH (loss of heterozygosity: a chromosomal deletion found in one of two genes) is frequently detected as a region on the chromosome where a tumor suppressor gene is likely to be present. Although the identification of the region is progressing, such a region and the chromosomal position where the KPC1 gene exists are If the positions match, KPC1 may be involved in the development of cancers with L0H in this region. In this case, by analyzing the KPC1 gene mutation and the expression level of KPC1 in such cancer, if the relationship between KPC1 and cancer is clarified, polynucleotides derived from MA encoding KPC1 may be used. Using an antibody that specifically binds to KPC1, such a cancer can be diagnosed or treated based on the method described in 9., 11., or 13.
6. KPC1をコードする DNAを導入した非ヒトトランスジエニック動物および KPC1遺 伝子欠損非ヒト動物の作製  6. Production of transgenic non-human animals and non-human animals deficient in the KPC1 gene into which DNA encoding KPC1 has been introduced
(l)KPClをコードする DNAを導入した非ヒトトランスジヱニック動物の作製  (l) Preparation of non-human transgenic animal into which DNA encoding KPCl has been introduced
KPC1をコードする DNAを導入した非ヒトトランスジエニック動物 （以下、 単に トランスジエニック動物ともいう） は、 文献 〔Proc. Natl. Acad. Sci . USA, 77, 7380 ( 1980) ; Nature, 344, 541 (1990) ; Nature, 315, 680 ( 1985) ; Immunol. Immunopathol . , 17, 303 ( 1987)〕 に記載の方法に準じて、 非ヒ卜哺乳 動物の受精卵へ直接、 該動物の細胞内で KPC1を発現させうるプロモーターの下流 に KPC1をコ一ドする DNAを連結した遺伝子構築物をィンジヱクシヨンすることに より作製することができる。 また、 非ヒト哺乳動物の胚性幹細胞を公知の方法 〔 Nature, 292, 154 ( 1981 ) ; Proc. Natl . Acad. Sci. USA, 78, 7634 ( 1981 ) ; US5453357 US5670372; Dev. Biol. , 163, 288 (1994) ; Reprod. Fertil. Dev. , 6, 563 (1994) ; Proc. Natl. Acad. Sci . USA, 92, 7844 (1996)〕 に基づいて樹 立し、 該胚性幹細胞へ通常の培養細胞への遺伝子導入の場合と同様にして該動物 の細胞内で KPC1を発現させうるプロモーターの下流に KPC1をコードする DNAを連 結した遺伝子構築物を導入後、 集合キメラ法や注入キメラ法等の手法によりトラ ンスジエニック動物を作出することができる （Manipulating the Mouse Embryo： A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) ; Gene Targeting, A Practical Approach, IRL Press at Oxford University Press ( 1993)〕 0 Non-human transgenic animals into which DNA encoding KPC1 has been introduced (hereinafter, also simply referred to as transgenic animals) are described in Proc. Natl. Acad. Sci. USA, 77, 7380 (1980); Nature, 344, 541 (1990); Nature, 315, 680 (1985); Immunol. Immunopathol., 17, 303 (1987)], directly into a fertilized egg of a non-human mammal, in the cells of the animal. Thus, a gene construct in which DNA encoding KPC1 is linked downstream of a promoter capable of expressing KPC1 can be prepared. A non-human mammalian embryonic stem cell can be obtained by a known method [Nature, 292, 154 (1981); Proc. Natl. Acad. Sci. USA, 78, 7634 (1981); US5453357 US5670372; Dev. Biol., 163 , 288 (1994); Reprod. Fertil. Dev., 6, 563 (1994); Proc. Natl. Acad. Sci. USA, 92, 7844 (1996)]. After introducing a gene construct in which KPC1-encoding DNA is linked downstream of a promoter capable of expressing KPC1 in the cells of the animal in the same manner as in the case of gene transfer into cultured cells of Transgenic animals can be produced by such techniques as (Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993) )) 0
プロモー夕—として、 3. (1 )に記載の動物細胞での発現ベクターに用いること のできるプロモ一夕一を同様に用いることができる。 適切なプロモー夕一を使用 することにより、 全身あるいは組織特異的に KPC1を高発現するトランスジヱニッ ク動物を得ることができる。 得られた該トランスジエニック動物は、 KPC1の活性により、 細胞内の p27Kiplの ュビキチン化および分解が促進するため、 細胞周期の異常が引き起こされ、 細胞 周期の異常を原因とする疾患、 例えば癌等の疾患モデルとなると考えられる。 ま た、 該トランスジヱニック動物に抗癌剤等の薬剤を投与し、 該トランスジェニヅ ク動物の疾患症状や病理を観察することにより、 薬剤の評価を行なうことができ る o As the promoter, a promoter that can be used for the expression vector in animal cells described in 3. (1) can be used in the same manner. By using an appropriate promoter, transgenic animals that express KPC1 at high levels systemically or tissue-specifically can be obtained. In the obtained transgenic animal, the activity of KPC1 promotes the ubiquitination and degradation of p27 Kipl in cells, thereby causing cell cycle abnormalities and causing diseases caused by abnormal cell cycles, such as cancer. It is considered to be a disease model such as. Further, the drug can be evaluated by administering a drug such as an anticancer drug to the transgenic animal and observing the disease symptoms and pathology of the transgenic animal.o
(2)KPC1遺伝子欠損非ヒト動物の作製  (2) Preparation of non-human animal deficient in KPC1 gene
遺伝子欠損をさせる非ヒト哺乳動物の KPC1遺伝子含むゲノム DNAを 2. (2)に記 載の方法で単離する。 該ゲノム DNAのェキソン部分の全部または一部を欠失させ た不活性型 KPC1遺伝子を含むターゲッティングベクターを作製する。 公知の手法 (Nature, 326, 295 ( 1987)、 Cell, 51, 503 (1987)〕 により、 夕ーゲヅティン グベクターを胚性幹細胞に導入し、 染色体上の KPC1遺伝子と導入した不活性型 KPC1遺伝子が相同組換えを起こした胚性幹細胞を作製することができる 〔 Nature, 3503 243 ( 1991)〕 。 Isolate the genomic DNA containing the KPC1 gene of the non-human mammal causing the gene deletion by the method described in 2. (2). A targeting vector containing an inactive KPC1 gene in which all or part of the exon portion of the genomic DNA has been deleted is prepared. Using a known method (Nature, 326, 295 (1987), Cell, 51, 503 (1987)), the evening-targeting vector was introduced into embryonic stem cells, and the KPC1 gene on the chromosome and the introduced inactive KPC1 gene were homologous. Recombinant embryonic stem cells can be produced [Nature, 350 3 243 (1991)].
選択した胚性幹細胞と非ヒト哺乳動物の受精卵を用い集合キメラ法あるいは注 入キメラ法を用い生殖系列キメラ個体を作出することができる。 このキメラ個体 と正常個体の掛け合わせにより、 全身の細胞の KPC1遺伝子が不活性型 KPC1遺伝子 に置換された個体を得ることができ、 さらにその個体の掛け合わせにより相同染 色体の双方の KPC1遺伝子が不活性型に変異したホモ個体を得ることができる。 夕一ゲヅティングベクターは、 Gene Targeting, A Practical Approach, IRL Press at Oxford University Press ( 1993)等に記載の方法にしたがって作製す ることができる。 夕ーゲヅティングベクターは、 リプレースメント型、 インサ一 ション型いずれでも用いることができる。  Using the selected embryonic stem cells and fertilized eggs of a non-human mammal, a germ-line chimeric individual can be produced using the collective chimera method or the injection chimera method. By crossing the chimeric individual with a normal individual, it is possible to obtain an individual in which the KPC1 gene of whole body cells has been replaced with an inactive KPC1 gene. Can be obtained as inactive individuals. The evening targeting vector can be prepared according to the method described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993) and the like. The evening targeting vector can be used in either a replacement type or an insertion type.
相同組換え体を効率的に選別する方法として、 例えば、 Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993) .等に記載 のポジティブ選択、 プロモータ一選択、 ネガティブ選択、 ポリ A選択などの方法 を用いることができる。 選別した細胞株の中から目的とする相同組換え体を選択 する方法としては、 ゲノム DNAに対するサザンハイブリダィゼーシヨン （モレキ ユラ一.クローニング第 3版） や PCR等があげられる。 Methods for efficiently selecting homologous recombinants include, for example, positive selection, promoter selection, negative selection, poly A selection described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993). The following method can be used. Methods for selecting the target homologous recombinant from the selected cell lines include Southern hybridization (Molecular I. Cloning 3rd Edition) for genomic DNA, PCR, and the like.
7. p27Kiplをュビキチン化する活性および p27Kiplの分解の測定 ( 1 ) KPC1または KPC1- KPC2複合体の p27Kiplをュビキチン化する活性の測定 7. Determination of degradation of the active and p27 Kipl to Yubikichin the p27 Kipl (1) Measurement of the activity of KPC1 or KPC1-KPC2 complex to ubiquitinate p27 Kipl
ュビキチン化反応のインビトロ再構成系 〔Proc . Natl . Acad. Sci . USA, 92, Acad. Sci. USA, 92, Proc. Natl. Acad. Sci.
2563 ( 1995 ) ; Science, 269, 682 ( 1995 ) ; Cell, 91 , 221 ( 1997)〕 を利用し、 KPC1または KPC1- KPC2複合体を用いることにより、 以下のようにして KPC1または KPC1- KPC2複合体が有する p27Kiplをュビキチン化する活性を測定することができ る o 2563 (1995); Science, 269, 682 (1995); Cell, 91, 221 (1997)], and using a KPC1 or KPC1-KPC2 complex, a KPC1 or KPC1-KPC2 complex as follows. The ability of the body to ubiquitinate p27 Kipl can be measured o
例えば上記 3.に記載の方法により得られる精製した KPC1または KPC1- KPC2複合 体を、 ρ27κίρ1、 Elおよび Ε2と試験管内で混合したものにュビキチンを添加し反応 を行なう。 SDS-PAGE等により、 ュビキチン化された p27Kiplを単離し、 p27Kiplに取 り込まれたュビキチンの量を測定する。 p27Kiplに取り込まれたュビキチンの量は 、 反応時に添加するュビキチンを蛍光、 ビォチン、 あるいは放射性同位体で標識 したものを使用し、 該標識に基づいたシグナルを測定する、 あるいは抗ュビキチ ン抗体を用いてュビキチンを検出することにより測定することができる 〔 Nature, 373, 81 ( 1995 ) ; FEBS Lett. , 377, 193 ( 1995 ) ; Science, 269, 682 ( 1995)〕 o また、 SDS-PAGE後に、 p27Kiplを認識する抗体 （p27Kiplと特異的に結合 する抗体、 または p27Kiplに付加したタグと特異的に結合する抗体） を利用したィ ムノブロット解析を行ない、 標的蛋白質の分子量の増加を指標にュビキチンの量 を測定することもできる 〔J. Biol . Chem. , 276, 48937 (2001 )〕。 For example, ubiquitin is added to a mixture of the purified KPC1 or KPC1-KPC2 complex obtained by the method described in the above item 3 and ρ27κίρ1 , El and Ε2 in a test tube, followed by the reaction. The ubiquitinated p27 Kipl is isolated by SDS-PAGE or the like, and the amount of ubiquitin incorporated into the p27 Kipl is measured. The amount of ubiquitin incorporated into p27 Kipl can be determined by using ubiquitin added during the reaction, labeled with fluorescence, biotin, or radioisotope, and measuring a signal based on the label, or by using an anti-ubiquitin antibody. Can be measured by detecting ubiquitin [Nature, 373, 81 (1995); FEBS Lett., 377, 193 (1995); Science, 269, 682 (1995)] o After SDS-PAGE, An immunoblot analysis using an antibody that recognizes p27 Kipl (an antibody that specifically binds to p27 Kipl or an antibody that specifically binds to a tag added to p27 Kipl ) was performed, and the increase in the molecular weight of the target protein was used as an index. The amount of ubiquitin can also be measured [J. Biol. Chem., 276, 48937 (2001)].
また、 例えば KPC1または KPC1- KPC2複合体を発現する形質転換体を 3. ( 1 )、 3. (4)に記載の方法に準じて作製し、 該形質転換体あるいは細胞抽出液等の該形質 転換体の処理物にュビキチンおよびプロテアソ一ム阻害剤を添加し、 適当な温度 で培養または保温することにより、 ュビキチン一プロテアソーム系の反応をポリ ュビキチン化までで停止させる系を、 上記のィンビト口再構成系の代わりに利用 して測定することもできる。 形質転換体の宿主としては、 動物細胞等、 内在性に p27Kipl、 Elおよび KPC1または KPC1- KPC2複合体が利用できる E2を発現しているも のを用いる。 この系において p27Kiplに取り込まれたュビキチンは、 上記のインビ ト口再構成系と同様の方法により測定することができる。 In addition, for example, a transformant expressing the KPC1 or KPC1-KPC2 complex is prepared according to the method described in 3. (1), 3. (4), and the transformant or the cell extract or the like is used for the transformant. By adding ubiquitin and a proteasome inhibitor to the processed product of the transformant and culturing or keeping the mixture at an appropriate temperature, the system for stopping the reaction of the ubiquitin-proteasome system until the ubiquitination is replaced with the above-mentioned invitro-retrofitting method. It can also be used instead of the component system for measurement. As a host of the transformant, an animal cell or the like expressing p2 Kipl , El and E2 which can use KPC1 or KPC1-KPC2 complex endogenously, such as an animal cell, is used. In this system, ubiquitin incorporated into p27 Kipl can be measured by the same method as in the above-mentioned in vitro reconstitution system.
(2) KPC1または KPC1- KPC2複合体に依存した p27Kiplの分解の測定 (2) Measurement of p27 Kipl degradation depending on KPC1 or KPC1-KPC2 complex
3. ( 1 )、 3. (4)に記載の方法に準じて作製した KPC1または KP -KPC2複合体を 発現する形質転換体を培養して、 経時的に一部を回収し、 それそれの細胞の p27Kiplの含量を測定し、 形質転換体の代わりに KPC1または KPC1- KPC2複合体を発 現しない宿主を用いた場合の p27Kiplの含量と比較することにより、 KPC1または KPC卜 KPC2複合体に依存した p27Kiplの分解を測定することができる。 形質転換体 の宿主としては、 動物細胞等、 内在性に p27Kipl、 El KPC1または KPC1- KPC2複合 体が利用できる E2、 およびプロテアソ一ムを発現しているものを用いる。 3.Culture the transformant expressing the KPC1 or KP-KPC2 complex prepared according to the methods described in (1) and 3. (4), collect a part of it over time, Cellular Depends on KPC1 or KPC2 KPC2 complex by measuring p27 Kipl content and comparing with p27 Kipl content when using a host that does not express KPC1 or KPC1-KPC2 complex instead of transformant The degradation of p27 Kipl can be measured. As a host for the transformant, an animal cell or the like which expresses p27 Kipl , E2 capable of utilizing El KPC1 or KPC1-KPC2 complex endogenously, and a proteasome , such as animal cells, is used.
ρ27 Ρΐの含量は、 形質転換体の培養時に培地に放射性同位体で標識したァミノ 酸を添加し、 p27Kiplを標識後、 SDS-PAGEを行ない放射活性を測定する、 あるいは 、 p27Kiplを認識する抗体を用いてィムノブロット解析を行なうことにより測定す ることができる。 Genes Dev. , 12, 2587 ( 1998) ; Mol . Cell, 1, 565 (1998) ; Genes Dev. , 11, 3046 ( 1997) ; Genes Dev. , 11, 1548 ( 1997)〕。 The content of ρ 27 Ρΐ is added Amino acid labeled with a radioactive isotope to the medium during the culturing of the transformant, after labeling the p27 Kipl, measuring the radioactivity subjected to SDS-PAGE, or recognize p27 Kipl It can be measured by performing an immunoblot analysis using an antibody to be tested. Genes Dev., 12, 2587 (1998); Mol. Cell, 1, 565 (1998); Genes Dev., 11, 3046 (1997); Genes Dev., 11, 1548 (1997)].
また、 細胞抽出液等の KPC1または KPC1- KPC2複合体を発現する上記の形質転換 体の処理物、 あるいは（ 1 )に記載したインビトロ再構成系から p27Kiplを除きュビ キチンおよびプロテアソ一ムを添加した系に、 ρ27κίρ1を添加して反応させ、 経時 的に一部を回収し、 p27Kiplの含量を測定し、 KPC1または KPC1- KPC2複合体を発現 しない宿主の処理物、 あるいは KPC1または KPC1- KPC2複合体を含まない上記のィ ンビトロ再構成系の系を用いた場合の p27Kiplの含量と比較することにより KPC1ま たは KPC1-KPC2複合体に依存した p27Kiplの分解を測定することができる。 p27Kiplの 含量の測定は、 上記と同様にして行なうことができる。 In addition, ubiquitin and proteasome were obtained by removing the p27 Kipl from the above-mentioned transformed product expressing the KPC1 or KPC1-KPC2 complex, such as a cell extract, or the in vitro reconstitution system described in (1). Add ρ27 κίρ1 to the added system and allow it to react.Collect a part over time, measure the content of p27 Kipl , and treat the host that does not express KPC1 or KPC1-KPC2 complex, or KPC1 or KPC1 -To determine the degradation of p27 Kipl depending on KPC1 or KPC1-KPC2 complex by comparing with the content of p27 Kipl using the above in vitro reconstitution system without KPC2 complex. Can be. The measurement of the content of p27 Kipl can be performed in the same manner as described above.
8. p27Kiplのュビキチン化を阻害する物質のスクリーニング方法 8. Screening method for substances that inhibit p27 Kipl ubiquitination
p27Kiplのュビキチン化を阻害する物質は、 以下（1 )および (2)に示すスクリー二 ング方法により、 取得することができる。 A substance that inhibits ubiquitination of p27 Kipl can be obtained by the screening methods described in (1) and (2) below.
(1 )KPC1または KPC1-KPC2複合体による p27Kiplのュビキチン化を測定することに基 づくスクリーニング方法 (1) Screening method based on measuring ubiquitination of p27 Kipl by KPC1 or KPC1-KPC2 complex
被験試料の存在下および非存在下で p27Kipl、 El、 E2およびュビキチンを含む系 で p27Kiplのュビキチン化反応を行い、 p27Kiplに取り込まれたュビキチンの量を測 定し、 比較することにより、 p27Kiplのュビキチン化を阻害する物質をスクリー二 ングすることができる。 By performing a ubiquitination reaction of p27 Kipl in a system containing p27 Kipl , El, E2, and ubiquitin in the presence and absence of the test sample, measuring the amount of ubiquitin incorporated into p27 Kipl , and comparing the results. Substances that inhibit ubiquitination of p27 Kipl can be screened.
すなわち、 上記 7. ( 1 )に記載した p27Kiplをュビキチン化する活性を測定する方 法に準じて、 KPC1もしくは KPC1-KPC2複合体、 p27Kipl、 El、 E2およびュビキチン を含むインビト口再構成系または KPC1もしくは KPC1- KPC2複合体を発現する形質 転換体もしくは該形質転換体の細胞抽出液等の処理物にュビキチンおよびプロテ ァソーム阻害剤を添カ卩した系に被験試料を添加した場合、 添加しなかった場合そ れそれで p27Kiplのュビキチン化反応を行い、 p27Kiplに取り込まれたュビキチンの 量を測定し、 比較する。 That is, according to the method for measuring the activity of ubiquitinating p27 Kipl described in 7. (1) above, an in vitro reconstitution system containing KPC1 or KPC1-KPC2 complex, p27 Kipl , El, E2 and ubiquitin Or a trait that expresses KPC1 or KPC1-KPC2 complex When a test sample is added to a system obtained by adding ubiquitin and a proteinase inhibitor to a transformant or a processed product such as a cell extract of the transformant, or not added, the ubiquitination reaction of p27 Kipl is performed. The amount of ubiquitin incorporated into p27 Kipl is measured and compared.
被験試料を添加しない場合と比較して、 被験試料を添加した場合に、 p27Kiplに 取り込まれたュビキチンの量が低下した場合は、 被験試料を p27Kiplのュビキチン 化を阻害する物質として選択する。 If the amount of ubiquitin incorporated into p27 Kipl decreases when the test sample is added as compared to when no test sample is added, the test sample is selected as a substance that inhibits ubiquitination of p27 Kipl .
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、 抗体、 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、 ラヅト、 モルモット、 ハムスター、 ブ夕、 ヒヅジ、 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、 更に、 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。 被験試料が単一の物質でなく組織抽出物や細胞培養上 清、 発酵生産物のように多数の物質の混合物の場合は、 選択された被験試料をさ らに精製し、 各精製画分についてスクリーニングをすることにより、 p27Kiplをュ ビキチン化する活性を阻害する物質を単離、 同定することができる。 Test samples include synthetic compounds, naturally occurring proteins, artificially synthesized proteins, antibodies, peptides, carbohydrates, lipids, modified forms and derivatives thereof, and mammals (eg, mouse, rat, guinea pig). Urine, body fluid, tissue extract, cell culture supernatant, and cell extract of hamsters, hamsters, bush, olive, olive, poma, dog, cat, monkey, human, etc., as well as non-peptide compounds, Examples include fermentation products, extracts of plants and other organisms, and the like. If the test sample is not a single substance but a mixture of many substances such as tissue extracts, cell culture supernatants, and fermentation products, the selected test sample is further purified, and the By screening, a substance that inhibits the activity of ubiquitinating p27 Kipl can be isolated and identified.
(2 )KPC1または KPC1- KPC2複合体と p27Kiplとの結合を阻害する物質をスクリーニン グする方法 (2) Screening method for substances that inhibit the binding of KPC1 or KPC1-KPC2 complex to p27 Kipl
被験試料を添加した状態で、 KPC1または KPC1- KPC2複合体を p27Kiplと接触させ た後、 KPC1または KPC1-KPC2複合体と ρ27κίρ1との結合量を測定する。 被験試料を 添加しなかった場合の KPC1または KPC1-KPC2複合体と ρ27κ の結合量と比較し、 結合量が低下した場合には、 KPC1または KPC1- KPC2複合体、 および p27Kiplの結合 を阻害する物質として選択する。 被験試料は（1 )と同様のものがあげられる。 ま た、 被験試料が混合物の場合は該被験試料をさらに精製し、 再スクリーニングす ることにより、 KPC1または KPC1- KPC2複合体と p27Kiplの結合を阻害する物質を単 離、 同定することができる。 After the KPC1 or KPC1-KPC2 complex is brought into contact with p27 Kipl with the test sample added, the amount of binding between KPC1 or KPC1-KPC2 complex and ρ27 κίρ1 is measured. Compared to KPC1 or KPC1-KPC2 complex and amount of binding of Ro27 kappa when not adding the test sample, if the amount of binding decreases, inhibits binding of KPC1 or KPC1- KPC2 complex, and p27 Kipl Select the substance to be used. The test sample is the same as in (1). If the test sample is a mixture, the test sample can be further purified and rescreened to isolate and identify substances that inhibit the binding of KPC1 or KPC1-KPC2 complex to p27 Kipl. .
KPC1または KPC1- KPC2複合体を p27Kiplと接触させるには、 3. ( 3 )に記載の方法 により得られる精製した KPC1、 あるいは 1.または 3. (4)に記載の方法により得ら れる精製した KPC1- KPC2複合体と p27Kiplをインビトロで混合することで行なえる 。 また、 内在性に p27Kiplを発現している宿主を用いて 3. ( 1 )または 3. (4)に記載 の方法により得られる KPC1または KPCl- KPC2複合体を発現する形質転換体を作製 する、 あるいは 3. (2)に記載の方法に準じて p27Kipl発現用ベクターを作製し、 KPC1または KPCl- KPC2複合体の発現用ベクターと共導入した形質転換体を作製す ることにより、 細胞内で接触させることができる。 被験試料の添加は、 上記のィ ンビトロの混合物または形質転換体の細胞抽出液等の処理物に被験試料を添加す るか、 被験試料を添加した培地で形質転換体を培養することにより行なうことが できる。 To bring the KPC1 or KPC1-KPC2 complex into contact with p27 Kipl , purified KPC1 obtained by the method described in 3. (3), or purified by the method described in 1. or 3. (4) This can be achieved by mixing the KPC1-KPC2 complex and p27 Kipl in vitro. Also, as described in 3. (1) or 3. (4), using a host endogenously expressing p27 Kipl. Prepare a transformant that expresses the KPC1 or KPCl-KPC2 complex obtained by the method described in 3., or 3.Create a p27 Kipl expression vector according to the method described in (2), and prepare a KPC1 or KPCl-KPC2 complex. By producing a transformant co-transfected with an expression vector for the body, the cells can be brought into contact in a cell. The test sample is added by adding the test sample to the above-mentioned in vitro mixture or a processed product such as a cell extract of the transformant, or by culturing the transformant in a medium containing the test sample. Can be done.
結合量の測定は、 抗 p27Kipl抗体を用いて免疫沈降を行ない、 免疫沈降物の中に 含まれる KPC1または KPC1-KPC2複合体を、 4.に記載の方法で作製した KPC1と特異 的に結合する抗体を用いたィムノブロット解析により検出することによって、 測 定することができる。 または、 KPC1と特異的に結合する抗体を用いて免疫沈降を 行ない、 免疫沈降物の中に含まれる p27Kiplを、 抗 p27Kipl抗体を用いたィムノブ口 ヅト解析により検出することによって、 測定することができる。 抗 p27Kipl抗体お よび KPC1と特異的に結合する抗体はそれそれ、 p27Kiplあるいは KPC1に付加した夕 グと特異的に結合する抗体を用いることもできる。 For the measurement of the binding amount, immunoprecipitation was performed using an anti-p27 Kipl antibody, and the KPC1 or KPC1-KPC2 complex contained in the immunoprecipitate was specifically bound to KPC1 prepared by the method described in 4. The measurement can be carried out by detecting by immunoblot analysis using an antibody to be used. Alternatively, immunoprecipitation is performed using an antibody that specifically binds to KPC1 , and p27 Kipl contained in the immunoprecipitate is detected by Immunob port analysis using an anti-p27 Kipl antibody. be able to. Antibodies specifically binding to anti-p27 Kipl antibody and KPC1 may be antibodies that specifically bind to p27 Kipl or KPC1-added antibodies.
以上の方法で得られた KPC1または KPC1-KPC2複合体と p27Kiplとの結合を阻害す る物質は、 KPC1または KPC1-KPC2複合体による p27Kiplのュビキチン化を阻害する ことができる。 The substance that inhibits the binding between KPC1 or KPC1-KPC2 complex and p27 Kipl obtained by the above method can inhibit ubiquitination of p27 Kipl by KPC1 or KPC1-KPC2 complex.
( 1 )または（2)のスクリーニング方法で選択された p27Kiplのュビキチン化を阻害 する物質は、 細胞内で、 p27Kiplの分解を抑制することができる。 さらに、 このよ うな物質は、 p27Kiplの分解を抑制することにより細胞周期の進行を抑制できるの で、 細胞周期の異常を原因とする疾患、 細胞周期を調節することにより症状を軽 減できるような疾患の治療薬として用いることができる。 細胞周期の異常を原因 とする疾患、 細胞周期を調節することにより症状を軽減できるような疾患として は、 癌、 動脈硬化、 慢性関節リウマチ、 前立腺肥大症、 経皮的経血管的冠動脈形 成術後の血管再狭窄、 肺線維症、 糸球体腎炎、 自己免疫疾患等をあげることがで き、 上記 p27Kiplのュビキチン化を阻害する物質は、 特に癌の治療に有効である。 p27Kiplのュビキチン化を阻害する物質の例として、 RINGフィンガ一ドメインが 欠失した KPC1、 例えば配列番号 2の 1〜； 1253番目のアミノ酸配列からなる蛋白質 をあげることができる。 9. KPC1をコードする mRNAの検出あるいは定量を行う方法 The substance that inhibits p27 Kipl ubiquitination selected by the screening method (1) or (2) can suppress the degradation of p27 Kipl in cells. Furthermore, such substances can suppress the progression of the cell cycle by suppressing the degradation of p27 Kipl , so that diseases caused by abnormal cell cycles and the symptoms can be reduced by regulating the cell cycle. It can be used as a therapeutic agent for various diseases. Diseases caused by abnormal cell cycles and diseases whose symptoms can be reduced by regulating the cell cycle include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, percutaneous transvascular coronary angioplasty. Subsequent vascular restenosis, pulmonary fibrosis, glomerulonephritis, autoimmune disease, etc. can be mentioned. The above-mentioned substance that inhibits ubiquitination of p27 Kipl is particularly effective for treating cancer. Examples of the substance that inhibits ubiquitination of p27 Kipl include KPC1 in which the RING finger domain has been deleted, for example, a protein consisting of the amino acid sequence at positions 1 to 1253 of SEQ ID NO: 2. 9. Method for detecting or quantifying mRNA encoding KPC1
(a)KPClをコードする DNAが有する塩基配列と相補的な配列の連続する 20塩基以 上の配列を含むポリヌクレオチド、 または (b )KPC1をコードする DNAが有する塩基 配列と相補的な配列の連続する 20〜100塩基の配列を含む DNAおよび KPC1をコード する MAが有する塩基配列の連続する 20〜100塩基の配列を含む DNAを用いて KPC1 をコードする mRNAの検出あるいは定量を行うことができる。 ポリヌクレオチドは DNAでも RNAでもよい。 .  (a) a polynucleotide containing a continuous sequence of 20 or more bases complementary to the base sequence of the DNA encoding KPC1, or (b) a polynucleotide complementary to the base sequence of the DNA encoding KPC1 KPC1-encoding mRNA can be detected or quantified using DNA containing a continuous 20-100 base sequence and DNA containing a continuous 20-100 base sequence of the nucleotide sequence of MA encoding KPC1. . The polynucleotide may be DNA or RNA. .
