WO2005030805A1 - Novel protein complex and use thereof - Google Patents

Novel protein complex and use thereof Download PDF

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Publication number
WO2005030805A1
WO2005030805A1 PCT/JP2004/014527 JP2004014527W WO2005030805A1 WO 2005030805 A1 WO2005030805 A1 WO 2005030805A1 JP 2004014527 W JP2004014527 W JP 2004014527W WO 2005030805 A1 WO2005030805 A1 WO 2005030805A1
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seq
protein
amino acid
acid sequence
sequence represented
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PCT/JP2004/014527
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French (fr)
Japanese (ja)
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Takafumi Ishii
Shuji Sato
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Takeda Pharmaceutical Company Limited
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Publication of WO2005030805A1 publication Critical patent/WO2005030805A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention relates to novel binding proteins and the like. More specifically, a complex of a TACT427 protein and a protein capable of binding to the protein, an antibody against the complex (such as an antibody against the TACT427 protein that inhibits or promotes the formation of the complex), the complex
  • the present invention relates to screening of a compound or a salt thereof that inhibits or promotes body formation, a compound or a salt thereof obtainable by the screening, an agent for preventing or treating cancer or a neurodegenerative disease, a diagnostic agent, and the like.
  • TACT427 is a one-time transmembrane protein highly expressed in human cancer tissues such as breast cancer and lung cancer.It is expressed on the cytoplasmic membrane of cancer cells, and antisense oligonucleotides induce cancer cell apoptosis. Therefore, it has been reported that it is involved in the suppression of apoptosis of cancer cells (Patent Document 1 Japanese Patent Application No. 2003-296081). In addition, it is known that the rat ortholog of TACT427 is most frequently expressed in the brain and suppresses apoptosis of neuroblastoma cell lines induced by serum starvation (J. Biol. Chem. 278 , 10531-10537, 2003).
  • human Filamin A (GenBank NP_001447; J. Cell Biol. Ill, 1089-1105, 1990), human Filamin B (GenBank NP-001448; J. Biol. Chem. 273, 17531) Pp. 17538, 1998) and human Filamin C (GenBank NPJ01449; Biochem. Biophys. Res. Conunun. 251: 914-919, 1998) are referred to as FLNA, FLNB, and FLNC, respectively.
  • Filamin consisting of FLNA, FLNB and FLNC was initially found as an actin-binding protein (J. Biol. Chem. 250, 5696-5705, 1975).
  • Filamin has a structure in which an actin-binding domain consisting of 275 amino acids at the N-terminus and a ⁇ -sheet structure consisting of about 96 amino acids at the C-terminal side are repeated 24 times, and in the middle of the repeating structure.
  • Fi 1 amin forms a dimer and is involved in the cross-linking of actin filaments
  • Filamin shows that Fc receptor It binds to receptors such as integrin or dopamine D2 receptor, low-molecular-weight G proteins such as Rho, Rac or CDC42, or signaling molecules such as TRAF2 or SEK1 via the C-terminus to stimulate extracellular stimulation. It has been reported that it works as a scaffold that transmits the inside of cells (Non-Patent Document 1 Nature Rev. Mol. Cell Biol. 2, 138-145, 2001). Also characterized by impaired movement of cerebral cortical neurons
  • NAV2 human Neuron navigator 2
  • GenBank NPJ92009 Gene 290, pp. 73-94, 2002; also known as P0MFIL2 or HELAD1
  • NAV2 human Neuron navigator 2
  • NAV2 is a protein highly homologous to UNC-53 in C. elegans, and UNC-53 is involved in muscle cell migration and elongation of nerve axons (Development Vol. 129, pp. 3367-3379, 2002 Year) .
  • NAV2 is a protein consisting of 2428 amino acids, consisting of a potent luponin homology domain consisting of amino acids 89 to 190, an ATP / GTP binding site consisting of amino acids 2098 to 2105, and amino acids 2091 to 2208. Consists of the ATPase domain (Genomics 80, 21-30, 2002; Proc. Natl. Acad. Sci. USA 99, 3422-3427, 2002). Marauders have been found to be localized in the cytoplasm or nucleus in cells, to be up-regulated in human colorectal cancer, to have ATP / GTP binding sites and
  • BTBD2 human BTB domain-containing 2
  • BTBD2 is a protein consisting of 525 amino acids and having a BTB domain consisting of amino acids 110 to 217 (Piam accession No. PF00651). Analysis by the yeast two-hybrid method revealed that it binds to DNA topoisomerase I (hereinafter referred to as T0P1). It has been reported that the DNA supercoil relaxing activity and the DNA-cleaving activity are inhibited (Non-Patent Document 3 BMC Genomics Vol. 3, pp. 1-9, 2002).
  • NPJ37533 is referred to as RAB3IL1.
  • RAB3IL1 is registered as a protein showing 59% homology with RAB3A interacting protein (RAB3IP, GenBank NP-071901). Based on sequence homology with RAB3 IP, it is predicted that Rab3A has a GTP / GDP nucleotide exchange activity and is involved in vesicle trafficking exocytosis I ⁇ cis (Public HumanPSD TM volume of the BioKnow ledge Library: www. incyte. com / control / researchproducts / insilico / proteome). Disclosure of the invention
  • Targets a protein complex that is specifically expressed in cancer cells and nerve cells, and is a safe cancer prevention and treatment agent that induces cancer cell growth inhibition, and a safe and superior nerve that suppresses nerve cell death. Prevention and treatment of degenerative diseases.
  • TACT427 By regulating the anti-apoptotic signal mediated by TACT427, it can be expected to kill cancer cells and induce an antitumor effect, or suppress neuronal cell death and prevent or treat neurodegenerative diseases. There are few detailed reports on the intracellular binding protein or intracellular signaling pathway of TACT427 at this time.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, it has been confirmed that the binding of FLNA, FLNB, FLN NAV2, BTBD2 and RAB3IL1 to the intracellular region of TACT427 by the yeast two hybrid method (Trends In Genet. 10, 286-292, 1994; Annu. Rev. Genet. 31, 663-704, 1997; The Yeast Two-Hybrid System, Oxford University Press, Bartel and Fields, 1997), and further, a method for evaluating the activity of regulating a TACT427-dependent anti-apoptotic signal was found. Based on these findings, studies were repeated, and the present invention was completed.
  • [2] a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45 is represented by SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, The complex according to the above [1], which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 25 or SEQ ID NO: 27,
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45 is represented by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45 is obtained as follows: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, The antibody according to the above [4] or [5], which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 25 or SEQ ID NO: 27,
  • the medicament according to the above (7) which is an agent for promoting apoptosis of cancer cells or an agent for preventing or treating cancer;
  • the medicament according to the above (8) which is a neuronal apoptosis inhibitor or a preventive / therapeutic agent for a neurodegenerative disease
  • a diagnostic agent comprising the antibody of (4) or (5) above,
  • SEQ ID NO: 50 or identical or real amino acid sequence to SEQ ID NO: 51 (13)
  • a partial peptide of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 46 is the second peptide of the amino acid sequence represented by SEQ ID NO: 46.
  • a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 47 is the first peptide of the amino acid sequence represented by SEQ ID NO: 47
  • a partial peptide of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 48 is the second peptide of the amino acid sequence represented by SEQ ID NO: 48
  • a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49 is the sixth peptide of the amino acid sequence represented by SEQ ID NO: 49.
  • a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 50 is the second peptide of the amino acid sequence represented by SEQ ID NO: 50. 0
  • a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 51 is the second peptide of the amino acid sequence represented by SEQ ID NO: 51.
  • [16a] a compound or a salt thereof that inhibits the formation of the complex according to [1], obtained by using the screening method according to [13] or the screening kit according to [16],
  • the medicament according to the above (16d) which is a neuronal apoptosis inhibitor or a prophylactic / therapeutic agent for a neurodegenerative disease.
  • an agent for promoting apoptosis of cancer cells or an agent for preventing or treating cancer comprising a compound or a salt thereof that inhibits the formation of the complex according to [1];
  • a neuronal apoptosis inhibitor or a prophylactic or therapeutic agent for a neurodegenerative disease comprising the compound or a salt thereof that promotes the formation of the complex according to the above [1];
  • [18a] a compound or a salt thereof that promotes the formation of the complex according to the above [1], [19] SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 Characterized by using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 50 or SEQ ID NO: 51, a partial peptide thereof, or a salt thereof, A method for screening a compound or a salt thereof having a prophylactic or therapeutic effect on cancer or neurodegenerative disease
  • [20] a method for promoting apoptosis of cancer cells or a method for preventing or treating cancer, which comprises inhibiting the formation of the complex according to [1].
  • [21] a method for inhibiting apoptosis of neurons or preventing and treating neurodegenerative diseases, which comprises promoting the formation of the complex according to [1] above;
  • (23) a method for inhibiting apoptosis of neurons or preventing and treating neurodegenerative diseases, comprising administering to a mammal an effective amount of the antibody according to (5).
  • FIG. 1 is a diagram showing a hydrophobicity plot of TACT427-A.
  • FIG. 2 is a diagram showing a hydrophobicity plot of TACT427-A2.
  • FIG. 3 is a diagram showing a hydrophobicity plot of TACT427-B.
  • FIG. 4 is a diagram showing a hydrophobicity plot of TACT427-2B.
  • FIG. 5 is a diagram showing a hydrophobicity plot of TACT427-C.
  • FIG. 6 is a diagram showing a hydrophobicity plot of TACT427-C2. BEST MODE FOR CARRYING OUT THE INVENTION
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45 is referred to as TACT427 protein.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 46 is referred to as FLNA protein.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 47 is referred to as FLNB protein.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 48 is referred to as FLNC protein.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49 is referred to as NAV2 protein.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 50 is referred to as BTBD2 protein.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 51 is referred to as RAB3IL1 protein.
  • the TACT427 protein, FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein and RAB3IL1 protein may be collectively referred to as the protein of the present invention or the protein used in the present invention.
  • the protein of the present invention can be obtained from cells of human warm-blooded animals (eg, guinea pig, rat, mouse, chicken, rabbit, egret, bush, sheep, horsetail, monkey, etc.) (eg, liver cells, spleen cells, nerve cells) , Glial cells, kidney jS cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, lines Fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, ⁇ cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocyte
  • the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling.
  • Proteins of the present invention C-terminal, carboxyl group (-C00H), carboxylate (-COO-), amide (-C0NH 2) or ester - may be either (C00R).
  • R in the ester e.g., methyl, Echiru, n- propyl, isopropyl, C HJ alkyl group such as n- heptyl, for example, cyclopentyl, C 3. 8 cycloalkyl groups such as cyclohexyl shea click port, e.g.
  • phenyl, alpha-naphthyl of which C 6 _ 12 Ariru group, e.g., benzyl, C 7 _ 14 such as flying one Nafuchiru C Bok 2 alkyl groups, such as phenyl -CH ⁇ alkyl group or alpha-naphthylmethyl, such as phenethyl Ararukiru And a pivaloyloxymethyl group.
  • the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus
  • the protein of the present invention includes those in which the carboxyl group is amidated or esterified.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the protein of the present invention has an N-terminal amino acid residue (eg, a methionine residue). Is protected by a protecting group (for example, a CJsacyl group such as a CHJ alkanol such as a formyl group or an acetyl group), and an N-terminal glutamine formed by cleavage in vivo.
  • a protecting group for example, a CJsacyl group such as a CHJ alkanol such as a formyl group or an acetyl group
  • an N-terminal glutamine formed by cleavage in vivo.
  • Appropriate protection of the residue in which the residue is pyroglutamine-oxidized, or a substituent on the side chain of the amino acid in the molecule eg, 1H, —SH, amino, imidazole, indole, guanidino, etc.
  • a substituent on the side chain of the amino acid in the molecule eg, 1H, —SH, amino, imidazole, indole, guanidino, etc.
  • Protected by a group for example, C ⁇ such as formyl group or acetyl group; CHJ acetyl group such as alkanoyl group
  • complex protein such as so-called sugar protein to which a sugar chain is bonded. And so on.
  • the homology of the following amino acid sequences is calculated using the homology calculation algorithm NCBI BLAST.
  • TACT427 protein examples include: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25 or a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 is used. Furthermore, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 7 is also included in the TACT427 protein.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 about 70% or more, preferably about 80% or more, particularly preferably about 70% or more of the amino acid sequence represented by SEQ ID NO: 1 Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, may be mentioned.
  • Examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by the aforementioned SEQ ID NO: 1 And a protein having substantially the same activity as a protein containing the amino acid sequence represented by SEQ ID NO: 1.
  • the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4 is about 70% or more, preferably about 80% or more, particularly preferably the amino acid sequence represented by SEQ ID NO: 4. Have a homology of about 90% or more, and most preferably about 95% or more. And amino acid sequences.
  • amino acid sequences having about 95% or more homology are particularly preferred.
  • Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 4 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 4 However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 4 is preferable.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 10 is about 70% or more, preferably about 80% or more, particularly preferably the amino acid sequence represented by SEQ ID NO: 10. Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 10 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 10 described above. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 10 is preferable.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 15 includes about 70% or more, preferably about 80% or more, particularly preferably, the amino acid sequence represented by SEQ ID NO: 15 Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology.
  • Amino acid sequences having a homology of 90% or more, most preferably about 95% or more may also be used.
  • Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 15 include, for example, an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 15 And a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 15 is preferable.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 17 includes about 70% or more, preferably about 80% or more, particularly preferably the amino acid sequence represented by SEQ ID NO: 17 Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology.
  • amino acid sequence represented by SEQ ID NO: 17 is about 70% or more, preferably about 80% or more, particularly preferably about 70% or more with respect to the 43rd to 292nd amino acid sequence.
  • Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, may also be used.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 17 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 17 described above. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 17 is preferable.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 20 includes about 70% or more, preferably about 80% or more, particularly preferably, the amino acid sequence represented by SEQ ID NO: 20. Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology. For example, about 70% or more, preferably about 80% or more, particularly preferably about 70% or more of the amino acid sequence from the 47th to the 296th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20 Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, may also be used.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 20 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 20 described above. And a protein having substantially the same activity as that of the protein having the amino acid sequence represented by SEQ ID NO: 20 is preferable.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 22 includes about 70% or more, preferably about 80% or more, particularly preferably, the amino acid sequence represented by SEQ ID NO: 22. Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology. For example, about 70% or more of the amino acid sequence represented by SEQ ID NO: 22 from amino acid sequence 43 to 292 in the amino acid sequence represented by SEQ ID NO: 22 is preferable. Preferably, an amino acid sequence having about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology is also included.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 22 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 22 described above. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 22 is preferable.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25 about 70% or more, preferably about 80% or more, particularly preferably the amino acid sequence represented by SEQ ID NO: 25 Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology.
  • the amino acid sequence represented by SEQ ID NO: 25 is about 70% or more, preferably about 80% or more, particularly preferably about 70% or more of the amino acid sequence from the 47th to the 296th amino acid sequence. Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, may also be used.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25 described above. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 25 is preferable.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 27 includes about 70% or more, preferably about 80% or more, particularly preferably, the amino acid sequence represented by SEQ ID NO: 27. Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology.
  • the amino acid sequence represented by SEQ ID NO: 27 is at least about 70%, preferably at least about 80%, particularly preferably at least about 80% with respect to the amino acid sequence from the 43rd to 29th amino acid sequence. Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, and the like can also be mentioned.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 27 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 27 described above.
  • An amino acid represented by SEQ ID NO: 27 Proteins having substantially the same activity as the protein containing the sequence are preferred.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 7 about 70% or more, preferably about 80% or more, particularly preferably about 70% or more of the amino acid sequence represented by SEQ ID NO: 7
  • Amino acid sequences having a homology of 90% or more, most preferably about 95% or more may be mentioned.
  • an amino acid sequence having about 95% or more homology is exemplified.
  • Examples of the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 7 include, for example, the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 7
  • a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 7 is preferred.
  • Examples of substantially the same activity include chloropoxidase activity.
  • Substantially identical indicates that the properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, the activity of the black mouth peroxidase is equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). However, quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the measurement of black mouth peroxidase activity may be performed according to a known method, for example, Journal of Biological Chemistry (J. Biol. Cem.) Vol.241, pp.1763-1768 (1966), etc. Can be measured according to the method described in (1) or a method analogous thereto.
  • TACT427 protein examples include: (i) SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 2 0, SEQ ID NO: 22, SEQ ID NO: 25 or SEQ ID NO: 27, 1 or 2 or more amino acids in the amino acid sequence (for example, about 1 to 100, preferably 1 to 30) Amino acid sequence in which about 1, preferably about 1 to 10, and more preferably (1 to 5) amino acids have been deleted, (ii) SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: No .: 7, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, 1 or more in the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 27 (For example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably the number (1 to 5) of amino acid sequences; (iii) SEQ ID NO
  • mucins such as proteins containing amino acid sequences combining them are also included.
  • the position of the insertion, deletion or substitution is not particularly limited.
  • TACT427 protein examples include, for example, a protein containing an amino acid sequence represented by SEQ ID NO: 1, a protein containing an amino acid sequence represented by SEQ ID NO: 4, and a protein represented by SEQ ID NO: 7.
  • an amino acid sequence containing the amino acid sequence represented by SEQ ID NO: 27 examples include, for example, a protein containing an amino acid sequence represented by SEQ ID NO: 1, a protein containing an amino acid sequence represented by SEQ ID NO: 4, and a protein represented by SEQ ID NO: 7.
  • the partial peptide of the TACT427 protein is the partial peptide of the TACT427 protein described above, and preferably has the same properties as the TACT427 protein described above. Any of them may be used.
  • a peptide having at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more amino acid sequences among the constituent amino acid sequences of the TACT427 protein is used.
  • Specific examples include a peptide having the amino acid sequence represented by SEQ ID NO: 45.
  • the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 includes SEQ ID NO: : 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 and about 70% or more, preferably about 80% or more, and particularly preferably about 80% or more Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology.
  • SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 Is, for example, an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51
  • Substantially homogenous indicates that the properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, it is preferable that the potent activities are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). Quantitative factors such as degree and molecular weight of the protein may vary.
  • Examples of the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein include, for example, (i) SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: : 50 Or one or more in the amino acid sequence represented by SEQ ID NO: 51 (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably the number (1 to (Ii) SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51
  • the position of the insertion, deletion or substitution is not particularly limited.
  • FLNA protein FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein
  • partial peptides of FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3 IL1 protein the aforementioned FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein Or a partial peptide of the RAB3IL1 protein, preferably any of the above-mentioned FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein. It may be one of.
  • FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3 IL1 protein at least 20 or more, preferably 50 or more, more preferably 70 or more of the amino acid sequence, More preferably, peptides having an amino acid sequence of 100 or more, most preferably 200 or more are used.
  • the partial peptide of the FLNA protein includes, for example, SEQ ID NO:
  • a partial peptide having the amino acid sequence at positions 2162 to 2412, 2206 to 2355, or 2141 to 2351 of the amino acid sequence represented by 46 As a partial peptide of the FLNB protein, for example, it has the amino acid sequence of the 1960th to 2299th, the 2156th to the 2314th or the 2174th to the 2565th of the amino acid sequence represented by SEQ ID NO: 47 And partial peptides.
