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WO2003100064A1 - Nouvelle ubiquitine-ligase - Google Patents

Nouvelle ubiquitine-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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Prior art keywords
sequence
protein
kipl
dna
kpc1
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English (en)
Japanese (ja)
Inventor
Keiichi Nakayama
Takumi Kamura
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KH Neochem Co Ltd
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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/ja
Publication of WO2003100064A1 publication Critical patent/WO2003100064A1/fr
Anticipated expiration legal-status Critical
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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
    • G01N33/57595—
    • 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

L'invention concerne une nouvelle protéine présentant une activité d'ubiquitination de p27Kipl, un ADN codant pour cette protéine, un procédé permettant de produire cette protéine, un procédé de criblage permettant d'identifier une substance inhibant l'ubiquitination de p27Kipl à l'aide de cette protéine, et une préparation de diagnostic et une préparation thérapeutique pour une pathologie causée par un cycle cellulaire anormal, telle qu'un cancer, contenant un polynucléotide ou un oligonucléotide de l'ADN ou de l'anticorps décrits.
PCT/JP2003/006749 2002-05-29 2003-05-29 Nouvelle ubiquitine-ligase Ceased WO2003100064A1 (fr)

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JP2008517278A (ja) * 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のユビキチン化アッセイ及びその使用方法
WO2009054439A1 (fr) 2007-10-23 2009-04-30 Institute Of Medicinal Molecular Design, Inc. Inhibiteur de la production de pai-1
WO2016116922A1 (fr) * 2015-01-19 2016-07-28 Rappaport Family Institute For Research In The Medical Sciences Promotion de la transformation de nf-kappab1 p105 en p50 par l'ubiquitine ligase kpc1, déclenchant un fort effet antitumoral

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WO2000055320A1 (fr) * 1999-03-12 2000-09-21 Human Genome Sciences, Inc. Sequences de genes et polypeptides associees au cancer du pancreas chez l'homme
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008517278A (ja) * 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のユビキチン化アッセイ及びその使用方法
WO2009054439A1 (fr) 2007-10-23 2009-04-30 Institute Of Medicinal Molecular Design, Inc. Inhibiteur de la production de pai-1
WO2016116922A1 (fr) * 2015-01-19 2016-07-28 Rappaport Family Institute For Research In The Medical Sciences Promotion de la transformation de nf-kappab1 p105 en p50 par l'ubiquitine ligase kpc1, déclenchant un fort effet antitumoral
EP3265112A4 (fr) * 2015-01-19 2018-10-03 Rappaport Family Institute For Research In The Medical Sciences Promotion de la transformation de nf-kappab1 p105 en p50 par l'ubiquitine ligase kpc1, déclenchant un fort effet antitumoral
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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