KPC1をコードする DNAが有する塩基配列と相補的な配列の連続する 20塩基以上 の配列を含む DNAは、 KPC1をコ一ドする二本鎖 DNAあるいはその連続する 20塩基以 上の配列を含む部分断片のアンチセンス鎖 DNAとして得られる。 2本鎖 DNAを 100 °Cで 5分間熱した後、 氷上で急速に冷却することにより、 センス鎖とアンチセン ス鎖を分離することができる。 また、 KPC1をコードする DNAあるいはその連続す る 20塩基以上の配列を含む部分断片の 3'端に T7プロモー夕一、 SP6プロモーター などのプロモー夕一配列を結合し、 RNAポリメラーゼを用いたイン ·ビトロの転 写系により、 KPC1をコ一ドする DNAが有する塩基配列と相補的な配列の連続する 20塩基以上の配列を含む RNAを得ることができる。 KPC1をコードする DNAの連続す る 20塩基以上の配列を含む部分断片は、 KPC1をコ一ドする DNAを適当な制限酵素 で切断することにより、 または KPC1をコードする DNAを錶型とし、 作製したい部 分断片の 5'端 20~40塩基の配列を 3'端に含む DNA、 3'端 20〜40塩基と相補的な配 列を 3，端に含む DNAをプライマ一とした PCI こより得ることができる。 プライマー は DNA合成機で合成できる。 KPC1をコ一ドする DNAが有する塩基配列と相補的な配 列の連続する 20〜100塩基の配列を含む DNA、 KPC1をコ一ドする DNAが有する塩基 配列の連続する 20〜； 100塩基の配列を含む DNAは MA合成機により調製することが できる。  A DNA containing a sequence of 20 or more consecutive nucleotides complementary to the nucleotide sequence of the DNA encoding KPC1 is a double-stranded DNA encoding KPC1 or a portion containing a sequence of 20 or more consecutive nucleotides thereof. Obtained as fragment antisense strand DNA. Heating the double-stranded DNA at 100 ° C for 5 minutes and then rapidly cooling it on ice can separate the sense and antisense strands. In addition, a promoter sequence such as T7 promoter and SP6 promoter is ligated to the 3 'end of DNA encoding KPC1 or a partial fragment containing a continuous sequence of 20 bases or more, and an in polymerase chain reaction using RNA polymerase is performed. By the in vitro transcription system, it is possible to obtain RNA containing a continuous sequence of 20 bases or more complementary to the base sequence of the DNA encoding KPC1. A partial fragment containing a sequence of 20 or more consecutive bases of the DNA encoding KPC1 is prepared by cleaving the DNA encoding KPC1 with an appropriate restriction enzyme, or by converting the DNA encoding KPC1 into a 錶 type. DNA that contains the sequence at the 5 'end 20 to 40 bases at the 3' end of the desired fragment, 3 at the 3 'end complementary to the sequence at the 20 to 40 bases, and DNA containing the DNA at the end as the primer be able to. Primers can be synthesized on a DNA synthesizer. DNA containing a continuous 20 to 100 base sequence with a sequence complementary to the base sequence of the DNA encoding KPC1, 20 to 100 continuous base sequences of the DNA having the DNA encoding KPC1; DNA containing the sequence can be prepared using an MA synthesizer.
KPC1をコードする mRNAの発現量を検出する方法としては、 例えば（1)ノーザン ハイブリダイゼーション、 （2)ドヅトブロッ 卜ハイブリダイゼ一ション、 （3)ln situハイプリダイゼ一ション、 (4)RT-PCR、 （5)デフアレンシャル 'ハイプリダイ ゼ一シヨン、 （6)DNAチヅプ、 （7)リボヌクレアーゼ保護アツセィ等があげられる 上記方法に供する検体としては、 生体から採取した細胞あるいは各種組織等の 生体試料、 生体試料から調製した初代培養細胞、 各種の培養細胞株、 3. (1 )に記 載した形質転換体等から取得した mRNAあるいは全 MAが用いられる。 以後、 該 mRNAおよび全 RNAを検体由来 RNAと称する。 検体由来 Aの調製は、 モレキュラー -クロ一ニング第 3版に記載の方法により行なうことができる。 （3)の situハ ィブリダイゼーシヨンでは、 検体由来 Aではなく、 組織の切片や細胞が用いら れる。 Methods for detecting the expression level of mRNA encoding KPC1 include, for example, (1) Northern hybridization, (2) Dot blot hybridization, (3) In situ hybridization, (4) RT-PCR, (5) ) Differential hybridization, (6) DNA chip, (7) ribonuclease-protected assay, etc. Samples to be subjected to the above method include cells collected from a living body or various tissues. Biological samples, primary cultured cells prepared from biological samples, various cultured cell lines, mRNAs obtained from the transformants described in 3. (1), or all MA are used. Hereinafter, the mRNA and total RNA are referred to as sample-derived RNA. Preparation of specimen-derived A can be performed by the method described in Molecular-Cloning Third Edition. In the situ hybridization of (3), tissue sections and cells are used instead of specimen-derived A.
ノ一ザンハイブリダイゼーションでは、 検体由来 RNAをゲル電気泳動で分離後 、 ナイロンフィル夕一等の支持体に転写し、 KPC1をコードする DNAが有する塩基 配列と相補的な配列の連続する 20塩基以上の配列を含むポリヌクレオチドを標識 したプローブを用いて、 ハイブリダイゼーシヨンならびに洗浄を行うことで、 KPC1をコードする m Aをバンドとして検出することができる。 ハイプリダイゼー シヨンならびに洗浄工程はストリンジェントな条件で行うことが望ましい。 標識プロ一ブは、 例えば、 ニック ' トランスレーション、 ランダム 'プライミ ングまたは 5，末端のリン酸ィ匕等の方法により放射性同位体、 ビォチン、 ジゴキシ ゲニン、 蛍光基、 化学発光基等を、 上記プローブとするポリヌクレオチドに取り 込ませることで調製できる。 標識プロ一ブの結合量は KPC1をコードする mRNAの量 を反映することから、 結合した標識プロ一ブの量を定量することで KPC1をコード する mRNAを定量することができる。 電気泳動、 メンプレンの移行、 プローブの調 製、 ハイブリダィゼ一シヨン、 mRNAの検出については、 モレキュラー 'クロ一二 ング第 3版に記載の方法により行なうことができる。  In Northern hybridization, RNA derived from a sample is separated by gel electrophoresis, transcribed onto a support such as Nylon Fil, and 20 or more consecutive nucleotides complementary to the nucleotide sequence of the DNA encoding KPC1 By performing hybridization and washing using a probe labeled with a polynucleotide containing the sequence of the above, mAA encoding KPC1 can be detected as a band. The hybridization and the washing step are desirably performed under stringent conditions. The labeled probe may be, for example, a nick 'translation, a random' priming, or a method such as 5, phosphorylation at the terminal, or the like, in which the radioactive isotope, biotin, digoxygenin, fluorescent group, Can be prepared by incorporation into a polynucleotide. Since the amount of labeled probe reflects the amount of mRNA encoding KPC1, the amount of mRNA bound to KPC1 can be quantified by quantifying the amount of bound labeled probe. Electrophoresis, transfer of membranes, preparation of probes, hybridization, and detection of mRNA can be performed by the methods described in Molecular 'Clothing Third Edition.
MAのドットプロットハイプリダイゼーシヨンは、 組織や細胞から抽出した RNA をメンブラン上に点状にスポヅトして固定し、 プローブとなる標識したポリヌク レオチドとハイブリダイゼーシヨンを行ない、 プローブと特異的にハイプリダイ ズする mRNAを検出する方法である。 プローブとしてはノーザンハイブリダイゼ一 シヨンと同様のものの用いることができる。 RNAの調製、 RNAのスポヅト、 ハイブ リダィゼ一シヨン、 mRNAの検出については、 モレキュラー 'クローニング第 3版 に記載の方法により行なうことができる。  MA dot-plot hybridization is performed by spot-fixing RNA extracted from tissues or cells onto a membrane in a dot-like manner, hybridizing with a labeled polynucleotide as a probe, and performing hybridization specifically with the probe. This is a method for detecting mRNA that hybridizes. As the probe, the same probe as that of Northern hybridization can be used. Preparation of RNA, spotting of RNA, hybridization, and detection of mRNA can be performed by the methods described in Molecular 'Cloning, Third Edition.
in situハイブリダイゼ一シヨンは、 生体から取得した組織のパラフィンまた はクリオス夕ヅト切片、 あるいは固定化した細胞を検体として用い、 標識したプ ローブとハイプリダイゼ一シヨンならびに洗浄の工程を行い、 顕微鏡観察により 、 m Aの組織や細胞内での分布や局在を調べる方法である 〔Methods in Enzymology, 254, 419 (1995)〕 。 プローブとしてはノーザンハイブリダィゼ一 シヨンと同様のものも用いることができる。 偽陽性を防ぐためには、 ハイブリダ ィゼ一シヨンならびに洗浄工程はストリンジェントな条件で行うことが望ましい In situ hybridization is performed by using a paraffin or cryo-isotope section of tissue obtained from a living body or immobilized cells as a sample, performing labeled probe, hybridization, and washing steps, and then performing microscopic observation. This is a method for examining the distribution and localization of mA in tissues and cells [Methods in Enzymology, 254, 419 (1995)]. As the probe, those similar to those in Northern hybridization can be used. Hybridization and washing steps should be performed under stringent conditions to prevent false positives
RT-PCRゃデファレンシャル ·ハイブリダィゼ一シヨンあるいは DNAチヅプでは 、 検体由来 RNAからオリゴ dTブライマ一またはランダムブラィマーおよび逆転写 酵素を用いて合成した cDNA (以後、 該 cDNAを検体由来 cDNAと称する） を測定に用 いる。検体由来 RNAが niRNAの場合は、 上記いずれのプライマ一も用いることがで きるが、 該検体由来 Aが全 RNAである場合は、 オリゴ dTプライマ一を用いること が必要である。 cDNAの合成は、 モレキュラー 'クロ一ニング第 3版に記載の方法 により行なうことができる。 In the RT-PCR differential hybridization or DNA chip, cDNA synthesized from an RNA derived from a sample using an oligo dT primer or a random primer and a reverse transcriptase (hereinafter referred to as a cDNA derived from the sample) is used. Used for measurement. When the sample-derived RNA is niRNA, any of the above primers can be used. However, when the sample-derived RNA is total RNA, it is necessary to use an oligo dT primer. cDNA can be synthesized by the method described in Molecular 'Cloning Third Edition.
RT-PCIま、 検体由来 cDNAを踌型とし、 KPC1をコードする cDNAの塩基配列から設 計した KPC1特異的なプライマーを用いて PCRを行ない、 KPC1をコードする cDNAの 断片を増幅することにより mRNAを検出する方法である。 KPC1特異的なプライマ一 としては、 KPC1をコードする cDNAのポリ A鎖を除いた適当な領域を選択し、 その 領域の塩基配列の 55端 20〜； 100塩基の配列からなる DNAおよび 3'端 20〜； 100塩基と 相補的な配列からなる DNAの組を用いることができる。 プライマーの配列は、 プ ライマ一間の結合やプライマ一内の結合を起こさず、 ァニーリング温度で標的 cDNAと特異的に結合して、 変性条件で標的 cMAからはずれる等の条件に基づき設 計するのが好ましい。 ァクチンゃグリセルアルデヒド一 3—リン酸デヒドロゲナ —ゼ （以下 G3PDHと略す） 等の細胞の種類や培養条件による発現量の変化がほと んどない蛋白質をコードする mRNAを内部コントロールとして置くことで、 KPC1を コードする mRNAの量を定量することが可能である。 mRNAの定量を行なう場合は、 増幅産物が指数関数的に増加している反応サイクルの回数内で PCRを行うことが 必要である。 この回数は、 反応サイクルの回数を段階的に増加させた PCRを行な V、、 各 PCRで増幅する DNA断片を回収してゲル電気泳動で定量することで知ること ができる。 PCRは、 モレキュラー ·クローニング第 3版に記載の方法により行な うことができる。 RT-PCI or cDNA derived from the sample was converted to type II, and PCR was performed using KPC1-specific primers designed from the nucleotide sequence of the cDNA encoding KPC1, to amplify a fragment of the cDNA encoding KPC1 to obtain mRNA. Is a method for detecting KPC1 The specific primers one, select the appropriate region excluding the poly A chain of cDNA encoding KPC1, its 5 5 ends 20 of the nucleotide sequence of regions; 100 DNA and 3 comprising the nucleotide sequence ' A set of DNAs having a sequence complementary to the terminal 20 to 100 bases can be used. The primer sequence should be designed based on conditions such as no binding between primers or within the primer, specific binding to the target cDNA at the annealing temperature, and removal from the target cMA under denaturing conditions. Is preferred. By using, as an internal control, mRNA encoding a protein such as actin-glyceraldehyde-13-phosphate dehydrogenase (hereinafter abbreviated as G3PDH) that rarely changes in expression level depending on cell types or culture conditions. However, it is possible to quantify the amount of mRNA encoding KPC1. When quantifying mRNA, it is necessary to perform PCR within the number of reaction cycles in which the amplification product increases exponentially. This number can be determined by performing PCR in which the number of reaction cycles is increased stepwise, collecting DNA fragments to be amplified in each PCR, and quantifying by gel electrophoresis. PCR can be performed by the method described in Molecular Cloning, Third Edition.
検体由来 cDNAをプロ一プとして、 KPC1をコードする DNAが有する塩基配列と相 補的な配列の連続する 20塩基以上の配列を含むポリヌクレオチドを固定化させた フィルターあるいはスライ ドガラスやシリコンなどの基盤に対してハイブリダィ ゼ一シヨンならびに洗浄を行うことで、 KPC1をコードする m Aの量の変動を検出 することができる。 このような原理に基づく方法には、 ディファレンシャルハイ プリダイゼ一シヨン 〔Trends Genet. , 7, 314 (1991)〕 や DNAチップ 〔Genome Res. , 6, 639 ( 1996)〕 がある。 いずれの方法もフィルターあるいは基盤上にァ クチンや G3PDHなどの内部コントロールを固定化することで、 対照検体と標的検 体の間での KPC1をコードする mRNAの量の違いを正確に検出することができる。 ま た対照検体と標的検体由来の RNAをもとにそれぞれ異なる標識の dNTP (dATP、 dGTP 、dCTP、dTTPの混合物） を用いて標識 cDNA合成を行い、 1枚のフィル夕一あるいは 1枚の基盤に 2つの標識 cDNAプローブを同時にハイブリダィズさせることで正確 な KPC1をコードする mRNAの定量を行うことができる。 Using the cDNA derived from the sample as a probe, it matches the nucleotide sequence of the DNA encoding KPC1. MAb encoding KPC1 by performing hybridization and washing on a filter or a base such as slide glass or silicon on which a polynucleotide containing a complementary sequence of consecutive 20 or more bases has been immobilized. Can be detected. Methods based on such a principle include a differential high prescription (Trends Genet., 7, 314 (1991)) and a DNA chip (Genome Res., 6, 639 (1996)). Both methods can immobilize internal controls such as actin and G3PDH on a filter or a substrate to accurately detect the difference in the amount of mRNA encoding KPC1 between the control sample and the target sample. it can. In addition, labeled cDNA was synthesized using different labeled dNTPs (a mixture of dATP, dGTP, dCTP, and dTTP) based on RNA from the control sample and the target sample. By simultaneously hybridizing two labeled cDNA probes, accurate quantification of mRNA encoding KPC1 can be performed.
リボヌクレァ一ゼ保護ァヅセィでは、 まず KPC1をコードする DNAの 3，端に T7プ 口モー夕—、 SP6プロモ一夕一などのプロモーター配列を結合し、 標識した NTP ( ATP、GTP、CTP、UTPの混合物） および RNAポリメラーゼを用いたィン ·ビトロの転写 系により、 標識したアンチセンス RNAを合成する。 該標識アンチセンス RNAを、 検 体由来 Aと結合させて、 A-RNAノヽィブリッドを形成させた後、 1本鎖 RNAのみ を分解するリボヌクレア一ゼ Αで消化する。 該消化物をゲル電気泳動し、 UNA- RNA ハイブリツドを形成することにより消化から保護された RNA断片を、 KPC1をコー ドする mRNAとして、 検出あるいは定量する。  In the ribonuclease protection assay, first, a promoter sequence such as T7 promoter, SP6 promoter, etc. is linked to the 3 and 3 ends of the DNA encoding KPC1, and labeled NTP (ATP, GTP, CTP, UTP). Mixture) and an in vitro transcription system using RNA polymerase to synthesize labeled antisense RNA. The labeled antisense RNA is combined with sample-derived A to form an A-RNA hybrid, and then digested with ribonuclease that degrades only single-stranded RNA. The digest is subjected to gel electrophoresis, and an RNA fragment protected from digestion by forming a UNA-RNA hybrid is detected or quantified as mRNA encoding KPC1.
上記の方法を用いて、 KPC1をコ一ドする mRNAの量の変動を検出し KPC1をコード する mRNAの量が健常人と比較して減少あるいは増加している疾患の判定または診 断を行うことができる。  Using the methods described above, detect fluctuations in the amount of mRNA encoding KPC1 and judge or diagnose a disease in which the amount of mRNA encoding KPC1 is decreased or increased compared to a healthy individual. Can be.
疾患の判定または診断に供する検体としては患者より取得した組織、 血液等の 生体試料あるいは該生体試料から細胞を取得して試験管内の適当な培地中で培養 した初代培養細胞試料から取得した mRNAあるいは全 RNAが用いられる。 また、 生 体試料から取得した組織の切片も用いることもできる。  Samples to be used for the determination or diagnosis of disease include biological samples such as tissues and blood obtained from patients or mRNA obtained from primary culture cell samples obtained from cells obtained from the biological samples and cultured in an appropriate medium in a test tube. Total RNA is used. In addition, a tissue section obtained from a biological sample can also be used.
KPC1の発現量が mRNAレベルで減少あるいは増加している疾患は以下のようにし て判定または診断できる。 まず、 複数の患者および健常者の検体について KPC1を コ一ドする] nRNAの量を上記にあげた検出方法で測定して比較し、 患者および健常 者の該 mRMの量の範囲を決定する。 被験者の検体の該 mMAの量を、 健常者の量の 範囲および患者の量の範囲とそれそれ比較し、 どちらの発現レベルの範囲に入る かを調べることにより判定または診断を行う。 Diseases in which the expression level of KPC1 decreases or increases at the mRNA level can be determined or diagnosed as follows. First, code for KPC1 in samples from multiple patients and healthy subjects.] Measure the amount of nRNA using the above-mentioned detection methods and compare the results. Determine the range of amounts of the mRM for the individual. The determination or diagnosis is performed by comparing the amount of the mMA in the subject's sample with the range of the amount of the healthy subject and the range of the amount of the patient, and examining which expression level falls into the range.
KPC1の発現量が mRNAレベルで増加している疾患としては、 細胞周期の異常を原 因とする疾患をあげることができる。 細胞周期の異常を原因とする疾患としては 、 癌、 動脈硬化、 慢性関節リウマチ、 前立腺肥大症、 肺線維症、 糸球体腎炎、 自 己免疫疾患等をあげることができ、 上記の方法は、 特に癌の判定または診断に有 効である。  Diseases in which the expression level of KPC1 is increased at the mRNA level include diseases caused by abnormal cell cycles. Diseases caused by cell cycle abnormalities include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, pulmonary fibrosis, glomerulonephritis, autoimmune diseases, and the like. Effective for determining or diagnosing cancer.
また、 KPC1をコードする mRNAを定量する方法は、 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。  Further, the method of quantifying mRNA encoding KPC1 can be used for predicting 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 mRNA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. The drug can be evaluated by comparing the expression level of the mRNA depending on the presence or absence of the drug.
10. 疾患と関連する KPC1遺伝子の変異および多型を検出する方法 10. Methods for detecting KPC1 gene mutations and polymorphisms associated with disease
KPG1をコードする遺伝子座中に存在する疾患の原因となる変異の存在の有無を 評 するための最も明確な試験は、 対照集団からの遺伝子と疾患患者からの遺伝 子とを直接比較することである。  The clearest test to assess for the presence of a disease-causing mutation in the KPG1-encoding locus is to directly compare genes from a control population with genes from diseased patients. is there.
具体的には疾患患者ならび健常者から、 生体試料あるいは該生体試料から樹立 した初代培養細胞由来の試料を集め、 該生体試料ならびに該初代培養細胞由来試 料中から MAまたは RNAを抽出する。 該 DNAあるいは、 該 DNAまたは該 RNAより合成 した cDNAを錄型にして KPC1造伝子が有する塩基配列に基づき設計したプライマ一 を用いて PCRにより増幅した KPC1をコードする DNA (これらの MAを以下、 検体由 来 DNAと称する） を試料 DNAとして用いることができる。  Specifically, a biological sample or a sample derived from primary cultured cells established from the biological sample is collected from a disease patient and a healthy person, and MA or RNA is extracted from the biological sample and the sample derived from the primary cultured cell. DNA encoding KPC1 that has been amplified by PCR using the DNA or a cDNA synthesized from the DNA or the RNA into a type I primer and designed based on the nucleotide sequence of the KPC1 gene (these MAs are referred to as , Which is referred to as sample-derived DNA) can be used as sample DNA.
KPC1遺伝子に疾患の原因となる変異があるかどうかを検出する方法として、 野 生型対立遺伝子を有する DNA鎖と変異対立遺伝子を有する DNA鎖とのハイプリダイ ズにより形成されるへテロ二本鎖を検出する方法を用いることができる。  As a method for detecting whether there is a disease-causing mutation in the KPC1 gene, a heteroduplex formed by hybridizing a DNA strand having a wild-type allele and a DNA strand having a mutant allele is used. A detection method can be used.
ヘテロ二本鎖を検出する方法には、 （1 )ポリアクリルアミドゲル電気泳動によ るへテロ二本鎖検出法 〔Trends Genet. , 7, 5 (1991 )〕、 （2)—本鎖コンフオメ ーション多型解析法 ( SSCP解析； single strand conformation polymorphism analysis) 〔 Genomics, 16, 325 ( 1993)〕 、 （3)ミスマッチの化学的切断法 (CCM 法、 chemical cleavage of mismatches ) Human Molecular Genetics, BIOS Scientific Publishers Limited (1996)〕 、 （4)ミスマッチの酵素的切断法 〔 Nat. Genet. , 9, 103 (1995)〕 、 （5)変性ゲル電気泳動法 (denaturing gradient gel electrophoresis DGGE法） 〔Mutat. Res. , 288, 103 (1993)〕 等の方法が 挙げられる。 Methods for detecting heteroduplexes include (1) heteroduplex detection method by polyacrylamide gel electrophoresis [Trends Genet., 7, 5 (1991)], and (2) —conformation conformation. Single strand conformation polymorphism analysis (SSCP analysis) [Genomics, 16, 325 (1993)], (3) Chemical cleavage of mismatches (CCM Method, chemical cleavage of mismatches) Human Molecular Genetics, BIOS Scientific Publishers Limited (1996)], (4) Enzymatic cleavage method of mismatch [Nat. Genet., 9, 103 (1995)], (5) Denaturing gel electrophoresis Method (denaturing gradient gel electrophoresis DGGE method) [Mutat. Res., 288, 103 (1993)].
ポリァクリルアミ ドゲル電気泳動によるへテロ二本鎖検出法は、 検体由来 DNA を鎵型とし'、 KPC1遺伝子の塩基配列に基づき設計したブラィマーを用いた PCIUこ より、 200bpよりも小さい断片として該遺伝子断片を増幅し、 ポリアクリルアミ ドゲル電気泳動を行う。 KPC1遺伝子の変異によりへテロ二本鎖が形成された場合 は、 変異を持たないホモ二本鎖よりも移動度が遅く、 それらは余分なバンドとし て検出することができる。 Hydrolink、 MDE等の特製のゲルを用いた方が分離度は よい。 200bpよりも小さい断片の検索ならば、 揷入、 欠失、 ほとんどの 1塩基置 換を検出可能である。 ヘテロ二本鎖解析は、 次に述べる一本鎖コンフオメーショ ン多型解析と組み合わせた一枚のゲルで行うことが望ましい。  Heteroduplex detection by polyacrylamide gel electrophoresis is based on the use of a DNA derived from a sample as type II, and a PCIU using a primer designed based on the nucleotide sequence of the KPC1 gene. Amplify and perform polyacrylamide gel electrophoresis. When a heteroduplex is formed due to a mutation in the KPC1 gene, the mobility is lower than that of a homoduplex having no mutation, and these can be detected as extra bands. Separation is better with special gels such as Hydrolink and MDE. Searching for fragments smaller than 200 bp can detect insertions, deletions, and most single base substitutions. Heteroduplex analysis is preferably performed on a single gel combined with the single-stranded conformation polymorphism analysis described below.
SSCP解析では、 検体由来 DNAを錄型とし、 KPC1遺伝子の塩基配列に基づき設計 したプライマ一を用いた PCRにより、 200bpよりも小さい断片として該遺伝子断片 を増幅し、 変性後、 未変性ポリアクリルアミドゲル中で泳動する。 PCRを行う際 にブラィマーを放射性同位体あるいは蛍光色素で標識するか、 または未標識の増 幅産物を銀染色することにより、 KPC1遺伝子断片をバンドとして検出することが できる。 野生型のパターンとの相違を明らかにするために、 コントロールの検体 も同時に泳動すると、 変異を持った断片を移動度の違いから検出できる。  In the SSCP analysis, the DNA derived from the sample was type- 、, and the gene fragment was amplified as a fragment smaller than 200 bp by PCR using a primer designed based on the base sequence of the KPC1 gene. Electrophoresis in When PCR is performed, the KPC1 gene fragment can be detected as a band by labeling the primer with a radioisotope or a fluorescent dye, or by staining an unlabeled amplified product with silver. If a control sample is also run to clarify the difference from the wild-type pattern, the mutated fragment can be detected from the difference in mobility.
CCM法では、 検体由来 DNAを鎢型とし、 KPC1遺伝子の塩基配列に基づき設計した プライマーを用いた PCIUこより増幅した該遺伝子断片を、 KPC1をコードする DNAに 放射性同位体あるいは蛍光色素をとり込ませた標識 DNAとハイブリダイズさせ、 四酸化ォスミゥムで処理することでミスマッチしている場所の DNAの一方の鎖を 切断させ変異を検出することができる。 CCM法は最も感度の高い検出法の 1つであ り、 キロベースの長さの検体にも適応できる。  In the CCM method, the DNA derived from the specimen is type III, and the gene fragment amplified from the PCIU using primers designed based on the nucleotide sequence of the KPC1 gene is incorporated into the DNA encoding KPC1 with a radioisotope or fluorescent dye. By hybridizing with the labeled DNA and treating with osmium tetroxide, one strand of the DNA at the mismatched site can be cleaved to detect the mutation. The CCM method is one of the most sensitive detection methods and can be applied to kilobase-length samples.
ミスマツチの酵素的切断法は、 上記四酸化ォスミゥムの代わりに T4ェンドヌク レアーゼ VIIのような細胞内でミスマヅチの修復に関与する酵素とリボヌクレア —ゼ Aと組み合わせることで、 酵素的にミスマッチを切断する方法である。 DGGE法では、 検体由来 DNAを鎵型とし、 KPC1遺伝子の塩基配列に基づき設計し たプライマーで増幅した該遺伝子断片を化学的変性剤の濃度勾配や温度勾配を有 するゲルを用いて電気泳動する。 該遺伝子断片はゲル内を一本鎖に変性する位置 まで移動し、 変性後は移動しなくなる。 該遺伝子断片に変異がある場合とない場 合ではゲル内での移動度が異なることから、 変異の存在を検出することが可能で ある。 検出感度を上げるにはそれそれのプライマーにポリ （G:C) 端末を付ける とよい。 The enzymatic cleavage method of Misumi is a method of enzymatically cleaving mismatches by combining ribonuclease A with an enzyme such as T4 endonuclease VII, which is involved in the repair of mischid in cells, instead of osmium tetroxide. It is. In the DGGE method, sample-derived DNA is converted into type II, and the gene fragment amplified with primers designed based on the base sequence of the KPC1 gene is electrophoresed on a gel with a concentration gradient or temperature gradient of a chemical denaturant. . The gene fragment moves in the gel to a position where it is denatured into a single strand, and does not move after denaturation. Since the mobility in the gel differs depending on whether the gene fragment has a mutation or not, the presence of the mutation can be detected. To increase the detection sensitivity, a poly (G: C) terminal should be attached to each primer.