  • Examples of the partial peptide of the FLNC protein include a partial peptide having the amino acid sequence at positions 2295 to 2705 of the amino acid sequence represented by SEQ ID NO: 48.
  • the partial peptide of the NAV2 protein includes, for example, a partial peptide having the 68th to 286th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49.
  • SEQ ID NO: 49 As a partial peptide of the BTBD2 protein, for example, SEQ ID NO:
  • Partial peptides having the 201st to 525th or the 215th to 506th amino acid sequence of the amino acid sequence represented by 50 are exemplified.
  • Examples of the partial peptide of RAB3 IL-1 protein include a partial peptide having the 282nd to 356th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51.
  • the partial peptide of the protein of the present invention has one or more (preferably about 1 to 10, more preferably 1 to 10) amino acids in the amino acid sequence. 5) amino acids are deleted, or one or more (preferably about 1-20, more preferably about 1-10, and more preferably a number (1-5 ) Amino acids are added or One or two or more amino acids (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably a number (1 to 5)) are inserted into the amino acid sequence. One or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the amino acid sequence may be substituted with another amino acid.
  • the partial peptide used in the present invention the C-terminus force Rupokishiru group (-C00H), carboxylate (-C00-), amide (-C0NH 2) or ester - may be either (C00R).
  • the partial peptide used in the present invention includes, as in the above-mentioned protein of the present invention, those having a carbonyl group (or carboxylate) other than the C-terminal, and the N-terminal amino acid residue (Eg, methionine residue) whose amino group is protected with a protecting group, N-terminal cleavage in vivo, glutamine residue generated by pyroglutamine oxidation, substitution of amino acid in the molecule on the side chain Also included are those in which the group is protected by an appropriate protecting group, and those in which a sugar chain is bonded, such as a complex peptide such as a so-called glycopeptide.
  • the partial peptide used in the present invention can also be used as an antigen for producing an antibody.
  • salt of the protein of the present invention or its partial peptide a salt with a physiologically acceptable acid (eg, an inorganic acid, an organic acid) or a base (eg, an alkali metal salt) is used. Acceptable acid addition salts are preferred.
  • a physiologically acceptable acid eg, an inorganic acid, an organic acid
  • a base eg, an alkali metal salt
  • salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid
  • Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid and the like are used.
  • the protein of the present invention or a partial peptide thereof or a salt thereof can be produced according to the below-mentioned Reference Examples, or from the above-described method for purifying a protein known per se from human or warm-blooded animal cells or tissues, It can also be produced by culturing a transformant containing DNA encoding the protein. It can also be produced according to the peptide synthesis method described below.
  • human or mammalian tissues or cells human or mammalian tissues or cells are homogenized, and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography, or the like. Purification and isolation can be achieved by combining chromatography such as mouth chromatography.
  • a commercially available resin for protein synthesis can be usually used.
  • a resin for protein synthesis examples include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxy resin.
  • the condensation of the above protected amino acids various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable.
  • carpoimides DCC, N, N'-diisopropylcarpoimide, N-ethyl N,-(3-dimethylaminoprolyl) carpoimide, and the like are used.
  • the protected amino acid may be added directly to the resin with a racemization inhibitor additive (eg, HOBt, HOOBt) or may be pre-protected as a symmetric anhydride or HOBtester or HOOBtester.
  • the amino acid can be added to the resin after activation.
  • the solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction.
  • solvents known to be usable for the protein condensation reaction for example, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpi Acid amides such as mouth ridone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; sulfoxides such as dimethyl sulfoxide; ethers such as pyridine, dioxane and tetrahydrofuran; and acetonitrile And nitriles such as propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof.
  • the reaction temperature is appropriately selected from the range that can be used for the protein bond formation reaction, and is usually appropriately selected from the range of about ⁇ 20 ° C. (: to 50 ° C.)
  • Activated amino The acid derivative is usually used in an excess of 1.5 to 4 times.As a result of the test using the ninhydrin reaction, if the condensation is insufficient, it is sufficient to repeat the condensation reaction without removing the protecting group. If sufficient condensation cannot be obtained even after repeating the reaction, the unreacted amino acid is acetylated with acetic anhydride or acetylimidazole so that the subsequent reaction is not affected. Can be
  • Examples of the protecting group for the starting amino group include Z, Boc, t-pentyloxycarbonyl, isopolnyoxycarbonyl, 4-methoxybenzyloxycarponyl, C1-Z, Br-Z, and adaman.
  • Tyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the lipoxyl group can be, for example, a linear, branched or cyclic alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Alkyl esterification), aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxy It can be protected by carbonylation, tert-butoxycarbonylhydrazide, tritylhydrazide and the like.
  • alkyl esterified eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl,
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • Suitable groups for this esterification include, for example, lower (C ⁇ 6 ) alkanol groups such as acetyl group, aroyl groups such as benzoyl group, and benzyloxy group.
  • Groups derived from carbonic acid such as a carbonyl group and an ethoxycarbonyl group are used.
  • Examples of a group suitable for etherification include a benzyl group, a tetrahydrobiranyl group, and a t-butyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, Bz l, C 1 2 - B zl, 2- two Torobenjiru, B r- Z, such as t one-butyl is used.
  • a protecting group for imidazole of histidine for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • activated raw propyloxyl groups include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5_trichloromouth phenol, 2,4-dinitrophenol) Phenol, cyanomethyl alcohol, para-nitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt).
  • active esters eg, pentachlorophenol, 2,4,5_trichloromouth phenol, 2,4-dinitrophenol
  • Phenol cyanomethyl alcohol
  • para-nitrophenol HONB
  • N-hydroxysuccinimide N-hydroxyphthalimide
  • esters with HOBt esters with HOBt
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or trifluoromethane.
  • a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or trifluoromethane.
  • Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, etc. Is also used.
  • the elimination reaction by the above-mentioned acid treatment is generally performed at a temperature of about 120 ° C to 40 ° C.
  • a force-thione scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol and the like.
  • a force-thione scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol and the like.
  • the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributofan is substituted with 1,2-ethanedithiol, 1,4-dithiol.
  • alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc. Therefore, it is also removed.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • Another method for obtaining an amide form of a protein or partial peptide is, for example, first protecting the carboxy-terminal amino acid with amidation of the amino group, and then adding a peptide (protein) chain on the amino group side to the desired length. After the elongation, a protein or partial peptide from which only the protecting group for the N-terminal amino group of the peptide chain has been removed and a protein or partial peptide from which only the protecting group for the C-terminal carboxyl group has been removed. Is produced and these proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above.
  • the crude protein or peptide can be purified using various known purification means, and the main fraction can be freeze-dried to obtain the desired protein or peptide amide.
  • an ester of a protein or peptide for example, after condensing the carboxy-terminal amino acid with a desired alcohol to form an amino acid ester, the desired protein is synthesized in the same manner as in the amide of a protein or peptide.
  • An ester of a protein or a peptide can be obtained.
  • the partial peptide or a salt thereof used in the present invention can be produced according to a peptide synthesis method known per se or by cleaving the protein of the present invention with an appropriate peptidase.
  • a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the target peptide. can do.
  • Examples of the known condensation method and elimination of the protecting group include the methods described in the following ( ⁇ ) to (V).
  • the polynucleotide encoding the protein of the present invention may be any polynucleotide as long as it contains the above-described nucleotide sequence encoding the protein of the present invention.
  • it is DNA.
  • the DNA may be any one of a genomic DNA, a genomic DNA library, the above-described cell and tissue-derived cDNA, the above-described cell and tissue-derived cDNA library, and a synthetic DNA.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
  • plasmid plasmid
  • cosmid plasmid
  • phagemid a preparation of a total RNA or mRNA fraction from the cells and tissues described above, direct reverse
  • RT-PCR method transcriptase polymerase chain reaction
  • TACT427 protein examples include:
  • DNA containing the nucleotide sequence represented by SEQ ID NO: 5, or SEQ ID NO: 5 Encodes a protein having a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 4, and having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 4.
  • a protein comprising a nucleotide sequence which hybridizes under high stringent conditions to the nucleotide sequence represented by 16 and having substantially the same properties as the protein comprising the amino acid sequence represented by SEQ ID NO: 15 D NA, which codes
  • DNA containing the nucleotide sequence represented by SEQ ID NO: 21 or a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 21 A DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 20;
  • DNA containing the nucleotide sequence represented by SEQ ID NO: 23 or a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 23 A DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 22;
  • DNA containing the nucleotide sequence represented by SEQ ID NO: 26, or hybridizing with the nucleotide sequence represented by SEQ ID NO: 26 under high stringent conditions DNA encoding a protein having a base sequence and having substantially the same properties as a protein having an amino acid sequence represented by SEQ ID NO: 25;
  • (x) a DNA containing the nucleotide sequence represented by SEQ ID NO: 28, or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 28 under high stringent conditions, SEQ ID NO: 27
  • Any DNA may be used as long as it encodes a protein having substantially the same properties as the protein having the amino acid sequence represented by
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 under high stringency conditions include, for example, about 70% or more, preferably about 80% or more of the nucleotide sequence represented by SEQ ID NO: 2. Particularly preferably, DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used. It is highly stringent with the nucleotide sequence represented by SEQ ID NO: 5.
  • Examples of the DNA that can hybridize under the conditions include, for example, about 70% or more, preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 90% or more of the nucleotide sequence represented by SEQ ID NO: 5.
  • DNA containing a nucleotide sequence having a homology of 95% or more is used, for example, about 70% or more, preferably, about 139 to 888 of the nucleotide sequence represented by SEQ ID NO: 5. Is about 80% or more, particularly preferred Is used, such as DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more.
  • Examples of a DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 8 under high stringency conditions include, for example, about 70% or more, preferably about 80% or more of the nucleotide sequence represented by SEQ ID NO: 8 , particularly preferably about 90%, most preferably rather t is a DNA containing the base sequence having about 95% homology, for example, is used, SEQ ID NO: from 1728 th nucleotide sequence represented by 8 About 70% or more, preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more of a base sequence having a homology of about 1782 with the nucleotide sequence, etc. Is also used.
  • DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 11 under high stringency conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 11
  • a DNA containing a nucleotide sequence having a homology of about 70% or more, preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more is used.
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 16 under high stringency conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 16 and about 70% or more, preferably about 80% or more. Particularly preferably, a DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used.
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 18 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 18 and about 70% or more, preferably about 80% or more. Particularly preferably, a DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used.
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 21 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 21 and about 70% or more, preferably about 80% or more. Particularly preferably, a DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used.
  • Examples of the DNA which can hybridize with the nucleotide sequence represented by SEQ ID NO: 23 under high stringent conditions include, for example, about 70% or more, preferably about 80% or more of the nucleotide sequence represented by SEQ ID NO: 23 Particularly preferably, a DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used.
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 26 under high stringent conditions include, for example, about 70% or more, preferably about 80% or more of the nucleotide sequence represented by SEQ ID NO: 26 Particularly preferably, a DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used.
  • DNA that can be resized include a homology of about 70% or more, preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more with the nucleotide sequence represented by SEQ ID NO: 28.
  • DNA containing a base sequence having a property is used.
  • Hybridization can be carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be performed under high stringency conditions.
  • High stringency conditions are, for example, when the sodium concentration is about 19 to 40 m
  • M preferably about 19-20 mM and a temperature of about 50-70 ° (preferably about 60
  • ⁇ 651 Indicates the condition. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.
  • the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 includes a DNA containing the base sequence represented by SEQ ID NO: 2 or a DNA containing the amino acid sequence represented by SEQ ID NO: 2 And (ii) a protein containing the amino acid sequence represented by SEQ ID NO: 4.
  • Examples of the DNA include a DNA containing the nucleotide sequence represented by SEQ ID NO: 5 or a DNA containing the nucleotide sequence represented by SEQ ID NO: 6;
  • the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 7 is a DNA containing the base sequence represented by SEQ ID NO: 8 or the base represented by SEQ ID NO: 9 DNA containing the sequence,
  • the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 10 may be a DNA containing the base sequence represented by SEQ ID NO: 11 or DNA containing the base sequence represented by SEQ ID NO: 12, etc.
  • the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 17 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 18 or the nucleotide sequence represented by SEQ ID NO: 19 DNA containing
  • the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 20 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 21 or the nucleotide sequence represented by SEQ ID NO: 24 DNA containing
  • the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 22 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 23 or the nucleotide sequence represented by SEQ ID NO: 24 DNA containing
  • DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 25 DNA containing the nucleotide sequence represented by SEQ ID NO: 26 or the nucleotide sequence represented by SEQ ID NO: 29 Such as DNA containing
  • the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 27 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 28 or the nucleotide sequence represented by SEQ ID NO: 29 And the like containing DNA.
  • Examples of the DNA encoding the FLNA protein include a DNA containing the nucleotide sequence represented by SEQ ID NO: 52, or a base that hybridizes with the nucleotide sequence represented by SEQ ID NO: 52 under high stringent conditions. DNA encoding a protein having a sequence and having substantially the same properties as a protein having the amino acid sequence represented by SEQ ID NO: 46.
  • Examples of the DNA encoding the FLNB protein include a DNA containing the base sequence represented by SEQ ID NO: 53, or a base sequence represented by SEQ ID NO: 53 and a string that hybridizes under simple conditions. DNA encoding a protein having a sequence and having substantially the same properties as a protein having the amino acid sequence represented by SEQ ID NO: 47.
  • Examples of the DNA encoding the FLNC protein include, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 54 or the nucleotide sequence represented by SEQ ID NO: 54 under stringent conditions. Examples include DNA encoding a protein containing a hybridizing base sequence and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 48.
  • Examples of the DNA encoding the NAV2 protein include, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 55 or the nucleotide sequence represented by SEQ ID NO: 55 under highly stringent conditions. And a DNA encoding a protein having a property substantially the same as that of the protein having the amino acid sequence represented by SEQ ID NO: 49.
  • Examples of the DNA encoding the BTBD2 protein include, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 56 or a DNA comprising the nucleotide sequence represented by SEQ ID NO: 56 in a highly stringent condition.
  • Examples include a DNA encoding a protein containing a soybean base sequence and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 50.
  • Examples of the DNA encoding the MB3IL1 protein include, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 57 or a DNA which hybridizes with the nucleotide sequence represented by SEQ ID NO: 57 under high stringent conditions.
  • Examples include a DNA encoding a protein containing a soybean base sequence and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 51.
  • the DNA that can be hybridized with, for example, is represented by SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57
  • DNA having a nucleotide sequence having a homology of about 70% or more, preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more with the nucleotide sequence is used.
  • Hybridization can be carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be performed under high stringency conditions.
  • the high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° (:, preferably, about 60 ° C.).
  • the condition is about 65 ° C.
  • the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
  • the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 46 includes the DNA containing the base sequence represented by SEQ ID NO: 52
  • Examples of the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 47 include a DNA having the nucleotide sequence represented by SEQ ID NO: 53.
  • DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 48 includes, for example, DNA having the nucleotide sequence represented by SEQ ID NO: 54, and (iv) SEQ ID NO:
  • the DNA encoding the protein having the amino acid sequence represented by 49 is, for example, the DNA having the nucleotide sequence represented by SEQ ID NO: 55.
  • the DNA encoding the protein containing the represented amino acid sequence includes SEQ ID NO:
  • the DNA containing the base sequence represented by 56 is, for example, (vi) the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 50 is represented by SEQ ID NO: 56.
  • DNA containing the base sequence to be used is used.
  • the polynucleotide (eg, DNA) encoding the partial peptide used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the partial peptide used in the present invention.
  • genomic DNA, cDNA, the above-mentioned cell-derived tissue-derived cDNA, and the above-mentioned cells may be either a tissue-derived cDNA library or a synthetic DNA.
  • DNAs encoding partial peptides of the TACT427 protein include, for example, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21, DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 23, SEQ ID NO: 26 or SEQ ID NO: 28, or SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11. SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 26 or hybridizes with the nucleotide sequence represented by SEQ ID NO: 28 under high stringent conditions.
  • a DNA containing a base sequence and a part of a DNA encoding a protein having substantially the same activity as the TACT427 protein may be used.
  • SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 26 or SEQ ID NO: 28 DNA that can hybridize with the base sequence represented by has the same significance as described above.
  • Hybridization methods and high stringency conditions are the same as described above.
  • Examples of the DNA encoding the partial peptide of the FLNA protein include, for example, DNA having a portion of DNA containing the nucleotide sequence represented by SEQ ID NO: 52, or the nucleotide sequence represented by SEQ ID NO: 52 and high string Contains a nucleotide sequence that hybridizes under mild conditions and contains a portion of DNA encoding a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 46. DNA or the like is used.
  • Examples of the DNA encoding the partial peptide of the FLNB protein include, for example, a DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 53, or the base sequence represented by SEQ ID NO: 53 and high stringency. Contains a nucleotide sequence that hybridizes under simple conditions, and contains a portion of DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 47. DNA or the like is used.
  • DNA encoding a partial peptide of FLNC protein includes, for example, SEQ ID NO: No .: a DNA having a portion of the DNA containing the base sequence represented by 54, or a base sequence which hybridizes with the base sequence represented by SEQ ID NO: 54 under high stringent conditions; For example, a DNA containing a part of a DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by 48 may be used.
  • Examples of the DNA encoding the partial peptide of the NAV2 protein include, for example, a DNA having a part of the DNA containing the nucleotide sequence represented by SEQ ID NO: 55, or the nucleotide sequence represented by SEQ ID NO: 55 and high stringency Containing a portion of DNA encoding a protein having a nucleotide sequence which hybridizes under simple conditions and having substantially the same properties as a protein having an amino acid sequence represented by SEQ ID NO: 49 Are used.
  • Examples of the DNA encoding the partial peptide of the BTBD2 protein include, for example, a DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 56, or the base sequence represented by SEQ ID NO: 56 and high stringency. DNA containing a portion of DNA encoding a protein having a nucleotide sequence that hybridizes under simple conditions and having substantially the same properties as a protein having an amino acid sequence represented by SEQ ID NO: 50, etc. Is used.
  • Examples of the DNA encoding the partial peptide of the RAB3IL1 protein include a DNA having a portion of the DNA containing the nucleotide sequence represented by SEQ ID NO: 57, or a DNA having a nucleotide sequence represented by SEQ ID NO: 57. Contains a nucleotide sequence that hybridizes under stringent conditions and contains a portion of DNA encoding a protein having substantially the same properties as a protein having the amino acid sequence represented by SEQ ID NO: 51 DNA or the like is used.
  • SEQ ID NO: 52 SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57 .
  • the protein of the present invention and its partial peptide (hereinafter, DNs encoding them) In the description of the cloning and expression of A, these may be simply abbreviated as the protein of the present invention.)
  • DNs partial peptide
  • the nucleotide sequence encoding the protein of the present invention Amplification by the PCR method using a synthetic DNA primer having a portion, or DNA integration into an appropriate vector, DNA fragment encoding a part or all of the protein of the present invention, or synthetic DNA Can be selected by hybridization with those labeled using Hybridization methods are described, for example, in Molecular Cloning 2nd (J. Sambrook et al., Cold
  • the DNA base sequence can be converted using PCR, a known kit, for example, Mutan TM -Super Express Km (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.), etc., using the 0M-LA PCR method.
  • the method can be carried out according to a method known per se, such as the gapped duplex method or the Kunkel method, or a method analogous thereto.
  • the DNA encoding the cloned protein can be used as it is depending on the purpose, or it can be used after digesting with a restriction enzyme or adding a linker, if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
  • the expression vector for the protein of the present invention can be prepared, for example, by (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) converting the DNA fragment into a promoter in an appropriate expression vector. It can be manufactured by connecting downstream.
  • vectors examples include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15) bacteriophage such as ⁇ phage, retrovirus, vaccinia virus, In addition to animal viruses such as baculovirus, pAl-11, pXTl, pRc / CMV, pRcZRSV, pcDNA IZNeo and the like are used.
  • Escherichia coli-derived plasmids eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis-derived plasmids eg, pUB110, pTP5, pC194
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SRa promoter when animal cells are used as host, SRa promoter, SV40 promoter, LTR motor, CMV promoter, HSV-TK promoter, etc. are included.c Among them, CMV (cytomegalovirus) promoter, SRo! It is preferable to use a promoter overnight.
  • tr When the host is Eshierihia genus bacteria, tr [rho promoter Isseki one, lac promoter, re cA promoter, AP L promoter one evening one, 1 pp promoter, T 7 promoter one is Ru host Bacillus der When the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. For example, SP ⁇ 1 promoter, SP02 promoter, penP promoter, etc. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the expression vector may further contain, if desired, an enhancer, a splicing signal, a poly-A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like.
  • an enhancer e.g., a splicing signal
  • a poly-A addition signal e.g., a selection marker
  • SV40 ori SV40 replication origin
  • the selectable marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance], an ampicillin resistance gene (hereinafter sometimes abbreviated as Amp f), neomycin resistance gene (hereinafter sometimes abbreviated as Ne o r, G418 resistance).
  • dh fr gene is used as a selection marker using dh fr gene-deficient Chinese hamster cells
  • the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal of the protein of the present invention.
  • the PhoA ′ signal sequence and the 0-A signal sequence are included.
  • the ⁇ -amylase signal sequence and the subtilisin signal sequence are included.
  • the host is yeast, MFo; signal sequence, SUC2, signal sequence, etc.
  • an insulin signal sequence, a single interferon / signal sequence, an antibody molecule / signal sequence, etc. can be used.
  • a transformant can be produced.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia include, for example, Escherichia coli.
  • Bacillus bacteria examples include, for example, Bacillus subtilis (Bacillus).
  • subtilis M I 1 14 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)] and the like are used.
  • yeast examples include, for example, Saccharomyces cerevisiae AH22, AH22R—, NA87-11A, DKD—5D, 20B—12, and Scliizosaccharomyces pombe NC YC 1913, NCYC 2036, Pichia pastoris (Pichia pastoris) K M71 or the like is used.
  • Insect cells include, for example, when the virus is AcNPV, a cell line derived from a larva of night roth moth (Spodoptera frugiperda cell; S f cell), in Tricoplusia ni. High Five TM cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • Sf cells for example, Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, J.L., et al., In Vivo, 13, 213-217, (1977)) and the like are used.
  • insects examples include silkworm larvae [Maeda et al., Nature, 315 Vol., 592 (1985)].
  • animal cells examples include monkey cells COS-7, Vero, Chinese Hams Yuichi cell CHO (hereinafter abbreviated as CHO cells), dh fr gene-deficient chicks, and Chinese hamster cell CHO (hereinafter CHO (dh fr ⁇ ) Cells), mouse L cells, mouse At T-20, mouse myeoma cells, mouse ATDC5 cells, rat GH3, human FL cells, etc. are used.
  • Transformation of yeast can be performed, for example, according to the method described in Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). it can.
  • Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, Ai-SS (1988).
  • Transformation of animal cells can be performed, for example, according to the method described in Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). Can do it.
  • a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, potato zein, meat extract, soybean meal, and potato extract.
  • the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like.
  • yeast extract, vitamins, growth promoting factors, etc. May be added.
  • the pH of the medium is preferably about 5-8.
  • an M9 medium containing glucose and casamino acid As a medium for cultivating a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in
  • a drug such as 3 / 3-indolylacrylic acid can be added to make the promoter work efficiently if necessary.
  • the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.
  • the cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours. If necessary, aeration and stirring may be applied.
  • the culture medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology , Volume 8, 396 (1959)], RPM I 1640 medium [The Journal of the American Medical Association Volume 199, 519 (1967) 3, 199 medium
  • the pH is about 6-8.
  • Culture is usually performed at about 30 ° (: ⁇ 40 for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • the protein of the present invention is placed inside the cell, in the cell membrane, or outside the cell of the transformant. It can produce protein.
  • the protein of the present invention can be separated and purified from the above culture by, for example, the following method.
  • the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme, and / or freeze-thawing. After the cells or cells are destroyed by the method described above, a method of obtaining a crude protein extract by centrifugation or filtration is used as appropriate.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X100 TM .
  • Purification of the protein contained in the culture supernatant or extract thus obtained can be carried out by appropriately combining known separation and purification methods.
  • known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • a method utilizing a difference in hydrophobicity, a method utilizing a difference in isoelectric point such as isoelectric focusing, and the like are used.
  • the protein thus obtained when the protein thus obtained is obtained as a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when the protein is obtained as a salt, a method known per se or The compound can be converted into a free form or another salt by an analogous method.
  • the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein-modifying enzyme before or after purification.
  • an appropriate protein-modifying enzyme for example, trypsin, chymotoribsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the presence of the protein of the present invention thus produced can be measured by, for example, enzymatic immunoassay western blotting using a specific antibody.
  • the TACT427 protein or its partial peptide or its salt simply referred to as the TACT427 protein
  • the FLNA protein or its partial peptide or its salt simply referred to as the FLNA protein
  • the FLNB protein or its partial peptide or its salt simply referred to as the FLNB protein FLNC protein or its partial peptide or its salt
  • RAB3IL1 protein or its partial peptide or a salt thereof is simply abbreviated as RAB3IL1 protein, respectively.
  • a complex containing one or more selected from (a) TACT427 protein and (b) FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein and RAB3IL1 protein is abbreviated as the complex of the present invention.
  • FLNA protein FLNB protein
  • FLNC protein FLNC protein
  • NAV2 protein BTBD2 protein
  • RAB3IL1 protein RAB3IL1 protein
  • the complex of the present invention comprises, in addition to TACT427 protein, FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or (and) RAB3IL1 protein, proteins, peptides, RNAs, nucleic acids, lipids, and saccharides other than those described above. , An amide group, a phosphate group and the like.
  • TACT427 protein, FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein and RAB3IL1 protein may be amidated, may contain phosphorylated amino acids, and may contain lipids (eg, , Myristic acid, etc.), and may be bound by any post-translational modification.
  • Examples of the region subjected to post-translational modification include a region corresponding to the amino acid sequence represented by SEQ ID NO: 45, and the like.
  • Examples of the complex of the present invention include (a) a cytoplasmic domain of TACT427 protein (eg, a peptide having the amino acid sequence represented by SEQ ID NO: 45) and (b) FLNA protein, FLNB protein, FLNC Complex containing one or more selected from proteins, NAV2 protein, BTBD2 protein and RAB3IL1 protein, for example, (a) cytoplasmic domain of TACT427 protein (eg, Such as a peptide having an amino acid sequence represented by SEQ ID NO: 45) and (b) one or more selected from FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein and RAB3IL1 protein Complex.
  • TACT427 protein eg, a peptide having the amino acid sequence represented by SEQ ID NO: 45
  • FLNA protein, FLNB protein, FLNC Complex containing one or more selected from proteins, NAV2 protein, BTBD2 protein and RAB3IL1 protein
  • FLNA protein eg, a peptide
  • Specific examples include (1) a complex in which the cytoplasmic domain of TACT427 protein (eg, a peptide having the amino acid sequence represented by SEQ ID NO: 45) and FLNA protein are bound; A complex in which the cytoplasmic domain of the protein (eg, a peptide having the amino acid sequence represented by SEQ ID NO: 45) binds to the FLNB protein; (3) a cytoplasmic domain of the TACT427 protein (eg, A complex in which FLNC protein is bound to a peptide having the amino acid sequence represented by SEQ ID NO: 45) and (4) a cytoplasmic domain of TACT427 protein (eg, represented by SEQ ID NO: 45) A complex in which an NAV2 protein is bound to a peptide having an amino acid sequence, etc .; (5) a cytoplasmic domain of TACT427 protein (eg, the amino acid sequence represented by SEQ ID NO: 45 (6) Cytoplasmic domain of TACT427 protein (eg, peptide having the amino
  • Examples of the region of the FLNA protein having a binding activity to the TACT427 protein include, for example, the 2162nd to 2412th, the 2206th to the 2355th or the 2141th to the amino acid sequence represented by SEQ ID NO: 46. No. 2351 is mentioned.
  • Examples of a region of the FLNB protein having a binding activity with the TACT427 protein include, for example, the 1960th to 2299th, the 2156th to the 2314th or the 2174th to the amino acid sequence represented by SEQ ID NO: 47.
  • the 2565th is mentioned.
  • Examples of a region of the FLNC protein having a binding activity with the TACT427 protein include the 2295th to 2705th amino acids of the amino acid sequence represented by SEQ ID NO: 48.
  • Examples of the region having binding activity to the TACT427 protein of the NAV2 protein include the 68th to 286th amino acids of the amino acid sequence represented by SEQ ID NO: 49.
  • the region of the BTBD2 protein having a binding activity with the TACT427 protein includes: Examples include the 201st to 525th or the 215th to 506th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50.
  • Examples of the region of the RAB3IL1 protein having a binding activity to the TACT427 protein include the 282nd to 356th amino acids of the amino acid sequence represented by SEQ ID NO: 51.
  • an antibody against the TACT427 protein having the activity of inhibiting or promoting the formation of the complex of the present invention has the activity of inhibiting or promoting the formation of the complex of the present invention.
  • any antibody that can recognize the TACT427 protein may be a polyclonal antibody or a monoclonal antibody.
  • the antibody of the present invention can be produced by using the TACT427 protein as an antigen (hereinafter sometimes abbreviated as antigen protein) according to a method for producing an antibody or antiserum known per se.
  • an antibody that recognizes the complex of the present invention can also be produced using the protein of the present invention as an antigen according to a method for producing an antibody or antiserum known per se.
  • the antigen protein is administered to a warm-blooded animal by itself or together with a carrier or a diluent at a site capable of producing an antibody upon administration.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • the warm-blooded animals used include, for example, monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
  • a monoclonal antibody-producing hybridoma When preparing monoclonal antibody-producing cells, select a warm-blooded animal immunized with the antigen, for example, an individual with an antibody titer from a mouse, collect the spleen or lymph nodes 2 to 5 days after the final immunization, and include them in By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared.
  • the measurement of the antibody titer in the antiserum can be performed, for example, by the following labeling method. After reacting the protein with the antiserum, the activity can be measured by measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
  • myeloma cells examples include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and the concentration of PEG (preferably PEG 1000 to PEG6000) S is about 10 to 80%.
  • PEG preferably PEG 1000 to PEG6000
  • the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which protein antigens are directly or adsorbed together with a carrier. Then, an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is mouse) or protein A labeled with a radioactive substance or an enzyme is added, and the mixture is bound to the solid phase.
  • a solid phase eg, microplate
  • an anti-immunoglobulin antibody anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is mouse
  • protein A labeled with a radioactive substance or an enzyme is added, and the mixture is bound to the solid phase.
  • Monoclonal antibody detection method hybridoma supernatant was added to a solid phase to which anti-immunoglobulin antibody or protein A was adsorbed, proteins labeled with radioactive substances, enzymes, etc. were added and bound to the solid phase. Examples
  • the selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or hybrid
  • a serum-free culture medium for culture SFM-101, Nissui Pharmaceutical Co., Ltd.
  • SFM-101 Nissui Pharmaceutical Co., Ltd.
  • the culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C.
  • the culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • culture Can be carried out usually under 5% carbon dioxide gas.
  • the antibody titer of the culture supernatant of the hybridoma can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)) Absorption / desorption method, ultracentrifugation method, gel filtration method, antigen binding Solid phase or specific purification method of collecting antibody only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody) You can do it.
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)
  • Absorption / desorption method eg, ultracentrifugation method, gel filtration method, antigen binding Solid phase or specific purification method of collecting antibody only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody
  • the polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, an immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting the antibody-containing substance and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of the carrier and the hapten depend on the antibody against the hapten immunized by cross-linking with the carrier. If it can be efficiently performed, any kind may be crosslinked at any ratio.
  • any kind may be crosslinked at any ratio.
  • serum serum albumin, thyroglobulin, hemocyanin, etc. may be used in a weight ratio of about 0 to 1 for hapten.
  • a method of coupling at a rate of 1 to 20, preferably about 1 to 5 is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • an active ester reagent containing a daltaraldehyde-carpoimide, a maleimide active ester, a thiol group, or a dithioviridyl group is used.
  • the condensation product is administered to a warm-blooded animal at a site where antibody production is possible, by itself or together with a carrier or diluent.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the dose is usually given about every 2 to 6 weeks, about 3 to 10 times in total.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same method for separation and purification of immunoglobulin as in the above-described separation and purification of the monoclonal antibody.
  • the protein or partial peptide of the present invention or a salt thereof hereinafter, sometimes abbreviated as the protein of the present invention
  • the DNA encoding the protein or partial peptide of the present invention hereinafter referred to as D
  • D The use of the antibody of the present invention and the complex of the present invention will be described.
  • TACT427 protein is increased in cancer tissues, and when the function (activity, expression, etc.) of TACT427 protein is inhibited, cancer cells undergo apoptosis.
  • the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein binds to the cytoplasmic domain of TACT427 protein and plays a role of TACT427 protein.
  • compounds that inhibit the formation of the complex of the present invention eg, peptides, proteins, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc.
  • a compound that inhibits the formation of a complex by (a) TACT427 protein and (b) one or more selected from FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein, and RAB3IL1 protein Preferably, the binding between (a) the TACT427 protein and (b) one or more selected from the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein and RAB3IL1 protein [eg, (1)
  • TACT427 protein and FLNA protein Binding between TACT427 protein and FLNA protein, (2) binding between TACT427 protein and FLNB protein, (3) binding between TACT427 protein and FLNC protein,
  • cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, ovarian cancer, thyroid cancer
  • cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, ovarian cancer, thyroid cancer
  • It can be used as a prophylactic / therapeutic agent for kidney cancer, brain tumor, blood tumor, etc., and as an apoptosis promoter for cancer cells.
  • neurodegenerative diseases eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.)
  • It can be used as an agent for inhibiting (suppressing) apoptosis of nerve cells.
  • the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that inhibits or promotes the formation of the complex of the present invention.
  • the present invention provides a method for screening a compound or a salt thereof that inhibits or promotes the formation of the complex of the present invention (eg, the binding of the above (1) to (6)), which comprises using the protein of the present invention.
  • a method for screening a compound or a salt thereof that inhibits or promotes the formation of the complex of the present invention eg, the binding of the above (1) to (6)
  • the protein of the present invention e.g., the binding of the above (1) to (6)
  • the protein of the present invention may be used in the screening method of the present invention, and a peptide corresponding to the cytoplasmic domain of TACT427 protein may be used. (Preferably, transformed with a DNA encoding the protein of the present invention). Transformants (eg, cells such as yeast and animal cells) may be used. Examples of such cells include (a) DNA encoding TACT427 protein (eg, DNA encoding a peptide corresponding to the cytoplasmic domain of TACT427 protein) and (b) FLNA protein, FLNB protein, FLNC protein, NAV2 protein. A transformant transformed with DNA encoding the protein, BTBD2 protein or RAB3IL1 protein is used.
  • FLNA protein FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein, FLNA protein, FLNB protein,
  • a solid phase eg, EIA plate
  • antibodies against FLNC, NAV2, BTBD2, or RAB3IL1 protein or use FLNA, FLNB, FLNC, NAV2, BTBD2 proteins
  • the RAB3IL1 protein is fused to a Tag protein (eg, His-Tag, GST (Dalphinthione-S-transferase), etc.) and then immobilized on a solid phase.
  • a Tag protein eg, His-Tag, GST (Dalphinthione-S-transferase), etc.
  • a partial peptide having a binding activity to TACT427 protein eg, SEQ ID NO: : A partial peptide having the amino acid sequence at positions 2162 to 2412, 2206 to 2355 or 2141 to 2351 of the amino acid sequence represented by 46, represented by SEQ ID NO: 47 1960th to 2299th amino acid sequence of the amino acid sequence.
  • Partial peptide having a sequence Partial peptide having the 201st to 525th or 215th to 506th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50, represented by SEQ ID NO: 51
  • TACT427 protein eg, a partial peptide corresponding to the cytoplasmic domain (eg, a partial peptide having the amino acid sequence represented by SEQ ID NO: 45) labeled with a labeling agent (eg, biotin)
  • a labeling agent eg, biotin
  • TACT427 protein labeled with a labeling agent eg, biotin
  • FLNA protein, FLNB protein, FLNC protein in which a partial peptide corresponding to the cytoplasmic domain (eg, a partial peptide having the amino acid sequence represented by SEQ ID NO: 45) and a test compound are immobilized.
  • the binding amount is measured by a known method, for example, using a solid-phased TACT427 protein or a partial peptide thereof using a commercially available kit for detecting a labeling agent (eg, biotin or the like) or an antibody against the TACT427 protein.
  • a labeling agent eg, biotin or the like
  • Test compounds include, for example, peptides, proteins, antibodies, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.
  • a test compound that reduces the amount of binding in the case (ii) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case (i) As a compound that inhibits the formation of the complex of the invention (hereinafter sometimes abbreviated as a binding inhibitor), the activity in the case of the above (ii) is about 20% as compared with the case of the above (i).
  • a test compound that increases the concentration by preferably 30% or more, more preferably about 50% or more is selected as a compound that promotes the formation of the complex of the present invention (hereinafter sometimes abbreviated as a binding promoter). .
  • TACT427 protein (eg, a partial peptide corresponding to the cytoplasmic domain of TACT427 protein) is immobilized on a solid phase, and FLNA, FLNB, FLNC, NAV2, BTBD2, or RAB3IL1 protein is immobilized on a solid phase. After contact, the amount of binding may be measured in the same manner.
  • a TACT427 protein or a partial peptide thereof labeled with a labeling agent (eg, biotin) or a solid phase (eg, a plate) labeled with avidin is preferably used.
  • a labeling agent eg, biotin
  • a solid phase eg, a plate
  • the binding amount is measured by a known method, for example, by immobilizing FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein using an antibody against the protein.