疾患の原因遺伝子を検出する別の方法として、 蛋白質短縮試験 （protein truncation test： PTT法) (Genomics, 20, 1 (1994)〕 がある。 該試験により蛋 白質の欠損を生み出すフレームシフト突然変異、 スプライス部位突然変異、 ナン センス突然変異を特異的に検出することができる。 PTT法では、 KPC1 cDNAの KPC1 をコードする領域の 5'末端から 20〜40塩基の配列に T7プロモー夕一配列と真核生 物翻訳開始配列をつないだ特殊なプライマーを設計し、 該プライマーを用いて検 体由来 RNAより RT- PCR法で cDNAを作製する。 該 cDNAを用い、 イン · ビトロ転写、 翻訳を行うと cDNAがコ一ドする蛋白質が生産される。 該蛋白質をゲルに泳動して 、 該蛋白質の泳動位置が完全長蛋白質に相当する位置にあれば欠損を生み出す変 異は存在せず、 該蛋白質に欠損がある場合は、 完全長蛋白質より短い位置に該蛋 白質は泳動され、 該位置より欠損の程度を知ることができる。  Another method for detecting the disease-causing gene is a protein truncation test (PTT method) (Genomics, 20, 1 (1994)). The PTT method can specifically detect splice site mutations and nonsense mutations.The PPC method uses the T7 promoter sequence as a sequence 20 to 40 bases from the 5 'end of the KPC1 coding region of the KPC1 cDNA. Design a special primer to which the nuclear translation initiation sequence is linked, and use the primer to produce cDNA from the RNA derived from the sample by RT-PCR.If the cDNA is used for in vitro transcription and translation, A protein encoded by cDNA is produced, and when the protein is electrophoresed on a gel and the electrophoresis position of the protein is at a position corresponding to the full-length protein, there is no mutation that produces a defect. Where there is a defect In this case, the protein is electrophoresed at a position shorter than the full-length protein, and the degree of deletion can be determined from the position.
検体由来 DNAならびに検体由来 cDNAの塩基配列を決定するために本発明の DNAが 有する塩基配列に基づいて設計したプライマーを用いることが可能である。 決定 された塩基配列を解析することにより、 検体由来 DNAあるいは検体由来 cDNAにの 原因となる変異があるか否かを判別できる。  In order to determine the nucleotide sequence of the sample-derived DNA and the sample-derived cDNA, it is possible to use a primer designed based on the nucleotide sequence of the DNA of the present invention. By analyzing the determined base sequence, it can be determined whether or not there is a causative mutation in the sample-derived DNA or the sample-derived cDNA.
KPC1遺伝子のコード領域以外の変異は、 該遺伝子の付近またはその中のィント ロンおよび調節配列のような、 非コード領域を検査することによって検出し得る 。 非コード領域中の変異に起因する疾患は、 上記に記載した方法に従い対照検体 と比較した場合の、 疾患患者における異常なサイズの、 または異常な生産量の mRNAを検出することで確認することができる。  Mutations other than the coding region of the KPC1 gene can be detected by examining non-coding regions, such as introns and regulatory sequences near or in the gene. Diseases caused by mutations in noncoding regions can be confirmed by detecting abnormally sized or abnormally produced mRNA in diseased patients when compared to control samples according to the method described above. it can.
このようにして非コード領域における変異の存在が示唆された該遺伝子につい ては、 2. (2)に記載の方法により、 KPC1をコ一ドする DNAをハイプリダイゼ一シ ョンのプローブとして用いてクローン化することができる。 非コード領域におけ る変異は上述のいずれかの方法に準じて探索することができる。 For the gene for which the presence of a mutation in the non-coding region was suggested in this manner, DNA encoding KPC1 was used as a probe for hybridization by the method described in 2. (2). Can be cloned. In non-coding areas Mutations can be searched for according to any of the methods described above.
見い出された変異は、 Handbook of Huian Genetics Linkage. The John Hopkins University Press, Baltimore (1994) に記載された方法に従い統計処 理を行うことで、 疾患との連鎖がある SNPs (シングル ·ヌクレオチド ·ポリモル フイズム） として同定することができる。 また、 疾患の病歴を持つ家族から、 先 に示した方法に従い DNAを取得し、 変異および多型を検出することで、 疾患の原 因遺伝子を同定することができる。  Statistical processing according to the method described in the Handbook of Huian Genetics Linkage. The John Hopkins University Press, Baltimore (1994) was carried out to find SNPs (single nucleotide polymorphism) linked to a disease. ). In addition, by acquiring DNA from a family member who has a history of the disease according to the method described above and detecting mutations and polymorphisms, the causative gene of the disease can be identified.
11. KPC1遺伝子に変異を有する疾患を判定または診断する方法  11. A method for determining or diagnosing a disease having a mutation in the KPC1 gene
KPC1遺伝子に変異を有する疾患を判定または診断する方法に用いられるオリゴ ヌクレオチドとしては、 KPC1をコ一ドする DNAが有する塩基配列の連続する 20〜 100塩基の配列を含むオリゴヌクレオチドおよび KPC1をコードする DNAが有する塩 基配列と相補的な配列の連続する 20〜： 100塩基の配列を含むォリゴヌクレオチド をあげることができる。 オリゴヌクレオチドは、 オリゴ DNAが好ましい。 オリゴ ヌクレオチドは、 DNA合成機により、 合成することができる。  Oligonucleotides used in the method for determining or diagnosing a disease having a mutation in the KPC1 gene include oligonucleotides containing a continuous 20 to 100 nucleotide sequence of the nucleotide sequence of DNA encoding KPC1, and KPC1 Oligonucleotides containing a sequence of 20 to 100 bases consecutive to a sequence complementary to the base sequence of DNA can be given. Oligonucleotides are preferably oligo DNAs. Oligonucleotides can be synthesized by a DNA synthesizer.
KPC1遺伝子に変異を有する疾患は、 ヒトのいずれかの組織における遺伝子の変 異を検出することによって判定または診断し得る。例えば、 生殖細胞系に変異が ある場合、 当該変異を遺伝した個人は、 疾患を発症し易い傾向である可能性があ る。 当該変異は、 該個人の体のいずれかの組織からの DNAを試験することによつ て検出し得る。 例えば、 採血しその血液の細胞から DMを抽出し、 この MAを用い 、 遺伝子の変異を試験することにより、 疾患を判定または診断することができる 。 また、 胎児細胞、 胎盤細胞または羊膜細胞を用い、 遺伝子の変異を試験するこ とにより、 出生前診断を行うことができる。  Diseases having a mutation in the KPC1 gene can be determined or diagnosed by detecting a mutation in the gene in any of human tissues. For example, if a germline mutation is present, individuals who inherit the mutation may be more likely to develop the disease. The mutation can be detected by testing DNA from any tissue of the individual's body. For example, a disease can be determined or diagnosed by collecting blood, extracting DM from cells of the blood, and testing gene mutation using this MA. In addition, prenatal diagnosis can be performed by using fetal cells, placental cells, or amniotic cells to test gene mutations.
また疾患を発症した患者から、 病巣部位の生体組織を取得して DMを試験する ことにより、 疾患の種類を判定または診断し、 投与する薬物の選択などに利用す ることができる。 組織中の遺伝子の変異を検出するためには、 周囲の正常組織か ら遊離した病巣部位の組織を単離することが有用である。 取得した組織をトリプ シンなどで処理し、 得られた細胞を適当な培地で培養する。 培養した細胞からは 染色体 DNAならびに RNAを抽出することができる。 RNAから cDNAを合成できる。 以後、 判定または診断を目的としてヒト検体から上記いずれかの方法で取得し た DNAまたは cDNAを診断検体由来 DNAと称する。 診断検体由来 DNAを用い、 KPC1をコードする DNAが有する塩基配列の連続する 20 ~100塩基の配列を含むォリゴヌクレオチドおよび KPC1をコ一ドする DNAが有する 塩基配列と相補的な配列の連続する 20〜100塩基の配列を含むオリゴヌクレオチ ドのうちの少なくとも 1つを利用した疾患の判定または診断を行うには、 （1)制 限酵素部位の検出、 （2)対立遺伝子特異的なォリゴヌクレオチドプローブを利用 する方法 (AS0： allele specific oligonucleotide hybridization )、 (3)対立 遺伝子特異的なオリゴヌクレオチドを'用いた PCR (ARMS ： amplification refractory mutation system )、 (4)オリゴヌクレオチド ·ライゲ一ション ·ァ ッセィ (OLA)、 (5)PCR-PHFA (PCR -preferential homoduplex iormation assay )、 (6) オリゴ DNAアレイを用いる方法 〔蛋白質核酸酵素、 i^, 2004 (1998)〕 等の方法を用いることができる。 In addition, by obtaining biological tissue at the site of a lesion from a patient who has developed the disease and testing DM, the type of the disease can be determined or diagnosed and used for selecting a drug to be administered. In order to detect a mutation in a gene in a tissue, it is useful to isolate a tissue at a focus site released from surrounding normal tissues. The obtained tissue is treated with trypsin or the like, and the obtained cells are cultured in an appropriate medium. Chromosomal DNA and RNA can be extracted from the cultured cells. CDNA can be synthesized from RNA. Hereinafter, DNA or cDNA obtained from a human specimen by any of the above methods for the purpose of determination or diagnosis is referred to as diagnostic specimen-derived DNA. Using a DNA derived from a diagnostic sample, a continuous nucleotide sequence containing 20 to 100 nucleotides in the base sequence of the DNA encoding KPC1 and a sequence complementary to the nucleotide sequence in the DNA encoding KPC1 are used. To determine or diagnose a disease using at least one of the oligonucleotides containing a 20 to 100 base sequence, (1) detection of a restriction enzyme site, (2) allele-specific oligonucleotide Method using nucleotide probe (AS0: allele specific oligonucleotide hybridization), (3) PCR using allele-specific oligonucleotide (ARMS: amplification refractory mutation system), (4) Oligonucleotide ligation (5) PCR-PHFA (PCR-preferential homoduplex iormation assay), (6) Method using oligo DNA array [Protein nucleic acid enzyme, i ^, 2004 (1998)] Can be.
単一塩基変化により制限酵素部位が消失あるいは発生する場合は、 診断検体由 来 DNAを、 KPC1 cDNAが有する配列に基づき設計したプライマーで増幅し、 該制限 酵素で消化し、 得られた制限酵素切断 DNA断片を正常人の場合と比較することで 簡便に変異を検出することができる。 しかし単一塩基変化が起こることはまれで あるので、 判定または診断目的には、 KPC1 cDNAが有する配列情報ならびに別途 同定された変異の情報を組合せることでォリゴ DNAプロ一ブを設計し、 該ォリゴ DNAプローブをフィル夕一に結合させハイブリダィズを行うリバ一スドヅトブロ ット法で変異を検出する。  If the restriction site disappears or occurs due to a single base change, the DNA derived from the diagnostic sample is amplified with primers designed based on the sequence of KPC1 cDNA, digested with the restriction enzyme, and the resulting restriction enzyme digested. Mutation can be easily detected by comparing the DNA fragment with that of a normal person. However, since single base changes rarely occur, for the purpose of determination or diagnosis, an Oligo DNA probe is designed by combining the sequence information of KPC1 cDNA and the information of a mutation identified separately. Mutations are detected by reverse dot blot method, in which an Oligo DNA probe is bound to the primer and hybridized.
30塩基以下の短いオリゴ DNAプロ一ブは、 完全に対合する配列とだけハイプリ ダイズするので、 この特徴を利用して、 対立遺伝子特異的なオリゴ DNAプローブ を用いて、 1塩基の変異を容易に検出することができる。 判定または診断目的に は、 ヒト KPC1 cDNAが有する配列と同定された変異に基づき設計したオリゴ DNAを フィル夕一に結合させ、 診断検体由来 DNAから KPC1 cDNAが有する配列を用いて設 計したプライマーと標識した dNTPを用いた PCRで作製したプロ一ブを用いてハイ ブリダィズを行うリバースドットブロットが用いられることが好ましい。 スライ ドガラスやシリコンなどの基盤に直接、 KPC1 cDNAが有する配列と該変異に基づ き設計したオリゴ DNAを合成して、 高密度のアレイを作ることからなる DNAチヅプ 法は、 少量の診断検体由来 DNAあるいは診断検体由来 cDNAについて多様な変異を より簡便に検出できるため大規模な診断目的に適した変異検出法である。 塩基変異は、 以下のオリゴヌクレオチド 'ライゲ一シヨン 'アツセィ （0LA) によっても検出できる。 Short oligo DNA probes of 30 bases or less hybridize only to perfectly matched sequences, and this feature is used to facilitate the mutation of one base using an allele-specific oligo DNA probe. Can be detected. For the purpose of judgment or diagnosis, an oligo DNA designed based on the mutation identified as the sequence contained in human KPC1 cDNA was bound to the primer, and a primer designed using the sequence contained in KPC1 cDNA from the DNA from the diagnostic sample was used. It is preferable to use a reverse dot blot in which hybridization is performed using a probe prepared by PCR using labeled dNTP. The DNA chip method, which consists of directly synthesizing the sequence of KPC1 cDNA and the oligo DNA designed based on the mutation on a substrate such as slide glass or silicon to create a high-density array, is based on a small amount of diagnostic sample. It is a mutation detection method suitable for large-scale diagnostic purposes because various mutations can be detected more easily in DNA or cDNA derived from diagnostic samples. Base mutations can also be detected with the following oligonucleotide 'ligation' Atsushi (0LA).
KPC1をコ一ドする遺伝子が有する配列より、 変異部位を挟んで該変異部位を 3' 末端にもつ配列からなるオリゴ DNAとその変異部位の 3'側に隣接する配列からな るオリゴ DNAを 2本作製する。 該診断検体由来 DNAと上記ォリゴ DNAとをハイプリ ダイズさせる。 ハイプリダイズ後に、 DNAリガ一ゼで 2本のオリゴ DNAを連結させ る。 該診断検体由来 DNAの変異部位に相当する配列がオリゴ DNAの配列と一致して いれば、 2本のオリゴ DNAは連結するが、 異なっていれば、 連結されない。 例え ば、 一方のオリゴ DNAにはピオチンを、 他方のオリゴ DNAにジゴキシゲニンのよう な異なる標識をつけると、 連結反応が起こったかどうかを速やかに検出すること が可能である。 0LAは電気泳動や遠心分離操作が不要なために、 多くのサンプル を効率的に短期間で判定または診断するのに適した変異検出法である。  From the sequence possessed by the gene encoding KPC1, the oligo DNA consisting of the sequence having the mutation site at the 3 'end with the mutation site in between and the oligo DNA consisting of the sequence adjacent to the 3' side of the mutation site is 2 This is made. The diagnostic sample-derived DNA and the oligo DNA are hybridized. After hybridization, ligate the two oligos with DNA ligase. If the sequence corresponding to the mutation site in the DNA derived from the diagnostic sample matches the sequence of the oligo DNA, the two oligo DNAs are linked, but if they are different, they are not linked. For example, if one oligo DNA is labeled with biotin and the other oligo DNA is labeled with a different label, such as digoxigenin, it is possible to quickly detect whether a ligation reaction has occurred. OLA is a mutation detection method that is suitable for efficiently judging or diagnosing many samples in a short period of time because electrophoresis and centrifugation are not required.
また、 以下の PCR- PHFA法 〔Br. J. Haematol . , 95, 198 (1996)〕 により微量な 変異遺伝子を定量的かつ容易に検出することができる。  In addition, a small amount of a mutant gene can be quantitatively and easily detected by the following PCR-PHFA method [Br. J. Haematol., 95, 198 (1996)].
PCR- PHFA法は、 PCR、 非常に高い特異性を示す液相でのハイブリダィゼ一ショ ン、 ELISAと同様の操作で PCR産物を検出する ED-PCR (enzymatic detection of PCR product) の 3つを組み合わせたものである。 ジニトロフエニル （DNP) 標識 およびビォチン標識したプライマ一セヅトを用いて、 KPC1をコードする DNAをテ ンプレートに PCR増幅を行い、 両末端標識増幅物を調製する。 プライマーセット としては、 KPC1をコードする DNAが有する塩基配列中の変異部位よりも 5'側に存 在する連続する 20〜100塩基の配列を 5'端に含むオリゴ MAおよび変異部位よりも 3'側に存在する連続する 20〜100塩基の配列と相補的な配列を 5'端に含むオリゴ DNAを用いることができる。 これに対して、 標識を持たない同じ配列を有するプ ライマーセヅ卜と診断検体由来 DNAあるいは診断検体由来 cDNAをテンプレートに 増幅して得た非標識増幅物を 20~100倍の大過剰量混合する。 そして熱変性後、 1 °C/ 5分〜 10分程度の緩やかな温度勾配で冷却し、 完全な相補鎖を優先的に形 成させる。 こうして再形成された標識 DNAはピオチンを介してストレブトァビジ ン固定化ゥエルに捕獲吸着し、 DNPを介して酵素標識抗 DNP抗体を結合させて酵素 による発色反応により検出する。 検体中に標識 DNAと同じ配列の遺伝子が存在し ない場合は、 元の二本鎖の標識 DNAが優先的に再形成されて発色を示す。 これに 対し、 同じ配列の遺伝子が存在する場合は、 相補鎖の置換がランダムに生じるた め再形成される標識 DNAは減少するので、 発色は著しく低下する。 これにより、 既知の変異 ·多型遺伝子の検出および定量が可能となる。 The PCR-PHFA method combines PCR, hybridization in a liquid phase showing extremely high specificity, and ED-PCR (enzymatic detection of PCR product), which detects PCR products in the same manner as ELISA. It is a thing. Using a primer set labeled with dinitrophenyl (DNP) and biotin, the DNA encoding KPC1 is subjected to PCR amplification on a template to prepare an amplified product with both ends labeled. As a primer set, an oligo MA having a sequence of 20 to 100 bases at the 5 'side of the mutation site in the nucleotide sequence of the DNA encoding KPC1 at the 5' end and 3 'more than the mutation site at the mutation site An oligo DNA containing a sequence complementary to a continuous 20 to 100 base sequence existing on the 5 'side at the 5' end can be used. In contrast, a primer set having the same sequence without a label and a non-labeled amplified product obtained by amplifying a DNA derived from a diagnostic specimen or a cDNA derived from a diagnostic specimen into a template are mixed in a large excess of 20 to 100 times. After heat denaturation, the mixture is cooled with a gentle temperature gradient of 1 ° C for 5 to 10 minutes to form a complete complementary strand preferentially. The labeled DNA thus re-formed is captured and adsorbed on streptavidin-immobilized gel via biotin, and bound with an enzyme-labeled anti-DNP antibody via DNP, and detected by a color reaction with an enzyme. If the sample does not contain a gene with the same sequence as the labeled DNA, the original double-stranded labeled DNA is preferentially reformed and shows color. to this On the other hand, when genes having the same sequence are present, the color development is remarkably reduced because the replacement of the complementary strand occurs randomly and the amount of the labeled DNA to be regenerated is reduced. This enables the detection and quantification of known mutation and polymorphism genes.
KPC1遺伝子に変異を有する疾患としては、 細胞周期の異常を原因とする疾患を あげることができる。 細胞周期の異常を原因とする疾患としては、 癌、 動脈硬化 、 慢性関節リウマチ、 前立腺肥大症、 肺線維症、 糸球体腎炎、 自己免疫疾患等を あげることができ、 本発明の判定または診断方法は特に癌の判定または診断に有 効である。  The disease having a mutation in the KPC1 gene includes a disease caused by an abnormality in the cell cycle. Diseases caused by cell cycle abnormalities include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, pulmonary fibrosis, glomerulonephritis, autoimmune diseases, and the like. Is particularly effective for determining or diagnosing cancer.
12. KPC1の発現を抑制する方法  12. Methods to suppress KPC1 expression
(l)RNA干渉により KPC1の発現を抑制する方法  (l) Method of suppressing KPC1 expression by RNA interference
KPC1をコ一ドする DNAの塩基配列中の連続する 19~25塩基に相当する配列を含 む二本鎖 RNA 〔以下 siENA (short interfering UNA) とよぶ〕 または該配列およ び該配列と相補的な配列を含みヘアビン構造を形成する RNA 〔以下、 shRNA ( short hairpin RNA ) とよぶ〕 を用いて、 RNA干渉 （以下、 RNAiとよぶ） 〔 Nature, 411, 94 (2001 )〕 により、 KPC1の発現を抑制することができる。 KPC1 の発現の抑制に用いる siRNA、 shRNA (以下それそれ、 KPC1 siRNA、 KPC1 shRNAと もよぶ） は、 以下のようにして作製することができる。 KPC1をコードする cDNAの 塩基配列中から AAで始まる 21〜27塩基の配列 〔AA(N)19-25、 Nは任意の塩基〕 、 好 ましくは AA(N)19TTからなる配列を選択する。 選択する配列は、 翻訳領域中の開 始コドンより 50塩基以上下流の領域中にあり、 GC含量が 30〜70%、 好ましくは 50 %前後の配列であって、 他の遺伝子の塩基配列と一致せず、 KPC1 cDNAに特異的 な配列がさらに好ましい。 選択した配列の末端の AAを除いた 19〜25塩基の配列ま たは AAおよび TTを除いた 19塩基の配列 （ただし配列中の Tは RNAでは Uとする、 以 下、 配列 Xとよぶ） 、 および該配列 Xと相補的な配列それそれの 35端に 2〜4個のヌ クレオチド、 好ましくは 2個の dT (デォキシ体の T)または ϋを付加した配列を有す る 2本の RNAを作製する。 配列 Xとしては、 ヒト KPC1 cDNAおよびマウス KPC1 cDNA の塩基配列の 1852〜； 1882番目に相当する、 配列番号 36に示す配列をあげることが できる。 作製した 2本の をァ二一リングすることにより siRNAを作製できる。 アニーリングは、 2本の RNAを適当なバッファー （例えば、 lOminol/L Tris-HC 50麵 ol/L NaCl, lmmol/L EDTA、 pH 7.5 ) に溶解した後、 ヒートプロヅクまたは サーマサイクラ一で 90〜95°Cで 1~5分間加熱した後、 25°Cまで 45〜60分間かけて 冷却することにより行うことができる。 KPC1の発現を抑制する s iRNAとしては、 配列番号 36で表される配列および該配列と相補的な配列の 3'端にそれそれ 2〜4個 のヌクレオチドを付加した配列からなる 2本鎖 RNA、 配列番号 34および 35でそれそ れ表される配列からなる 2本鎖 RNAをあげることができる。 これらの siRNAは、 ヒ ト KPC1およびマウス KPC1の発現を抑制することができる。 Double-stranded RNA containing a sequence corresponding to 19 to 25 consecutive nucleotides in the nucleotide sequence of DNA encoding KPC1 (hereinafter referred to as siENA (short interfering UNA)) or the sequence and its complement RNA (hereinafter referred to as RNAi) using RNA (hereinafter referred to as shRNA (short hairpin RNA)) that contains a specific sequence and forming a hairbin structure, Expression can be suppressed. SiRNAs and shRNAs (hereinafter also referred to as KPC1 siRNAs and KPC1 shRNAs) used to suppress the expression of KPC1 can be prepared as follows. From the nucleotide sequence of the cDNA encoding KPC1, select a sequence of 21 to 27 bases starting with AA (AA (N) 19-25 , where N is any base), preferably a sequence consisting of AA (N) 19 TT I do. The sequence to be selected is located in a region at least 50 bases downstream from the start codon in the translation region and has a GC content of 30 to 70%, preferably around 50%, and matches the nucleotide sequence of another gene. Instead, a sequence specific to KPC1 cDNA is more preferred. A sequence of 19 to 25 bases excluding AA at the end of the selected sequence or a sequence of 19 bases excluding AA and TT (where T in the sequence is U for RNA, hereinafter referred to as sequence X) , and two to four nucleotide sequence complementary thereto that of 35 ends with the sequence X, 2 pieces of that preferably have a sequence obtained by adding two dT (T of Dokishi body) or ϋ Make RNA. Examples of the sequence X include the sequence shown in SEQ ID NO: 36, which corresponds to the 1852th to 1882th nucleotides of the human KPC1 cDNA and mouse KPC1 cDNA. An siRNA can be prepared by chaining the two prepared DNAs. Annealing is performed by dissolving the two RNAs in an appropriate buffer (for example, lOminol / L Tris-HC 50 l / L NaCl, lmmol / L EDTA, pH 7.5), and then heat This can be done by heating at 90-95 ° C for 1-5 minutes in a Thermacycler and then cooling to 25 ° C over 45-60 minutes. The iRNA that suppresses the expression of KPC1 includes a double-stranded RNA comprising a sequence represented by SEQ ID NO: 36 and a sequence complementary to the sequence and having 2 to 4 nucleotides added to the 3 ′ end. And double-stranded RNAs consisting of the sequences represented by SEQ ID NOs: 34 and 35, respectively. These siRNAs can suppress the expression of human KPC1 and mouse KPC1.
shRNAとしては、 上記配列 Xと該配列 Xと相補的な配列とを、 3〜15塩基からなる 適当なスぺ一サ一配列でつないで、 3，末端に 2〜4個のヌクレオチド、 好ましくは 2個の dTまたは ϋを付加した配列を有する RNAを作製する。 スぺ一サー配列の 5'端 は dTdTまたは UUが好ましい。 配列 Xと配列 Xと相補的な配列の位置はどちらが先で もよい。 KPC1の発現を抑制する shRNAとして、 配列番号 38で表される配列からな る RNAをあげることができる。 該 shRNAは、 ヒト KPC1およびマウス KPC1の発現を抑 制することができる。 shRNAは細胞内で切断を受けて、 siRNAに変換される。  As the shRNA, the above-mentioned sequence X and a sequence complementary to the sequence X are connected by a suitable spacer sequence consisting of 3 to 15 bases, and 3, 2 to 4 nucleotides at the terminal, preferably An RNA having a sequence to which two dTs or ϋ are added is prepared. The 5 'end of the spacer sequence is preferably dTdT or UU. Either of the sequence X or the position of the sequence complementary to the sequence X may be located first. An example of shRNA that suppresses the expression of KPC1 is an RNA consisting of the sequence represented by SEQ ID NO: 38. The shRNA can suppress the expression of human KPC1 and mouse KPC1. shRNA is cleaved in the cell and converted to siRNA.
上記の s iRNAに用いる 2本の RNAおよび shRNAは DNA合成機を用いて化学合成する ことができる。 また、 サイレンサー （Silencer) siRNA作製キット等を利用して 、 T7プロモーター配列おょぴ作製する RNAの配列を有する 2本鎖 DNAを作製し、 こ の MAを錡型とした T7 ポリメラ一ゼを用いたインビトロ転写系によっても、 調製することができる。 あるいは、 以下のようにして作製できる KPC1 siRNA発現 用ベクターを、 培養細胞または生体内の細胞に導入することにより、 細胞内で siRNAを発現することができる。 KPC1 siRNA発現べクタ一は、 U6プロモーターあ る 、は HIプロモー夕一等 RNAポリメラーゼ IIIのプロモーターを含む s iRNA発現用 ベクターに、 上記配列 X、 TTからはじまる 3〜15塩基からなるスぺ一サー配列、 配 列 Xと相補的な配列および RNAポリメラーゼ 111夕一ミネ一夕一となる 4〜6個の Tか らなる配列を含む DNAを揷入して作製することができる。 細胞内で、 U6プロモー 夕一からの RNAポリメラーゼ III反応により、 配列 Xおよび相補的な配列を含む shRNAが合成され、 この sMNAが細胞内で切断を受けて siRNAに変換される。 siRNA 発現用べクタ一としては、 pSilencer 1.0-U6 (Ambion社製） 、 pSilencer 3.0 ( Ambion社製） 、 pSUPER (OligoEngine社製） 等をあげることができる。 レトロゥ ィルスベクターやレンチウィルスベクタ一を利用した siRNA発現用ベクター 〔 Science, 296, 550 (2002) ; Proc. Natl. Acad. Sci USA, 100, 1844 (2003) ; Nat. Genet. , 33. 401 (2003)〕 を用いることもできる。 KPC1 siRNA発現用べク 夕一としては、 レトロウイルスベクター pMX-puro I Iの Not I/Sal Iサイ ト間に、 U6 プロモーターおよびその下流に配列番号 37に示す配列をつなげた DNAを挿入して 作製した KPC1 siRNA発現用レトロウイルスベクターをあげることができる。 この ベクタ一をヒトまたはマウス細胞に導入することにより、 配列番号 34および 35で 表される配列を有する siRNAが発現し、 ヒト KPC1またはマウス KPC1の発現を抑制 することができる。 The two RNAs and shRNAs used for the above siRNA can be chemically synthesized using a DNA synthesizer. In addition, using a silencer siRNA production kit or the like, a double-stranded DNA having the sequence of the T7 promoter sequence and the RNA to be produced is produced, and a T7 polymerase having the MA as a type II is used. It can also be prepared using an in vitro transcription system. Alternatively, the siRNA can be expressed in cells by introducing the KPC1 siRNA expression vector prepared as follows into cultured cells or cells in a living body. The KPC1 siRNA expression vector contains a U6 promoter or a HI promoter, etc. A sirRNA expression vector containing an RNA polymerase III promoter, a primer consisting of 3 to 15 bases starting from the above sequences X and TT It can be prepared by inserting a DNA containing a sequence, a sequence complementary to sequence X, and a sequence consisting of 4 to 6 Ts, which is equivalent to RNA polymerase 111 min. In the cell, shRNA containing sequence X and a complementary sequence is synthesized by the RNA polymerase III reaction from the U6 promoter, and this sMNA is cleaved in the cell and converted to siRNA. Examples of the siRNA expression vector include pSilencer 1.0-U6 (manufactured by Ambion), pSilencer 3.0 (manufactured by Ambion), pSUPER (manufactured by OligoEngine) and the like. A vector for siRNA expression using a retrovirus vector or a lentivirus vector [Science, 296, 550 (2002); Proc. Natl. Acad. Sci USA, 100, 1844 (2003); Nat. Genet., 33. 401 (2003)]. The KPC1 siRNA expression vector was prepared by inserting the U6 promoter and the DNA linked to the sequence shown in SEQ ID NO: 37 downstream of the U6 promoter between Not I / Sal I sites of the retroviral vector pMX-puro II. Retrovirus vectors for expressing KPC1 siRNA. By introducing this vector into human or mouse cells, siRNA having the sequences represented by SEQ ID NOs: 34 and 35 is expressed, and the expression of human KPC1 or mouse KPC1 can be suppressed.