  • Test compounds include, for example, peptides, proteins, antibodies, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.
  • the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein may be a protein fused with a Tag protein, in which case the FLNA protein, FLNB protein, The FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein may be detected and quantified using an antibody against the protein, or may be detected and quantified using an antibody against the Tag protein.
  • the binding amount in the case of the above (iv) is about 20% as compared with the case of the above (iii).
  • a test compound that preferably reduces by 30% or more, more preferably about 50% or more is referred to as a compound that inhibits the formation of the complex of the present invention (hereinafter, may be abbreviated as a binding inhibitor).
  • the activity in the case of (iv) is increased by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii).
  • a test compound is selected as the binding promoter.
  • a encodes a chimeric protein in which a partial peptide corresponding to the cytoplasmic domain of TACT427 protein (for example, a partial peptide containing the amino acid sequence represented by SEQ ID NO: 45) and a repo overnight-gene binding domain are fused DNA
  • DNA is converted to yeast (eg,
  • the phenotype of the reporter gene ⁇ -galactosidase gene and the histidine synthesis gene HIS3 is expressed by the action of the two hybrids.
  • the yeast strain is cultured in the presence of the test compound for a certain period of time, and a test compound that reduces the ⁇ -galactosidase activity of the yeast strain or converts the yeast strain to histidine requirement is selected as a binding inhibitor.
  • the yeast strain can be cultured in the same manner as in the culture of a transformant whose host is yeast as described above.
  • ⁇ -galactosidase activity can be measured using X-Gal (5-bromo-4-chloro-3-indolyl-pD-galactopyranoside), ONPG (o-nitrophenyl ⁇ -D-galactopyranoside) or CPRG (clorop enyl red).
  • -pD-galactopyranoside can be measured according to a known method as a substrate.
  • Expression of the HIS3 phenotype can be measured by culturing the yeast in a minimal medium lacking histidine.
  • cytotoxic compounds and compounds that inhibit the activity of the reporter gene product itself due to interaction with the reporter gene product are excluded as false positive compounds.
  • Reporter genes eg, chloramphenicol acetylacetyltransferase (CAT) gene, chromium luciferase gene
  • animal cells eg, CH0 cells
  • the transcriptional regulatory region of the reporter gene is, for example, a promoter that functions in animal cells (eg, a minimal promoter (TATA box) derived from the adenovirus Elb, etc.) downstream of which a transcriptional activation sequence (UAS) of GAL1 is linked.
  • TATA box minimal promoter derived from the adenovirus Elb, etc.
  • UAS transcriptional activation sequence
  • a GAL4-DNA binding domain was fused to a partial peptide corresponding to the cytoplasmic domain of TACT427 protein (for example, a partial peptide containing the amino acid sequence represented by SEQ ID NO: 45).
  • DNA encoding the chimeric protein and (b) a chimeric protein obtained by fusing a simple virus-derived VP16 protein to the FLNA, FLNB, FLNC, NAV2, BTBD2, or MB3IL1 protein.
  • an animal cell line expressing the repo-all-one gene is obtained by the action of the single hybrid.
  • This cell line is cultured for a certain period of time in the presence of the test compound, the activity of the repo overnight gene product is measured, and the compound that reduces the activity is selected as a binding inhibitor.
  • the animal cell strain can be cultured in the same manner as the culture of the above-mentioned transformant whose host is an animal cell.
  • the activity of a reporter gene product eg, CAT, luciferase, etc.
  • a reporter gene product eg, CAT, luciferase, etc.
  • cytotoxic compounds and compounds that inhibit the activity of the repo overnight gene product itself due to interaction with the repo overnight gene product are excluded as false positive compounds.
  • Test compounds include, for example, peptides, proteins, antibodies, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.
  • the screening kit of the present invention contains FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein, and further contains TACT427 protein or a partial peptide thereof (eg, a partial peptide corresponding to a cytoplasmic domain). It may be contained.
  • the screening kit of the present invention includes cells capable of producing the protein of the present invention (preferably, a transformant transformed with a DNA encoding the protein of the present invention (eg, Cells such as yeast and animal cells)).
  • the cells encode FLNA protein, FLNB protein, FLNC protein, DNA encoding NAV2 protein, BTBD2 protein or RAB3IL1 protein, and TACT427 protein (eg, a partial peptide corresponding to the cytoplasmic domain).
  • the transformant may be a transformant transformed with DNA.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention may be a test compound (eg, peptide, protein, antibody, non-peptide compound, synthetic compound, fermentation product, cell extract, Plant extract, animal tissue extract, plasma, etc.).
  • a test compound eg, peptide, protein, antibody, non-peptide compound, synthetic compound, fermentation product, cell extract, Plant extract, animal tissue extract, plasma, etc.
  • TACT427 protein, FLNA protein, FLNB protein, FLNC protein, NAV2 protein e.g, TACT427 protein, FLNA protein, FLNB protein, FLNC protein, NAV2 protein,
  • a compound that inhibits or promotes binding to BTBD2 protein or RAB3IL1 protein is A compound that inhibits or promotes binding to BTBD2 protein or RAB3IL1 protein.
  • salt of the compound those similar to the aforementioned salts of the protein of the present invention are used.
  • Compounds that inhibit the formation of the complex of the present invention include, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterus) It is useful as a preventive-therapeutic agent for cancer, testicular cancer, ovarian cancer, thyroid cancer, knee cancer, brain tumor, blood tumor, etc.) or as an apoptosis promoter for cancer cells.
  • cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterus
  • the compound that promotes the formation of the complex of the present invention may be used as a prophylactic / therapeutic agent for neurodegenerative diseases (eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.)) Or as an agent for inhibiting (suppressing) apoptosis of nerve cells.
  • neurodegenerative diseases eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.)
  • an agent for inhibiting (suppressing) apoptosis of nerve cells eg, apoptosis of nerve cells.
  • a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method.
  • compositions for oral administration include solid or liquid dosage forms, such as tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). ), Syrups, emulsions, suspensions and the like.
  • Such a composition is produced by a method known per se, and is commonly used in the pharmaceutical field. It contains a carrier, diluent or excipient.
  • a carrier diluent or excipient.
  • lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
  • compositions for parenteral administration include injections, suppositories, and the like.
  • Injections are intravenous, subcutaneous, intradermal, intramuscular, intravenous, intraarticular. Dosage forms such as agents.
  • Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above compound or a salt thereof in a sterile aqueous or oily liquid usually used for injections.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants is used, and a suitable solubilizing agent, for example, alcohol (eg, ethanol), polyalcohol (eg, , Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyetylene (50mol) adduct of hydrogenated catalyst)], etc. Good.
  • a suitable solubilizing agent for example, alcohol (eg, ethanol), polyalcohol (eg, , Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyetylene (50mol) adduct of hydrogenated catalyst)], etc. Good.
  • the oily liquid for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a
  • compositions are conveniently prepared in dosage unit forms to be compatible with the dosage of the active ingredient.
  • dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually 5 to 500 mg, especially It is preferable that the injection contains 5 to 100 mg of the above compound, and other dosage forms contain 10 to 25 mg of the above compound.
  • compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above compound.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or in warm-blooded animals (for example, mice, rats, puppies, higgs, bushus, puppies, pumas, birds, cats, dogs). , Monkeys, chimpanzees, etc.) orally or parenterally.
  • the dose of the compound (preferably a binding inhibitor) or a salt thereof varies depending on its action, target disease, subject to be administered, administration route and the like.
  • the compound is orally administered for the purpose of treating breast cancer.
  • about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 mg of the compound or a salt thereof per day is used.
  • Omg is administered.
  • the single dose of the compound or a salt thereof varies depending on the subject to be administered, the target disease, and the like, but, for example, the compound is in the form of an injection for the purpose of treating breast cancer.
  • the compound or a salt thereof is administered in an amount of about 0.01 to 3 Omg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 0 Omg per day. It is convenient to administer 1 Omg to the cancerous lesion by injection. In the case of other animals, the dose can be administered in terms of the weight per 6 O kg of body weight.
  • An antibody that specifically recognizes the complex of the present invention can be used for quantification of the complex of the present invention in a test solution, particularly for quantification by a sandwich immunoassay.
  • a method for quantifying the complex of the present invention in a test solution which comprises:
  • the complex of the present invention can be quantified using a monoclonal antibody against the complex of the present invention (hereinafter, sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like.
  • the antibody molecule itself may be used, or F (ab ′) 2 , Fab ′, or Fab fraction of the antibody molecule may be used.
  • the method for quantifying the complex of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody corresponding to the amount of antigen (eg, the amount of protein) in the test solution.
  • any method that detects the amount of the antigen complex by chemical or physical means and calculates this from a standard curve prepared using a standard solution containing a known amount of antigen can be used. Is also good. For example, nephrometry, a competition method, an immunometric method and a sandwich method are suitably used, but it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, [125 1], [131 1], [3 ⁇ 4], and the like are used [14 c].
  • a stable enzyme having a large specific activity is preferable. For example, / 3-galactosidase,] 3-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • luminescent substance for example, luminol, luminol derivative, reluciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction another labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or staggered.
  • the labeling agent and the method of insolubilization may be the same as those described above. . Also,
  • the type of antibody used is not necessarily one type, and a mixture of two or more types of antibodies may be used for the purpose of improving measurement sensitivity and the like.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site of the complex of the present invention.
  • the antibody used in the primary reaction and the secondary reaction is used in the primary reaction, for example, when the antibody used in the secondary reaction recognizes the complex portion formed by the C-terminal of TACT427.
  • the antibody an antibody that recognizes a site other than the site, for example, a complex formed by the N-terminal of TACT427 is used.
  • an antigen in a test solution and a labeled antigen are used for the antibody.
  • the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), and the amount of labeled B or F is measured. Then, the amount of the antigen in the test solution is determined.
  • a soluble antibody is used as an antibody
  • polyethylene glycol is used for B / F separation
  • a liquid phase method using a second antibody against the antibody is used as the first antibody.
  • an immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of the label in either phase is measured to determine the amount of the antigen in the test solution.
  • the amount of insoluble sediment resulting from the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
  • a measurement system for the complex of the present invention may be constructed by adding ordinary technical considerations to those skilled in the art to the operation method. For details of these general technical means, reference can be made to reviews and written documents.
  • cancer eg, colon cancer, breast cancer
  • lung cancer prostate cancer
  • esophageal cancer stomach cancer
  • liver cancer biliary tract cancer
  • spleen cancer kidney cancer
  • bladder cancer uterine cancer
  • testicular cancer ovarian cancer
  • thyroid cancer knee cancer, brain tumor, blood tumor, etc.
  • Etc. can be diagnosed as likely to be affected in the future.
  • a decrease in the concentration of the complex of the present invention is detected by quantifying the concentration of the complex of the present invention using the antibody of the present invention, for example, a neurodegenerative disease (eg, Alzheimer's disease (family (Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.) or the likelihood of future disease is high.
  • a neurodegenerative disease eg, Alzheimer's disease (family (Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.
  • it can be used to detect the complex of the present invention present in the antibody, and the production of an antibody column used for purifying the complex of the present invention, and the present invention in each fraction at the time of purification.
  • Complex detection, test can be used for analysis of the behavior of the complex of the present invention in cells.
  • An antibody against the TACT427 protein having an activity of inhibiting the formation of the complex of the present invention has an apoptosis-promoting effect and includes, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver). Cancer, biliary tract, spleen, kidney, bladder, uterus, testes, ovary, thyroid, knee, brain, blood, etc.) It can also be used as a monocis accelerator.
  • cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver.
  • Cancer biliary tract, spleen, kidney, bladder, uterus, testes, ovary, thyroid, knee, brain, blood, etc.
  • It can also be used as a monocis accelerator.
  • an antibody against the TACT427 protein having an activity of promoting the formation of the complex of the present invention has an apoptosis-suppressing effect, and is used for, for example, neurodegenerative diseases (eg, Alhaima disease (familial Alzheimer disease, juvenile Alzheimer disease, It can also be used as a prophylactic / therapeutic agent for diseases such as Alzheimer's disease and sporadic Alhaima disease, and as an inhibitor of neuronal apoptosis (suppression).
  • neurodegenerative diseases eg, Alhaima disease (familial Alzheimer disease, juvenile Alzheimer disease, It can also be used as a prophylactic / therapeutic agent for diseases such as Alzheimer's disease and sporadic Alhaima disease, and as an inhibitor of neuronal apoptosis (suppression).
  • the above-mentioned prophylactic / therapeutic agents, accelerators, inhibitors and the like for the above-mentioned diseases containing the antibody of the present invention have low toxicity, and are used as they are as liquids or as pharmaceutical compositions in appropriate dosage forms. It can be administered orally or parenterally (eg, intravascular, subcutaneous, etc.) to rats, rabbits, rabbits, sheep, pigs, mice, cats, dogs, monkeys, etc.). It can also be administered as a vaccine according to the usual methods.
  • the antibody of the present invention may be administered as it is, or may be administered as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain the antibody of the present invention or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.
  • compositions for parenteral administration include, for example, injections, suppositories, vaccines, and the like. May be included.
  • Such an injection can be prepared according to a known method.
  • Injection preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody of the present invention or a salt thereof in a sterile aqueous or oily liquid commonly used for injections.
  • Aqueous liquids for injection include, for example, saline, bud Isotonic solutions containing sugar and other adjuvants are used, and suitable solubilizers, for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants Agents [eg, polysorbate 80, HCO-50 (.polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
  • the prepared injection solution is preferably filled in a suitable ampoule.
  • a suppository for rectal administration may be prepared by mixing the antibody or a salt thereof with a conventional suppository base.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets
  • compositions are prepared by known methods and may contain carriers, diluents or excipients commonly used in the field of formulation.
  • carriers and excipients for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • the above-mentioned parenteral or oral pharmaceutical composition is conveniently prepared in the form of a dosage unit so as to match the dosage of the active ingredient.
  • dosage unit forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories.
  • the antibody content is usually about 5 to 50 mg per dosage unit dosage form, especially about 5 to 100 mg for injections, and about 10 to 250 mg for other dosage forms. Is preferably contained.
  • the dosage of the above-mentioned prophylactic / therapeutic agent, promoter, or inhibitor containing the antibody of the present invention is administered.
  • the amount varies depending on the subject, target disease, symptoms, administration route, and the like.
  • the antibody of the present invention is usually about 0.01 to 2 Omg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 10 mg / kg body weight. It is convenient to administer 0.1-5 mg / kg body weight by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent dose can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.
  • the antibody of the present invention can be administered by itself or as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • a composition is provided as a dosage form suitable for oral or parenteral administration (eg, intravascular injection, subcutaneous injection, etc.).
  • compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
  • TACT427 protein The expression of TACT427 protein is increased in cancer tissues, and when the function (activity, expression, etc.) of TACT427 protein is inhibited, cancer cells undergo apoptosis. Further, it is considered that FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein binds to the cytoplasmic domain of TACT427 protein and plays a role of TACT427 protein.
  • cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus
  • stomach cancer liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testis cancer, ovarian cancer, thyroid cancer, thyroid cancer, brain cancer, blood tumor, etc.
  • neurodegenerative diseases eg, Alhaima
  • bases, amino acids and the like are indicated by abbreviations based on the abbreviations by IUPAC-IUB Communication on Biochemical Nomenclature or commonly used abbreviations in the field, and examples thereof are described below.
  • optical isomer for an amino acid the L-form is indicated unless otherwise specified.
  • HONB 1-Hydroxy-5-norporene-2,3-dicarboxide The sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • the nucleotide sequence of a DNA containing the full length gene encoding hCP50177 is shown.
  • the nucleotide sequence of the DNA containing the full length gene encoding MP1762319 is shown.
  • [SEQ ID NO: 1 2] Shows the nucleotide sequence of DNA containing the full-length gene encoding FU13515.
  • DNA encoding TACT427-B2 having the amino acid sequence represented by SEQ ID NO: 22 Shows the base sequence.
  • the nucleotide sequence of a DNA containing the full-length gene encoding TACT427-B and TACT427-B2 is shown.
  • the nucleotide sequence of the DNA containing the full-length gene encoding TACT427-C and TACT427-C2 is shown.
  • the base sequence of the primer 8 used in Reference Examples 6, 7, 8, and 20 is shown.
  • TanMan probe 1 used in Reference Example 6, Reference Example 7, Reference Example 8, and Reference Example 20 is shown.
  • [SEQ ID NO: 48] 2 shows the amino acid sequence of human FLNC.
  • 26 shows the nucleotide sequence of primer 111 used in Reference Example 26.
  • the base sequence of the primer 14 used in Reference Example 26 is shown.
  • the experiment method was in accordance with the Aifymetrix experiment manual (Expression analysis technical manual).
  • breast cancer tissue (lot.0009-192-00101, lot.0009-192-00120, lot.0009—192—00153, lot.0009-192-00178) and lung cancer tissue (lot.0009-192-00122, lot.0011-192-01293, lot.0011-192-01297), compared to normal breast tissue and normal lung tissue, respectively.
  • FU20539 gene (SEQ ID NO: 2)
  • the hCP50177 gene (SEQ ID NO: 5), which is a FLJ20539-related gene, the MP1762319 gene (SEQ ID NO: 8), which is a F20539-related gene, and the FLJ13515 gene (SEQ ID NO: 11), and (2) Reference Example 4 described below Alternatively, TACT427-A gene (SEQ ID NO: 16), TACT427-A2 gene (SEQ ID NO: 18), TACT427-B gene (SEQ ID NO: 21), TACT427- Enhanced expression of B2 gene (SEQ ID NO: 23), TACT427-C gene (SEQ ID NO: 26) and TACT427-C2 gene (SEQ ID NO: 28) were detected (Tables 2 and 4). table
  • the gene expression level was normalized using the median expression level of all genes whose expression was detected by the oligonucleotide microarray as 1, ND; not detected.Table 3
  • Lung cancer tissue (lot. 0009-192-00122) BioCl inical Partners Lung cancer tissue (lot. 0011 (lot. 0011 -192-01297) Normal lung tissue from BioCl inical Partners (lot.0009 -192-00150) Normal lung tissue from BioCl inical Partners (lot. Tissue (lot.0011.-192-01283) BioCl inical Partners Normal lung tissue (lot.0011 ⁇ -192-01285) BioCl inical Partners Normal lung tissue (lot.0011 Table 4
  • the gene expression level was determined by ol igonucleotide microarray.
  • NCI-H460 a human non-small cell lung cancer cell line purchased from the American Type Culture Collection (ATCC), was added to RPMI-1640 medium (containing 25 mM HEPES) (Invitrogen) and supplemented with 10% bovine fetal serum (ATCC). And seeded on a 96-well flat bottom tissue culture plate (BD Falcon) at a cell density of 4000 cells / well. After culturing overnight at 37 ° C. in a 5% carbon dioxide gas stream, antisense oligonucleotides were transfected.