(2)アンチセンス配列を有するオリゴヌクレオチドを利用して KPC1の発現を抑制 する方法  (2) Method of suppressing KPC1 expression using an oligonucleotide having an antisense sequence
アンチセンス RNA/DNA技術 〔バイォサイエンスとィンダス トリー， 50, 322 (1992) ; ィ匕学， 681 (1991) ; Biotechnology, 9, 358 (1992) ; Trends BiotechnoL , 10, 87 ( 1992) ; Trends Biotechnol. , 10, 152 ( 1992) ; 細胞工学 ， 16, 1463 (1997)〕 、 トリプル ·ヘリヅクス技術 〔Trends Biotechnol . , 10, 132 ( 1992)〕 に基づき、 KPC1をコードする DNAが有する塩基配列と相補的な配列 (アンチセンス配列） の連続する 20〜； 100塩基の配列を含むオリゴヌクレオチド または該ォリゴヌクレオチドの誘導体を細胞あるいは生体に投与することにより 、 細胞あるいは生体内での KPC1遺伝子の転写または翻訳を抑制し、 その結果、 KPC1の発現を抑制することができる。 特に KPC1をコードする領域の開始コドンを 含む 20〜； 100塩基と相補的な塩基配列が好ましい。 また、 デォキシリポヌクレア ーゼ、 リボヌクレアーゼによる分解を受けないオリゴヌクレオチド誘導体が好ま しい。  Antisense RNA / DNA technology [Bioscience and Industry, 50, 322 (1992); Gidani, 681 (1991); Biotechnology, 9, 358 (1992); Trends BiotechnoL, 10, 87 (1992); Trends Biotechnol., 10, 152 (1992); Cell engineering, 16, 1463 (1997)], based on triple helix technology [Trends Biotechnol., 10, 132 (1992)]. Transcription of the KPC1 gene in a cell or in vivo by administering an oligonucleotide containing a sequence of 20 to 100 nucleotides complementary to the complementary sequence (antisense sequence) or a derivative of the oligonucleotide to the cell or in vivo Alternatively, translation can be suppressed, and as a result, expression of KPC1 can be suppressed. In particular, a nucleotide sequence complementary to 20 to 100 nucleotides including the start codon of the region encoding KPC1 is preferable. Oligonucleotide derivatives that are not subject to degradation by deoxyliponuclease or ribonuclease are preferred.
該ォリゴヌクレオチド誘導体としては、 ォ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オリゴヌクレオチド誘導体、 オリゴヌクレオチド中のリン酸ジエステル結合が N3， -P5'ホスフォアミデ一ト結 合に変換されたォリゴヌクレオチド誘導体、 ォ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ォリゴヌクレオチド誘導 体、 オリゴヌクレオチド中のゥラシルが C- 5プロピニルゥラシルで置換されたォ リゴヌクレオチド誘導体、 ォリゴヌクレオチド中のゥラシルが C- 5チアゾールゥ ラシルで置換されたオリゴヌクレオチド誘導体、 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ォリゴヌクレオチド誘導体、 ォリゴヌ 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)で置換されたォリゴヌクレオチド誘導体、 ォ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ォリゴヌクレオチド誘導体、 ある いはオリゴヌクレオチド中のリボースが ーメ トキシエトキシリボースで置換さ れたオリゴヌクレオチド誘導体等をあげることができる 〔細胞工学， ， 1463 (1997)〕。 Examples of the oligonucleotide derivative include an oligonucleotide derivative in which a phosphoric ester bond in an oligonucleotide is converted into a phosphorothioate bond, and a phosphoric diester bond in an oligonucleotide in which an N3, -P5 'phosphoramide bond is formed. Oligonucleotide derivative, Oligonucleotide derivative in which ribose and phosphodiester bond in Oligonucleotide are converted to Peptide Nucleic Acid bond, Peracyl in Oligonucleotide is replaced by C-5 propynyl Peracyl Oligonucleotide derivatives, Oligonucleotide derivatives in which peracyl in the oligonucleotide is substituted with C-5 thiazole peracyl, Cytosine in the oligonucleotide is substituted with C-5 propynylcytosine, Oligonu Oligonucleotide derivatives in which cytosine in the nucleotide is substituted with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2, -0-propylribose, or Oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with -methoxyethoxyribose can be mentioned [Cell Engineering,, 1463 (1997)].
上記のォリゴヌクレオチドまたはォリゴヌクレオチド誘導体は MA合成機を用 いて合成することができる。  The above-mentioned oligonucleotides or oligonucleotide derivatives can be synthesized using an MA synthesizer.
siRNAs shRNA、 アンチセンスオリゴヌクレオチド、 該オリゴヌクレオチドの誘 導体はオリゴフエクトァミン （Oligofectanine ) 試薬 （インビトロジェン社製） 、 リポフエクトァミン （Lipofectamine ) 2000 (インビトロジェン社製） 、 トラ ンスメッセンジャー . トランスフエクシヨン (TransMessenger Transf ection ) 試薬 （キアゲン社製） 等のリボソーム系トランスフエクシヨン用試薬を用いて、 細胞に導入することができる。 また、 KPC1 siRNA発現べクタ一は、 2.に記載した 発現べクタ一を動物細胞に導入する方法と同様にして、 細胞に導入することがで きる。 ウィルスベクタ一を利用した KPC1 siRNA発現ベクターの場合は、 該ベクタ —を利用して作製した組換えウィルスを投与し、 細胞に感染させることにより、 導入することができる。 動物やヒトに投与する場合は、 siRNA、 shRNA、 アンチセ ンスオリゴヌクレオチド、 該オリゴヌクレオチドの誘導体、 あるいは KPC1 siRNA 発現べク夕一をそのままあるいはリポソーム調製物として、 静脈注射等の方法で 投与することができる。  siRNAs shRNAs, antisense oligonucleotides, and derivatives of the oligonucleotides include oligofectamine reagent (manufactured by Invitrogen), lipofectamine (Lipofectamine) 2000 (manufactured by Invitrogen), and transmessenger transfection. (TransMessenger Transfection) It can be introduced into cells using a reagent for ribosome transfection such as a reagent (manufactured by Qiagen). Further, the KPC1 siRNA expression vector can be introduced into cells in the same manner as the method for introducing the expression vector into animal cells described in 2. In the case of a KPC1 siRNA expression vector using a virus vector, the recombinant virus prepared using the vector can be administered and the cells can be infected to introduce the virus. When administered to animals or humans, the siRNA, shRNA, antisense oligonucleotide, derivative of the oligonucleotide, or KPC1 siRNA expression vector can be administered as it is or as a liposome preparation by intravenous injection or the like. it can.
KPC1は、 細胞内の p27Kiplをュビキチン化し、 その分解を促進するので、 （1 )お よび（2)に示した方法によって、 KPC1の発現を抑制することにより、 p27Kiplの分 解を抑制することができる。 さらに、 p27Kiplの分解を抑制することにより細胞周 期の進行を抑制できるので、 該方法は、 細胞周期の異常を原因とする疾患、 細胞 周期を調節することにより症状を軽減できるような疾患の治療に用いることがで きる。 細胞周期の異常を原因とする疾患、 細胞周期を調節することにより症状を 軽減できるような疾患としては、 癌、 動脈硬化、 慢性関節リウマチ、 前立腺肥大 症、 経皮的経血管的冠動脈形成術後の血管再狭窄、 肺線維症、 糸球体腎炎、 自己 免疫疾患等をあげることができ、 特に該方法は癌の治療に有効である。 13. KPC1と特異的に結合する抗体を用いた KPC1の検出および定量法Since KPC1 ubiquitinates p27 Kipl in cells and promotes its degradation, it suppresses p27 Kipl degradation by suppressing KPC1 expression by the methods described in (1) and (2). be able to. Furthermore, since the progression of the cell cycle can be suppressed by suppressing the degradation of p27 Kipl, the method is used for diseases caused by abnormal cell cycles and diseases in which the symptoms can be alleviated by regulating the cell cycle. Can be used for treatment. Diseases caused by abnormal cell cycle and diseases whose symptoms can be reduced by regulating the cell cycle include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, and percutaneous transvascular coronary angioplasty. Vascular restenosis, pulmonary fibrosis, glomerulonephritis, autoimmune diseases, etc., and the method is particularly effective for treating cancer. 13. Detection and quantification of KPC1 using an antibody that specifically binds to KPC1
4.で得られる KPC1と特異的に結合する抗体を用い、 抗原抗体反応を行わせるこ とにより、 KPC1または KPC1を含む細胞または組織を免疫学的に検出および定量す ることができる。 測定試料としては、 細胞や組織の抽出液、 培養上清、 血液、 尿 、 唾液等の体液、 あるいは組織のパラフィン切片ゃクリオス夕ット切片等が用い られる。 By performing an antigen-antibody reaction using the antibody that specifically binds to KPC1 obtained in step 4, KPC1 or a cell or tissue containing KPC1 can be immunologically detected and quantified. As the measurement sample, cell or tissue extract, culture supernatant, body fluids such as blood, urine, saliva, etc., or paraffin section or cryo-section section of the tissue are used.
免疫学的に検出および定量する方法としては、 放射免疫測定法 （RIA) 、 免疫 染色法、 免疫蛍光染色法、 ィムノブロット法、 ドットプロット法、 免疫沈降法、 サンドイッチ ELISA 〔単クローン抗体実験マニュアル （講談社サイェンティフィ ック） （1987)、 続生化学実験講座 5， 免疫生化学研究法 （東京化学同人） （1986) 〕等が挙げられる。  Methods for immunological detection and quantification include radioimmunoassay (RIA), immunostaining, immunofluorescent staining, immunoblotting, dot plotting, immunoprecipitation, sandwich ELISA [monoclonal antibody experiment manual (Kodansha) Scientific) (1987), Lectures on Sequential Chemistry, 5, Immunobiochemical Research (Tokyo Kagaku Doujinshi) (1986)].
MAとは、 測定試料に、 KPC1と特異的に結合する抗体を反応させ、 さらに放射 性同位体で標識され、 かつ該抗体と結合する抗体を反応させた後、 抗原抗体複合 体を分離してシンチレ一シヨンカウン夕一などで放射能を測定し、 測定試料中の KPC1を検出および定量する方法である。 該抗体と結合する抗体としては、 KPC1と 特異的に結合する抗体の作製時の免疫動物の IgGと結合する抗体、 例えば KPC1と 特異的に結合する抗体がラットを免疫して作製したラット抗体であれば、 抗ラッ ト IgG抗体があげられる。  MA refers to the reaction of an antibody that specifically binds to KPC1 with a measurement sample, and then reacts with an antibody that is labeled with a radioisotope and binds to the antibody.Then, the antigen-antibody complex is separated. This is a method of measuring radioactivity at scintillation counters and detecting and quantifying KPC1 in measurement samples. Examples of the antibody that binds to the antibody include an antibody that binds to IgG of an immunized animal at the time of producing an antibody that specifically binds to KPC1, for example, a rat antibody that is prepared by immunizing a rat with an antibody that specifically binds to KPC1. If present, anti-rat IgG antibodies can be mentioned.
免疫染色法とは、 組織切片や細胞等の測定試料に、 KPC1と特異的に結合する抗 体を反応させ、 さらにペルォキシダ一ゼ等の酵素、 ピオチン等で標識した該抗体 と結合する抗体を反応 せた後、 標識物質に応じた発色反応を行い、 顕微鏡観察 により、 測定試料中の KPC1を検出する方法である。  The immunostaining method involves reacting an antibody that specifically binds to KPC1 with a measurement sample such as a tissue section or cell, and then reacting with an antibody that binds to an enzyme such as peroxidase or an antibody that binds to the antibody labeled with biotin. After that, a color reaction according to the labeling substance is performed, and KPC1 in the measurement sample is detected by microscopic observation.
免疫蛍光染色法は、 組織切片や細胞等の測定試料に、 KPC1と特異的に結合する 抗体を反応させ、 さらにフルォレセインイソチオシァネート （FITC) 、 Alexa 546、 テトラメチルローダミンイソチオシァネート等の蛍光物質で標識した該抗 体と結合する抗体を反応させた後、 蛍光顕微鏡観察により、 測定試料中の KPC1を 検出する方法である。  The immunofluorescent staining method involves reacting an antibody that specifically binds to KPC1 with a measurement sample such as a tissue section or a cell, and then reacts with fluorescein isothiocyanate (FITC), Alexa 546, and tetramethylrhodamine isothiocyanate. This is a method in which KPC1 in a measurement sample is detected by reacting an antibody that binds to the antibody labeled with a fluorescent substance such as a phosphate and then observing the sample with a fluorescence microscope.
ィムノブロッ ト （ウエスタンプロヅ ト） 法とは、 測定試料を SDS-PAGE 〔 Antibodies- A Laboratory Manual, Cold Spring Harbor Laboratory, 、丄988) 〕 で分画した後、 PVDF膜あるいはニトロセルロース膜にブロッテイングし、 該膜 に KPClと特異的に結合する抗体を反応させ、 さらにペルォキシダ一ゼなどの酵素The immunoblotting (Western plot) method is a method in which a sample to be measured is fractionated by SDS-PAGE (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 丄 988), and then blotted on a PVDF membrane or nitrocellulose membrane. And the membrane With an antibody that specifically binds to KPCl, and an enzyme such as peroxidase.
、 ビォチン、 放射性同位体等で標識した該抗体と結合する抗体を反応させた後、 標識物質に応じた方法で測定試料中の KPC1をバンドとして検出および定量する方 法である。 This is a method in which, after reacting an antibody that binds with the antibody labeled with biotin, radioisotope, or the like, KPC1 in the measurement sample is detected and quantified as a band by a method according to the labeling substance.
ドットプロット法とは、 測定試料を二トロセルロース膜にドヅト状にプロッテ イングし、 該膜に KPC1と特異的に結合する抗体を反応させ、 さらにペルォキシダ ーゼなどの酵素、 ビォチン、 放射性同位体等で標識した該抗体と結合する抗体を 反応させた後、 標識物質に応じた方法で測定試料中の KPC1を検出および定量する 方法である。  The dot plot method is a method in which a measurement sample is printed in a dot form on a nitrocellulose membrane, an antibody that specifically binds to KPC1 is reacted with the membrane, and enzymes such as peroxidase, biotin, radioisotope, etc. After reacting the antibody that binds to the antibody labeled with, the KPC1 in the measurement sample is detected and quantified by a method according to the labeling substance.
免疫沈降法とは、 測定試料を KPC1と特異的に結合する抗体と反応させた後、 プ ロティン A—セファロ一ス等ィムノグロブリンと特異的に結合する担体を加えて 反応させ、 遠心分離等により、 抗原抗体複合体と結合した担体を単離する方法で ある。 担体から抗原抗体複合体を溶出させ、 ィムノブロット法と同様にして測定 試料中の KPC1を検出する。  The immunoprecipitation method involves reacting a measurement sample with an antibody that specifically binds to KPC1, then adding a carrier that specifically binds to immunoglobulin such as protein A-cepharose, and reacting it. Is a method for isolating a carrier bound to an antigen-antibody complex. The antigen-antibody complex is eluted from the carrier, and KPC1 in the measurement sample is detected in the same manner as in the immunoblot method.
サンドィツチ ELISAとは、 KPC1と特異的に結合する抗体を吸着させたプレート に、 測定試料を反応させた後、 上記抗体とはェピトープが異なる KPC1と特異的に 結合する抗体を反応させ、 さらに該抗体と結合するペルォキシダーゼ等の酵素で 標識した抗体を反応させた後、 酵素に応じた発色反応を行い、 測定試料中の KPC1 を検出および定量する方法である。  A sandwich ELISA is a method in which a measurement sample is reacted with a plate on which an antibody that specifically binds to KPC1 is adsorbed, and then an antibody that specifically binds to KPC1 having a different epitope from the above antibody is reacted. After reacting an antibody labeled with an enzyme such as peroxidase that binds to the enzyme, a color reaction is performed according to the enzyme to detect and quantify KPC1 in the measurement sample.
14. KPC1と特異的に結合する抗体の診断および治療への利用  14. Use of antibodies that specifically bind to KPC1 for diagnosis and therapy
KPC1と特異的に結合する抗体を用いて、 KPC1の発現量の変化を検出し、 KPC1の 発現量が減少または増加している疾患の判定または診断を行なうことができる。 Using an antibody that specifically binds to KPC1, a change in the expression level of KPC1 can be detected to determine or diagnose a disease in which the expression level of KPC1 decreases or increases.
KPC1の発現量が減少している疾患としては、 細胞周期の異常を原因とする疾患、 例えば、 癌、 動脈硬化、 慢性関節リウマチ、 前立腺肥大症、 肺線維症、 糸球体腎 炎、 自己免疫疾患等をあげることができ、 特に癌に有効である。 Diseases in which the expression level of KPC1 is decreased include diseases caused by abnormal cell cycles, such as cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, pulmonary fibrosis, glomerulonephritis, and autoimmune diseases. Etc., and is particularly effective for cancer.
抗体を用いた KPC1の発現量を定量して判定または診断する方法としては、 13. にあげた KPC1を検出および定量する方法をあげることができる。  Methods for quantifying and diagnosing or diagnosing the expression level of KPC1 using an antibody include the methods for detecting and quantifying KPC1 described in 13.
上記方法による判定または診断に供する検体としては、 疾患の患者より取得し た組織、 血液、 血清、 尿、 便、 唾液等の生体試料そのものあるいは、 該生体試料 から取得した細胞ならびに細胞抽出液が用いられる。 また、 生体試料から取得し た組織のパラフィンあるいはクリオスタツト切片を用いることもできる。 As a sample to be used for determination or diagnosis by the above method, a biological sample itself such as tissue, blood, serum, urine, stool, saliva, or a cell or cell extract obtained from the biological sample obtained from a patient with a disease is used. Can be It can also be obtained from biological samples A paraffin or cryostat section of the tissue can also be used.
KPC1の発現量の変化を伴う疾患は以下のようにして判定または診断できる。 ま ず、 複数の患者および健常者の検体について KPC1の発現量を上記にあげた検出方 法で測定して比較し、 患者および健常者の KPC1の発現レベルの範囲を決定する。 被験者の検体の KPC1の発現レベルを、 健常者の発現レベルおよび患者の発現レベ ルとそれそれ比較し、 どちらの発現レベルの範囲に入るかを調べることにより判 定または診断を行う。  A disease associated with a change in the expression level of KPC1 can be determined or diagnosed as follows. First, the expression levels of KPC1 in multiple samples of patients and healthy subjects are measured and compared by the detection methods described above, and the range of the expression levels of KPC1 in patients and healthy subjects is determined. Judgment or diagnosis is made by comparing the expression level of KPC1 in the subject's sample with the expression level of a healthy person and the expression level of a patient, and examining which expression level falls within the range.
KPC1と特異的に結合する抗体のうち、 p27Kiplと KPC1との結合を阻害する抗体、 あるいは KPC1が有する p27Kiplをュビキチン化する活性を阻害する作用を示す抗体 は、 細胞内の KPC1のュビキチン化を抑制し、 その分解を抑制することができる。 さらに該抗体は、 p27Kiplの分解を抑制することにより、 細胞周期の進行を抑制で きるので、 細胞周期の異常を原因とする疾患、 細胞周期を調節することにより症 状を軽減できるような疾患の治療に用いることができる。 細胞周期の異常を原因 とする疾患、 細胞周期を調節することにより症状を軽減できるような疾患として は、 癌、 動脈硬化、 慢性関節リウマチ、 前立腺肥大症、 経皮的経血管的冠動脈形 成術後の血管再狭窄、 肺線維症、 糸球体腎炎、 自己免疫疾患等をあげることがで き、 該抗体は特に癌の治療に有効である。 Among the antibodies that specifically bind to KPC1 , antibodies that inhibit the binding between p27 Kipl and KPC1, or antibodies that inhibit KPC1's activity to ubiquitinate p27 Kipl, are ubiquitination of intracellular KPC1 And its decomposition can be suppressed. Furthermore, the antibody can suppress the progression of the cell cycle by suppressing the degradation of p27 Kipl , so that diseases caused by abnormalities in the cell cycle and diseases in which the symptoms can be alleviated by regulating the cell cycle. Can be used for the treatment of Diseases caused by abnormal cell cycles and diseases whose symptoms can be reduced by regulating the cell cycle include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, percutaneous transvascular coronary angioplasty. The latter can be exemplified by vascular restenosis, pulmonary fibrosis, glomerulonephritis, autoimmune disease and the like, and the antibody is particularly effective for treating cancer.
15. KPC1をコードする配列に由来するポリヌクレオチドもしくはオリゴヌクレオ チド、 または KPC1と特異的に結合する抗体を含有する医薬  15. A drug containing a polynucleotide or oligonucleotide derived from the sequence encoding KPC1, or an antibody that specifically binds to KPC1
12. に記載した KPC1の発現を抑制するオリゴヌクレオチドまたはオリゴヌクレ ォチド誘導体、 14. に記載した KPC1と特異的に結合する抗体であって、 p27Kiplと KPC1との結合を阻害する抗体、 あるいは KPC1が有する p27Kiplをュビキチン化する 活性を阻害する作用を示す抗体は、 治療薬として単独で投与することも可能では あるが、 通常は薬理学的に許容される 1つあるいはそれ以上の担体と一緖に混合 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 として提供するのが望ましい。 The oligonucleotide or oligonucleotide derivative that suppresses the expression of KPC1 described in 12., the antibody that specifically binds to KPC1 described in 14., which is an antibody that inhibits the binding of p27 Kipl to KPC1, or the KPC1 is An antibody that has the activity of inhibiting the activity of ubiquitinating p27 Kipl can be administered alone as a therapeutic agent, but is usually combined with one or more pharmacologically acceptable carriers. And provided as a pharmaceutical preparation produced by any method well-known in the field of pharmacology.
投与経路は、 治療に際し最も効果的なものを使用するのが望ましく、 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などの非経口投与 をあげることができ、 望ましくは静脈内投与をあげることができる。  It is desirable to use the most effective route for treatment, including oral administration or parenteral administration such as buccal, respiratory, rectal, subcutaneous, intramuscular and intravenous, Desirably, intravenous administration can be mentioned.
投与形態としては、 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、 軟膏、 テープ剤などがあげられる。 Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, Suppositories, injections, ointments, 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 、 顆粒剤などがあげられる。  Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.
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 糖などの糖類、 ポリエチレングリコール、 プロピレングリコールなどのグリコー ル類、 ごま油、 ォリーブ油、 大豆油などの油類、 P-ヒドロキシ安息香酸エステル 類などの防腐剤、 ストロベリーフレーバー、 ペパーミントなどのフレーバー類な どを添加剤として用いて製造できる。  Liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, soybean oil, P- It can be manufactured using preservatives such as hydroxybenzoic acid esters and flavors such as strawberry flavor and peppermint as additives.
カプセル剤、 錠剤、 散剤、 顆粒剤などは、 乳糖、 ブドウ糖、 ショ糖、 マンニト —ルなどの賦形剤、 デンプン、 アルギン酸ナトリウムなどの崩壊剤、 ステアリン 酸マグネシウム、 タルクなどの滑沢剤、 ポリビニルアルコール、 ヒドロキシプロ ピルセルロース、 ゼラチンなどの結合剤、 脂肪酸エステルなどの界面活性剤、 グ リセリンなどの可塑剤などを添加剤として用いて製造できる。  Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl alcohol It can be produced using additives such as a binder such as hydroxypropylcellulose and gelatin, a surfactant such as a fatty acid ester, and a plasticizer such as glycerin.
非経口投与に適当な製剤としては、 注射剤、 座剤、 噴霧剤などがあげられる。 注射剤は、 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などを用 いて調製される。 座剤はカカオ脂、 水素化脂肪またはカルボン酸などの担体を用 いて調製される。 また、 噴霧剤は受容者の口腔および気道粘膜を刺激せず、 かつ 有効成分を微細な粒子として分散させ吸収を容易にさせる担体などを用いて調製 される。  Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. The injection is prepared using a carrier comprising a salt solution, a glucose solution or a mixture of both. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. Sprays are prepared using a carrier which does not irritate the oral and respiratory mucosa of the recipient and which disperses the active ingredient as fine particles to facilitate absorption.
担体として具体的には乳糖、 グリセリンなどが例示される。 該 DNAまたはオリ ゴヌクレオチド、 さらには用いる担体の性質により、 エアロゾル、 ドライパウダ 一などの製剤が可能である。 また、 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もできる。  Specific examples of the carrier include lactose and glycerin. Formulations such as aerosols and dry powders can be made depending on the properties of the DNA or oligonucleotide and the carrier used. Also, in these parenteral preparations, the components exemplified as additives in the oral preparation 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、 体重などにより異なるが、 通常成人 1日当たり 10〃g/kg〜20mg/kg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 20 mg / kg per adult per day.
KPC1をコ一ドする DNAの塩基配列の 20bp以上の連続した配列と一致する、 ある いは相補的な配列を含むポリヌクレオチドもしくはォリゴヌクレオチド、 または KPC1と特異的に結合する抗体を含有する診断薬の場合は、 目的の診断法に応じて 、 KPC1をコ一ドする mfiNAあるいは KPC1の定量あるいは KPC1遺伝子の変異の検出を 行うのに必要な試薬、 例えば緩衝剤、 塩、 反応用酵素、 本発明の抗体と結合する 標識された抗体、 検出用発色剤等を含んでもよい。 Diagnostics containing polynucleotides or oligonucleotides containing a sequence that is identical or complementary to a continuous sequence of 20 bp or more of the nucleotide sequence of the DNA encoding KPC1, or an antibody that specifically binds to KPC1 In the case of drugs, reagents required for quantifying mfiNA or KPC1 encoding KPC1 or detecting mutations in the KPC1 gene, such as buffers, salts, reaction enzymes, Binds to the antibody of the invention It may contain a labeled antibody, a color former for detection, and the like.
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 図面の簡単な説明  Hereinafter, the present invention will be described with reference to specific examples, but this does not limit the scope of the present invention.
第 1図 第 1図は、 ゥサギ網状赤血球抽出液からの KPCの精製プロトコ一ルを 示す。  Fig. 1 Fig. 1 shows a protocol for purifying KPC from a heron reticulocyte extract.
第 2図 第 2図の上段は、 Superose 6ゲルろ過カラムで得られた各フラクショ ンの、 インビト口再構成系による ρ27κίρ1をュビキチン化する活性の測定を示す。 左の p27、 p27-(GST-Ub) p27-(GST- Ub)2、 p27- (GST- Ub)3、 p27- (GST- Ub)nはそ れそれ、 p27Kipl、 GST- Ubが 1個付加した p27Kipl、 GST-Ubが 2個付加した p27Kipl、 GST- Ubが 3個付加した p27Kip GST- Ubが 3個以上付加した p27Kiplの SDS-PAGE上の 位置を示す （以下の図面でも同様） 。 フラクションの上に、 分子量マーカ一がゲ ルろ過で溶出されるフラクションの位置を示す。 下段は、 同じフラクションを、 SDS- PAGE後、 クマシ一染色した結果を示す。 左に分子量マ一カーの位置、 右に KPC1および KPC2の位置を示す。 Fig. 2 The upper part of Fig. 2 shows the measurement of the activity of each fraction obtained by the Superose 6 gel filtration column to ubiquitinate ρ27κίρ1 by the in vitro reconstitution system. P27 , p27- (GST-Ub) p27- (GST-Ub) 2, p27- (GST-Ub) 3, p27- (GST-Ub) n on the left , p27 Kipl and GST-Ub are 1 P27 Kipl with two GST-Ub added, p27 Kipl with two GST-Ub added, p27 Kip with three GST-Ub added p27 Kipl with three or more GST-Ub indicates the position on SDS-PAGE The same applies to drawings). Above the fraction, the molecular weight marker indicates the position of the fraction eluted by gel filtration. The lower panel shows the results of the same fraction stained with Komasi after SDS-PAGE. The position of the molecular weight marker is shown on the left, and the positions of KPC1 and KPC2 are shown on the right.
第 3図 第 3図は、 第 2図と同様に、 上段は、 ミニ Qカラムで得られた各フラ クシヨンの、 インビト口再構成系による p27Kiplをュビキチン化する活性の測定を 、 下段は同じフラクションを、 SDS- PAGE後、 クマシ一染色した結果を示す。 左に 分子量マーカーの位置、 右に KPC 1および KPC2の位置を示す。 Fig. 3 Like Fig. 2, Fig. 3 shows the measurement of the activity of each fraction obtained with the mini-Q column to ubiquitinate p27 Kipl by the in vitro reconstitution system. The result of fractionation of the fractions after SDS-PAGE and coomassie staining is shown. The position of the molecular weight marker is shown on the left, and the positions of KPC1 and KPC2 are shown on the right.
第 4図 第 4図は、 ゥサギ網状赤血球抽出液から精製した KPCを SDS-PAGE後、 クマシ一染色した結果を示す。 左に分子量マ一カーの位置、 右に KPC1および KPC2 の位置および、 それそれから得られた部分ペプチドのァミノ酸配列を 1文字表記 で示す。  FIG. 4 FIG. 4 shows the results of SDS-PAGE and Komasi-stained KPC purified from a perch reticulocyte extract. The position of the molecular weight marker is shown on the left, the positions of KPC1 and KPC2 are shown on the right, and the amino acid sequence of the partial peptide obtained therefrom is shown in one-letter code.
第 5図 第 5図 aは KPC1および KPC2の構造を示す。 SPRAYは SPRAYドメインを、 RINGは RINGフィンガ一ドメインを、 UBUまュビキチン様 （ubiquitin- like) ドメ インを、 また UBAはュビキチン結合 （ubiquitin- associated) ドメインをそれそ れ示している。 第 5図 bは、 ヒト KPC1のアミノ酸配列を示す。 精製サンプルのぺ プチド解析から得られた配列と一致する部分を下線で、 SPRYドメイン、 RINGフィ ンガードメインをそれそれ白黒反転および枠囲みで示し、 RINGフインガ一ドメイ ン中の亜鉛結合部位と予測される配列に *を記す。 Fig. 5 Fig. 5a shows the structure of KPC1 and KPC2. SPRAY stands for SPRAY domain, RING stands for RING finger domain, UBU stands for ubiquitin-like domain, and UBA stands for ubiquitin-associated domain. FIG. 5b shows the amino acid sequence of human KPC1. The part corresponding to the sequence obtained from peptide analysis of the purified sample is underlined, the SPRY domain and the RING finger domain are shown in black and white inverted and boxed, respectively, and the RING finger domain is shown. * Is added to the sequence predicted to be the zinc binding site in the protein.
第 6図 第 6図は、 昆虫細胞で発現させ、 精製した組換え KPC1-KPC2複合体を SDS-PAGE後、 クマシ一染色した結果を示す。 左に分子量マ一カーの位置、 右に発 現させた His6/FLAG夕グ付加 KPC1および His6/HSV夕グ付加 KPC2の位置を示す。  Fig. 6 Fig. 6 shows the results of SDS-PAGE of the purified recombinant KPC1-KPC2 complex expressed and expressed in insect cells, followed by coomassie staining. The position of the molecular weight marker is shown on the left, and the positions of the His6 / FLAG-added KPC1 and His6 / HSV-added KPC2 are shown on the right.
第 7図 第 7図は、 昆虫細胞で発現させた KPC1および KPCl( AR)と KPC2の細胞 内での会合を免疫沈降により解析した結果を示す。 WTは野生型 KPC1、 ΔΙΙは KPC1( . Δί を、 +は KPC2をそれぞれ発現させたこと、 一は発現させなかったことを示す 。 左は、 細胞抽出液の SDS-PAGE、 右は抗 FLAG抗体 （KPC1および KPC1( ΔΙΙ)と結合 ) による免疫沈降物についてそれそれ、 上段は抗 FLAG抗体、 下段は抗 HSV抗体 （ KPC2と結合） によるィムノブロヅト解析を示す。  Fig. 7 Fig. 7 shows the results of immunoprecipitation analysis of intracellular association of KPC1 and KPC1 (AR) expressed in insect cells with KPC2. WT indicates wild-type KPC1, ΔΙΙ indicates KPC1 (.Δί, + indicates KPC2, and one indicates no expression. Left: SDS-PAGE of cell extract, right: anti-FLAG antibody Each of the immunoprecipitates by (binding to KPC1 and KPC1 (ΔΙΙ)) shows the immunoblot analysis using an anti-FLAG antibody, and the lower panel using an anti-HSV antibody (binding to KPC2).
第 8図 第 8図は、 昆虫細胞で発現させた KPC1- KPC2複合体と p27Kiplのインビト 口での結合を免疫沈降により解析した結果を示す。 +は添加、 一は非添加を示す 。 左は、 KPC1-KPC2複合体と p27Kiplの混合物の SDS- PAGE、 右は抗 p27Kipl抗体による 免疫沈降物についてそれそれ、 上段は抗 FLAG抗体 （KPC1と結合） 、 中段は抗 HSV 抗体 （KPC2と結合） 、 下段は抗 p27Kipl抗体によるィムノブロット解析を示す。 Fig. 8 Fig. 8 shows the results of immunoprecipitation analysis of the binding of the KPC1-KPC2 complex expressed in insect cells to p27 Kipl at the in vivo opening. + Indicates addition, and 1 indicates no addition. Left: SDS-PAGE of a mixture of KPC1-KPC2 complex and p27 Kipl , right: immunoprecipitation by anti-p27 Kipl antibody, upper: anti-FLAG antibody (binding to KPC1), middle: anti-HSV antibody (KPC2) The lower part shows an immunoblot analysis using an anti-p27 Kipl antibody.
第 9図 第 9図は、 昆虫細胞で発現させた KPC1- KPC2複合体の p27Kiplおよびリン 酸化 Sicl (Sicl-P) をュビキチン化する活性を測定した結果を示す。 +はインビ トロ再構成系の各成分、 KPC1-KPC2複合体、 SCFede4 (図中で SCF Cdc4と表記） の 添加、 一は非添加を示す。 左は p27Kiplを基質とした場 、 右は Sicl- Pを基質とし て添加し、 それそれ抗 p27Kipl抗体、 抗 HPC4抗体 （Sicl-Pと結合） によりィムノブ ロット解析した場合を示す。 Sicl-Pヽ Sicl-P-(GST-Ub)ls Sic卜 P- (GST- Ub)nはそ れそれ、 Sicl-P、 GST- Ubが 1個付加した Sicl- P、 GST- Ubが 2個以上付加した Sicl-Pの SDS- PAGE上の位置を示す。 FIG. 9 FIG. 9 shows the results of measuring the activity of the KPC1-KPC2 complex expressed in insect cells to ubiquitinate p27 Kipl and phosphorylated Sicl (Sicl-P). + Indicates addition of each component of the in vitro reconstitution system, KPC1-KPC2 complex, and SCF ede4 (denoted as SCF Cdc4 in the figure), and one indicates no addition. The left shows the case where p27 Kipl was used as the substrate, and the right shows the case where Sicl-P was added as the substrate and the immunoblotting analysis was performed using anti-p27 Kipl antibody and anti-HPC4 antibody (binding to Sicl-P). Sicl-P ヽ Sicl-P- (GST-Ub) l s Sic P- (GST-Ub) n, Sicl-P and GST-Ub with one Sicl-P and GST-Ub added The positions on the SDS-PAGE of two or more Sicl-Ps are shown.
第 10図 第 10図は、 昆虫細胞で発現させた KPC1、 PCl( AR), KPC1-KPC2複合体 、 KPCl( AR)- KPC2複合体の p27Kiplをュビキチン化する活性を測定した結果を示す 。 WTは野生型 KPC1、 ΔίΙは KPC1( /\R)を、 +は KPC2をそれそれ発現させたこと、 ― は発現させなかったことを示す。 反応の +はインビトロ再構成系の反応を行った 場合、 —はインビト口再構成系の構成成分を添加せず反応を行わなかったことを 示す。 FIG. 10 FIG. 10 shows the results of measuring the activity of ubiquitinating p27 Kipl of KPC1, PCl (AR), KPC1-KPC2 complex, and KPCl (AR) -KPC2 complex expressed in insect cells. WT indicates wild-type KPC1, ΔίΙ indicates KPC1 (/ \ R), + indicates KPC2, and-indicates no. In the reaction, + indicates that the reaction was performed in the in vitro reconstitution system, and-indicates that the reaction was not performed without adding the components of the in vitro reconstitution system.
第 11図 第 11図は、 E2として各レーンの上に示す種々の蛋白質を用いたときに 、 KPC1- KPC2複合体が p27Kiplをュビキチン化する活性を測定した結果を示す。 （-） は、 E2を添加しなかった場合の結果を示す。 Fig. 11 Fig. 11 shows the results when various proteins shown above each lane were used as E2. FIG. 10 shows the results of measuring the activity of the KPC1-KPC2 complex to ubiquitinate p27 Kipl . (-) Shows the results when E2 was not added.
第 12図 第 12図は、 KPC1-KPC2複合体の、 p27Kiplおよび ρ27κίρ1のリン酸化部位変 異体 （S10A、 T187A, S10E、 T187E) をュビキチン化する活性を測定した結果を示 す。 反応の +はインビトロ再構成系の反応を行った塲合、 —はインビトロ再構成 系の構成成分を添加せず反応を行わなかったことを示す。 FIG. 12 shows the results of measuring the activity of the KPC1-KPC2 complex to ubiquitinate p27 Kipl and ρ27 κίρ1 phosphorylation site variants (S10A, T187A, S10E, T187E). In the reaction, + indicates that the reaction of the in vitro reconstitution system was performed, and-indicates that the reaction was not performed without adding the components of the in vitro reconstitution system.
第 13図 第 13図は、 KPC1- KPC2複合体、 KPC1( ΔΙΙ)- KPC2複合体を高発現させた 細胞における p27Kiplの分解を解析した結果を示す。 第 13図 aは leptomycin B非存 在下、 第 13図 bは leptomycin B存在下で蛋白質の核外移行を阻害した場合を示す 。 左は SDS- PAGEによる解析で上段は抗 p27Kipl抗体、 下段は抗 GSK- 3^抗体による ィムノブロットを示す。 右は、 p27Kiplの量の経時的な分解を表すグラフで、 横軸 は時間、 縦軸は 0時間を 100 %としたときの残存する p27Kiplの量 （％) を示し、 〇 は KPC1- KPC2複合体、 口は KPCl( AIl)- KPC2複合体をそれそれ発現させた細胞、 ▲ はコントロールのベクターのみを導入した細胞での結果を示す。 KPCl(WT)- KPC2 は KPC1- KPC2複合体、 KPC1( AR)- KPC2は KPC1( AR)-KPC2複合体をそれそれ発現さ せた細胞、 コントロールはコントロールのベクタ一のみを導入した細胞を表す。 第 14図 第 14図は、 RNAiによる KPC1の発現の抑制と p27Kiplの分解を解析した結 果を示す。 上段は、 KPC1 siRNA発現べクタ一を導入した細胞における KPC1および KPC2の発現の解析を示す。 左は抗 KPC1抗体、 右は抗 KPC2抗体によるィムノブロヅ トを示し、 KPC1は KPC1 siRNA発現べクタ一を導入した細胞、 EGFPは、 コント口一 ルの EGFP siRNA発現ベクターを導入した細胞における解析を示す。 下段は、 KPC1 siRNA発現ベクターを導入した細胞における p27Kiplの経時的な分解の解析を示す 。 上の 2つの段は抗 p27Kipl抗体、 下の 2つの段は抗 GSK- 3 5抗体を用いたィムノ ブロッ トを示し、 RNAi :KPClは KPC1 siRNA発現べクタ一を導入した細胞、 RNAi :EGFPは、 コントロールの ECTP siRNA発現べクタ一を導入した細胞における 解析を示す。 FIG. 13 FIG. 13 shows the results of analyzing the degradation of p27 Kipl in cells overexpressing the KPC1-KPC2 complex and the KPC1 (ΔΙΙ) -KPC2 complex. Fig. 13a shows the case where the nuclear export of the protein was inhibited in the absence of leptomycin B, and Fig. 13b shows the case where the nuclear export of the protein was inhibited in the presence of leptomycin B. The left panel shows the results of SDS-PAGE analysis, and the upper panel shows the anti-p27 Kipl antibody and the lower panel shows the immunoblot using the anti- GSK -3 ^ antibody. On the right is a graph showing the decomposition of the amount of p27 Kipl over time. The horizontal axis is time, the vertical axis is the amount (%) of p27 Kipl remaining when 0 hour is 100%, and 〇 is KPC1- The results of the KPC2 complex, the results of the cells in which the KPCl (AIl) -KPC2 complex was expressed, and the results of the cells in which the control vector alone was introduced are shown. KPCl (WT) -KPC2 indicates KPC1-KPC2 complex, KPC1 (AR) -KPC2 indicates cells expressing KPC1 (AR) -KPC2 complex, and control indicates cells into which only one control vector was introduced. . FIG. 14 shows the results of analyzing the suppression of KPC1 expression by RNAi and the degradation of p27 Kipl . The upper part shows the analysis of KPC1 and KPC2 expression in cells into which the KPC1 siRNA expression vector has been introduced. Left shows anti-KPC1 antibody and right shows immunoblotting with anti-KPC2 antibody. . The lower part shows an analysis of the time-dependent degradation of p27 Kipl in cells into which the KPC1 siRNA expression vector has been introduced. The upper two rows show the anti-p27 Kipl antibody, and the lower two rows show the immunoblots using the anti- GSK - 35 antibody. Indicates analysis in cells into which a control ECTP siRNA expression vector was introduced.
第 15図 第 15図は、 RNAiにより KPC1の発現を抑制した細胞の、 各種蛋白質の量を 経時的に解析した結果を示す。 上から 2段ずつそれぞれ、 抗 p27Kipl抗体、 抗サイ クリン A抗体、 抗 GSK- 3 ?抗体、 抗 KPC1抗体、 抗 KPC2抗体を用いたィムノブロット による、 レーンの上に示す各時間における蛋白質量 0解析を示す。 KPC1は KPC1 siMA発現べクタ一を導入した細胞、 EOTPは、 コントロールの EGFP siRNA発現べ クタ一を導入した細胞における解析を示す。 FIG. 15 FIG. 15 shows the results of a time-course analysis of the amounts of various proteins in cells in which KPC1 expression was suppressed by RNAi. Analysis of protein mass at each time on lane by immunoblot using anti-p27 Kipl antibody, anti- cyclin A antibody, anti-GSK-3? Antibody, anti-KPC1 antibody, and anti-KPC2 antibody Is shown. KPC1 is KPC1 The cells transfected with the siMA expression vector and EOTP show the analysis in the cells transfected with the control EGFP siRNA expression vector.
第 16図 第 16図は、 RNAiにより KPC1の発現を抑制した細胞の、 細胞増殖を示すグ ラフで、 横軸は時間 （h ) 、 縦軸は細胞数を示す。 園は KPC1 siRNA発現ベクター を導入した細胞、 秦は、 コントロールの EGFP siRNA発現ベクターを導入した細胞 における細胞数を示す。 発明を実施するための最良の形態 FIG. 16 FIG. 16 is a graph showing cell proliferation of cells in which KPC1 expression was suppressed by RNAi, wherein the horizontal axis indicates time (h) and the vertical axis indicates the number of cells. Garden shows the number of cells transfected with the KPC1 siRNA expression vector, and Hata shows the number of cells transfected with the control EGFP siRNA expression vector. BEST MODE FOR CARRYING OUT THE INVENTION
[実施例 1 ] p27Kiplに対する新規なュビキチンリガーゼ複合体 KPCの単離、 精製 とその構成成分の解析 [Example 1] Isolation and purification of a novel ubiquitin ligase complex KPC for p27 Kipl and analysis of its components
ゥサギ網状赤血球抽出液より、 p27Kiplをュビキチン化する活性を指標にして新 規ュビキチンリガーゼ複合体を分離、 精製した。 ゥ A new ubiquitin ligase complex was separated and purified from heron reticulocyte extract using the activity of ubiquitination of p27 Kipl as an index.
( 1 )p27Kiplのュビキチン化反応のィンビトロ再構成系の構築および p27Kiplをュビキ チン化する活性の測定 (1) Construction of an in vitro reconstitution system for p27 Kipl ubiquitination and measurement of p27 Kipl ubiquitination activity
以下に記載する、 ュビキチン化反応に必要な分子については、 既報の方法 〔J. Biol . Chem.， 276, 48937 (2001)〕 に従い大腸菌で発現、 精製した組換え蛋白質 を利用した。 ュビキチン化反応の E1分子である酵母 (Saccharomyces cerevisiae ) Ubalは、 N末、 C末にそれそれ Mycタグ、 His6タグを融合した蛋白質として、 E2 分子であるヒト UbcH5Aは N末、 C末にそれそれ His6タグ、 FLAGタグを融合した蛋白 質として、 またマウスュビキチンはグル夕チオン S-トランスフェラーゼ （GST) 融合蛋白質 （以下、 GST-Ubと略す） として、 いずれも大腸菌 BL21 (DE3) pLysS株 (ノバジェン社製） で発現し、 ProBond resine (インビトロジェン社製）あるいは グル夕チオンーセファロース CL- 4B (アマシャム 'バイオサイェンシズ社製） に て精製を行った。 反応の基質になる p27Kiplは、 マウス p27Kiplを GST融合蛋白質とし て上記大腸菌で発現し、 グル夕チオンビーズ （アマシャム 'バイオサイェンシズ 社製） で精製を行った後、 さらに PreScissionプロテアーゼ （アマシャム 'バイ ォサイエンシズ社） で GST部分を切断、 除去し、 ミニ Qカラム （アマシャム 'バイ ォサイエンシズ社) にて精製した標品を利用した。 For the molecules required for the ubiquitination reaction described below, recombinant proteins expressed and purified in Escherichia coli according to the previously reported method [J. Biol. Chem., 276, 48937 (2001)] were used. The yeast (Saccharomyces cerevisiae) Ubal, which is the E1 molecule of the ubiquitination reaction, is a N-terminal, C-terminal, Myc-tag, and His6-tag-fused protein, and the E2 molecule, human UbcH5A, is an N-terminal, C-terminal As a protein fused with His6 tag and FLAG tag, and mouse ubiquitin as glutathione S-transferase (GST) fusion protein (hereinafter abbreviated as GST-Ub), both E. coli BL21 (DE3) pLysS strain (Novagen) ), And purified using ProBond resine (Invitrogen) or Guruya Thion-Sepharose CL-4B (Amersham's Biosciences). P27 Kipl , which serves as a substrate for the reaction, was expressed in the above Escherichia coli using mouse p27 Kipl as a GST fusion protein, purified with glutathione beads (Amersham, manufactured by Biosciences), and further purified with PreScission protease (Amersham). The GST portion was cleaved and removed with Biosciences, Inc., and a sample purified using a mini Q column (Amersham's Biosciences) was used.
反応は、 活性を測定する試料、 50 ngの Ubal、 100 ngの UbcH5A、 3 〃gの GST- Ub 、 50 ngの p27Kiplを含む 10 〃Lの反応緩衝液 〔40 nnnol/L Hepes-NaOH (pH 7.9)、 60 腿 ol/L酢酸カリウム、 0.5 腿 ol/L EGTA、 腿 ol/いジチオスレィトール （以下 DTTと略す） 、 5 腦 ol/L塩化マグネシウム、 10% (ν/ν)グリセロール、 1.5 雇 ol/L ATP) 中で、 26°Cで 30分間行った。 The reaction was performed using a 10 μL reaction buffer (40 nnnol / L Hepes-NaOH containing 50 ng of Ubal, 100 ng of UbcH5A, 3 μg of GST-Ub, and 50 ng of p27 Kipl ). pH 7.9), 60 thigh ol / L potassium acetate, 0.5 thigh ol / L EGTA, thigh ol / dithiothreitol (hereinafter abbreviated as DTT), 5 brain ol / L magnesium chloride, 10% (ν / ν) glycerol, 1.5 employment ol / L ATP) for 30 minutes at 26 ° C.
p27KiPlをュビキチン化する活性は、 反応サンプルの SDS-PAGEを行った後、 抗 p27Kipl抗体 （モノクローナル抗体、 トランスダクシヨン 'ラボラトリ一社製） を 用いたィムノブロット解析により、 ュビキチン化された p27Kipl分子を検出するこ とにより測定した。 本方法は、 以下に述べる精製フラクションのァヅセィだけで なく、 組換え KPC1、 KPC2、 KPC1- KPC2複合体の p27Kiplをュビキチン化する活性を 評価するすべての実験に共通する方法として利用した。 activity Yubikichin the p 27 Ki Pl, after SDS-PAGE was carried out in the reaction sample, the anti-p27 Kipl antibodies by Imunoburotto analysis using (monoclonal antibodies, trans duct Chillon 'one company Ltd. Laboratory) were Yubikichin of It was determined by detecting the p27 Kipl molecule. This method was used as a method common to all experiments for evaluating the activity of the recombinant KPC1, KPC2, and KPC1-KPC2 complex to ubiquitinate p27 Kipl, as well as the purified fraction described below.
(2)p27KipIに対する新規なュビキチンリガーゼ複合体 KPCの精製 (2) Purification of a novel ubiquitin ligase complex KPC for p27 KipI
Hershkoらの方法 〔J. Biol. Chem.， 258, 8206 ( 1983)〕 で調製したゥサギ網 状赤血球抽出液 （約 20 gの蛋白質量） を緩衝液 A 〔50 腿 ol/L Tris-HCl (7.4)、 0.1 腿 ol/L DTT、 10% (v/v)グリセロール〕 で処理した DE52レジン 700 mLと 45分 間混合した。 直径 10cmのカラムに充填後、 緩衝液 Aで洗浄、 100および 300 雇 ol/L 塩化カリウムを含む緩衝液 Aにて段階溶出を行い、 それそれ 140 mLずつを集めた  A heron reticulocyte extract (approximately 20 g of protein) prepared by the method of Hershko et al. [J. Biol. Chem., 258, 8206 (1983)] was used in buffer A [50 t / l Tris-HCl ( 7.4), 0.1 t ol / L DTT, 10% (v / v) glycerol] and mixed with 700 mL of DE52 resin for 45 minutes. After packing into a 10 cm diameter column, the column was washed with buffer A, and stepwise eluted with buffer A containing 100 and 300 ol / L potassium chloride, and 140 mL each was collected.
( 1)で記載した方法にて、 各フラクションの p27Kiplをュビキチン化する活性を 測定し、 活性のあるフラクションを集め、 60%硫酸アンモニゥム添加による濃縮 を行い、 得られた沈殿物を 150 纖 ol/L塩化カリウムを含む lOmLの緩衝液 Aに懸濁 した。 緩衝液 Aに対し透析を行い、 残留する硫酸アンモニゥムを除去後、 遠心分 離 （12, 000 xg、 15分） にて回収された上清を 150 腿 ol/L塩化カリウムを含む緩 衝液 Aにて前処理したスーパ一デゾクス （Superdex) 200ゲルろ過カラム （アマシ ャム バイオサイェンシズ社製) に 10 mL/hの速度で通塔した。 p27Kiplをュビキチ ン化する活性を示すフラクションを集め、 60, 000 xgで 20分間遠心分離後、 得ら れた上清を緩衝液 P [5 雇 ol/Lリン酸カリウム（pH7.4)、 50 麗 ol/L塩化ナトリゥ ム、 0.1 藤 ol/L DTT、 10% (v/v)グリセロール] で前処理したセラミック 'ハイ ドロキシァパタイト 'タイプ 1カラム （バイオラッド社製） に通塔した。 5-300 顧 ol/Lのリン酸カリウムを含む 60 mLの緩衝液 Pによる連続的溶出を行い、 2 mLず つのフラクションを回収した。 According to the method described in (1), the activity of each fraction to ubiquitinate p27 Kipl was measured, and the active fractions were collected, concentrated by adding 60% ammonium sulfate, and the resulting precipitate was collected using 150 fiber ol. The suspension was suspended in 10 mL of buffer A containing 1 / L potassium chloride. After dialysis against buffer A to remove residual ammonium sulfate, the supernatant collected by centrifugation (12,000 xg, 15 minutes) was added to buffer A containing 150 t ol / L potassium chloride. The column was passed through a Superdex 200 gel filtration column (manufactured by Amersham Biosciences) at a rate of 10 mL / h. Fractions showing the activity to ubiquitinate p27 Kipl were collected, centrifuged at 60,000 xg for 20 minutes, and the resulting supernatant was added to buffer P [5 ol / L potassium phosphate (pH 7.4), 50 l ol / L sodium chloride, 0.1 w / ol / L DTT, 10% (v / v) glycerol] and passed through a ceramic 'hydroxyapatite' type 1 column (Bio-Rad). . Continuous elution was performed with 60 mL of buffer P containing 5-300 ol / L potassium phosphate, and 2 mL fractions were collected.
活性を示すフラクション （20-50 腿 ol/Lのリン酸カリウムを含む溶出部分に相 当） を集め、 40 靈 ol/L塩化カリウムを含む緩衝液 Aに対して透析を行った。 60， 000 xgで 20分間遠心分離して得られた上清を回収し、 40 顧 ol/L塩化カリウム を含む緩衝液 Aで前処理したモノ Qカラム （アマシャム 'バイオサイェンシス'社製 ) に通塔した。 40-350 腿 ol/Lの塩化カリウムを含む 30mLの緩衝液 Aによる連続的 溶出を行い、 1 mLずつのフラクションを回収した。 活性を示すフラクション （ 100-160 删 ol/Lの塩化カリウムを含む溶出部分に相当） を集め、 150 腿 ol/L塩化 カリウムを含む緩衝液 Aで前処理したスーパ一ロース （Superose) 6ゲルろ過カラ ム （アマシャム -バイオサイェンシズ社製） に通塔し、 l mLずつのフラクション を回収した。 活性を示すフラクションを集め 80 雇 ol/L塩化カリウムを含む緩衝 液 Aに対して透析後、 60, 000 X gで 20分間遠心分離し、 上清を 80 雇 ol/L塩化カリ ゥムを含む緩衝液 Aで前処理したミニ Q (Mini Q) カラム （アマシャム ·バイオサ イエンシズ社製） に通塔した。 80-350 腿 ol/Lの塩化カリウムを含む 4.5 mLの緩 衝液 Aによる連続的溶出を行い、 150 / 1ずつのフラクションを回収した。 活性は 塩化力リゥム濃度 130-180 腦 ol/Lのフラクションに検出された。 Active fractions (equivalent to the elution area containing 20-50 t ol / L potassium phosphate) This was collected and dialyzed against buffer A containing 40 μl / L potassium chloride. The supernatant obtained by centrifugation at 60,000 xg for 20 minutes is collected, and a mono-Q column (Amersham 'Bioscience') pretreated with buffer A containing 40 ol / L potassium chloride Passed through the tower. Continuous elution was performed with 30 mL of buffer A containing 40-350 t / l of potassium chloride, and fractions of 1 mL each were collected. Collect the active fractions (corresponding to the elution portion containing 100-160 / ol / L potassium chloride) and pretreat with buffer A containing 150 t ol / L potassium chloride. 6 Superfiltration (Superose) 6 gel filtration The mixture was passed through a column (Amersham-Biosciences) and fractions of 1 mL were collected. Collect fractions showing activity, dialyze against Buffer A containing 80 ol / L potassium chloride, centrifuge at 60,000 X g for 20 minutes, and discard supernatant containing 80 ol / L potassium chloride The solution was passed through a Mini Q column (Amersham Biosciences) pretreated with buffer A. Continuous elution was performed with 4.5 mL of Buffer A containing 80-350 t / l of potassium chloride, and 150/1 fractions were collected. The activity was detected in the fraction with a chloride concentration of 130-180 mol / L.
第 1図には精製プロトコ一ルを、 第 2図にはスーパ一ロース 6ゲルろ過カラム、 第 3図にはミニ Qカラムでそれそれ得られた各フラクションの SDS- PAGEによる蛋白 質の解析および ρ27κίρ1をュビキチン化する活性の測定の結果を示している。 活性 と一致する蛋白質として 140kDaと 50kDaのバンドが確認され、 それそれを KPC1お よび KPC2とした。 Fig. 1 shows the purified protocol, Fig. 2 shows a Superose 6 gel filtration column, and Fig. 3 shows the protein analysis by SDS-PAGE of each fraction obtained using the Mini Q column. The result of measurement of the activity of ubiquitinating ρ27κ ρ1 is shown. 140 kDa and 50 kDa bands were identified as proteins that corresponded to the activity, which were designated as KPC1 and KPC2.
(3)KPC1および KPC2の部分アミノ酸配列の決定  (3) Determination of partial amino acid sequences of KPC1 and KPC2
KPC1および KPC2を含むフラクシヨンを 10%SDS-PAGEで分離し、 クマシ一染色で 可視化した後、 各バンドを切り出した （第 4図） 。 ゲル中での還元、 S-カルボキ シアミドメチル化、 トリプシン消化の各処理後、 n RPC C2/C18カラム （アマシ ャム .バイオサイェンシズ社製） にてペプチドを分離回収し、 エドマン分解によ るアミノ酸配列解析を行った。 KPC1のバンドから得られた 11種のぺプチドのアミ ノ酸配列を配列番号 9〜19に、 KPC2のバンドから得られた 2種のぺプチドのァミノ 酸配列を配列番号 20および 21に記載した。  Fractions containing KPC1 and KPC2 were separated by 10% SDS-PAGE, visualized by coomassie staining, and the bands were cut out (Fig. 4). After each reduction, S-carboxamidomethylation and trypsin digestion in the gel, the peptides are separated and recovered on nRPC C2 / C18 column (Amersham Biosciences), and subjected to Edman degradation. Amino acid sequence analysis was performed. The amino acid sequences of 11 peptides obtained from the band of KPC1 are shown in SEQ ID NOS: 9 to 19, and the amino acid sequences of two peptides obtained from the band of KPC2 are shown in SEQ ID NOs: 20 and 21. .