  • ATCC American Type Culture Collection
  • the FU20539 gene (SEQ ID NO: 2), the hCP50177 gene (SEQ ID NO: 5), which is a FLJ20539-related gene, the MP1762319 gene (SEQ ID NO: 8), which is a FLJ20539-related gene, and the FLJ13515 gene ( SEQ ID NOs: 1 1)
  • TACT427-A gene (SEQ ID NO: 16), TACT427-A2 gene (SEQ ID NO: 18), TACT427-B gene (SEQ ID NO: 21) obtained in Reference Example 4 or Reference Example 5 described below
  • Antibodies that hybridize to the protein coding region sequence or 3 'untranslated region of the TACT427-B2 gene (SEQ ID NO: 23), TACT427-C gene (SEQ ID NO: 26) and TACT427-C2 gene (SEQ ID NO: 28)
  • control oligonucleotide As a control, the reverse sequence (SEQ ID NO: 14) of the nucleotide sequence represented by SEQ ID NO: 13 was similarly phosphorothioated and purified by HPLC (hereinafter abbreviated as control oligonucleotide).
  • Antisense oligonucleotides or control oligonucleotides were diluted with Opti-MEM (Invitrogen), and mixed with FuGENE6 reagent (Roche Diagnostics) at a ratio of 4 times (4 / 2L / zg oligonucleotide). The solution was left at room temperature for 30 minutes. This oligonucleotide solution was added to the plate at a rate of 1 ⁇ l. The final concentration of the oligonucleotide was adjusted to 140 nM. After culturing for another 3 days under the above conditions, Cell Death Detection ELISA PLUS kit
  • the apoptosis-inducing activity of the above oligonucleotides was measured using (Roche Diagnostics) according to the attached protocol.
  • Table 5 apo Bok one cis-inducing activity (A 4 5 -. A 49 mean standard deviation
  • the human non-small cell lung cancer cell line NCI-H460 used in Reference Example 2 was suspended in the same medium as in Reference Example 2, and a 24-well flat bottom tissue culture plate (BD Falcon) was used at a cell density of 24,000 cells / well. Seeded. After culturing overnight at 37 ° C. in a 5% carbon dioxide gas stream, antisense oligonucleotide and control oligonucleotide were transfected. The addition amount of the oligonucleotide solution was adjusted to 390 xL / well, and the final concentration of the oligonucleotide was adjusted to 200 nM.
  • a cDNA corresponding to 10 ng of total RNA was designated as type II, and two types of primers (primer 1 (SEQ ID NO: 30) and primer 1 (SEQ ID NO: 31)) and SYBR Green PCR Master Mix (Applied Biosystems) ),
  • the FLJ20539 gene (SEQ ID NO: 2), the hCP50177 gene (SEQ ID NO: 5), which is a FU20539-related gene, the P1762319 gene (SEQ ID NO: 8), which is a FLJ20539-related gene, and the FU13515 gene (SEQ ID NO: 8) No .: 11)
  • TACT427-A gene SEQ ID NO: 16
  • TACT427-A2 gene SEQ ID NO: 18
  • TACT427-B gene (SEQ ID NO: 18) obtained in Reference Example 4 described below.
  • TACT427-B2 gene (SEQ ID NO: 23), TACT427-C gene (SEQ ID NO: 26),
  • the expression amount of ⁇ -actin gene contained in the same amount of type III cDNA was measured using TaaMan ⁇ -act in Control Reagents (Applied Biosystems) and used as an internal standard. 7
  • the expression level of the 10 genes was 0.10% of the expression level of the i3-actin gene, whereas the antisense oligonucleotide represented by SEQ ID NO: 13 was administered. In the group, the expression level was 0.046%, indicating a statistically significant (P ⁇ 0.05) decrease in the expression level.
  • control oligonucleotide (SEQ ID NO: 14) administration group was 0.088%, and no statistically significant decrease in the expression level was observed as compared with the non-transfection group.
  • the results showed that a decrease in the expression level of the above 10 genes induced apoptosis of the human lung cancer cell line.
  • PCR was carried out using two types of primers (primer 3 (SEQ ID NO: 32) and primer 4 (SEQ ID NO: 33)).
  • the composition of the reaction solution used in the reaction was as follows. Using cDNAl xl as type I, 6 ⁇ 25 U of PfuTurbo DNA Polymerase (STRATAGENE), primer 3 (SEQ ID NO: 32) and primer 4 (SEQ ID NO: 33) 1.0 xM of each, 200 M of dNTPs, and 51 of Pfu Buffer (STRATAGENE) were added to make a liquid volume of 50 il.
  • STRATAGENE PfuTurbo DNA Polymerase
  • PCR reaction product was purified using a PCR Purification Kit (QIAGEN). This was subcloned into a plasmid vector pCR-BluntII-T0P0 (Invitrogen) according to the prescription of Zero Blunt T0P0 PCR Cloning Kit (Invitrogen). This was introduced into E. coli T0P10, and a clone having cDNA was selected on an LB agar medium containing kanamycin.
  • the sequence The nucleotide sequences of the cDNAs represented by No. 16 and SEQ ID No. 21 were obtained.
  • the nucleotide sequence at positions 1 to 160 and 2483 to 2755 of the full length gene sequence of FU20539 represented by SEQ ID NO: 3 is added to the 5 'end and 3' end of the nucleotide sequence represented by SEQ ID NO: 16, respectively.
  • the base sequence thus obtained is shown in SEQ ID NO: -19.
  • the nucleotide sequence at positions 1 to 160 and 2483 to 2755 of the FLJ20539 full-length gene represented by SEQ ID NO: 3 is added to the 5 'end and 3' end of the nucleotide sequence represented by SEQ ID NO: 21, respectively.
  • the base sequence thus obtained is shown in SEQ ID NO: 24.
  • a plasmid having a DNA fragment having the nucleotide sequence represented by SEQ ID NO: 16 is called TACT427_A / pCR_Blimt II-TOP0
  • a plasmid having a DNA fragment having the nucleotide sequence represented by SEQ ID NO: 21 is called TACT427-B / PCR-Blunt l-T0P0.
  • a protein containing the amino acid sequence (SEQ ID NO: 15) encoded by the nucleotide sequence represented by SEQ ID NO: 16 was designated as TACT427-A.
  • a protein containing the amino acid sequence (SEQ ID NO: 20) encoded by the nucleotide sequence represented by SEQ ID NO: 21 was designated as TACT427-B.
  • the transformant into which the plasmid TACT427-A / pCR-Blunt II-T0P0 was introduced was transformed into Escherichia col i T0P10 / 47427A / pCR-Bluntll-T0P0 and plasmid TACT427-B / pCR-BluntIl-T0P0.
  • the transformed transformant is transformed into Escherichia coli
  • the 278th Arg of the TACT427-A amino acid sequence (SEQ ID NO: 15) is Gin, the 825th Glu is Lys, and the 826th Ala is In Pro, the 970th Val has been replaced with Ala, respectively, and the 340th Ser has been deleted.
  • the 833th g of the DNA encoding TACT427-A (SEQ ID NO: 16) is a and the 1482th g is In c, the 1590th a is replaced with a, the 2473th g is replaced with a, the 2476th g is replaced with c, and the 2909th t is replaced with c, and the 1015-1017 age is deleted.
  • SEQ ID NO: 17 shows a sequence in which the first to fourth amino acid sequences of the amino acid sequence of TACT427-A are deleted.
  • the protein having the amino acid sequence represented by SEQ ID NO: 17 is designated as TACT427-.
  • TACT427- The nucleotide sequence of the DNA encoding A2 No .: See 18
  • SEQ ID NO: 22 shows a sequence in which the first to fourth amino acid sequences of TACT427-B are deleted.
  • the protein having the amino acid sequence represented by SEQ ID NO: 22 is designated as TACT427-B2.
  • the nucleotide sequence of the DNA encoding TACT427-B2 is shown in SEQ ID NO: 23.
  • the nucleotide sequence of the DNA encoding TACT427-A (SEQ ID NO: 16) showed the highest homology to the nucleotide sequence of the DNA encoding FU20539 (SEQ ID NO: 2) in humans. With respect to the nucleotide sequence at positions 1 to 138 and 889 to 3072 of the nucleotide sequence represented by SEQ ID NO: 16, Correspond to each other, and show 99.3% and 100% homology in each partial sequence, respectively.
  • the nucleotide sequence of the DNA encoding FU20539 (SEQ ID NO: 2) lacks the nucleotide sequence corresponding to positions 139 to 888 of the nucleotide sequence of the DNA encoding TACT427-A (SEQ ID NO: 16). This sequence is specific to TACT427-A.
  • TACT427-A Compared to TACT427-A, TACT427-A2, TACT427-B and TACT427-B2 show the highest homology to FU20539, and have similar base substitutions and deletions of base sequences.
  • TACT427-A amino acid sequence (SEQ ID NO: 15) at amino acids 735 to 792; TACT427-A2 amino acid sequence (SEQ ID NO: 17) at amino acids 731 to 788;
  • a search on the motif search site SMART (ht tp: // smart. Embl-heidel erg. De /) indicates that it has chloropa-oxidase activity because it has a black mouth peroxidase chief.
  • Fig. 1 shows the hydrophobicity plot of TACT427-A
  • Fig. 2 shows the hydrophobicity plot of TACT427-A
  • Fig. 3 shows the hydrophobicity plot of TACT427-B2.
  • the sex plots are shown in Figure 4 respectively.
  • Reference example 5 Cloning and nucleotide sequencing of cDNAs encoding TACT427-C and TACT427-C2 from human lung cancer cell line NCI-H522
  • Human non-small cell lung cancer cell line NCI-H522 (purchased from ATCC) in RPMI-1640 medium
  • RNA was extracted using Reasy Mini Total RNA Kit (QIAGEN).
  • QIAGEN Reasy Mini Total RNA Kit
  • reverse transcription was performed using Ta Man Reverse Transcription Reagents (Applied Biosystems) according to the attached protocol to obtain cDNA.
  • the cDNA obtained here was designated as type III, and a PCR reaction was carried out using two types of primers [Primer 5 (SEQ ID NO: 34) and Primer 6 (SEQ ID NO: 35)].
  • the composition of the reaction solution for this reaction was prepared using the above cDNA as type II, PiuTurbo Hotstart DNA Polymerase.
  • nucleotide sequences at positions 1 to 160 and 2483 to 2755 of the FLJ20539 full length gene sequence represented by SEQ ID NO: 3 are added to the 5 'end and 3'. End of the nucleotide sequence represented by SEQ ID NO: 26, respectively.
  • the base sequence thus obtained is shown in SEQ ID NO: 29.
  • a plasmid having a DNA fragment having the base sequence of SEQ ID NO: 26 was designated as TACT427-C / pCR-BluntII-TOP0.
  • a protein containing the amino acid sequence (SEQ ID NO: 25) encoded by the nucleotide sequence of DNA represented by SEQ ID NO: 26 was designated as TACT427-C.
  • the base sequence of the DNA encoding TACT427-A (SEQ ID NO: 16) has the 504th a and the 939th a g, 1471th g to a, 1482th g to c, 1590th a to g, 2473th to a, 2476th g to c, 2909th t to c It has been replaced and age 1015-1017 has been deleted.
  • SEQ ID NO: 27 shows a sequence in which the first to fourth amino acid sequences of TACT427-C are deleted.
  • the protein having the amino acid sequence represented by SEQ ID NO: 27 is named TACT427-C2.
  • the nucleotide sequence of the DNA encoding TACT427-C2 is shown in SEQ ID NO: 28.
  • the nucleotide sequence of the DNA encoding TACT427-C (SEQ ID NO: 26) showed the highest homology to the nucleotide sequence of the DNA encoding FU20539 (SEQ ID NO: 2) in humans.
  • the base sequence represented by SEQ ID NO: 2 is represented by positions 1 to 138 and 139 to 2322.
  • the third base sequence corresponds, and shows 99.3% and 99.5% homology in each partial sequence, respectively.
  • the nucleotide sequence of the DNA encoding FU20539 (SEQ ID NO: 2) lacks the DNA base sequence corresponding to positions 139 to 885 represented by TACT427-C (SEQ ID NO: 26). , A sequence specific to TACT427-C.
  • TACT427-C2 shows the highest homology to FLJ20539 and has a similar nucleotide sequence deletion.
  • amino acid sequence of amino acids 734-791 of TACT427-C (SEQ ID NO: 25) and the amino acids 730-787 of amino acid sequence of TACT427-C2 (SEQ ID NO: 27) are both amino acid domains.
  • a motif search site, SMART (ht tp: // smart. Einbl-hei del berg, de /), indicates that it has a black mouth peroxidase activity because it has a mouth mouth peroxidase motif.
  • TACT427-A gene (SEQ ID NO: 16), the TACT427-A2 gene (SEQ ID NO: 18), the TACT427-B gene (SEQ ID NO: 21), the TACT427-B2 gene (SEQ ID NO: : 23), TACT427-C gene (SEQ ID NO: 26) and TACT427-C2 gene (SEQ ID NO: 28) may be collectively abbreviated as TACT427 gene.
  • TACT427-A protein SEQ ID NO: 15
  • TACT427-A2 protein SEQ ID NO: 17
  • TACT427-B protein SEQ ID NO: 20
  • TACT427-B2 protein SEQ ID NO: 22
  • the TACT427-C protein SEQ ID NO: 25
  • TACT427-C2 protein SEQ ID NO: 27
  • composition of the reaction solution in the reaction was determined using the cDNA
  • the total amount of the above genes was about 7 times and about 16 times that of the surrounding normal tissues in 2 out of 4 human breast cancer tissues, respectively, and in 2 out of 3 human lung cancer tissues compared to the surrounding normal tissues, respectively.
  • Two out of five human ovarian cancer tissues, 10-fold and about 5-fold showed about 3-fold and about 20-fold expression levels, respectively, compared to surrounding normal tissues.
  • a cDNA prepared from human lung cancer tissue (purchased from Direct Clinical Access and BioClinical Partners) was used as a type II cDNA in the same manner as in Reference Example 6, and the genes used in Reference Example 6 in cancer tissues and normal tissues were used.
  • the composition of the reaction solution in this reaction was the same as that in Reference Example 6, except that the above cDNA 1 / ⁇ was used as type III, and a PCR reaction was performed.
  • the copy number of the J3-actin gene contained in the cDNA 11 was calculated using TadMan TM Human i3-act in Control Reagents (Applied Biosystems) and used as an internal standard.
  • the total amount of the above genes was about 144 times, about 3 times, and about 3 times in 8 out of 10 human lung cancer tissues compared to normal lung tissues in the sample of Direct Clinical Access Co. It increased 5 times, about 4 times, about 4 times, about 13 times, about 3 times, and about 19 times, and markedly increased expression in human lung cancer tissues.
  • Human normal airway epithelial cells SAEC and human normal prostate epithelial cells HPrEC were purchased from Clonetics.
  • Human colon cancer cell line C0CM1, human non-small cell lung cancer cell line VMRC-LCD, and human prostate cancer cell line PC3 were purchased from JCRB. These cell lines may also be used in Reference Examples 9 and thereafter.
  • RNA was prepared from 91 cell lines described above using RNeasy Mini Total RNA Kit (QIAGEN). TaQMan Reverse
  • cDNA was prepared by a reverse transcription reaction using random primers.
  • the genes FLJ20539 (SEQ ID NO: 2), MP50177 (SEQ ID NO: 5), MP1762319 (SEQ ID NO: 8) and FLJ13515 (SEQ ID NO: 8) were obtained. : 1 1) and the expression level of the TACT427 gene were examined.
  • the reaction was carried out in the same manner as in Reference Example 6, using the cDNA obtained from 5 ng of the above total RNA as Form III.
  • the copy number of the i8-actin gene contained in 1 ng of the total RNA was calculated using TadMan TM Human i3-act in Control Reagents (Applied Biosystems) and used as an internal standard.
  • Expression vector for animal cells expressing a protein in which a 3xFLAG tag was fused to the C-terminal of TACT427-A and TACT427-B proteins was constructed.
  • TACT427-A / pCR-Bluntll-T0P0, TACT427-B / pC-B1un111-T0P0, and p3xFLAG-CMV-14 (Sigma) obtained in Reference Example 4 were treated with restriction enzymes EcoRI and Xbal, After separation by agarose gel electrophoresis, DNA fragments corresponding to TACT427-A, TACT427-B and p3xFLAG-CMV-14 were recovered and purified using Gel Extraction Kit (QIAGEN). Each DNA fragment was subjected to a ligation reaction using DNA Ligation Kit ver. 2 (Takara Bio), then introduced into E. coli TOP10, and selected in LB agar medium containing ampicillin.
  • an expression vector p3xFLAG-TACT427- for animal cells having a cDNA sequence encoding the TACT427-A protein (SEQ ID NO: 15) and the TACT427-B protein (SEQ ID NO: 20) was obtained.
  • a and p3xFLAG-TACT427-B were obtained.
  • An expression vector for animal cells expressing the TACT427-A protein (SEQ ID NO: 15) was constructed.
  • PCR was carried out using Primer 9 (SEQ ID NO: 39) and Primer 10 (SEQ ID NO: 40)-this reaction
  • the composition of the reaction mixture in p3xFLAG-TACT427-A 200 ng 2.5 U of Pfu Turbo Hotstart DNA Polymerase (STRATAGENE), primer 9
  • peptides 1 to 3 Based on the amino acid sequence of the TACT427 protein, the following three peptides (peptides 1 to 3) consisting of 15 amino acids were synthesized by Fmoc solid phase synthesis.
  • amino acid sequence of peptide 1 [Gly-Ser-Gly-Glu-Glu-Glu-Asn-Asp-Pro-Gly_Glu-Gin-Ala-Leu-Pro-Cys (SEQ ID NO: 41)] is the TACT427-A protein (sequence). This sequence has Cys added to the C-terminal of the amino acid sequence from position 220 to position 233 of No. 15).
  • the peptide 2 CGly-Pro-Ala-Glu-Gly-Pro-Ala-Glu-Pro-Ala-Ala-Glu-Ala-Ser-Cys (SEQ ID NO: 42)] has an amino acid sequence of TACT427-A protein ( This is a sequence in which Cys has been added to the C-terminal of the amino acid sequence from position 517 to position 530 of SEQ ID NO: 15).
  • the amino acid sequence of peptide 3 [Gly-Ser-Vat Gly-Gly-Asn-Thr-Gly-Vat Arg-Gly-Lys-Phe-Glu-Cys (SEQ ID NO: 43)] is the TACT427-A protein (SEQ ID NO: 1 This is a sequence in which Cys has been added to the C-terminal of the amino acid sequence from position 800 to position 813 in 5).
  • Mossinin (KLH) was bound to whole limpet as a carrier protein and used as an antigen to prepare a Perian polyclonal antibody as follows.
  • the serum thus obtained was concentrated by the ammonium sulfate salting-out method, and the entire amount of the obtained crude IgG fraction was purified using a protein A affinity column (Amersham-Bioscience) to obtain peptide 1, peptide 2 or About 223 mg, about 495 mg and about 390 mg of purified IgG were obtained from the egret immunized with peptide 3. Furthermore, IgG fractions that bind to the column on which each immunogenic peptide was immobilized were obtained using purified IgG 11 mg for peptide 1, 248 mg of purified IgG for peptide 2 and 195 mg of purified IgG for peptide 3 as materials.
  • TACT427-A protein SEQ ID NO: 15
  • the detection of the TACT427-A protein was performed using the rabbit serum containing the peptide antibody prepared in Reference Example 11.
  • Dulbecco's modified Eagle minimum culture containing 1.0 x 10 6 HEK293 cells from human fetal kidney containing 10% fetal bovine serum (JRH)
  • JRH fetal bovine serum
  • the suspension was suspended in 9 ml of ground (Invitrogen) and seeded on a 10 cm diameter petri dish. After culturing at 37 ° C in a 5% carbon dioxide gas stream, mix with FuGene6 Transfection Reagent 181 (Roche Diagnostics) and OPTI-MEM I (Invitrogen) and leave at room temperature for 15 minutes.
  • the previously added p3xFLAG-TACT427-A6 / g was added, and the culture was continued under the same conditions. After 2 days, the cells are washed with PBS and then ice-cold RIPA buffer [50 mM Tris, hydrochloric acid buffer, pH 7.5, 150 mM sodium chloride, l% Triton X-100, 0.1% SDS, 1% Deoxycholic acid, Complete TM tablets (Roche Diagnostics), Phosphatase Inhibitor Cocktail 1-2 (Sigma)] 8001 were added, and the mixture was allowed to stand at 4 ° C for 15 minutes.
  • RIPA buffer 50 mM Tris, hydrochloric acid buffer, pH 7.5, 150 mM sodium chloride, l% Triton X-100, 0.1% SDS, 1% Deoxycholic acid, Complete TM tablets (Roche Diagnostics), Phosphatase Inhibitor Cocktail 1-2 (Sigma)
  • the RIPA buffer was collected, and the supernatant obtained by centrifugation at 15, OOO rpm for 20 minutes was used as a cell-free extract, and 201 was subjected to SDS-PAGE on a 7.5% acrylamide gel.
  • the proteins separated by electrophoresis were transferred to a clear blot P membrane (ATT0) according to a conventional method, and then transferred to a blocking solution (Tris buffered saline, 0.1% Tween-20, 5% skim milk). It was left at room temperature for a time.
  • Protein G-Sepharose 4FF (Amersliam-Bioscience) suspension (equal volume) was added to 1 ml of the cell-free extract prepared by the same procedure as in Reference Example 12 using p3xFLAG-TACT427-A obtained in Reference Example 9. Suspended with RIPA buffer of 50) Stirring was performed overnight at 4 ° C.
  • RIPA buffer As the egret serum, any one of three kinds of egos serum including the peptide antibodies AS-2480, AS-2481 or AS-2482 prepared in Reference Example 11 was used. After washing the ProteinG-Sepharose 4FF coprecipitate fraction with RIPA buffer, suspend it in SDS-PAGE sample buffer (Bio Rad) containing 1% 2-mercaptoethanol and heat at 95 for 5 minutes.
  • peptide antibodies AS-2480, AS-2481 and AS-2482 were subjected to SDS-PAGE on 7.5% acrylamide gel. Detection was performed according to the method described in Reference Example 12. However, mouse anti-FLAGM2 antibody (Sigma) as the primary antibody was diluted to 10 g / ml with a blocking solution, and HRP-labeled anti-mouse IgG antibody (Amersham-Bioscience) as the secondary antibody was used in a blocking solution of 50,000. A one-fold dilution was used. When immunoprecipitation was performed using any of the three types of egret serum containing the peptide antibodies AS-2480, AS-2481 or AS-2482, a specific band derived from the TACT427-A protein with a molecular weight of around 150 kD. Thus, peptide antibodies AS-2480, AS-2481 and AS-2482
  • Human embryonic kidney-derived HEK293 cells (1 ⁇ 10 5 cells) were suspended in Dulbecco's modified Eagle's minimal medium (Invitrogen) containing 10% fetal bovine serum (JRH), and 2 ⁇ L-poly-D-lysine-coated culture slides (BD (Falcon).
  • Dulbecco's modified Eagle's minimal medium Invitrogen
  • JRH fetal bovine serum
  • BD L-poly-D-lysine-coated culture slides
  • 5 ⁇ 10 4 human non-small cell lung cancer cell lines NCI-H460 were suspended in RPMI1640 medium (Invitrogen) containing 10% fetal bovine serum (JRH) and 2 ⁇ L-poly-D-lysine-coated culture slide (BD Falcon). Company).
  • mouse anti-FLAG M2 antibody (Sigma) diluted to 10 g / ml with PBS containing 1% BSA was added, reacted at room temperature for 45 minutes, washed with PBS, and then 1% BSA was added.
  • Alexa488-labeled anti-mouse IgG antibody diluted to 10 ig / ml with PBS
  • TACT427-A protein was expressed on the cell membrane in both HEK293 and NCI-H460.
  • pcDNA3.1 (+)-TACT427-A plasmid was introduced into HEK293 cells in the same manner, and 10 oz / ml of lOig / ml AS-2480, AS-2481 and AS-2482 as primary antibodies.
  • TACT427-A protein Localization of TACT427-A protein (Piotin labeling)
  • the p3xFLAG_TACT427-A plasmid was introduced into HEK293 cells, and the protein exposed on the cell surface 48 hours later was subjected to Cellular Labeling and Immunoprecipitation tation Kit (Roche Diagnostics). A biotin marker was performed by using. Furthermore, SDS-PAGE was performed by immunoprecipitation using lml of the cell-free extract prepared according to the method of Reference Example 12 and mouse anti-FLAG ⁇ 2 antibody (Sigma) 3 / ig according to the method of Reference Example 13.
  • TACT427 protein Localization of TACT427 protein (Piotin labeling)
  • Biotin labeling was performed using Labeling and Immunoprecipitation Kit (Roche Diagnostics). Further, immunoprecipitation was performed using lml of the cell-free extract prepared according to the method of Reference Example 12 and the egret peptide antibody AS-24823 according to the method of Reference Example 13, and SDS-PAGE was performed. HRP-labeled streptavidin (Amersham-Bioscience) was used to detect TACT427-A, TACT427-A2, and TACT427-B proteins with a molecular weight of around 150 kD in all of A549, NCI-H226 and NCI-H522. Bands derived from protein, TACT427-B2 protein, TACT427-C protein and TACT427-C2 protein were observed.
  • A549 cells were suspended at a concentration of lx 10 6 cells / ml with AS-2482 or non-immune rabbits (Jackson, Inc.) to a final concentration of 5 / ig / ml. ) And left on ice for 5 hours. Subsequently, the cells were washed with buffer A, suspended in buffer A containing 10 g / ml of Alexa488-labeled anti-Peacock IgG antibody (Molecular Probes), and allowed to stand on ice for 1.5 hours. After washing again with buffer A, analysis was performed with FACScan (BD Biosciences). As a result, A549 cells were stained specifically for the egret peptide antibody AS-2482. It was revealed that it was expressed above. Reference Example 1 9
  • human non-small cell lung cancer cell lines other than NCI-H460 described in Reference Example 2 c human non-small cell lung cancer cell lines A549 and NCI-H226 were examined to determine whether apoptosis was induced by the introduction of antisense oligonucleotides.
  • Both purchased from ATCC and fetal calf serum (Layer) in Kaighn's modified F-12 Nutrient Mixture (Invitrogen) and RPMI-1640 medium (Invitrogen) containing 25 mM HEPES, respectively. It was suspended in 10% added medium, and seeded into 1 Ueru per IX 10 4 cells density in 96-well flat-bottom tissue culture plates (BD off. Alcon). After culturing overnight at 37 ° C. in a 5% carbon dioxide gas stream, oligonucleotides were introduced.
  • the antisense oligonucleotide obtained in Reference Example 2 was used.
  • the apoptosis-inducing activity of the above oligonucleotide was measured according to the protocol attached to Cell Death Detection ELISA PLUS (Roche Diagnostics).
  • the antisense oligonucleotide showed 1.65-fold and 3.03-fold apoptosis-inducing activities, respectively, as compared to the control oligonucleotide and the sense oligonucleotide used as negative controls, and a statistically significant difference (P ⁇ 0.01).
  • Reference Example 20
  • FLJ20539 gene (SEQ ID NO: 2), hCP50177 gene (SEQ ID NO: 5), hCP1762319 gene (SEQ ID NO: 8) and FLJ13515 in human non-small cell lung cancer cell lines A549 and NCI-H226 by introducing antisense oligonucleotides Gene (SEQ ID NO: 11) and mRNA expression of TACT427 gene decreased
  • Anti-serum was also detected in human non-small cell lung cancer cell lines other than NCI-H460 described in Reference Example 3. It was examined whether or not the administration of the oligonucleotide reduced the mRNA expression level of the above gene.
  • oligonucleotide was transfected.
  • each of the antisense oligonucleotide (SEQ ID NO: 13), the control oligonucleotide (SEQ ID NO: 14), and the sense oligonucleotide (SEQ ID NO: 44) is lipofector.
  • the mixture was mixed with 200 l of Opti-MEMI (Invitrogen) together with Min 2000 (Invitrogen) 3.21 and left at room temperature for 20 minutes. Add the entire volume of the mixture to ⁇ 549 cells that had been medium-exchanged to 0PTI-MEM I (Invitrogen) 2001 before continuing cultivation for 3 hours, then containing 10% fetal bovine serum (JRH). Kaiglm's Modified F-12 Nutrient Mixture
  • 1 ⁇ -tal RA was extracted from A549 cells and NCI-H226 cells using the RNeasyMini Total RNA Kit (QIAGEN). TaaMan TM Reverse Transcription Reagents
  • cDNA was prepared from total RNA by reverse transcription using random primers.
  • the cDNA prepared from 5 ng of total RA was designated as type I, and the FLJ20539 gene (sequence number No .: 2), hCP50177 gene (SEQ ID NO: 5), hCP1762319 gene (SEQ ID NO: 8), FLJ13515 gene (SEQ ID NO: 11), and expression level of TACT427 gene were measured.
  • the relative expression ratio (%) of the above gene to the i3-actin gene expression is based on the introduction of the control oligonucleotide (SEQ ID NO: 14) or the sense oligonucleotide (SEQ ID NO: 44) used as a negative control.
  • the expression level was significantly reduced when the antisense oligonucleotide (SEQ ID NO: 13) was introduced, and a statistically significant (P ⁇ 0.01) decrease in the expression level was observed (Table 8).
  • the human non-small cell lung cancer cell lines A549 and NCI-H226 also showed the FU20539 gene (SEQ ID NO: 2), hCP50177 gene (SEQ ID NO: 5),
  • TACT427 protein Human non-small cell lung cancer cell lines A549 and NCI-H226 were suspended in the same medium as each Example 1 9, the A549 cell line 2. 25 X 10 6 cells, in NCI H226 cell lines 1. 45X 10 6 pieces of Cells were seeded on a 10 cm diameter Petri dish (BD Falcon).
  • each of the antisense oligonucleotide (SEQ ID NO: 13), the control oligonucleotide (SEQ ID NO: 14) and the sense oligonucleotide (SEQ ID NO: 44) were Fectamine
  • the mixture was mixed with 3.75 ml of 0pt i_MEM I (Invitrogen) together with (Invitrogen) 60 / z1, and left at room temperature for 20 minutes.
  • the total volume of the mixture was added to NCI-H226 cells whose medium had been changed to 0 PTI-MEM I (Invitrogen) 3.75 ml in advance, and cultivation was continued for another 4 hours, followed by 30% fetal bovine serum (JRH).
  • 3.75 ml of RPMI-1640 medium (Invitrogen) containing 25 mM HEPES containing the following.
  • a cell-free extract was prepared according to the method of Reference Example 12.
  • the protein concentration of the obtained cell-free extract was measured using a BCAProtein Assay Kit (Pierce), and the protein concentration in each cell-free extract was adjusted.
  • 100 g of the cell-free extract of the A549 cell line and 140 g of the cell-free extract of the NCI-H226 cell line were subjected to SDS-PAGE and Western plotting according to the method of Reference Example 12 respectively.
  • AS-2482 prepared in Reference Example 11 was used as the primary antibody at a concentration of 3 g / ml, and HRP-labeled anti-Peagle IgG antibody was used as the secondary antibody.
  • A31 cells 1. 25 X 10 5 cells with 10% fetal bovine serum (JRH Co.) and 50 g / ml gentamicin (Invi trogen Inc.) Dulbecco's modified Eagle's medium containing (Invi trogen Inc.) were suspended in 2 ⁇ 5 ml After seeding in a 1-well of a 6-well plate, the cells were cultured overnight at 37 in a 5% carbon dioxide gas stream. Further, after changing the medium to 2.5 ml and continuing the culture for 4 hours, FUGENE6 transfection reagent 3.81 (Roche Diagnostics) and OPTI-MEM I
  • Cells were collected using (Invitrogen), suspended in 10 ml of the above-mentioned medium (G418 selective medium) containing 0.5 mg / ml of G418 (Promega), and seeded on a Petri dish having a diameter of 10 cm. Continue culturing with G418 selection medium and subculture twice, then prepare 12 series of 2-fold dilutions starting from 100 cells / well (medium volume: 0.1 ml) and inoculate them into 96-well plates respectively. The culture was continued while replacing the G418 selection medium every three days. Cells were harvested 11 days after the 0.8-3.2 cells grew and formed a colony per well, and seeded equally in 2 wells of a 24-well plate.
  • G418 selective medium the above-mentioned medium
  • G418 selection medium containing 0.5 mg / ml of G418 (Promega)
  • the TACT-1 and its parental A31 cells obtained in Reference Example 22 were transformed with Dulbecco's modified idal minimal medium (Invitrogen) containing 10% fetal bovine serum (JRH) and SO ⁇ g / ml gentamicin (Invitrogen).
  • the suspension was suspended in 0.1 ml and seeded on a 96-well plate for tissue culture at 6 x 10 3 cells / well. After culturing at 37 ° C in a 5% carbon dioxide gas stream, the above medium to which various concentrations of topoisomerase I inhibitor camptothecin (WakoPure Chemical) or protein synthesis inhibitor anisomycin (Wako Pure Chemical) is added. And the culture was continued.
  • MAP kinase involved in the anti-apoptotic action was compared to the A31 cell line.
  • Dulbecco's modified minimal medium Invitrogen
  • JRH fetal calf serum
  • 50 g / ml Genymycin Invitrogen
  • TACT-1 cells were used for TACT-1 cells.
  • the same medium supplemented with 0.6 mg / ml G418 (Promega)
  • the cells were cultured at 37 ° C.
  • the cell lysate was recovered using a scraper, and centrifuged at 15,000 rpm for 20 minutes at 4 ° C to remove the precipitate.
  • 20 1 of a 5-fold concentrated SDS-PAGE sample buffer (Bio Rad) containing 10% 2-mercaptoethanol was added, and the mixture was heated at 95 ° C for 5 minutes.
  • the 50-cell lysate was diluted 10-fold with distilled water, and the protein concentration was measured according to the prescription of Micro BCA protein assay reagent (Pierce).
  • TTBS washing for 10 minutes was performed four times.
  • HRP-labeled anti-Egret IgG antibody (Amersham Bioscience) diluted 10000-fold with TTBS containing 5% skim milk was added, and the mixture was incubated at room temperature for 1 hour, and then washed 10 times with TTBS for 10 minutes.
  • Bands corresponding to ERK1 / 2 protein and phosphorylated ERK1 / 2 protein were detected using ECLpIus reagent (Amersham Bioscience). Phosphorylation of ERK1 / 2 protein induced by the addition of anisomycin, ie, activation, was enhanced as compared to the vesicle strain.
  • ERK1 / 2 activation enhancement in TACT-1 cells To determine the degree of ERK1 / 2 activation enhancement in TACT-1 cells, read the developed film as an image with a lumino image analyzer LAS-LOOOOplus (FUJIFILM), and use the attached image gauge software to phosphorylate ERK1 and phosphorylation. The band intensity of ERK2 was quantified. The rate of phosphorylation enhancement of TACT-1 cells was calculated for each of the anisomicin treatment times (2 hours, 4 hours, and 8 hours), with the band intensity of A31 cells as 100%. Were 164%, 150% and 158%, and the phosphorylation rate of ER2 was 130%, 137% and 172%.
  • the cells were washed once with 5 ml of PBS containing lmM sodium orthovanadate (V) (Wako Pure Chemical) before or at 15 minutes, 30 minutes and 60 minutes after the addition of anisomycin, and described in Reference Example 12.
  • 0.2 ml of a cell lysis buffer obtained by adding Phosphatase Inhibitor Cocktail 1 to the RIPA buffer was added and left at 4 ° C for 15 minutes.
  • the cell lysate was collected with a scraper and centrifuged at 15,000 rpm at 4 ° C for 5 minutes to remove the precipitate.
  • 201 1 of a 5-fold concentrated SDS-PAGE sample buffer (Bio Rad) containing 5% 2-mercaptoethanol was added, and heated at 95 ° C for 5 minutes.
  • a cell lysate was prepared by diluting the above cell lysis buffer 20 times with distilled water. 15 Dilute 20 1-fold and measure the protein concentration according to the formulation of Micro BCA protein assay reagent (Pierce), and confirm that the protein concentration is almost the same. After subjecting to SDS-PAGE using a% polyacrylamide gradient gel, it was transferred to a PVDF membrane.
  • the transferred membrane was blocked with TTBS containing 5% skim milk (TBS containing 0.1% Tween 20) for 1 hour at room temperature, and then washed with TTBS three times for 5 minutes. Then, using a primary antibody solution of anti-P38 MAPK antibody (Cell lsignaling) or anti-phosphorylated P38 MAPK antibody (Cells ignaling) diluted 1000-fold with TTBS containing 5% BSA (Sigma). Reaction was carried out overnight at ° C.
  • an HRP-labeled anti-heron IgG antibody (Amersham Bioscience) diluted 5000 times with TTBS containing 5% skim milk was added, and the mixture was incubated at room temperature for 1 hour. Perform TTBS washing 3 times again for 5 minutes to remove excess antibody.
  • NAV2 protein An expression vector for animal cells expressing the NAV2 protein (SEQ ID NO: 49) was constructed. Using human brain Marathon-Ready cDNA (CL0NTECH) as type III, PCR was performed using two primers [Primer 11 (SEQ ID NO: 52) and Primer 12 (SEQ ID NO: 53)]. The cDNA encoding the amino acid sequence from the first to the 189th amino acid sequence of NAV2 (SEQ ID NO: 49) was cloned.
  • the composition of the reaction solution used in the reaction was as follows: using the above cDNA II as type I, PfuTurbo Hotstart DNA Polymerase (STRATAGENE) 2.5 ⁇ , primer 11 (SEQ ID NO: 58) and primer 12 ( SEQ ID NO: 59) was added to each of 1.0 M, dNTPs at 200 ⁇ l, and 2 L of 2XGC Buf fer I (TaKaRa Bio) to give a 50 ⁇ l volume.