[実施例 2 ] KP,C1および KPC2をコードする cDNAの単離 [Example 2] Isolation of cDNAs encoding KP, C1 and KPC2
実施例 1 (3)で得られた KPC1および KPC2の部分アミノ酸配列をコ一ドしうる塩 基配列を塩基配列デ一夕ペース ·ジェンバンクから検索した。 その結果、 KPC1 cDNAと考えられる塩基配列として、 ジェンパンク登録番号 BE885419およびExample 1 A salt capable of encoding the partial amino acid sequence of KPC1 and KPC2 obtained in (3) The base sequence was searched from the base sequence database GenBank. As a result, the nucleotide sequence considered to be KPC1 cDNA was Genpunk Accession No. BE885419 and
BE885914のヒト ESTの配列を見出した。 これらの ESTの配列を有する IMAGEコンソ ーシアム cDNAク ロ一ン IMAGE :3909169および IMAGE :3909203を、 ResearchThe sequence of human EST of BE885914 was found. The IMAGE consortium cDNA clones with these EST sequences were identified as IMAGE: 3909169 and IMAGE: 3909203 in Research.
Genetics社より入手した。 これらの cDNAクローンの cDNAの塩基配列を解析し、 ヒ ト KPC1 cDNAの塩基配列を明らかにした。 配列番号 1に、 両 cDNAクローンに含まれ るヒト KPC1 cDNAの KPC1をコードする領域の塩基配列を、 配列番号 2に、 配列番号Obtained from Genetics. The nucleotide sequences of these cDNA clones were analyzed, and the nucleotide sequence of human KPC1 cDNA was determined. SEQ ID NO: 1 shows the nucleotide sequence of the KPC1 coding region of the human KPC1 cDNA contained in both cDNA clones, and SEQ ID NO: 2 shows the nucleotide sequence of SEQ ID NO:
1で表される塩基配列がコードするヒト KPC1のアミノ酸配列を示した。 配列番号 1 で表される塩基配列および配列番号 2で表されるアミノ酸配列ともに、 塩基配列 デ—夕ベースおよびアミノ酸配列データベースには同一の配列はなく、 新規な配 列であった。 ヒト KPC1のアミノ酸配列中に実施例 1 (3)で得られた KPC1の部分ァ ミノ酸配列が存在することが確認された （第 5図） 。 The amino acid sequence of human KPC1 encoded by the nucleotide sequence represented by 1 is shown. Both the nucleotide sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2, the nucleotide sequence database and the amino acid sequence database had no identical sequences, and were novel sequences. It was confirmed that the amino acid sequence of human KPC1 contained the partial amino acid sequence of KPC1 obtained in Example 1 (3) (FIG. 5).
また、 ヒト KPC1の cDNAの塩基配列をクエリ一とした塩基配列データベースの相 同性解析から、 マウス KPC1 cDNAの KPC1をコードすると推定される領域の 5'端の 塩基配列と 3'端の塩基配列を見出した。 これらの配列に基づく配列番号 25および 26で表されるそれそれの塩基配列からなる DNA (KPC1-M1, KPC1-M2) をプライマ —とし、 マウス T細胞 cDNAライブラリ一 （クロンテック社製） を錡型とした PCRに より、 マウス KPC1をコードする cDNA断片を増幅し単離した。 この cDNA断片の塩基 配列を解析し、 マウス KPC1 cDNAの塩基配列を明らかにした。 配列番号 3にマウス KPC1 cDNAの KPC1をコードする領域の塩基配列を、 配列番号 4に配列番号 3で表さ れる塩基配列がコードするマウス KPC1のアミノ酸配列を示した。 配列番号 3で表 される塩基配列および配列番号 4で表されるアミノ酸配列ともに、 塩基配列デー 夕べ一スおよびアミノ酸配列データベースには同一の配列はなく、 新規な配列で あった。  Based on the homology analysis of the nucleotide sequence database using the nucleotide sequence of the human KPC1 cDNA as a query, the nucleotide sequence at the 5 'end and 3' end of the region predicted to encode KPC1 in the mouse KPC1 cDNA was determined. I found it. Based on these sequences, DNA (KPC1-M1, KPC1-M2) consisting of the nucleotide sequence represented by SEQ ID NO: 25 or 26 was used as a primer, and mouse T cell cDNA library (Clontech) was used as type II. The cDNA fragment encoding mouse KPC1 was amplified and isolated by PCR. By analyzing the nucleotide sequence of this cDNA fragment, the nucleotide sequence of mouse KPC1 cDNA was determined. SEQ ID NO: 3 shows the nucleotide sequence of the region encoding KPC1 of mouse KPC1 cDNA, and SEQ ID NO: 4 shows the amino acid sequence of mouse KPC1 encoded by the nucleotide sequence represented by SEQ ID NO: 3. Both the nucleotide sequence represented by SEQ ID NO: 3 and the amino acid sequence represented by SEQ ID NO: 4 were novel sequences, with no identical sequences in the nucleotide sequence database and the amino acid sequence database.
KPC1はヒト、 マウスとも 1314アミノ酸からなり、 132〜253番目に SPRYドメイン が、 C末に近い 1254〜1291番目に SCF複合体、 APC/C複合体、 VHL複合体等の RING型 ュビキチンリガ一ゼに共通して存在する RINGフィンガ一ドメインが存在した （第 5図 a ) 。  KPC1 is composed of 1314 amino acids in both human and mouse, and the SPRY domain is in the 132-253 position, and the RING-type ubiquitin ligase such as the SCF complex, APC / C complex and VHL complex is in the 1254-1291 position near the C-terminal There was a common RING finger domain (Figure 5a).
またゥサギ KPC2は、 その部分アミノ酸配列から、 ヒトの神経膠芽腫細胞分化因 子関連 (glioblastoma cell differentiati on factor-related protein、 GBDR1 ) 蛋白質 〔Li， Cつ et al .， Genomics 65, 243 (2000)〕 のオルソログと推定され た。 したがって、 ヒト KPC2をコードする cDNAはヒト GBDR1 cDNAと同一であり、 ヒ ト GBDR1 cDNAの塩基配列 （ジェンバンク登録番号 AF 176796 ) に基づく配列番号 27 および 28で表されるそれそれの塩基配列からなる DNA (KPC2-Hls KPC2-H2) をプ ライマーとし、 ヒト肝臓 cDNAライブラリー （クロンテック社製） を錶型として用 いた PCI こより、 その 0RFの領域を、 ヒト KPC2をコードする cDNA断片として取得し た。 なお、 この PCRにより得られた DNA断片は、 実施例 3に示した昆虫細胞での発 現に用いるため、 N末に、 プライマ一 KPC2- HIに由来する HSVタグが付加したヒト KPC2をコードし、 53端に lサイト、 3'端に lサイ トを有する。 From the partial amino acid sequence of Escherichia coli KPC2, human glioblastoma cell differentiation factor-related protein (GBDR1) It was estimated to be the ortholog of the protein [Li, C et al., Genomics 65, 243 (2000)]. Therefore, the cDNA encoding human KPC2 is identical to the human GBDR1 cDNA and consists of the respective nucleotide sequences represented by SEQ ID NOS: 27 and 28 based on the nucleotide sequence of human GBDR1 cDNA (GenBank Accession No. AF 176796). The DNA (KPC2-Hls KPC2-H2) was used as a primer, and a human liver cDNA library (Clontech) was used as a type II PCI. The 0RF region was obtained as a cDNA fragment encoding human KPC2. . The DNA fragment obtained by this PCR encodes human KPC2 with an HSV tag derived from primer KPC2-HI added to the N-terminus, for use in the expression in the insect cells shown in Example 3. 5 1 site at 3 end, 1 site at 3 'end.
また、 マウス KPC2.については、 塩基配列デ一夕ベース 'ジェンバンクからマウ ス GBDR1 cDNAの 5'側の ESTの配列 （ジェンバンク登録番号： BE137808) を見出し 、 この ESTの配列を有する IMAGEコンソーシアム cDNAクローン IMAGE： 1547481を、 Research Genetics社より入手した。 この cDNAクローンの cDNAの塩基配列を解析 し、 マウス KPC2 cDNAの塩基配列を明らかにした。 配列番号 7に、 この cDNAクロ一 ンに含まれるマウス KPC2 cDNAの KPC2をコードする領域の塩基配列を、 配列番号 8 に、 配列番号 7で表される塩基配列がコードするマウス KPC2のァミノ酸配列を示 した。 KPC2は N末側に ubiquitin- likeドメイ ン、 また 2ケ所の ubiquitin- associatedドメインを有していた （第 5図 a ) 。  In addition, for mouse KPC2., We found the EST sequence (GenBank accession number: BE137808) on the 5 'side of mouse GBDR1 cDNA from GenBank, and identified the IMAGE consortium cDNA containing this EST sequence. Clone IMAGE: 1547481 was obtained from Research Genetics. The nucleotide sequence of the cDNA of this cDNA clone was analyzed to determine the nucleotide sequence of mouse KPC2 cDNA. SEQ ID NO: 7 shows the nucleotide sequence of the KPC2 coding region of the mouse KPC2 cDNA contained in this cDNA clone. SEQ ID NO: 8 shows the amino acid sequence of mouse KPC2 encoded by the nucleotide sequence represented by SEQ ID NO: 7 showed that. KPC2 had a ubiquitin-like domain on the N-terminal side and two ubiquitin-associated domains (Fig. 5a).
[実施例 3 ] 組換え KPC1- KPC2複合体による p27Kiplのポリュビキチン化 [Example 3] Polyubiquitination of p27 Kipl by recombinant KPC1-KPC2 complex
実施例 2で取得した cDNAがコ一ドするヒト KPC1および KPC2が、 p27Kiplを基質と するュビキチンリガーゼ複合体を構成することを検証するため、 該 cDNAを用い組 換え KPC1、 KPC2を調製し、 KPC1- KPC2複合体の p27Kiplをュビキチン化する活性を 測定した。 To verify that human KPC1 and KPC2 encoded by the cDNA obtained in Example 2 constitute a ubiquitin ligase complex using p27 Kipl as a substrate, recombinant KPC1 and KPC2 were prepared using the cDNA. The activity of the KPC1-KPC2 complex to ubiquitinate p27 Kipl was measured.
( 1 )組換え KPC1- KPC2複合体の調製 ，  (1) Preparation of recombinant KPC1-KPC2 complex,
配列番号 29および 30で表される配列からなる DNA (KPCl-H-ls KPC1-H-Z) をプ ライマーとし、 ヒト KPC1 cDNAクローン IMAGE :3909169を錶型とした PCRにより、 N 末に FLAGタグを付加したヒト KPC1をコードする DNAを増幅した。 一方、 昆虫細胞 発現用トランスファーベクタ一 pBacPAK9 (クロンテック社製） の サイ ト間に、 ベクタ一 pRSET B (インビトロジェン社製） His6タグをコードする領域 含む Xbal- Xhol断片 （約 140bp) を挿入した。 このべクタ一の サイ ト間 に、 上記の KPC1増幅断片を 5'にある I、 3'端にある^ Iで切断後、 揷入するこ とにより、 pBacPAOに、 N末に配列番号 22で表されるアミノ酸配列からなる His6/FLAG夕グを付加したヒト KPC1をコードする DNAが揷入されたトランスファー ベクタ一を作製した。 Using a DNA consisting of the sequences represented by SEQ ID NOS: 29 and 30 (KPCl-H-ls KPC1-HZ) as a primer and adding a FLAG tag to the N-terminal by PCR using human KPC1 cDNA clone IMAGE: 3909169 as type III DNA encoding human KPC1 was amplified. On the other hand, the region encoding the vector pRSETB (Invitrogen) His6 tag is located between the transfer vector pBacPAK9 (Clontech) site for insect cell expression. Xbal-Xhol fragment (about 140 bp) was inserted. Between the sites of this vector, the above KPC1 amplified fragment was digested with I at the 5 'and ^ I at the 3' end, and then inserted into pBacPAO and SEQ ID NO: 22 at the N-terminus. A transfer vector into which DNA encoding human KPC1 to which His6 / FLAG fragment having the amino acid sequence represented was added was prepared.
また、 実施例 2で増幅したヒト KP02 cDNA断片は、 HSVタグを N末に付加したヒ ト KPC2をコードしているので、 KPC1増幅断片を 5，端にある I、 33端にある Xhol で切断後、 同様に、 上記の His6タグをコードする領域を挿入した pBacPA の M I/ i lサイト間に挿入することにより、 N末に配列番号 23で表されるアミノ酸 配列からなる His6/HSVタグを付加したヒト KPC2をコードする DNAが挿入されたト ランスファーべクタ一を作製した。 Also, human KP02 cDNA fragment amplified in Example 2, since the encoding human KPC2 added with HSV tag at the N-terminal, with Xhol with the KPC1 amplified fragment 5, the I, 3 3 ends at the end After cleavage, similarly, a His6 / HSV tag consisting of the amino acid sequence represented by SEQ ID NO: 23 is added to the N-terminus by inserting between the MI / il sites of pBacPA into which the region encoding the His6 tag described above has been inserted. A transfer vector into which DNA encoding human KPC2 was inserted was prepared.
得られたトランスファ一ベクターそれそれと BacPAKバキュロウィルス発現シス テム（クロンテック社製）とを用いて作製した組換えバキュロウィルスを Sf21細胞 に共感染させ、 既報の方法 CKamura et al. , Genes Dev. , 13, 2928 (1999)〕 に 従い、 KPC1-KPC2複合体の発現ならびに精製を行った。 第 6図には、 精製した KPC1 - KPC2複合体を SDS-PAGEにて分離しクマシ一染色した結果を示した。  Sf21 cells were co-infected with the recombinant baculovirus prepared using the obtained transfer vector and the BacPAK baculovirus expression system (manufactured by Clontech), and the method described in CKamura et al., Genes Dev., 13 , 2928 (1999)], and expressed and purified the KPC1-KPC2 complex. FIG. 6 shows the results of separating the purified KPC1-KPC2 complex by SDS-PAGE and staining with Kumashi.
(2) KPC1と KPC2の結合能の確認  (2) Confirmation of binding ability between KPC1 and KPC2
KPC1および KPC2を発現させた Sf21細胞の細胞抽出液について、 FLAG夕グに対す る抗体およびプロテイン A-セファロ一スビーズ （シグマ社製） を用いて KPC1を免 疫沈降し、 共沈する KPC2の有無を HSVタグに対する抗体で検出した。 その結果、 KPC1に対する免疫沈降物中に KPC2が検出され、 両蛋白質が細胞内で結合している ことが明らかになった （第 7図） 。  KPC1 is immunoprecipitated from the cell extract of Sf21 cells expressing KPC1 and KPC2 using antibodies against FLAG and protein A-Sepharose beads (manufactured by Sigma). Was detected with an antibody against the HSV tag. As a result, KPC2 was detected in the immunoprecipitate against KPC1, and it was revealed that both proteins were bound intracellularly (Fig. 7).
(3) KPC; - KPC2複合体と p27 plの結合 (3) KPC;-binding of KPC2 complex to p27 pl
実施例 1にて取得した大腸菌発現の組換え p27Kiplと、 （1)で調製した組換え KPC1- KPC2複合体を混ぜ合わせた。 p27Kipiに対する抗体で免疫沈降を行った後、 FLAG夕グに対する抗体および HSVタグに対する抗体をそれそれ用いて KPC1および KPC2を検出した。 その結果、 免疫沈降物中に KPC1- KPC2複合体が含まれることが 確認された （第 8図） 。 この結果は、 KPC1- KPC2複合体と p27Kiplが互いに結合する 分子であること示している。 The recombinant p27 Kipl expressed in Escherichia coli obtained in Example 1 and the recombinant KPC1-KPC2 complex prepared in (1) were mixed. After immunoprecipitation with an antibody against p27 Kipi, KPC1 and KPC2 were detected using an antibody against FLAG and an antibody against HSV tag, respectively. As a result, it was confirmed that the immunoprecipitate contained the KPC1-KPC2 complex (FIG. 8). This result indicates that the KPC1-KPC2 complex and p27 Kipl are molecules that bind to each other.
(4)組換 KPC1- KPC2複合体または KPC1による p27Kiplのポリュビキチン化 KPC1-KPC2複合体が実際に p27Kiplのポリュビキチン化におけるュビキチンリガ ーゼとして機能するかを以下のようにして調べた。 （1)で調製した組換え KPC1- KPC2複合体 200 ngと 50 ngの組換え p27Kiplを用い、 実施例 1 ( 1)に記載したュビキ チン化反応を行ない、 反応後のサンプルを SDS— PAGEにて解析した結果を第 9図左 に示した。 El(Ubal)および E2(UbcH5A)共存下で明らかなポリュビキチン化が検出 され、 KPC1- KPC2複合体が p27Kiplポリュビキチン化におけるュビキチンリガーゼ として働くことが確認された。 (4) Polyubiquitination of p27 Kipl by recombinant KPC1-KPC2 complex or KPC1 Whether or not the KPC1-KPC2 complex actually functions as ubiquitin ligase in polyubiquitination of p27 Kipl was examined as follows. Using 200 ng of the recombinant KPC1-KPC2 complex prepared in (1) and 50 ng of the recombinant p27 Kipl , the ubiquitination reaction described in Example 1 (1) was performed, and the sample after the reaction was subjected to SDS-PAGE. The results of the analysis in Fig. 9 are shown on the left of Fig. 9. Apparent polyubiquitination was detected in the presence of El (Ubal) and E2 (UbcH5A), confirming that the KPC1-KPC2 complex acts as a ubiquitin ligase in p27 Kipl polyubiquitination.
また、 （1)で作製した KPC1発現用トランスファ一ベクタ一に由来する組換えバ キュロウィルスのみを感染させた Sf21細胞から精製した KPC1を、 KPC1-KPC2複合 体の代わりに用いて同様の反応を行なった。 その結果、 KPC1単独では KPC1- KPC2 複合体と比較して、 ュビキチンが 1つだけ付加した p27Kiplの量はほとんど変わら ないが、 ポリュビキチン化した p27Kiplの量は明らかに多く、 効率的に p27Kiplがポ リュビキチン化を受けることがわかった （第 10図） 。 したがって KPC1は、 単独で p27 plをュビキチン化する活性を有するュビキチンリガーゼであることがわかつ た。 また、 KPC2は、 1つだけュビキチンが付加した p27Kiplをさらにポリュビキチ ン化する反応を阻害する可能性がある。 The same reaction was performed using KPC1 purified from Sf21 cells infected with only the recombinant baculovirus derived from the transfer vector for KPC1 expression prepared in (1), instead of the KPC1-KPC2 complex. Done. As a result, the KPC1 alone compared to KPC1- KPC2 complex, but Yubikichin is hardly changes the amount of p27 Kipl was added only one, the amount of p27 Kipl that Poryubikichin of apparently much efficiently p27 Kipl Was found to undergo polyubiquitination (Fig. 10). Therefore, it was found that KPC1 was a ubiquitin ligase having an activity of ubiquitinating p27 pl by itself. In addition, KPC2 may inhibit the reaction to further polyubiquitinate p27 Kipl to which only one ubiquitin has been added.
KPC1 -KPC2複合体の ρ27Κίρ1に対する特異性を検証するため、 Saccharomyces serevisiaeの CDKインヒビ夕一である Siclに対するポリュビキチン化作用の有無 を検討した。 Siclは His6/HPC4タグ化蛋白質として既報の方法 〔Ka ira et al.， Science 284, 657 (1999)〕 にて大腸菌で発現した蛋白質を用いた。 p27Kiplの代 わりに Siclを用いて上記と同様の反応を行い、 解析した。 その結果、 p27Kiplと異 なり Siclのポリュビキチン化は見られないことから （第 9図右） 、 KPC1-KPC2複合 体は p27Kiplに選択性を有するュビキチンリガーゼ複合体であることが確認された In order to verify the specificity of the KPC1-KPC2 complex for ρ27 有無 ρ1, the presence or absence of polyubiquitination of Saccharomyces serevisiae on Sicl, a CDK inhibitor, was examined. Sicl used was a protein expressed in Escherichia coli according to a previously reported method [Ka ira et al., Science 284, 657 (1999)] as a His6 / HPC4-tagged protein. The same reaction as above was performed using Sicl instead of p27 Kipl and analyzed. As a result, since polyubiquitination of Sicl was not observed unlike p27 Kipl (Fig. 9, right), it was confirmed that the KPC1-KPC2 complex is a ubiquitin ligase complex having selectivity for p27 Kipl .
(5)p27Kiplのポリュビキチン化における KPC1の RINGフィンガ一ドメインの重要性(5) Importance of the RING finger domain of KPC1 in polyubiquitination of p27 Kipl
KPC1の C末に存在する RINGフィンガ一ドメインの機能を調べるため、 RINGフィ ンガ一ドメインを欠損した変異型 KPC1 〔以下 KPCl( AR)と称す〕 を昆虫細胞で発 現させた。 配列番号 29および 31で表される塩基配列からなる DNA (KPCl-H-ls KPC1-H-3) をプライマ一とし、 ヒト KPC1 cDNAクローン IMAGE :3909169を錶型とし た PCRにより、 KPCl( AR) (配列番号 2の 1〜； 1253番目のアミノ酸配列からなる） の N末に FLAGタグを付加した蛋白質をコードする DNA断片を増幅した。 この DNA断 片を 5'にある Ι、 3'端にある 1で切断後、 （1 )で作製した His6タグをコード する領域を挿入した pBacPAOの li I/ klサイ ト間に挿入することにより、 pBacPAK9に、 N末に His6/FLAGタグを付加した KPC1 (厶 R)をコードする DNAが挿入さ れたトランスファーベクタ一を作製した。 このトランスファーベクタ一、 および ( 1 )で作製した、 N末にアミノ酸配列からなる His6/HSVタグを付加したヒト KPC2を コ一ドする DNAが揷入されたトランスファ一ベクタ一を用いて、 （1 )と同様にして 、 KPCl ( AR)- KPC2複合体を発現する Sf21細胞を作製した。 KPC1 ( AI -KPC2複合体 を発現する Sf21細胞の細胞抽出液について、 （2)と同様に、 FLAGタグに対する抗 体で KPCl( AR)を免疫沈降し、 共沈する KPC2の有無を検出した。 その結果、 KPC1( 厶 R)に対する免疫沈降物中に KPC2が検出され、 KPC2との結合には RINGフィンガ一 ドメインが関与していないことわかった （第 7図） 。 In order to examine the function of the RING finger domain present at the C-terminus of KPC1, mutant KPC1 lacking the RING finger domain [hereinafter referred to as KPCl (AR)] was expressed in insect cells. DNA (KPCl-H-ls KPC1-H-3) consisting of the nucleotide sequence represented by SEQ ID NO: 29 or 31 was used as a primer, and KPCl (AR) was obtained by PCR using human KPC1 cDNA clone IMAGE: 3909169 as type III. (1 ~ of SEQ ID NO: 2; consisting of the amino acid sequence at position 1253) A DNA fragment encoding a protein having a FLAG tag added to the N-terminus was amplified. After cutting this DNA fragment at the 5 'Ι and 1 at the 3' end, insert it into the liI / kl site of pBacPAO into which the region encoding the His6 tag prepared in (1) was inserted. Then, a transfer vector was prepared in which DNA encoding KPC1 (mR) having a His6 / FLAG tag added to the N-terminal was inserted into pBacPAK9. Using this transfer vector and the transfer vector into which DNA encoding human KPC2 added with a His6 / HSV tag consisting of an amino acid sequence at the N-terminus and inserted into the N-terminal was prepared, ), Sf21 cells expressing the KPCl (AR) -KPC2 complex were prepared. In the same manner as in (2), KPC1 (AR) was immunoprecipitated with an antibody against a FLAG tag in the cell extract of Sf21 cells expressing the KPC1 (AI-KPC2 complex, and the presence or absence of co-precipitated KPC2 was detected. As a result, KPC2 was detected in the immunoprecipitate against KPC1 (mR), indicating that the RING finger domain was not involved in binding to KPC2 (FIG. 7).
KPCl ( AR)- KPC2複合体を用いて、 （4)に記載した方法により p27Kiplのポリュビ キチン化反応を行った。 第 10図に示すように、 野生型 KPC1- KPC2複合体で見られ るュビキチン化が KPC1( ΔΙ - KPC2複合体では検出されず、 ポリュビキチン化反応 において KPC1の RINGフィンガ一ドメインが必須であることが確認された。 Using the KPCl (AR) -KPC2 complex, p27 Kipl was subjected to polyubiquitination by the method described in (4). As shown in Fig. 10, the ubiquitination observed in the wild-type KPC1-KPC2 complex was not detected in the KPC1 (ΔΙ-KPC2 complex, and the RING finger domain of KPC1 may be essential in the polyubiquitination reaction. confirmed.
(6) KPC1-KPC2複合体によるポリュビキチン化反応における E2の選択性  (6) Selectivity of E2 in polyubiquitination reaction by KPC1-KPC2 complex
文献 〔J. Biol . Chem. , 276, 33111 (2001 ) ; J. Biol . Chem. , 276, 48937 (2001 )〕 に記載の方法に従い、 N末に His6タグを付加した各種組換えヒト E2 ( Ubc2A、 Ubc3、 Ubc4、 UbcH6、 UbcH7、 UbcH8) を調製し、 それらを用いた p27Kiplの ポリュビキチン化反応により、 どの E2が KPC1- KPC2複合体の反応に関与するかを 解析した。 その結果、 KPC卜 KPC2複合体によるュビキチン化反応においては Ubc4 および UbcH5Aが関与することが明らかになった （第 11図） 。 Chem., 276, 33111 (2001); J. Biol. Chem., 276, 48937 (2001)]. Ubc2A, Ubc3, Ubc4, UbcH6, UbcH7, UbcH8) were prepared, and the E27 involved in the KPC1-KPC2 complex reaction was analyzed by porubiquitination of p27 Kipl using these. As a result, it was revealed that Ubc4 and UbcH5A are involved in the ubiquitination reaction by the KPC-KPC2 complex (Fig. 11).
(7) KPC1 -KPC2複合体によるポリュビキチン化反応に対する p27Kiplのリン酸化の影 響 (7) Effect of phosphorylation of p27 Kipl on polyubiquitination reaction by KPC1-KPC2 complex
p27Kiplの細胞内局在と分解調節は、 p27Kiplの 2個所のアミノ酸部位でのリン酸 化 （10番目のセリン、 187番目のスレオニン；それそれ Ser- 10, Thr- 187と略す） が重要であることが報告されている。 KPC1-KPC2複合体によるポリュビキチン化 反応に対するそれらアミノ酸のリン酸化の影響を見る目的で、 リン酸化部位のァ ミノ酸を置換した変異型 p27Kiplを作製し、 KPC卜 KPC2複合体によるポリュビキチ ン化を解析した。 degradation regulating intracellular localization of p27 Kipl is phosphorylation at amino acid position two places of p27 Kipl (10 serine, 187th threonine; it it Ser- 10, abbreviated as Thr- 187) is important Is reported. In order to examine the effect of phosphorylation of these amino acids on the polyubiquitination reaction by the KPC1-KPC2 complex, a mutant p27 Kipl in which the amino acid at the phosphorylation site was substituted was prepared, and polyubiquitination by the KPC-KPC2 complex was performed. Was analyzed.
変異型 p27Kiplとして、 Ser- 10、 Thr-187をそれぞれァラニンに置換したもの （ それそれ S10A、 T187Aと称す） とグルタミン酸に置換したもの （それそれ S10E、 T187Eと称す） を、 実施例 1 ( 1 )に記載した組換え p27Kiplの調製と同手法により調 製した。 これら変異型 p27Kiplを基質としてポリュビキチン化反応を行った結果、 すべての変異型において野生型 p27Kipl同様、 ュビキチン化が行われることが確認 された （第 12図） 。 この結果から、 従来の分解制御機構と異なり KPC1- KPC2複合 体による p27Kiplの分解調節においてはこれらアミノ酸のリン酸化は関与していな いことがわかり、 KPC1-KPC2複合体による反応が新規なメカニズムであることを 支持する結果といえる。 The mutant p27 Kipl was obtained by substituting Ser-10 and Thr-187 with alanine (referred to as S10A and T187A, respectively) and substituting with glutamic acid (respectively referred to as S10E and T187E) in Example 1 ( It was prepared by the same method as the preparation of recombinant p27 Kipl described in 1). As a result of performing a polyubiquitination reaction using these mutant p27 Kipls as substrates, it was confirmed that all mutants were ubiquitinated similarly to wild-type p27 Kipl (Fig. 12). These results show that unlike the conventional degradation control mechanism, phosphorylation of these amino acids is not involved in the regulation of p27 Kipl degradation by the KPC1-KPC2 complex, and the reaction by the KPC1-KPC2 complex is a novel mechanism. This is a result that supports this.