  • the PCR reaction is 94 After one minute, a cycle of 94 ° C for 20 seconds, 58 ° C for 15 seconds, and 72 ° C for 10 minutes was repeated 40 times, followed by a reaction at 72 ° C for 10 minutes.
  • the PCR reaction product was electrophoresed on an agarose gel and purified using a Gel Extraction Kit (QIAGEN). This was subcloned into a plasmid vector pCR4Blunt-TOP0 (Invitrogen), introduced into E. coli T0P10 (Invitrogen), and clones having cDNA were selected in LB agar medium containing kanamycin. As a result of analyzing the sequence of each clone, the nucleotide sequence of the cDNA encoding the amino acid sequence from the 1st to the 1889th of the NAV2 protein (SEQ ID NO: 49) was obtained. The plasmid vector obtained here was named pCR4Blunt-TOP0-NAV2-5 '.
  • the composition of the reaction solution used in the reaction was as follows: cDNA lzl was used as type I, 2.5 U of PfuTurbo Hotstart DNA Polymerase (STRATAGENE), Primer 13 (SEQ ID NO: 60) and Primer 14 (SEQ ID NO: 61) was added to each of 1.0 M, 200 ⁇ l of dNTPs, and 25 ⁇ l of 2 ⁇ GC Buffer I (TaKaRa Bio) to give a liquid volume of 50 ⁇ l.
  • a cycle of 94 ° C for 1 minute, 94T for 20 seconds, 58 ° C for 15 seconds, 72 ° C for 10 minutes was repeated 40 times, followed by a reaction at 72 ° C for 10 minutes. went.
  • the PCR reaction product was electrophoresed on an agarose gel and purified using a Gel Extraction Kit (QIAGEN). This was subcloned into the plasmid vector pCR4Blimt-TOP0 (Invitrogen) and introduced into Escherichia coli TOP10 (Invitrogen), and the clone having the cDNA was selected in LB agar medium containing kanamycin. As a result of analyzing the sequence of each clone, the nucleotide sequence of the cDNA encoding the amino acid sequence from position 1796 to position 2429 of the NAV2 protein (SEQ ID NO: 49) was obtained. The obtained plasmid vector was designated as pCR4Blunt-T0P0-NAV2-3 ′.
  • pCR4Blunt-TOP0-NAV2-3 'and p3XFLAG-CMV-14 were treated with restriction enzymes EcoRV and Xbal.
  • Each DNA fragment was subjected to electrophoresis on agarose gel, and then purified using Gel Extraction Kit (QIAGEN). Then, get here After performing a ligation reaction using DNA Ligat ion Kit ver. 2 (TaKaRa Bio), the obtained DNA fragment was introduced into E. coli T0P10 (Invitrogen) and selected in LB agar medium containing ampicillin. did.
  • a cDNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 45 was amplified from the plasmid p3xFLAG-TACT427-A obtained in the above Reference Example by PCR.
  • the resulting cDNA was recombinantly introduced into yeast expression vector pGBT.Q.
  • pGBT.Q is a derivative closely related to pGBT.C (Nat. Genet. 12, 72-77, 1996), which is obtained by modifying a polylinker at one position and introducing an M13 primer for sequence. is there.
  • This new construct was selected directly in the yeast strain PNY200, depending on its ability to synthesize tryptophan (genotype of this strain: MAT tr1-901 leu2-3, 112 ura3-52 his3-200 ade2 gal4A gal80 A ).
  • the protein was produced as a fusion protein bound to the C-terminal side of the DNA-binding domain (amino acids 1 to 147) of the transcription factor Gal4 protein (GenBank NPJ15076).
  • the play library was used for the yeast BK100 strain (genotype of this strain: MATa trp 901 leu2-3, 112 ura3-52 is3-200 gal4A gal80 A LYS2:: GAL- HIS3 GAL2-ADE2 met2:: GAL7 T lacZ) and selected for its ability to synthesize leucine.
  • each cDNA was produced as a fusion protein linked to the C-terminal side of the transcription promoting domain (amino acids 768 to 881) of the transcription factor Gal4 protein (GenBank NP-015076).
  • PNY200 cells expressing the bait protein (conjugated ⁇ !) Were conjugated to BK100 cells expressing the prey protein in the prey library (conjugated MATa).
  • the resulting diploid yeast cells expressing the prey protein interacting with the pate protein were selected for their ability to synthesize tryptophan, leucine, histidine and adenine.
  • DNA is prepared from each clone and transformed into the E. coli KC8 strain by the electoporation method. Cells are then transformed without tributofan (for bait plasmid selection) or without leucine (for plasmid selection in the pre-library library). For selection on ampicillin-containing medium.
  • the DNAs of both plasmids were prepared, and the sequences were determined by the dideoxynucleotide chain-initiation method. The identity of the bait cDNA insert was confirmed, and the cDNA insert obtained from the prey library plasmid was identified using the BLAST program, which checks against known nucleotide and protein databases.
  • a cDNA fragment encoding the amino acid sequence of nucleotides 2206 to 2355 or 2141 to 2351 of FLNA was obtained.
  • a cDNA fragment encoding the amino acid sequence at positions 2162 to 2412 of FLNA was obtained from the human hippocampus cDNA library.
  • a cDNA fragment encoding the amino acid sequence of positions 1960 to 2299 or 2156 to 2314 of FLNB was obtained from human.
  • a cDNA fragment encoding the amino acid sequence at positions 2174 to 2565 of FLNB was obtained.
  • a cDNA fragment encoding the amino acid sequence from position 201 to position 525 of BTBD2 was obtained from the human hippocampus cDNA library.
  • a cDNA fragment encoding the amino acid sequence from position 215 to position 506 of (SEQ ID NO: 50) was obtained.
  • TACT427-A protein and NAV2 protein (cell staining) 1.5 ⁇ 10 6 human fetal kidney-derived HEK293 cells were modified with Darbecco's modified Eagle's minimal medium containing 10% fetal bovine serum (JRH) ( (Invitrogen) and seeded on a 10-cm diameter dish. After culturing at 37 ° C in a 5% carbon dioxide gas stream, mix with FuGene6 transfection reagent 181 (Roche Diagnostics) and OPTI-MEM I (Invitrogen) in advance and leave at room temperature for 15 minutes.
  • JRH fetal bovine serum
  • TACT427-A was added, and the culture was continued under the same conditions.
  • 5 ⁇ 10 3 cells were seeded on an 8-well poly-D-lysine culture slide (BD Falcon). After culturing overnight at 37 ° C in a 5% carbon dioxide gas stream, the cells were washed with PBS, and 10% neutral formalin buffer was added, and fixed at room temperature for 30 minutes. Thereafter, TritonX-100 diluted to 0.1% with PBS was added, and the mixture was allowed to stand at room temperature for 5 minutes. After washing with PBS again, PBS containing 1% BSA was added, and the mixture was allowed to stand at 4 ° C for 24 hours.
  • a compound or a salt thereof that inhibits the formation of a complex containing a qualitatively identical amino acid sequence or a partial peptide thereof or a complex containing a salt thereof includes, for example, cancer
  • hematologic malignancies include, for example, neurodegenerative diseases (eg, Alzheimer's disease (familial Alzheimer disease, juvenile Alzheimer's disease, It is useful as an agent for prevention and treatment of Alzheimer's disease).
  • neurodegenerative diseases eg, Alzheimer's disease (familial Alzheimer disease, juvenile Alzheimer's disease, It is useful as an agent for prevention and treatment of Alzheimer's disease).
  • Proteins containing amino acid sequences or partial peptides or salts thereof are useful for screening preventive and therapeutic agents for cancer and neurodegenerative diseases and for diagnosing cancer and neurodegenerative diseases.

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Abstract

A novel conjugated protein; and a preventive/therapeutic agent for cancer or neurodegenerative disorder, etc. A compound, or its salt, capable of inhibiting conjugation between (a) protein containing an amino acid sequence identical or substantially identical with the amino acid sequence of SEQ ID NO. 45, or its partial peptide, or a salt thereof and (b) protein containing an amino acid sequence identical or substantially identical with the amino acid sequence of SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50 or SEQ ID NO. 51, or its partial peptide, or a salt thereof is useful as, for example, a preventive/therapeutic agent for cancer, etc. On the other hand, a compound, or its salt, capable of promoting the above conjugation is useful as, for example, a preventive/therapeutic agent for neurodegenerative disorder, etc.

Description

明細書 新規タンパク質複合体およびその用途 技術分野  Description Novel protein complexes and their uses
本発明は、 新規結合タンパク質などに関する。 さらに詳しくは、 TACT427 夕 ンパク質と、 該タンパク質と結合しうるタンパク質との複合体、 該複合体に対す る抗体 （該複合体の形成を阻害または促進する TACT427タンパク質に対する抗 体等） 、 該複合体の形成を阻害または促進する化合物またはその塩のスクリ一二 ング、 該スクリーニングで得られうる化合物またはその塩、 癌または神経変性疾 患の予防 ·治療剤、 診断薬などに関する。 背景技術  The present invention relates to novel binding proteins and the like. More specifically, a complex of a TACT427 protein and a protein capable of binding to the protein, an antibody against the complex (such as an antibody against the TACT427 protein that inhibits or promotes the formation of the complex), the complex The present invention relates to screening of a compound or a salt thereof that inhibits or promotes body formation, a compound or a salt thereof obtainable by the screening, an agent for preventing or treating cancer or a neurodegenerative disease, a diagnostic agent, and the like. Background art
TACT427は、 乳癌や肺癌などのヒト癌組織で高発現している 1回膜貫通型タンパ ク質であり、 癌細胞の細胞質膜上に発現し、 アンチセンスオリゴヌクレオチドで 癌細胞のアポトーシスが誘発されることから癌細胞のアポトーシス抑制に関与し ていることが報告されている （特許文献 1 特願 2003- 296081号公報） 。 また、 TACT427のラットオルソログが脳で最も多く発現しており、 かつ、 血清飢餓条件 で誘発された神経芽腫細胞株のアポトーシスを抑制することが知られている （J. Biol. Chem. 278巻、 10531頁 - 10537頁、 2003年） 。  TACT427 is a one-time transmembrane protein highly expressed in human cancer tissues such as breast cancer and lung cancer.It is expressed on the cytoplasmic membrane of cancer cells, and antisense oligonucleotides induce cancer cell apoptosis. Therefore, it has been reported that it is involved in the suppression of apoptosis of cancer cells (Patent Document 1 Japanese Patent Application No. 2003-296081). In addition, it is known that the rat ortholog of TACT427 is most frequently expressed in the brain and suppresses apoptosis of neuroblastoma cell lines induced by serum starvation (J. Biol. Chem. 278 , 10531-10537, 2003).
以下、 ヒト Fi lamin A (GenBank NP_001447； J. Cel l Biol. I l l巻、 1089頁- 1105頁、 1990年) 、 ヒト Fi lamin B (GenBank NP— 001448； J. Biol. Chem. 273巻、 17531頁- 17538頁、 1998年) 、 ヒ卜 Fi lamin C (GenBank NPJ01449； Biochem. Biophys. Res. Conunun. 251巻、 914頁 - 919頁、 1998年） を、 それぞれ FLNA、 FLNB、 および FLNCと称する。  Hereinafter, human Filamin A (GenBank NP_001447; J. Cell Biol. Ill, 1089-1105, 1990), human Filamin B (GenBank NP-001448; J. Biol. Chem. 273, 17531) Pp. 17538, 1998) and human Filamin C (GenBank NPJ01449; Biochem. Biophys. Res. Conunun. 251: 914-919, 1998) are referred to as FLNA, FLNB, and FLNC, respectively.
FLNA、 FLNBおよび FLNCからなる Fi laminは、 当初、 ァクチン結合ダンパク質と して見出された (J. Biol. Chem. 250巻、 5696頁- 5705頁、 1975年) 。 Fi laminは、 N末端の 275アミノ酸からなるァクチン結合ドメインと、 C末側に約 96アミノ酸か らなる βシート構造が 24回繰り返された構造を有し、 かつ、 繰り返し構造の途中 には 1ケ所または 2ケ所の ンジ領域が認められていること、 F i 1 aminは 2量体を形 成し、 ァクチン線維の架橋に関与していること、 Fi laminは、 Fcレセプ夕一、 ィ ンテグリンまたはド―パミン D2レセプターなどのレセプ夕一、 Rho、 Racまたは CDC42などの低分子量 Gタンパク質、 または TRAF2や SEK1などのシグナル伝達分子 と C末側を介して結合し、 細胞外の刺激を細胞内部に伝える足場として働いてい ることが報告されている （非特許文献 1 Nature Rev. Mol. Cel l Biol. 2巻、 138頁- 145頁、 2001年） 。 また、 大脳皮質神経細胞の移動障害を特徴とする Filamin consisting of FLNA, FLNB and FLNC was initially found as an actin-binding protein (J. Biol. Chem. 250, 5696-5705, 1975). Filamin has a structure in which an actin-binding domain consisting of 275 amino acids at the N-terminus and a β-sheet structure consisting of about 96 amino acids at the C-terminal side are repeated 24 times, and in the middle of the repeating structure. Shows that one or two enzyme regions are recognized, that Fi 1 amin forms a dimer and is involved in the cross-linking of actin filaments, and Filamin shows that Fc receptor It binds to receptors such as integrin or dopamine D2 receptor, low-molecular-weight G proteins such as Rho, Rac or CDC42, or signaling molecules such as TRAF2 or SEK1 via the C-terminus to stimulate extracellular stimulation. It has been reported that it works as a scaffold that transmits the inside of cells (Non-Patent Document 1 Nature Rev. Mol. Cell Biol. 2, 138-145, 2001). Also characterized by impaired movement of cerebral cortical neurons
Periventricular heterotopia病の原因が FLNA遺伝子の変異であることも報告さ れている (Neuron 21巻、 1315頁- 1325頁、 1998年） It has also been reported that the cause of Periventricular heterotopia disease is a mutation in the FLNA gene (Neuron 21, 1315-1325, 1998).
以下、 ヒト Neuron navigator 2 (GenBank NPJ92009； Gene 290巻、 73頁- 94頁、 2002年；別名、 P0MFIL2または HELAD1) を、 NAV2と称する。  Hereinafter, human Neuron navigator 2 (GenBank NPJ92009; Gene 290, pp. 73-94, 2002; also known as P0MFIL2 or HELAD1) is referred to as NAV2.
NAV2は、 線虫の UNC- 53に相同性の高いタンパク質であり、 UNC- 53は筋細胞の遊 走や神経軸索の伸長に関与している (Development 129巻、 3367頁- 3379頁、 2002 年） 。 NAV2は、 2428アミノ酸よりなるタンパク質で、 89番目〜 190番目のァミノ 酸よりなる力ルポニンホモロジ一ドメイン、 2098番目〜 2105番目のアミノ酸より なる ATP/GTP結合部位、 および 2091番目〜 2208番目のアミノ酸よりなる ATPaseド メインからなる （Genomics 80巻、 21頁- 30頁、 2002年； Proc. Nat l. Acad. Sci. USA 99巻、 3422頁- 3427頁、 2002年) 。 匪は、 細胞内では細胞質または核に局 在すること、 ヒト大腸癌で発現亢進していること、 ATP/GTP結合部位および  NAV2 is a protein highly homologous to UNC-53 in C. elegans, and UNC-53 is involved in muscle cell migration and elongation of nerve axons (Development Vol. 129, pp. 3367-3379, 2002 Year) . NAV2 is a protein consisting of 2428 amino acids, consisting of a potent luponin homology domain consisting of amino acids 89 to 190, an ATP / GTP binding site consisting of amino acids 2098 to 2105, and amino acids 2091 to 2208. Consists of the ATPase domain (Genomics 80, 21-30, 2002; Proc. Natl. Acad. Sci. USA 99, 3422-3427, 2002). Marauders have been found to be localized in the cytoplasm or nucleus in cells, to be up-regulated in human colorectal cancer, to have ATP / GTP binding sites and
ATPaseドメインを含む 1958番目〜 2234番目のアミノ酸からなる組換えタンパク質 がへリカーゼ活性を有すること、 および NAV2のアンチセンスオリゴヌクレオチド 導入により大腸癌細胞株 SW480にアポトーシスが誘導されることが報告されてい る （非特許文献 2 Oncogene 21巻、 6387頁- 6394頁、 2002年) 。 It has been reported that a recombinant protein consisting of amino acids 1958 to 2234 containing the ATPase domain has helicase activity, and that the introduction of NAV2 antisense oligonucleotide induces apoptosis in colon cancer cell line SW480. (Non-Patent Document 2 Oncogene 21, 6363-6394, 2002).
以下、 ヒト BTB domain-containing 2 (GenBank NP— 060267； Gene 262巻、 275 頁- 281頁、 2001年） を、 BTBD2と称する。  Hereinafter, human BTB domain-containing 2 (GenBank NP-060267; Gene, 262, 275-281, 2001) is referred to as BTBD2.
BTBD2は 525アミノ酸よりなるタンパク質で、 110番目〜 217番目のアミノ酸より なる BTBドメイン （Piam accession No. PF00651) を有している。 酵母ツーハイブ リツド法での解析で、 DNA topoisomerase I (以下、 T0P1と称する） と結合する こと、 BTBD2の 86番目〜 525番目のアミノ酸からなる組換えタンパク質が T0P1の DNAスーパーコイル弛緩活性、 DNA切断活性を阻害することが報告されている （非 特許文献 3 BMC Genomics 3巻、 1頁 - 9頁、 2002年) 。 BTBD2 is a protein consisting of 525 amino acids and having a BTB domain consisting of amino acids 110 to 217 (Piam accession No. PF00651). Analysis by the yeast two-hybrid method revealed that it binds to DNA topoisomerase I (hereinafter referred to as T0P1). It has been reported that the DNA supercoil relaxing activity and the DNA-cleaving activity are inhibited (Non-Patent Document 3 BMC Genomics Vol. 3, pp. 1-9, 2002).
以下、 ヒ卜画 A interact ing protein-l ike 1 (非特許文献 4 GenBank  Below, a human painting A interacting protein-like 1 (Non-patent document 4 GenBank
NPJ37533) を、 RAB3IL1と称する。 NPJ37533) is referred to as RAB3IL1.
RAB3IL1は RAB3A interact ing protein (RAB3IP, GenBank NP— 071901) と 59% の相同性を示すタンパク質として登録されている。 RAB3 IPとの配列相同性から、 低分子量 G夕ンパク質 Rab3Aの GTP/GDPヌクレオチド交換活性を有し小胞輸送ゃェ キソサイ I ^一シスに関与すると予測されている （Publ ic HumanPSD™volume of the BioKnow ledge Library： www. incyte. com/control/researchproducts/ ins i l ico/proteome) 。 発明の開示  RAB3IL1 is registered as a protein showing 59% homology with RAB3A interacting protein (RAB3IP, GenBank NP-071901). Based on sequence homology with RAB3 IP, it is predicted that Rab3A has a GTP / GDP nucleotide exchange activity and is involved in vesicle trafficking exocytosis I ^ cis (Public HumanPSD ™ volume of the BioKnow ledge Library: www. incyte. com / control / researchproducts / insilico / proteome). Disclosure of the invention
癌細胞や神経細胞に特異的に発現するタンパク質複合体を標的とし、 癌細胞の 増殖阻害を誘導する安全な癌の予防 ·治療剤、 および神経細胞の細胞死を抑制す る安全で優れた神経変性疾患の予防 ·治療剤が切望されている。  Targets a protein complex that is specifically expressed in cancer cells and nerve cells, and is a safe cancer prevention and treatment agent that induces cancer cell growth inhibition, and a safe and superior nerve that suppresses nerve cell death. Prevention and treatment of degenerative diseases.