[実施例 4 ] 免疫蛍光染色法による KPC1および KPC2の細胞内局在解析 [Example 4] Intracellular localization analysis of KPC1 and KPC2 by immunofluorescence staining
実施例 2 (1)で作製した KPC1の発現用トランスファーべクタ一の、 His6/FLAG夕 グを付加した KPC1をコードする DNAを含む^ 断片を、 レトロウィルス発 現用べクタ一 pMX-puro CProc. Natl. Acad. Sci. USA, 94, 3938 (1997)〕 の HI/^ Iサイ ト間に挿入し、 KPC1発現用レトロウイルスベクターを作製した。 また、 配列番号 32および 33で表される塩基配列からなる DNA (KPC2-H- 3、 KPC2-H- 4) をプライマ一とし、 実施例 2で増幅したヒト KPC2 cDNA断片を鎵型にした PCR により、 C末に配列番号 24で表されるアミノ酸配列からなる HAタグを付加した KPC2をコードする DNA断片を増幅した。 この MA断片を 5'端にある iHIおよび 3' 端にある^ Iで切断後、 同様に pMX- puroの BamHI/XhoIサイ ト間に挿入し、 KPC2発 現用レトロウィルスベクタ一を作製した。  The ^ fragment containing the DNA encoding KPC1 to which His6 / FLAG was added, of the transfer vector for expression of KPC1 prepared in Example 2 (1), was inserted into a retroviral expression vector pMX-puro CProc. Natl. Acad. Sci. USA, 94, 3938 (1997)] to construct a retroviral vector for KPC1 expression. In addition, PCR was performed by using DNA (KPC2-H-3, KPC2-H-4) consisting of the nucleotide sequences represented by SEQ ID NOs: 32 and 33 as a primer, and transforming the human KPC2 cDNA fragment amplified in Example 2 into a 鎵 type. As a result, a DNA fragment encoding KPC2 having an HA tag having the amino acid sequence represented by SEQ ID NO: 24 added at the C-terminus was amplified. This MA fragment was digested with iHI at the 5 'end and ^ I at the 3' end, and then similarly inserted between the BamHI / XhoI sites of pMX-puro to prepare a retroviral vector for KPC2 expression.
これらの組換えレ トロウイルス発現用べクタ一をそれそれ、 既報 〔Gene Ther. , 7, 1063 (2000)〕 に従い Plat E細胞にトランスフエクトすることにより 組換えレトロウイルスを作製した。 得られた組換えウィルスを NIH3T3細胞 （ATCC No. CRL-1658) に共感染させ、 10〃g/mLピューロマイシンを含む培地で選択する ことにより、 組換え KPC1および KPC2を発現する NIH3T3細胞を作製した。 細胞を、 抗 FLAG抗体 （シグマ社） および抗 HA抗体 （サン夕クルーズ社） を用いた免疫蛍光 染色法 〔J. Biol. Chem. , 276, 33111 (2001)〕 により、 KPC1および KPC2の細胞 内局在を解析した。 その結果、 両蛋白質は共通して細胞質に局在し、 核内には局在しないことが明 らかになつた。 p27Kiplは細胞周期 G0-G1期において核内から細胞質へと輸送され ること 〔EMB0 20, 6672 (2001)〕 、 また p27Kiplをュビキチン化する活性が細 胞質由来の粗精製分画に存在する 〔J. Biol . Chem.， 276, 48937 (2001 )〕 とい う、 従来の報告と一致しており、 本結果は、 KPC1、 KPC2が細胞質に局在し、 GO - G1期に核から細胞質に移行した p27Kiplをュビキチン化するという仮説を支持する 結果である。 Recombinant retroviruses were produced by transfecting these recombinant retrovirus expression vectors into Plat E cells according to the previously reported [Gene Ther., 7, 1063 (2000)]. NIH3T3 cells (ATCC No. CRL-1658) were co-infected with the obtained recombinant virus and selected in a medium containing 10 μg / mL puromycin to produce NIH3T3 cells expressing recombinant KPC1 and KPC2. did. The cells were cultured in KPC1 and KPC2 cells by immunofluorescence staining using anti-FLAG antibody (Sigma) and anti-HA antibody (Sanyu Cruise) [J. Biol. Chem., 276, 33111 (2001)]. The localization was analyzed. As a result, it became clear that both proteins were commonly localized in the cytoplasm but not in the nucleus. p27 Kipl is transported from the nucleus to the cytoplasm during the G0-G1 phase of the cell cycle [EMB0 20, 6672 (2001)], and the activity to ubiquitinate p27 Kipl is present in crude fractions derived from cytoplasm [J. Biol. Chem., 276, 48937 (2001)], which is consistent with the previous report, indicating that KPC1 and KPC2 are localized in the cytoplasm, This is a result that supports the hypothesis that p27 Kipl migrated to ubiquitination.
[実施例 5 ] KPC卜 KPC2複合体の過剰発現による p27Kiplの分解に対する影響 実施例 2 (1 )で作製した KPC1の発現用トランスファーぺク夕一の、 His6/FLAG夕 グを付加した KPCl( AR)をコードする MAを含む IMHI- 2^1断片を、 発現用レトロ ウィルスベクタ一 pMX- puroの i HI/Smlサイ ト間に挿入し、 KPCl( AR)発現用レ トロウィルスベクタ一を作製した。 実施例 4と同様の方法で、 KPCl( AR)および KPC2を高発現した NIH3T3細胞を作製した。 [Example 5] Effect of overexpression of the KPC2 complex on degradation of p27 Kipl KPCl with the His6 / FLAG fragment added to the transfer protein for expression of KPC1 prepared in Example 2 (1) Insert the IMHI-2 ^ 1 fragment containing MA encoding AR) into the expression retrovirus vector pMX-puro between the iHI / Sml sites to create a retrovirus vector for KPCl (AR) expression did. In the same manner as in Example 4, NIH3T3 cells overexpressing KPCl (AR) and KPC2 were produced.
この細胞、 実施例 4で作製した KPC1および KPC2を高発現した NIH3T3細胞、 およ びコントロール用の pMX-puroベクタ一から作製した組換えレトロウィルスを感染 させた NIH3T3細胞を用いて、 細胞内での p27Kiplの分解に対する KPCの機能を調べ た。接触阻害により細胞周期 GOに同調した細胞を細胞密度約 40 %で蒔き直すこと により細胞周期 G1へのェントリーを可能にし、 それ以後の p27Kipl量をィムノプロ ットにより経時的に解析した （第 13図 a ) 。 KPC1および KPC2を高発現した細胞で はコントロール細胞に比べ、 p27Kiplの分解が早く起きること、 また KPCl( AR)お よび KPC2の発現細胞では逆に分解が遅れることが確認された。 この結果は、 KPC1- KPC2複合体が、 細胞内での p27Kiplの GO- G1期における分解に関与することを 示すと同時に KPCl( A R)がドミナントネガティブ型分子として機能することを示 している。 Using these cells, NIH3T3 cells highly expressing KPC1 and KPC2 prepared in Example 4, and NIH3T3 cells infected with a recombinant retrovirus prepared from pMX-puro vector for control, intracellularly The function of KPC for the degradation of p27 Kipl was investigated. The cells synchronized with the cell cycle GO by contact inhibition were replated at a cell density of about 40% to enable entry into the cell cycle G1, and the amount of p27 Kipl thereafter was analyzed over time using an immo plot (No. 13 Figure a). It was confirmed that p27 Kipl degradation occurred earlier in cells expressing KPC1 and KPC2 at higher levels than in control cells, and that degradation was delayed in cells expressing KPCl (AR) and KPC2. This result indicates that the KPC1-KPC2 complex is involved in the degradation of p27 Kipl in the GO-G1 phase in cells, and that KPCl (AR) functions as a dominant negative molecule. .
同様の実験を、 蛋白質の核外移行を阻害する薬剤 leptomycin存在下で行った結 果、 p27Kiplの分解速度に差がないことがわかり、 KPC卜 KPC2複合体を介した p27Kipl の分解調節は、 p27Kiplの核から細胞質への移行が重要であることが確認されだ ( 第 13図 b ) 。 [実施例 6 ] KPC1と特異的に結合する抗体および KPC2と特異的に結合する抗体 マウス KPC1の N末端から 300アミノ酸を含む KPC1の部分断片、 マウス KPC2をそれ それ抗原として、 ゥサギを免役し、 抗血清を採取することにより、 KPC1と特異的 に結合するポリクロ一ナル抗体および KPC2と特異的に結合するポリクローナル抗 体を得た。 実施例 7および第 14図に示すように、 これらの抗体を用いたィムノブ ロヅトにより、 NIH3T3細胞で内在性に発現している KPC1および KPC2を、 それそれ 検出することができた。 A similar experiment was performed in the presence of leptomycin, an agent that inhibits protein nuclear export.As a result, it was found that there was no difference in the degradation rate of p27 Kipl , and the regulation of p27 Kipl degradation via the KPC-KPC2 complex was The translocation of p27 Kipl from the nucleus to the cytoplasm was confirmed to be important (Fig. 13b ). [Example 6] Antibody that specifically binds to KPC1 and antibody that specifically binds to KPC2 Mouse KPC1 partial fragment of KPC1 containing 300 amino acids from the N-terminus, mouse KPC2 each as an antigen, immunizing the egret, By collecting the antiserum, a polyclonal antibody that specifically binds to KPC1 and a polyclonal antibody that specifically binds to KPC2 were obtained. As shown in Example 7 and FIG. 14, it was possible to detect KPC1 and KPC2 endogenously expressed in NIH3T3 cells, respectively, by the immunoblotting using these antibodies.
[実施例 Ί ] RNAiによる KPC1の発現の抑制およびその ρ27Κίρ1の分解および細胞の 増殖に対する影響 [Example Ί] Suppression of KPC1 expression by RNAi and its effect on degradation of ρ27 Κίρ1 and cell proliferation
発現用レトロウイルスベクター pffl Exp. HematoL 24, 324 ( 1996 )〕 の SildIII/^ Iサイ ト間にピューロマイシン耐性遺伝子を挿入し、 ベクタ一 pMX- puro IIを作製した。 pMX- puro IIの Notl/Sallサイ ト間に、 マウス U6プロモー夕 一およびその下流に配列番号 37で表される配列をつなげた DNAを挿入し、 KPC1 siRNA発現用レトロウィルスベクタ一を作製した。 該ベクターを導入した細胞は 、 マウス KPC1遺伝子 （配列番号 3) の 1852〜1872番目の配列を標的とする si Aを 発現する。 また、 コントロールとして、 pMX- puro IIの Notl/Sal lサイト間に、 マ ウス U6プロモーターおよびその下流に配列番号 39に示す配列をつなげた DNAを揷 入し、 改良型のグリーン蛍光蛋白質である EGFP Gene, 173, 33 (1996) ) の siRNA発現用レトロウィルスベクターを作製した。 該ベクタ一を導入した細胞は 、 EGFP遺伝子のコード領域の 126〜146番目の配列を標的とする si Aを発現する 。 これらの発現用レトロウイルスベクタ一を用いて、 実施例 4と同様にして組換 ぇレトロウィルスを作製し、 得られた組換えウィルスをそれぞれ NIH3T3細胞に感 染させ、 10 /g/mLピューロマイシンを含む培地で選択することにより、 マウス KPC1の発現を抑制する siRNAおよび EGFP s iRNAをそれそれ発現する N IH3T3細胞を 作製した。 実施例 6で作製した抗マウス KPC1抗体を用いたィムノブロット解析に より、 コントロールの EGFP siRNAを発現する NIH3T3細胞と比較して、 KPC1 siRNA を発現する NIH3T3細胞では、 KPC1の発現が特異的に抑制されていることが確認さ れた。 なお、 KPC2の発現は、 どちらの NIH3T3細胞も同程度で、 抑制はみられなか つた (第 14図) 。 これらの NIH3T3細胞を、 接触阻害により細胞周期 GOに同調した後、 細胞密度約 40%で蒔き直すことにより細胞周期 G1へ進入させ、 以後の細胞の p27Kipl量をィム ノブロットにより経時的に解析した （第 14図） 。 コント口一ルとして、 GSK- 3 の蛋白質量も同時に解析した。 その結果、 コントロールの EGFP siMAを発現する NIH3T3細胞では、 時間の経過とともに、 p27Kiplの分解が見られるのに対し、 マウ ス KPC1 siRNAを発現する NIH3T3細胞では、 p27Kiplの分解が抑制されていることが 確認された。 コントロールの GSK- 3 ?の分解は、 どちらの細胞でも特に見られな かった。 The puromycin resistance gene was inserted between the SildIII / ^ I sites of the retrovirus vector for expression pffl Exp. HematoL 24, 324 (1996)] to prepare the vector pMX-puro II. Between the Notl / Sall sites of pMX-puro II, a DNA having the sequence represented by SEQ ID NO: 37 connected to mouse U6 promoter and downstream thereof was inserted to prepare a retrovirus vector for expressing KPC1 siRNA. Cells into which the vector has been introduced express siA targeting the 1852th to 1872nd sequence of the mouse KPC1 gene (SEQ ID NO: 3). As a control, a DNA having the mouse U6 promoter and the sequence shown in SEQ ID NO: 39 connected downstream thereof was inserted between the Notl / Sall sites of pMX-puro II, and EGFP, an improved green fluorescent protein, was inserted. Gene, 173, 33 (1996)). Cells into which the vector has been introduced express siA targeting the 126th to 146th sequence of the coding region of the EGFP gene. Using these expression retrovirus vectors, recombinant ぇ retroviruses were prepared in the same manner as in Example 4, and the resulting recombinant viruses were respectively infected into NIH3T3 cells, and 10 / g / mL puromycin was prepared. By selecting in a medium containing E. coli, NIH3T3 cells which express siRNA and EGFP siRNA which suppress the expression of mouse KPC1 respectively were prepared. By immunoblot analysis using the anti-mouse KPC1 antibody prepared in Example 6, KPC1 expression was specifically suppressed in NIH3T3 cells expressing KPC1 siRNA, compared to NIH3T3 cells expressing control EGFP siRNA. Was confirmed. The expression of KPC2 was similar in both NIH3T3 cells, and was not suppressed (Fig. 14). After synchronizing these NIH3T3 cells with the cell cycle GO by contact inhibition, they were re-plated at a cell density of about 40% to enter the cell cycle G1, and the amount of p27 Kipl in subsequent cells was analyzed over time by immunoblot. (Fig. 14). As a control, the amount of GSK-3 protein was also analyzed. As a result, NIH3T3 cells expressing control EGFP siMA show degradation of p27 Kipl over time, whereas NIH3T3 cells expressing mouse KPC1 siRNA inhibit degradation of p27 Kipl . Was confirmed. Degradation of the control GSK-3? Was not particularly observed in either cell.
また、 これらの NIH3T3細胞を、 0.1 %血清を含む培地で培養し、 p27Kipl、 サイ クリン A、 KPC1、 KPC2および GSK- 3 ?の蛋白質量ならびに細胞数を経時的に解析し た （第 15図および第 16図） 。 蛋白質量は、 ィムノブロットにより測定した。 その 結果、 コントロールの EGFP siRNAを発現する NIH3T3細胞と比較して、 KPC1 siRNA を発現する NIH3T3細胞では、 p27Kiplの分解が抑制されることにより、 時間の経過 とともに p27Kiplがより多く蓄積すること、 p27Kiplにより活性化および分解が阻害 されるサイクリン Aの分解が促進されていることが確認された。 KPC1、 KPC2およ びコントロールの GSK- 3 ^の蛋白質量の変化は、 どちらの細胞でも特に見られな かった。 また、 コントロールの EGFP siRNAを発現する NIH3T3細胞では、 細胞数が 経時的に増加し、 細胞周期が進行していると考えられるのに対し、 KPC1 siRNAを 発現する NIH3T3細胞では、 細胞数の増加が見られず、 細胞周期の進行が抑制され ていた。 したがって、 RNAiによって KPC1の発現を抑制することにより、 p27Kiplの 分解の抑制、 サイクリン Aの分解の促進、 細胞周期の進行の抑制、 および細胞の 増殖の抑制をすることができた。 産業上の利用可能性 These NIH3T3 cells were cultured in a medium containing 0.1% serum, and p27 Kipl , cyclin A, KPC1, KPC2, and GSK-3? Protein content and cell number were analyzed over time (Fig. 15). And Figure 16). Protein content was measured by immunoblot. As a result, compared to NIH3T3 cells expressing control EGFP siRNA, NIH3T3 cells expressing KPC1 siRNA suppress degradation of p27 Kipl , resulting in more accumulation of p27 Kipl over time, It was confirmed that p27 Kipl promoted the degradation of cyclin A, whose activation and degradation were inhibited. No change in the protein levels of KPC1, KPC2 and the control GSK-3 ^ was observed in any of the cells. In addition, the cell number of NIH3T3 cells expressing control EGFP siRNA is thought to increase over time and the cell cycle is progressing, whereas the cell number of NIH3T3 cells expressing KPC1 siRNA is increased. Not seen, cell cycle progression was suppressed. Therefore, by suppressing the expression of KPC1 by RNAi, it was possible to suppress the degradation of p27 Kipl , promote the degradation of cyclin A, suppress the progression of the cell cycle, and suppress the proliferation of cells. Industrial applicability
本発明により、 p27Kiplをュビキチン化する活性を有する新規なュビキチンリガ —ゼ蛋白質、 該蛋白質を含む p27Kiplをュビキチン化する活性を有するュビキチン リガーゼ複合体、 該蛋白質をコードする DNA、 該 DNAを含む組換え体 DNA、 該組換 え体 DNAで形質転換された形質転換体、 ·および該蛋白質を認識する抗体が提供さ れる。本発明は、 癌、 動脈硬化、 慢性関節リウマチ、 前立腺肥大症、 経皮的経血 管的冠動脈形成術後の血管再狭窄、 肺線維症、 糸球体腎炎、 自己免疫疾患等の細 胞周期の異常を原因とする疾患、 細胞周期を調節することにより症状を軽減でき るような疾患、 特に癌の診断、 治療等に有用である。 The present invention, novel Yubikichinriga has an activity of Yubikichin the p27 Kipl - DNA encoding peptidase protein, Yubikichin ligase complex having an activity of Yubikichin the p27 Kipl containing protein, the protein, the set comprising the DNA Provided are a recombinant DNA, a transformant transformed with the recombinant DNA, and an antibody recognizing the protein. The present invention relates to the treatment of cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, vascular restenosis after percutaneous transluminal coronary angioplasty, pulmonary fibrosis, glomerulonephritis, autoimmune diseases, etc. It is useful for diseases caused by abnormal cell cycle, diseases whose symptoms can be alleviated by regulating the cell cycle, and particularly for diagnosis and treatment of cancer.
「配列表フリーテキスト」 "Sequence List Free Text"
配列番号 1一発明者：中山敬一；嘉村巧 SEQ ID NO: 1 Inventor: Keiichi Nakayama; Takumi Kamura
配列番号 22—His6/FLAG夕グ SEQ ID NO: 22—His6 / FLAG
配列番号 23— His6/HSV夕グ SEQ ID NO: 23—His6 / HSV
配列番号 24— HAタグ SEQ ID NO: 24—HA tag
配列番号 25— PCRプラィマー KPCl- Ml SEQ ID NO: 25—PCR primer KPCl-Ml
配列番号 26— PCRプラィマー KPC1 - M2 SEQ ID NO: 26—PCR primer KPC1-M2
配列番号 27— PCRブラィマー KPC2-H1 SEQ ID NO: 27—PCR primer KPC2-H1
配列番号 28— PCRブラィマ一 KPC2 - H2 SEQ ID NO: 28—PCR primer KPC2-H2
配列番号 29— PCRブラィマー KPCl- H-1 SEQ ID NO: 29—PCR primer KPCl-H-1
配列番号 30— PCRプラィマー KPCl- H - 2 SEQ ID NO: 30—PCR primer KPCl-H-2
配列番号31—？(¾プラィマー ( 1-11-3 SEQ ID NO: 31—? (¾Primer (1-11-3
配列番号 32— PCRブラィマ一 KPC2-H-3 SEQ ID NO: 32—PCR primer KPC2-H-3
配列番号 33— PCRブラィマー KPC2- H-4 SEQ ID NO: 33—PCR primer KPC2-H-4
配列番号 34— KPCl siRNAセンス鎖 SEQ ID NO: 34—KPCl siRNA sense strand
配列番号 35— KPCl siMAアンチセンス鎖 SEQ ID NO: 35—KPCl siMA antisense strand
配列番号 36— KPCl siRNA用の配列 X SEQ ID NO: 36—Sequence X for KPCl siRNA
配列番号 37— KPCl siRNA発現ベクターに揷入した DM SEQ ID NO: 37—DM inserted into KPCl siRNA expression vector
配列番号 38— KPCl shRNA SEQ ID NO: 38— KPCl shRNA
配列番号 39— EGFP s iRNA発現べクタ一に揷入した DNA SEQ ID NO: 39—DNA inserted into EGFP s iRNA expression vector

Claims

請求の範囲 The scope of the claims
1. p27Kiplをュビキチン化する活性を有する複合体の構成成分であり、 分子 量が 140kDaである、 p27Kiplをュビキチン化する活性を有する蛋白質。 1. A protein having an activity of ubiquitinating p27 Kipl , which is a component of a complex having an activity of ubiquitinating p27 Kipl and having a molecular weight of 140 kDa.
2. 配列番号 2または 4で表されるァミノ酸配列を含む蛋白質。  2. A protein containing an amino acid sequence represented by SEQ ID NO: 2 or 4.
3. 配列番号 2または 4で表されるアミノ酸配列において 1つ以上のアミノ酸 が付加、 欠失あるいは置換したアミノ酸配列からなり、 かつ p27Kiplをュビキチン 化する活性を有する蛋白質。 3. A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4 in which one or more amino acids have been added, deleted or substituted, and has an activity of ubiquitinating p27 Kipl .
4. 配列番号 2または 4で表されるアミノ酸配列と 60%以上の相同性を有する アミノ酸配列からなり、 かつ p27Kiplをュビキチン化する活性を有する蛋白質。 4. A protein consisting of an amino acid sequence having at least 60% homology with the amino acid sequence represented by SEQ ID NO: 2 or 4, and having an activity of ubiquitinating p27 Kipl .
5. 請求項 1〜4のいずれか 1項に記載の蛋白質および以下の（a)〜（c )のいず れかに記載の蛋白質を構成成分として含む、 p27Kiplをュビキチン化する活性を有 する複合体。 5. It has an activity of ubiquitinating p27 Kipl , which comprises the protein according to any one of claims 1 to 4 and the protein according to any one of the following (a) to (c) as components. Complex.
( a)配列番号 6または 8で表されるァミノ酸配列を含む蛋白質  (a) a protein containing an amino acid sequence represented by SEQ ID NO: 6 or 8
(b)配列番号 6または 8で表されるアミノ酸配列において 1つ以上のアミノ酸が付 加、 欠失あるいは置換したアミノ酸配列からなり、 かつ配列番号 2または 4で表さ れるアミノ酸配列を含む蛋白質と会合する蛋白質  (b) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8 with one or more amino acids added, deleted or substituted, and comprising the amino acid sequence represented by SEQ ID NO: 2 or 4; Associated proteins
( c )配列番号 6または 8で表されるァミノ酸配列と 60 %以上の相同性を有するアミ ノ酸配列からなり、 かつ配列番号 2または 4で表されるァミノ酸配列を含む蛋白質 と会合する蛋白質  (c) an amino acid sequence having at least 60% homology with the amino acid sequence represented by SEQ ID NO: 6 or 8, and which associates with the protein containing the amino acid sequence represented by SEQ ID NO: 2 or 4 protein
6. 請求項 2〜4のいずれか 1項に記載の蛋白質をコ一ドする DNA。  6. A DNA encoding the protein of any one of claims 2 to 4.
7. 配列番号 1または 3で表される塩基配列を含む DNA。  7. DNA containing the base sequence represented by SEQ ID NO: 1 or 3.
8. 配列番号 1または 3で表される塩基配列と相補的な塩基配列からなる 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 p27Kiplをュビキチン化する 活性を有する蛋白質をコードする DNA。 8. A DNA that hybridizes with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 or 3 under stringent conditions and encodes a protein having an activity of ubiquitinating p27 Kipl .
9. 請求項 6〜8のいずれか 1項に記載の DNAをベクターに組み込んで得られる 組換え体舰。  9. A recombinant obtained by incorporating the DNA according to any one of claims 6 to 8 into a vector.
10. 請求項 9に記載の組換え体 DNAを宿主細胞に導入して得られる形質転換体  10. A transformant obtained by introducing the recombinant DNA according to claim 9 into a host cell.
11. 請求項 10に記載の形質転換体を培養液中で培養し、 請求項 2〜4のいずれ か 1項に記載の蛋白質を該培養物中に生成、 蓄積させ、 該培養物中より該蛋白質 を採取することを特徴とする請求項 2〜4のいずれか 1項に記載の蛋白質の製造法 11. The transformant according to claim 10 is cultured in a culture medium, and the protein according to any one of claims 2 to 4 is produced and accumulated in the culture. protein A method for producing the protein according to any one of claims 2 to 4, wherein the protein is collected.
12. 請求項 6〜8のいずれか 1項に記載の DNAおよび以下の（a)〜（e)のいずれ かに記載の DNAをべクタ一に組み込んで得られる組換え体 DNA。 12. A recombinant DNA obtained by incorporating the DNA according to any one of claims 6 to 8 and the DNA according to any one of the following (a) to (e) into a vector.
( a)配列番号 6または 8で表されるァミノ酸配列を含む蛋白質をコードする DNA (a) DNA encoding a protein containing an amino acid sequence represented by SEQ ID NO: 6 or 8
(b)配列番号 6または 8で表されるアミノ酸配列において 1つ以上のアミノ酸が付 加、 欠失あるいは置換したアミノ酸配列からなり、 かつ配列番号 2または 4で表さ れるアミノ酸配列を含む蛋白質と会合する蛋白質をコードする DNA (b) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8 with one or more amino acids added, deleted or substituted, and comprising the amino acid sequence represented by SEQ ID NO: 2 or 4; DNA encoding the associated protein
( c )配列番号 6または 8で表されるァミノ酸配列と 60 %以上の相同性を有するアミ ノ酸配列からなり、 かつ配列番号 2または 4で表されるァミノ酸配列を含む蛋白質 と会合する蛋白質をコ一ドする DNA  (c) an amino acid sequence having at least 60% homology with the amino acid sequence represented by SEQ ID NO: 6 or 8, and which associates with the protein containing the amino acid sequence represented by SEQ ID NO: 2 or 4 DNA that codes for proteins
(d)配列番号 5または 7で表される塩基配列を含む MA  (d) MA containing the base sequence represented by SEQ ID NO: 5 or 7
( e )配列番号 5または 7で表される塩基配列と相補的な塩基配列からなる DNAとスト リンジェントな条件下でハイブリダイズし、 かつ配列番号 2または 4で表されるァ ミノ酸配列を含む蛋白質と会合する蛋白質をコードする MA  (e) The amino acid sequence which hybridizes with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 5 or 7 under stringent conditions, and the amino acid sequence represented by SEQ ID NO: 2 or 4 MA encoding a protein that associates with the containing protein
13. 請求項 12に記載の組換え体 DNAを宿主細胞に導入して得られる形質転換 体。  13. A transformant obtained by introducing the recombinant DNA according to claim 12 into a host cell.
14. 請求項 9に記載の組換え体 DNAおよび以下の（a)~( e)のいずれかに記載の DNAをベクターに組み込んで得られる組換え体 MAを宿主細胞に導入して得られる 形質転換体。  14. A trait obtained by introducing a recombinant MA obtained by incorporating the recombinant DNA according to claim 9 and a DNA according to any one of the following (a) to (e) into a vector into a host cell: Convertible.
(a)配列番号 6または 8で表されるアミノ酸配列を含む蛋白質をコードする DNA (A) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8
(b)配列番号 6または 8で表されるアミノ酸配列において 1つ以上のアミノ酸が付 加、 欠失あるいは置換したアミノ酸配列からなり、 かつ配列番号 2または 4で表さ れるアミノ酸配列を含む蛋白質と会合する蛋白質をコードする MA (b) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8 with one or more amino acids added, deleted or substituted, and comprising the amino acid sequence represented by SEQ ID NO: 2 or 4; MA encoding the associated protein
(c )配列番号 6または 8で表されるアミノ酸配列と 60%以上の相同性を有するアミ ノ酸配列からなり、 かつ配列番号 2または 4で表されるァミノ酸配列を含む蛋白質 と会合する蛋白質をコードする DNA  (c) a protein consisting of an amino acid sequence having at least 60% homology with the amino acid sequence represented by SEQ ID NO: 6 or 8, and being associated with the protein containing the amino acid sequence represented by SEQ ID NO: 2 or 4 DNA encoding
( d)配列番号 5または 7で表される塩基配列を含む MA  (d) MA containing the nucleotide sequence represented by SEQ ID NO: 5 or 7
(e)配列番号 5または 7で表される塩基配列と相補的な塩基配列からなる DNAとスト リンジェントな条件下でハイブリダィズし、 かつ配列番号 2または 4で表されるァ ミノ酸配列を含む蛋白質と会合する蛋白質をコードする DM (e) hybridizes with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 5 or 7 under stringent conditions, and binds to the DNA represented by SEQ ID NO: 2 or 4 DM encoding a protein that associates with a protein containing a amino acid sequence
15. ,請求項 13または 14に記載の形質転換体を培養液中で培養し、 請求項 5に 記載の複合体を該培養物中に生成、 蓄積させ、 該培養物中より該複合体を採取す ることを特徴とする請求項 5に記載の複合体の製造法。  15., culturing the transformant according to claim 13 or 14 in a culture medium, producing and accumulating the complex according to claim 5 in the culture, and converting the complex from the culture. The method for producing a composite according to claim 5, wherein the composite is collected.
16. 請求項 6〜8のいずれかの 1項に記載の DNAが有する塩基配列と相補的な 配列を含むポリヌクレオチド。  16. A polynucleotide comprising a sequence complementary to the base sequence of the DNA according to any one of claims 6 to 8.
17. 以下の (a)または (b)を用いて、 請求項 2〜4のいずれか 1項に記載の蛋白 質をコードする mRNAの検出または定量を行う方法。  17. A method for detecting or quantifying mRNA encoding the protein according to any one of claims 2 to 4, using the following (a) or (b).
(a)請求項 6〜8のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列の 連続する 20塩基以上の配列を含むポリヌクレオチド  (a) a polynucleotide comprising a continuous sequence of 20 or more nucleotides complementary to the nucleotide sequence of the DNA according to any one of claims 6 to 8;
(b)請求項 6〜8のいずれか 1項に記載の DNAが有する塩基配列中の連続する 20〜 100塩基の配列を含む DNAおよび請求項 6~8のいずれかの 1項に記載の DNAが有す る塩基配列と相補的な配列中の連続する 20〜100塩基の配列を含む DNA  (b) a DNA comprising a continuous sequence of 20 to 100 nucleotides in the nucleotide sequence of the DNA according to any one of claims 6 to 8 and the DNA according to any one of claims 6 to 8 DNA containing a continuous 20-100 base sequence in the sequence complementary to the base sequence
18. 以下の (a)または (b)を用いて、 請求項 2〜4のいずれか 1項に記載の蛋白 質の発現量が健常人と比較して mRNAレベルで増加または減少している疾患の判定 または診断を行う方法。  18. A disease in which the expression level of the protein according to any one of claims 2 to 4 is increased or decreased in mRNA level as compared with a healthy person using the following (a) or (b): How to make a judgment or diagnosis.
(a)請求項 6〜8のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中 の連続する 20塩基以上の配列を含むポリヌクレオチド  (a) a polynucleotide comprising a sequence of 20 or more consecutive nucleotides in a sequence complementary to the nucleotide sequence of the DNA according to any one of claims 6 to 8;
(b)請求項 6〜8のいずれか 1項に記載の DNAが有する塩基配列中の連続する 20〜 100塩基の配列を含む DNAおよび請求項 6〜8のいずれかの 1項に記載の DNAが有す る塩基配列と相補的な配列中の連続する 20〜100塩基の配列を含む MA  (b) a DNA comprising a continuous sequence of 20 to 100 nucleotides in the nucleotide sequence of the DNA according to any one of claims 6 to 8 and the DNA according to any one of claims 6 to 8 MA containing a sequence of 20 to 100 bases in a sequence complementary to the base sequence of
19. 以下の (a)または (b)を含む、 請求項 2〜4のいずれか 1項に記載の蛋白質 の発現量が健常人と比較して mRNAレベルで増加または減少している疾患の診断薬  19. Diagnosis of a disease in which the expression level of the protein according to any one of claims 2 to 4 is increased or decreased in mRNA level as compared with a healthy person, including the following (a) or (b): Medicine
(a)請求項 6〜8のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中 の連続する 20塩基以上の配列を含むポリヌクレオチド (a) a polynucleotide comprising a sequence of 20 or more consecutive nucleotides in a sequence complementary to the nucleotide sequence of the DNA according to any one of claims 6 to 8;
(b)請求項 6〜8のいずれか 1項に記載の MAが有する塩基配列中の連続する 20〜 100塩基の配列を含む DNAおよび請求項 6〜8のいずれかの 1項に記載の DNAが有す る塩基配列と相補的な配列中の連続する 20~100塩基の配列を含む DNA  (b) a DNA comprising a continuous sequence of 20 to 100 nucleotides in the base sequence of the MA according to any one of claims 6 to 8, and a DNA according to any one of claims 6 to 8; DNA containing a continuous sequence of 20 to 100 nucleotides in the sequence complementary to the nucleotide sequence of
20. 請求項 6〜8のいずれか 1項に記載の DNAが有する塩基配列中の連続する 20〜； 100塩基の配列を含むォリゴヌクレオチドおよび請求項 6〜8のいずれかの 1 項に記載の DNAが有する塩基配列と相補的な配列中の連続する 20〜100塩基の配列 を含むオリゴヌクレオチドのうちの少なくとも 1つを用いて、 請求項 2〜4のいず れか 1項に記載の蛋白質をコ一ドする遺伝子の変異を検出する方法。 20. Contiguous in the base sequence of the DNA according to any one of claims 6 to 8 Oligonucleotides comprising 20 to 100 nucleotide bases and a continuous 20 to 100 base sequence in a sequence complementary to the nucleotide sequence of the DNA according to any one of claims 6 to 8 and the DNA according to any one of claims 6 to 8. A method for detecting a mutation in a gene encoding the protein according to any one of claims 2 to 4, using at least one of the nucleotides.
21. 請求項 6〜8のいずれか 1項に記載の MAが有する塩基配列中の連続する 20〜； 100塩基の配列を含むォリゴヌクレオチドおよび請求項 6~8のいずれかの 1 項に記載の MAが有する塩基配列と相補的な配列中の連続する 20〜100塩基の配列 を含むオリゴヌクレオチドのうちの少なくとも 1つを用いて、 請求項 2〜4のいず れか 1項に記載の蛋白質をコードする遺伝子が変異を有している疾患を判定また は診断する方法。  21. A continuous 20 to 20 nucleotides in the base sequence of the MA according to any one of claims 6 to 8; an oligonucleotide comprising a sequence of 100 nucleotides, and the oligonucleotide according to any one of claims 6 to 8. The method according to any one of claims 2 to 4, wherein at least one of oligonucleotides containing a continuous 20 to 100 base sequence in a sequence complementary to the base sequence of the MA is used. A method for determining or diagnosing a disease in which a gene encoding a protein has a mutation.
22. 請求項 6〜8のいずれか 1項に記載の DNAが有する塩基配列中の連続する 20〜； L00塩基の配列を含むオリゴヌクレオチドおよび請求項 6〜8のいずれかの 1 項に記載の DNAが有する塩基配列と相補的な配列中の連続する 20〜100塩基の配列 を含むオリゴヌクレオチドのうちの少なくとも 1つを含む、 請求項 2〜4のいずれ か 1項に記載の蛋白質をコードする遺伝子が変異を有している疾患の診断薬。  22. An oligonucleotide comprising a sequence of 20-L00 bases in the base sequence of the DNA according to any one of claims 6 to 8; and an oligonucleotide according to any one of claims 6 to 8; The protein according to any one of claims 2 to 4, which comprises at least one of oligonucleotides containing a continuous 20 to 100 nucleotide sequence in a sequence complementary to the nucleotide sequence of DNA. A diagnostic agent for a disease in which a gene has a mutation.
23. 以下の（a)〜（c)からなる群から選ばれる RNA。  23. An RNA selected from the group consisting of (a) to (c) below.
(a)配列番号 36で表される配列および該配列と相補的な配列の 3'端にそれぞれ 2〜 4個のヌクレオチドを付加した配列からなる 2本鎖 RNA  (a) a double-stranded RNA consisting of a sequence represented by SEQ ID NO: 36 and a sequence complementary to the sequence and having a sequence obtained by adding 2 to 4 nucleotides to the 3 ′ end,
(b)配列番号 34および 35で表される配列からなる 2本鎖 RNA  (b) a double-stranded RNA consisting of the sequences represented by SEQ ID NOs: 34 and 35
(c)配列番号 38で表される配列からなる A  (c) A consisting of the sequence represented by SEQ ID NO: 38
24. 以下の（a)〜（d)からなる群から選ばれる少なくとも 1つを用いて、 請求 項 2〜4のいずれか 1項に記載の蛋白質の発現を抑制する方法。  24. A method for suppressing the expression of the protein according to any one of claims 2 to 4, using at least one selected from the group consisting of the following (a) to (d).
(a)請求項 6〜8のいずれか 1項に記載の MAが有する塩基配列中の連続する 19〜25 塩基に相当する配列を含む二本鎖 RNA  (a) a double-stranded RNA comprising a sequence corresponding to 19 to 25 consecutive bases in the base sequence of the MA according to any one of claims 6 to 8;
(b)請求項 23に記載の A  (b) A according to claim 23
(c)請求項 6〜8のいずれか 1項に記載の DNAが有する塩基配列中の連続する 19〜25 塩基に相当する配列および該配列と相補的な配列を含みヘアピン構造を形成する RNA  (c) RNA comprising a sequence corresponding to 19 to 25 consecutive bases in the base sequence of the DNA according to any one of claims 6 to 8 and a sequence complementary to the sequence to form a hairpin structure
(d)請求項 6〜8のいずれかの 1項に記載の MAが有する塩基配列と相補的な配列中 の連続する 20〜； L00塩基の配列を含むォリゴヌクレオチドまたはォリゴヌクレオ チド誘導体 (d) 20- or more consecutive nucleotides in the sequence complementary to the nucleotide sequence of the MA according to any one of claims 6 to 8; an oligonucleotide or an oligonucleonucleotide containing a sequence of L00 bases; Tide derivatives
25. 以下の (a)〜(d)からなる群から選ばれる少なくとも 1つを用いて、 細胞 内の p27Kiplのュビキチン化を阻害する方法。 25. A method for inhibiting ubiquitination of p27 Kipl in a cell using at least one selected from the group consisting of the following (a) to (d).
(a)請求項 6〜8のいずれか 1項に記載の DNAが有する塩基配列中の連続する 19〜25 塩基に相当する配列を含む二本鎖 A  (a) a double-stranded A comprising a sequence corresponding to 19 to 25 consecutive bases in the base sequence of the DNA according to any one of claims 6 to 8;
(b)請求項 23に記載の RNA  (b) the RNA of claim 23;
(c)請求項 6〜8のいずれか 1項に記載の MAが有する塩基配列中の連続する 19〜25 塩基に相当する配列および該配列と相補的な配列を含みヘアピン構造を形成する MA  (c) a MA which comprises a sequence corresponding to consecutive 19 to 25 bases in the base sequence of the MA according to any one of claims 6 to 8 and a sequence complementary to the sequence, thereby forming a hairpin structure.
(d)請求項 6〜8のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中 の連続する 20〜； 100塩基の配列を含むオリゴヌクレオチドまたはオリゴヌクレオ チド誘導体  (d) an oligonucleotide or oligonucleotide derivative comprising a sequence of 20 to 100 consecutive nucleotides complementary to the nucleotide sequence of the DNA according to any one of claims 6 to 8;
26. 以下の（a)〜（ からなる群から選ばれる少なくとも 1つを用いて、 細胞 内の p27Kiplの分解を抑制する方法。 26. A method for suppressing the degradation of p27 Kipl in cells using at least one selected from the group consisting of the following (a) to ().
(a)請求項 6〜8のいずれか 1項に記載の MAが有する塩基配列中の連続する 19〜25 塩基に相当する配列を含む二本鎖 RNA  (a) a double-stranded RNA comprising a sequence corresponding to 19 to 25 consecutive bases in the base sequence of the MA according to any one of claims 6 to 8;
(b)請求項 23に記載の RNA  (b) the RNA of claim 23;
(c)請求項 6〜8のいずれか 1項に記載の DNAが有する塩基配列中の連続する 19〜25 塩基に相当する配列および該配列と相補的な配列を含みヘアピン構造を形成する RNA  (c) RNA comprising a sequence corresponding to 19 to 25 consecutive bases in the base sequence of the DNA according to any one of claims 6 to 8 and a sequence complementary to the sequence to form a hairpin structure
(d)請求項 6〜8のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中 の連続する 20〜 100塩基の配列を含むオリゴヌクレオチドまたはオリゴヌクレオ チド誘導体  (d) an oligonucleotide or oligonucleotide derivative comprising a continuous sequence of 20 to 100 nucleotides in a sequence complementary to the nucleotide sequence of the DNA according to any one of claims 6 to 8;
27. 以下の（a)〜（ からなる群から選ばれる少なくとも 1つを有効成分とし て含有する、 細胞周期の異常を原因とする疾患または細胞周期を調節することに より症状を軽減できる疾患の治療薬。  27. A disease caused by abnormal cell cycle or a disease whose symptoms can be alleviated by regulating the cell cycle, which contains at least one selected from the group consisting of the following (a) to () as an active ingredient: Therapeutic drugs.
(a)請求項 6〜8のいずれか 1項に記載の MAが有する塩基配列中の連続する 19〜25 塩基に相当する配列を含む二本鎖 RNA  (a) a double-stranded RNA comprising a sequence corresponding to 19 to 25 consecutive bases in the base sequence of the MA according to any one of claims 6 to 8;
(b)請求項 23に記載の A  (b) A according to claim 23
(c )請求項 6〜8のいずれか 1項に記載の DNAが有する塩基配列中の連続する 19〜25 塩基に相当する配列および該配列と相補的な配列を含みヘアピン構造を形成する(c) continuous 19 to 25 in the base sequence of the DNA according to any one of claims 6 to 8; Contains a sequence corresponding to a base and a sequence complementary to the sequence to form a hairpin structure
RNA RNA
(d)請求項 6〜8のいずれかの 1項に記載の DNAが有する塩基配列と相補的な配列中 の連続する 20〜100塩基の配列を含むォリゴヌクレオチドまたはォリゴヌクレオ チド誘導体 ' (d) an oligonucleotide or an oligonucleotide derivative comprising a continuous 20 to 100 nucleotide sequence in a sequence complementary to the nucleotide sequence of the DNA according to any one of claims 6 to 8;
28. 疾患が癌である請求項 26に記載の治療薬。 28. The therapeutic agent according to claim 26, wherein the disease is cancer.
29. 請求項 6〜8のいずれかの 1項に記載の DNAを導入することにより作製し た非ヒトトランスジヱニック動物。  29. A non-human transgenic animal produced by introducing the DNA according to any one of claims 6 to 8.
30. 請求項 29に記載の非ヒトトランスジエニック動物を細胞周期の異常を原 因とする疾患のモデル動物として用いる方法。  30. A method of using the non-human transgenic animal according to claim 29 as a model animal for a disease caused by abnormal cell cycle.
31. 疾患が癌である、 請求項 30に記載の方法。  31. The method of claim 30, wherein the disease is cancer.
32. 請求項 29に記載の非ヒトトランスジエニック動物を用いて薬剤を評価す る方法。  32. A method for evaluating a drug using the non-human transgenic animal according to claim 29.
33. 薬剤が抗癌剤である、 請求項 32に記載の方法。  33. The method of claim 32, wherein the agent is an anti-cancer agent.
34. 請求項 2〜4のいずれか 1項に記載の蛋白質をコードする遺伝子が欠損し た非ヒト動物。  34. A non-human animal deficient in a gene encoding the protein according to any one of claims 2 to 4.
35. 被験試料の存在下および非存在下で、 請求項 1〜4のいずれか 1項に記載 の蛋白質または請求項 5に記載の複合体と p27Kiplとを接触させる工程、 該蛋白質 または該複合体と p27Kiplとの結合量を測定する工程、 および被験試料の存在下お よび非存在下での該蛋白質または該複合体と p27Kiplとの結合量を比較する工程を 含む、 p27Kiplと請求項 1〜4のいずれか 1項に記載の蛋白質との結合を阻害する物 質のスクリーニング方法。 35. a step of contacting the protein according to any one of claims 1 to 4 or the complex according to claim 5 with p27 Kipl in the presence and absence of a test sample; measuring the amount of binding between the body and the p27 Kipl, and the step of comparing the amount of binding between the protein or conjugate and p27 Kipl in the presence Contact and absence of a test sample, p27 Kipl and billing Item 5. A method for screening a substance that inhibits binding to a protein according to any one of Items 1 to 4.
36. 被験試料の存在下および非存在下で、 請求項 1〜4のいずれか 1項に記載 の蛋白質または請求項 5に記載の複合体と p27Kiplを接触させる工程、 該蛋白質ま たは該複合体と p27Kiplとの結合量を測定する工程、 および被験試料の存在下およ び非存在下での該蛋白質または該複合体と p27Kiplとの結合量を比較する工程を含 む、 p27Kiplのュビキチン化を抑制する物質のスクリーニング方法。 36. a step of contacting the protein according to any one of claims 1 to 4 or the complex according to claim 5 with p27 Kipl in the presence and absence of a test sample; Measuring the amount of binding between the complex and p27 Kipl , and comparing the amount of binding between the protein or the complex and p27 Kipl in the presence and absence of the test sample. A screening method for a substance that inhibits ubiquitination of Kipl.
37. 被験試料の存在下および非存在下で、 請求項 1〜4のいずれか 1項に記載 の蛋白質または請求項 5に記載の複合体と p27Kiplを接触させる工程、 該蛋白質ま たは該複合体と p27Kiplとの結合量を測定する工程、 および被験試料の存在下およ び非存在下での該蛋白質または該複合体と p27Kiplとの結合量を比較する工程を含 む、 p27Kiplの分解を抑制する物質のスクリーニング方法。 37. a step of contacting the protein according to any one of claims 1 to 4 or the complex according to claim 5 with p27 Kipl in the presence and absence of a test sample; Measuring the amount of binding between the complex and p27 Kipl ; and A method of screening for a substance that inhibits the degradation of p27 Kipl , comprising a step of comparing the amount of binding of the protein or the complex with p27 Kipl in the absence and absence of p27 Kipl .
38. 被験試料の存在下および非存在下で、 請求項 1〜4のいずれか 1項に記載 の蛋白質または請求項 5に記載の複合体と p27Kiplを接触させる工程、 該蛋白質ま たは該複合体と p27Kiplとの結合量を測定する工程、 および被験試料の存在下およ び非存在下での該蛋白質または該複合体と p27Kiplとの結合量を比較する工程を含 む、 細胞周期の異常を原因とする疾患または細胞周期を調節することにより症状 を軽減できる疾患の治療薬のスクリーニング方法。 38. a step of contacting the protein according to any one of claims 1 to 4 or the complex according to claim 5 with p27 Kipl in the presence and absence of a test sample; A cell comprising a step of measuring the amount of binding between the complex and p27 Kipl , and a step of comparing the amount of binding between the protein or the complex and p27 Kipl in the presence and absence of a test sample. A method for screening a therapeutic agent for a disease caused by abnormal cycle or a disease whose symptom can be reduced by regulating the cell cycle.
39. 疾患が癌である、 請求項 38に記載の方法。  39. The method of claim 38, wherein the disease is cancer.
40. 請求項 1〜4のいずれか 1項に記載の蛋白質または請求項 5に記載の複合 体、 ュビキチン活性化酵素、 ュビキチン結合酵素、 ュビキチンおよび p27Kiplを含 む系において被験試料の存在下および非存在下で p27Kiplのュビキチン化を行うェ 程、 p27Kiplにとりこまれたュビキチンの量を測定する工程、 および被験試料の存 在下および非存在下での p27Kiplにとりこまれたュビキチンの量を比較する工程を 含む、 p27Kiplのュビキチン化を抑制する物質のスクリーニング方法。 40.The system according to any one of claims 1 to 4, or the complex according to claim 5, ubiquitin activating enzyme, ubiquitin conjugating enzyme, ubiquitin and p27 Kipl in the presence of a test sample and absence as E performing Yubikichin of p27 Kipl, measuring the amount of Yubikichin taken into p27 Kipl, and the amount of Yubikichin taken into p27 Kipl in existence under and absence of test sample A method for screening a substance that suppresses ubiquitination of p27 Kipl , comprising a step of comparing.
41 . 請求項 1〜4のいずれか 1項に記載の蛋白質または請求項 5に記載の複合 体、 ュビキチン活性化酵素、 ュビキチン結合酵素、 ュビキチンおよび p27Kiplを含 む系において被験試料の存在下および非存在下で p27Kiplのュビキチン化を行うェ 程、 および p27Kiplにとりこまれたュビキチンの量を測定する工程、 および被験試 料の存在下および非存在下での p27Kiplにとりこまれたュビキチンの量を比較する 工程を含む、 p27Kiplの分解を抑制する物質のスクリーニング方法。 41. The system according to any one of claims 1 to 4 or the complex according to claim 5, ubiquitin activating enzyme, ubiquitin conjugating enzyme, ubiquitin and p27 Kipl in the presence of a test sample. more E which in the absence performs Yubikichin of p27 Kipl, and p27 measuring the amount of Yubikichin taken into Kipl, and test specimen presence and in the absence of Yubikichin taken into p27 Kipl of A method for screening a substance that suppresses the degradation of p27 Kipl , comprising a step of comparing the amounts.
42. 請求項 1〜4のいずれか 1項に記載の蛋白質または請求項 5に記載の複合 体、 ュビキチン活性化酵素、 ュビキチン結合酵素、 ュビキチンおよび p27Kiplを含 む系において被験試料の存在下および非存在下で p27Kiplのュビキチン化を行うェ 程、 および p27Kiplにとりこまれたュビキチンの量を測定する工程、 および被験試 料の存在下および非存在下での p27Kiplにとりこまれたュビキチンの量を比較する 工程を含む、 細胞周期の異常を原因とする疾患または細胞周期を調節することに より症状を軽減できる疾患の治療 のスクリーニング方法。 42.The system according to any one of claims 1 to 4 or the complex according to claim 5, ubiquitin activating enzyme, ubiquitin conjugating enzyme, ubiquitin and p27 Kipl in the presence of a test sample and more E which in the absence performs Yubikichin of p27 Kipl, and p27 measuring the amount of Yubikichin taken into Kipl, and test specimen presence and in the absence of Yubikichin taken into p27 Kipl of A screening method for the treatment of a disease caused by an abnormality in the cell cycle or a disease whose symptoms can be alleviated by regulating the cell cycle, comprising a step of comparing the amounts.
43. 疾患が癌である、 請求項 38に記載の方法。  43. The method of claim 38, wherein the disease is cancer.
44. 請求項 1〜4のいずれかの 1項に記載の蛋白質、 または請求項 5に記載の 複合体と特異的に結合する抗体。 44. The protein according to any one of claims 1 to 4, or the protein according to claim 5 An antibody that specifically binds to the complex.
45. 抗体が、 p27Kiplと請求項 1〜4のいずれか 1項に記載の蛋白質または請求 項 5に記載の複合体との結合を阻害する抗体である、 請求項 44に記載の抗体。 45. The antibody according to claim 44, wherein the antibody is an antibody that inhibits binding of p27 Kipl to the protein according to any one of claims 1 to 4 or the complex according to claim 5.
46. 抗体が、 請求項 1〜4のいずれか 1項に記載の蛋白質または請求項 5に記 載の複合体が有する p27Kiplをュビキチン化する活性を阻害する作用を示す抗体で ある、 請求項 44に記載の抗体。 46. The antibody, wherein the antibody has an activity of inhibiting the activity of the protein according to any one of claims 1 to 4 or the complex according to claim 5 to ubiquitinate p27 Kipl. 44. The antibody according to 44.
47. 請求項 44〜46のいずれか 1項に記載の抗体を用いる、 請求項 1〜4のいず れか 1項に記載の蛋白質または請求項 5に記載の複合体を免疫学的に検出又は定 量する方法。  47. The antibody according to any one of claims 44 to 46, wherein the protein according to any one of claims 1 to 4 or the complex according to claim 5 is immunologically detected. Or the method of quantification.
48. 請求項 44〜46のいずれか 1項に記載の抗体を用いて、 請求項 1〜4のいず れか 1項に記載の蛋白質の発現量が、 健常人と比較して蛋白質レベルで増加また は減少している疾患を判定または診断する方法。  48. The antibody according to any one of claims 44 to 46, wherein the expression level of the protein according to any one of claims 1 to 4 is expressed at a protein level as compared with a healthy person. A method of determining or diagnosing a disease that is increasing or decreasing.
49. 請求項 44〜46のいずれか 1項に記載の抗体を含有する、 請求項 1〜4のい ずれか 1項に記載の蛋白質の発現量が、 健常人と比較して蛋白質レベルで増加ま たは減少している疾患の診断薬。  49. The expression level of the protein according to any one of claims 1 to 4 containing the antibody according to any one of claims 44 to 46 is increased at the protein level as compared to a healthy person. Or a diagnostic agent for a declining disease.
50. 請求項 45または 46に記載の抗体を用いて、 p27Kiplのュビキチン化を阻害 する方法。 50. A method for inhibiting ubiquitination of p27 Kipl using the antibody according to claim 45 or 46.
51 . 請求項 45または 46に記載の抗体を用いて、 p27Kiplの分解を抑制する方法 51. A method for suppressing the degradation of p27 Kipl using the antibody according to claim 45 or 46.
52. 請求項 45または 46に記載の抗体を有効成分として含有する細胞周期の異 常を原因とする疾患または細胞周期を調節することにより症状を軽減できる疾患 の治療剤。 52. A therapeutic agent for a disease caused by abnormal cell cycle or a disease whose symptoms can be alleviated by regulating the cell cycle, comprising the antibody according to claim 45 or 46 as an active ingredient.
53. 疾患が癌である、 請求項 52に記載の治療剤。  53. The therapeutic agent according to claim 52, wherein the disease is cancer.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008517278A (en) * 2004-10-15 2008-05-22 シグナル ファーマシューティカルズ，エルエルシー p27 ubiquitination assay and methods of use thereof
WO2009054439A1 (en) 2007-10-23 2009-04-30 Institute Of Medicinal Molecular Design, Inc. Pai-1 production inhibitor
WO2016116922A1 (en) * 2015-01-19 2016-07-28 Rappaport Family Institute For Research In The Medical Sciences Ubiquitin ligase kpc1 promotes processing of p105 nf-kappab1 to p50, eliciting strong tumor suppression

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999033981A2 (en) * 1997-12-31 1999-07-08 Incyte Pharmaceuticals, Inc. Human signal peptide-containing proteins
WO2000055320A1 (en) * 1999-03-12 2000-09-21 Human Genome Sciences, Inc. Human pancreas and pancreatic cancer associated gene sequences and polypeptides
WO2001032927A2 (en) * 1999-11-04 2001-05-10 Incyte Genomics, Inc. Tissue specific genes of diagnostic import

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999033981A2 (en) * 1997-12-31 1999-07-08 Incyte Pharmaceuticals, Inc. Human signal peptide-containing proteins
WO2000055320A1 (en) * 1999-03-12 2000-09-21 Human Genome Sciences, Inc. Human pancreas and pancreatic cancer associated gene sequences and polypeptides
WO2001032927A2 (en) * 1999-11-04 2001-05-10 Incyte Genomics, Inc. Tissue specific genes of diagnostic import

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] Database accession no. (BC021811) *
HARA T. ET AL.: "Degradation of p27Kip1 at the G0-G1 transition mediated by a Skp2-independent ubiquitination pathway", J. BIOL. CHEM., vol. 276, no. 52, December 2001 (2001-12-01), pages 48937 - 48943, XP002970261 *
LI C. ET AL.: "Identification of a glialblastoma cell differentiation factor-related gene mRNA in human microvascular endothelial cells", GENOMICS, vol. 65, no. 3, May 2000 (2000-05-01), pages 243 - 252, XP004439365 *
MALEK N.P. ET AL.: "A mouse knock-in model exposes sequential proteolytic pathways that regulate p27Kip1 in G1 and S phase", NATURE, vol. 413, September 2001 (2001-09-01), pages 323 - 327, XP002970262 *
SCHULMAN B.A. ET AL.: "Insights into SCF ubiquitin ligases from the structure of the Skp-1Skp2 complex", NATURE, vol. 408, November 2000 (2000-11-01), pages 381 - 386, XP002970263 *
STRAUSBERG R.L. ET AL.: "Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequence", PROC. NATL. ACAD. SCI. USA, vol. 99, December 2002 (2002-12-01), pages 16899 - 16903, XP002245220 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008517278A (en) * 2004-10-15 2008-05-22 シグナル ファーマシューティカルズ，エルエルシー p27 ubiquitination assay and methods of use thereof
WO2009054439A1 (en) 2007-10-23 2009-04-30 Institute Of Medicinal Molecular Design, Inc. Pai-1 production inhibitor
WO2016116922A1 (en) * 2015-01-19 2016-07-28 Rappaport Family Institute For Research In The Medical Sciences Ubiquitin ligase kpc1 promotes processing of p105 nf-kappab1 to p50, eliciting strong tumor suppression
EP3265112A4 (en) * 2015-01-19 2018-10-03 Rappaport Family Institute For Research In The Medical Sciences Ubiquitin ligase kpc1 promotes processing of p105 nf-kappab1 to p50, eliciting strong tumor suppression
US11452759B2 (en) 2015-01-19 2022-09-27 Technion Research & Development Foundation Limited Ubiquitin ligase KPC1 promotes processing of P105 NF-κB1 to p50, eliciting strong tumor suppression

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