TACT427が介在する抗アポトーシスシグナルを調節することにより、 癌細胞を 死滅させ抗腫瘍効果を導き出す、 または、 神経細胞の細胞死を抑制し神経変性疾 患の予防 ·治療に役立てることが期待できるものの、 TACT427の細胞内結合タン パク質または細胞内情報伝達経路に関する詳細な報告は現時点では殆どなされて いない。  By regulating the anti-apoptotic signal mediated by TACT427, it can be expected to kill cancer cells and induce an antitumor effect, or suppress neuronal cell death and prevent or treat neurodegenerative diseases. There are few detailed reports on the intracellular binding protein or intracellular signaling pathway of TACT427 at this time.
本発明者らは、 上記の課題を解決するために鋭意研究を重ねた結果、 FLNA、 FLNB、 FLN NAV2、 BTBD2および RAB3IL1が、 TACT427の細胞内領域と結合するこ とを酵母ツーハイブリッド法 (Trends in Genet. 10巻、 286頁- 292頁、 1994年； Annu. Rev. Genet. 31巻、 663頁- 704頁、 1997年； The Yeas t Two- Hybrid Sys tem, Oxford Univers i ty Press, Bartel and Fields, 1997年） を用いて見出し、 さら に、 TACT427依存性抗アポトーシスシグナルを調節する活性の評価方法を見出し、 これらの知見に基づき検討を重ね、 本発明を完成するに至った。  The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, it has been confirmed that the binding of FLNA, FLNB, FLN NAV2, BTBD2 and RAB3IL1 to the intracellular region of TACT427 by the yeast two hybrid method (Trends In Genet. 10, 286-292, 1994; Annu. Rev. Genet. 31, 663-704, 1997; The Yeast Two-Hybrid System, Oxford University Press, Bartel and Fields, 1997), and further, a method for evaluating the activity of regulating a TACT427-dependent anti-apoptotic signal was found. Based on these findings, studies were repeated, and the present invention was completed.
すなわち、 本発明は、  That is, the present invention
〔1〕 （a) 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に同 一のアミノ酸配列を含有するタンパク質もしくはその部分べプチドまたはその塩、 および （b) 配列番号： 4 6で表されるアミノ酸配列と同一もしくは実質的に同 一のアミノ酸配列を含有するタンパク質もしくはその部分べプチドまたはその塩、 配列番号： 4 7で表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸 配列を含有するタンパク質もしくはその部分ペプチドまたはその塩、 配列番号： 4 8で表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含有 するタンパク質もしくはその部分ペプチドまたはその塩、 配列番号： 4 9で表さ れるァミノ酸配列と同一もしくは実質的に同一のァミノ酸配列を含有する夕ンパ ク質もしくはその部分ペプチドまたはその塩、 配列番号： 5 0で表されるァミノ 酸配列と同一もしくは実質的に同一のァミノ酸配列を含有するタンパク質もしく はその部分ペプチドまたはその塩、 ならびに配列番号： 5 1で表されるアミノ酸 配列と同一もしくは実質的に同一のァミノ酸配列を含有する夕ンパク質もしくは その部分ペプチドまたはその塩から選ばれる一または二種以上を含有する複合体、 〔1 a〕 (a) 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に 同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはその 塩、 および （b) 配列番号： 4 6で表されるアミノ酸配列と同一もしくは実質的 に同一のァミノ酸配列を含有する夕ンパク質もしくはその部分べプチドまたはそ の塩を含有する上記 〔1〕 記載の複合体、 [1] (a) identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 45 A protein containing one amino acid sequence or a partial peptide thereof or a salt thereof; and (b) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 46, or a portion thereof A peptide or a salt thereof, a protein or a partial peptide thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 47, or a salt thereof; an amino acid sequence represented by SEQ ID NO: 48 A protein containing the same or substantially the same amino acid sequence as the above, or a partial peptide thereof or a salt thereof; a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49; Protein or a partial peptide thereof or a salt thereof, the same as the amino acid sequence represented by SEQ ID NO: 50 Or a protein containing a substantially identical amino acid sequence or a partial peptide thereof or a salt thereof, and containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 51 A complex containing one or more selected from protein or a partial peptide thereof or a salt thereof; [1a] (a) identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 45; A protein containing an amino acid sequence or a partial peptide thereof, or a salt thereof; and (b) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 46, or a partial protein thereof. The complex according to the above (1), which comprises a peptide or a salt thereof,
〔l b〕 (a) 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に 同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはその 塩、 および （b) 配列番号： 4 7で表されるアミノ酸配列と同一もしくは実質的 に同一のアミノ酸配列を含有する夕ンパク質もしくはその部分べプチドまたはそ の塩を含有する上記 〔1〕 記載の複合体、  [Lb] (a) a protein or a partial peptide thereof or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, and (b) a protein represented by SEQ ID NO: 47 The conjugate according to the above (1), which comprises a protein containing the same or substantially the same amino acid sequence as the amino acid sequence to be obtained, or a partial peptide or a salt thereof;
〔l c〕 （a) 配列番号： 4 5で表されるアミノ酸配列 同一もしくは実質的に 同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはその 塩、 および （b) 配列番号： 4 8で表されるアミノ酸配列と同一もしくは実質的 に同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはそ の塩を含有する上記 〔1〕 記載の複合体、  [Lc] (a) a protein having the same or substantially the same amino acid sequence as SEQ ID NO: 45 or a partial peptide thereof or a salt thereof, and (b) a protein represented by SEQ ID NO: 48 The conjugate according to the above (1), which comprises a protein containing the same or substantially the same amino acid sequence as the amino acid sequence, or a partial peptide thereof, or a salt thereof
〔l d〕 (a) 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に 同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはその 塩、 および （b) 配列番号： 4 9で表されるアミノ酸配列と同一もしくは実質的 に同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはそ の塩を含有する上記 〔1〕 記載の複合体、 [Ld] (a) identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 45 A protein containing the same amino acid sequence or a partial peptide thereof, or a salt thereof; and (b) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49, or a partial peptide thereof, or The complex according to the above (1), which comprises a salt thereof,
〔l e〕 (a) 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に 同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはその 塩、 および （b) 配列番号： 5 0で表されるアミノ酸配列と同一もしくは実質的 に同一のァミノ酸配列を含有するタンパク質もしくはその部分べプチドまたはそ の塩を含有する上記 〔1〕 記載の複合体、  [Le] (a) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45 or a partial peptide thereof or a salt thereof; and (b) a protein represented by SEQ ID NO: 50 The conjugate according to the above-mentioned (1), which comprises a protein containing the same or substantially the same amino acid sequence as the amino acid sequence to be obtained or a partial peptide or a salt thereof;
〔1 f ] (a) 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に 同一のアミノ酸配列を含有するタンパク質もしくはその部分べプチドまたはその 塩、 および （b) 配列番号： 5 1で表されるアミノ酸配列と同一もしくは実質的 に同一のァミノ酸配列を含有するタンパク質もしくはその部分べプチドまたはそ の塩を含有する上記 〔1〕 記載の複合体、  [1 f] (a) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, or a partial peptide thereof or a salt thereof; and (b) SEQ ID NO: 51 The conjugate according to the above-mentioned (1), which comprises a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by or a partial peptide thereof or a salt thereof,
〔2〕 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に同一の アミノ酸配列を含有するタンパク質が、 配列番号： 1、 配列番号： 4、 配列番 号： 1 0、 配列番号： 1 5、 配列番号： 1 7、 配列番号： 2 0、 配列番号： 2 2 . 配列番号： 2 5または配列番号： 2 7で表されるアミノ酸配列からなるタンパク 質である上記 〔1〕 記載の複合体、  [2] a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22. The protein according to the above [1], which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 27 Complex,
〔2 a〕 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に同一 のアミノ酸配列を含有するタンパク質が、 配列番号： 1 5、 配列番号： 1 7、 配 列番号： 2 0、 配列番号： 2 2、 配列番号： 2 5または配列番号： 2 7で表され るアミノ酸配列からなるタンパク質である上記 〔1〕 記載の複合体、  [2a] A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45 is represented by SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, The complex according to the above [1], which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 25 or SEQ ID NO: 27,
〔3〕 (a) 配列番号： 1 5、 配列番号： 1 7、 配列番号： 2 0、 配列番号： 2 2、 配列番号： 2 5または配列番号： 2 7で表されるアミノ酸配列と同一もしく は実質的に同一のアミノ酸配列を含有するタンパク質、 および （b) 配列番号： 4 6、 配列番号： 4 7、 配列番号： 4 8、 配列番号： 4 9、 配列番号： 5 0また は配列番号： 5 1で表されるアミノ酸配列と同一もしくは実質的に同一のァミノ 酸配列を含有するタンパク質もしくはその部分ペプチドまたはその塩を含有する 上記 〔1〕 記載の複合体、 [3] (a) SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25 or amino acid sequence represented by SEQ ID NO: 27 Or (b) SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or sequence No. 51 Contains a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 1, or a partial peptide thereof or a salt thereof The composite according to the above (1),
〔4〕 上記 〔1〕 記載の複合体の形成を阻害する活性を有する、 配列番号： 4 5で表されるァミノ酸配列と同一もしくは実質的に同一のァミノ酸配列を含有す るタンパク質もしくはその部分ペプチドまたはその塩に対する抗体、  [4] a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, which has the activity of inhibiting the formation of the complex according to [1], or a protein thereof; An antibody against a partial peptide or a salt thereof,
〔5〕 上記 〔1〕 記載の複合体の形成を促進する活性を有する、 配列番号： 4 5で表されるァミノ酸配列と同一もしくは実質的に同一のァミノ酸配列を含有す るタンパク質もしくはその部分ペプチドまたはその塩に対する抗体、  [5] a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, which has the activity of promoting the formation of the complex according to [1], or a protein thereof; An antibody against a partial peptide or a salt thereof,
〔6〕 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に同一の アミノ酸配列を含有するタンパク質が、 配列番号： 1、 配列番号： 4、 配列番 号： 1 0、 配列番号： 1 5、 配列番号： 1 7、 配列番号： 2 0、 配列番号： 2 2 . 配列番号： 2 5または配列番号： 2 7で表されるアミノ酸配列からなるタンパク 質である上記 〔4〕 または 〔5〕 記載の抗体、  [6] A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45 is represented by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22. The above-mentioned [4] or [4] which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 27 5) the antibody described above;
〔6 a〕 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に同一 のアミノ酸配列を含有するタンパク質が、 配列番号： 1 5、 配列番号： 1 7、 配 列番号： 2 0、 配列番号： 2 2、 配列番号： 2 5または配列番号： 2 7で表され るアミノ酸配列からなるタンパク質である上記 〔4〕 または 〔5〕 記載の抗体、 [6a] A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45 is obtained as follows: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, The antibody according to the above [4] or [5], which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 25 or SEQ ID NO: 27,
〔7〕 上記 〔4〕 記載の抗体を含有してなる医薬、 (7) a medicine comprising the antibody according to (4),
'〔8〕 上記 〔5〕 記載の抗体を含有してなる医薬、  '(8) a medicine comprising the antibody of the above (5),
〔9〕 癌細胞のアポトーシス促進剤または癌の予防 ·治療剤である上記 〔7〕 記載の医薬、  (9) the medicament according to the above (7), which is an agent for promoting apoptosis of cancer cells or an agent for preventing or treating cancer;
〔1 0〕 神経細胞のアポトーシス阻害剤または神経変性疾患の予防 ·治療剤で ある上記 〔8〕 記載の医薬、  (10) the medicament according to the above (8), which is a neuronal apoptosis inhibitor or a preventive / therapeutic agent for a neurodegenerative disease;
〔1 1〕 上記 〔4〕 または 〔5〕 記載の抗体を含有してなる診断薬、  (11) a diagnostic agent comprising the antibody of (4) or (5) above,
〔1 2〕 癌または神経変性疾患の診断薬である上記 〔1 1〕 記載の診断薬、 〔1 3〕 (a) 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に 同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはその ' 塩、 および （b) 配列番号： 4 6、 配列番号： 4 7、 配列番号： 4 8、 配列番 号： 4 9、 配列番号： 5 0または配列番号： 5 1で表されるアミノ酸配列と同一 もしくは実質的に同一のアミノ酸配列を含有するタンパク質もしくはその部分べ プチドまたはその塩を用いることを特徴とする、 上記 〔1〕 記載の複合体の形成 を阻害または促進する化合物またはその塩のスクリーニング方法、 [12] the diagnostic agent of the above-mentioned [11], which is a diagnostic agent for cancer or a neurodegenerative disease; [13] (a) identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 45 A protein containing an amino acid sequence or a partial peptide thereof or a salt thereof, and (b) SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 Protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 51 or a portion thereof. A method for screening a compound or a salt thereof that inhibits or promotes the formation of the complex according to the above (1), which comprises using a peptide or a salt thereof,
〔1 4〕 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に同一 のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはその塩を 産生する能力を有する細胞を用いることを特徴とする上記 〔1 3〕 記載のスクリ 一二ング方法、  [14] using a cell capable of producing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, a partial peptide thereof, or a salt thereof The screening method according to the above (13),
〔1 5〕 配列番号： 4 6、 配列番号： 4 7、 配列番号： 4 8、 配列番号： 4 9 . 配列番号： 5 0または配列番号： 5 1で表されるアミノ酸配列と同一もしくは実 質的に同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまた はその塩を産生する能力を有する細胞を用いることを特徴とする上記 〔1 3〕 記 載のスクリーニング方法、  [15] SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49. SEQ ID NO: 50 or identical or real amino acid sequence to SEQ ID NO: 51 (13) The screening method according to the above (13), which comprises using a cell having the ability to produce a protein containing the same amino acid sequence or its partial peptide or a salt thereof.
〔1 5 a〕 配列番号： 4 6で表されるアミノ酸配列と同一もしくは実質的に同 一のアミノ酸配列を含有するタンパク質の部分ペプチドが、 配列番号： 4 6で表 されるアミノ酸配列の第 2 1 6 2番目〜第 2 4 1 2番目、 第 2 2 0 6番目〜第 2 3 5 5番目または第 2 1 4 1番目〜第 2 3 5 1番目のアミノ酸配列を有する部分 ペプチドである上記 〔1 3〕 記載のスクリーニング方法、  [15a] A partial peptide of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 46 is the second peptide of the amino acid sequence represented by SEQ ID NO: 46. The partial peptide having the amino acid sequence of the 16th to the 24th second, the 220th to the 23rd fifth or the 21st to the 23rd first amino acid 1 3) described screening method,
〔1 5 b〕 配列番号： 4 7で表されるアミノ酸配列と同一もしくは実質的に同一 のアミノ酸配列を含有するタンパク質の部分ペプチドが、 配列番号： 4 7で表さ れるアミノ酸配列の第 1 9 6 0番目〜第 2 2 9 9番目、 第 2 1 5 6番目〜第 2 3 1 4番目または第 2 1 7 4番目〜第 2 5 6 5番目のアミノ酸配列を有する部分べ プチドである上記 〔1 3〕 記載のスクリーニング方法、  [15b] A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 47 is the first peptide of the amino acid sequence represented by SEQ ID NO: 47 A partial peptide having an amino acid sequence from the 60th to the 229th, the 215th to the 231st or the 217th to the 256th amino acid 1 3) described screening method,
〔1 5 c〕 配列番号： 4 8で表されるアミノ酸配列と同一もしくは実質的に同 一のアミノ酸配列を含有するタンパク質の部分ペプチドが、 配列番号： 4 8で表 されるアミノ酸配列の第 2 2 9 5番目〜第 2 7 0 5番目のアミノ酸配列を有す ¾ 部分ペプチドである上記 〔1 3〕 記載のスクリーニング方法、  [15c] A partial peptide of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 48 is the second peptide of the amino acid sequence represented by SEQ ID NO: 48 The screening method according to the above (13), which is a partial peptide having an amino acid sequence of the 295th to the 275th amino acid,
〔1 5 d〕 配列番号： 4 9で表されるアミノ酸配列と同一もしくは実質的に同 一のアミノ酸配列を含有するタンパク質の部分ペプチドが、 配列番号： 4 9で表 されるアミノ酸配列の第 6 8番目〜第 2 8 6番目のアミノ酸配列を有する部分べ プチドである上記 〔1 3〕 記載のスクリーニング方法、 〔1 5 e〕 配列番号： 5 0で表されるアミノ酸配列と同一もしくは実質的に同 一のアミノ酸配列を含有するタンパク質の部分ペプチドが、 配列番号： 5 0で表 されるアミノ酸配列の第 2 0 1番目〜第 5 2 5番目または第 2 1 5番目〜第 5 0 6番目のアミノ酸配列を有する部分ペプチドである上記 〔1 3〕 記載のスクリー ニング方法、 [15d] A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49, is the sixth peptide of the amino acid sequence represented by SEQ ID NO: 49. The screening method according to the above (13), which is a partial peptide having an amino acid sequence of the 8th to 28th amino acids. [15e] A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 50 is the second peptide of the amino acid sequence represented by SEQ ID NO: 50. 0 The screening method according to the above-mentioned (13), which is a partial peptide having an amino acid sequence of the 1st to 525th or the 215th to 506th amino acid sequence,
〔1 5 f ) 配列番号： 5 1で表されるアミノ酸配列と同一もしくは実質的に同 一のアミノ酸配列を含有するタンパク質の部分ペプチドが、 配列番号： 5 1で表 されるアミノ酸配列の第 2 8 2番目〜第 3 5 6番目のアミノ酸配列を有する部分 ペプチドである上記 〔1 3〕 記載のスクリーニング方法、  [15 f) A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 51 is the second peptide of the amino acid sequence represented by SEQ ID NO: 51. The screening method according to the above (13), which is a partial peptide having the 8th to 36th amino acid sequence.
〔1 5 g〕 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に同 一のアミノ酸配列を含有するタンパク質の部分ペプチドが、 配列番号： 4 5で表 されるアミノ酸配列からなるペプチドである上記 〔1 3〕 記載のスクリーニング 方法、  [15 g] a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, a peptide comprising the amino acid sequence represented by SEQ ID NO: 45 The screening method according to the above (13), wherein
〔1 6〕 (a) 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に 同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはその 塩、 および （b) 配列番号： 4 6、 配列番号： 4 7、 配列番号： 4 8、 配列番 号： 4 9、 配列番号： 5 0または配列番号： 5 1で表されるアミノ酸配列と同一 もしくは実質的に同一のァミノ酸配列を含有するタンパク質もしくはその部分べ プチドまたはその塩を含有することを特徴とする、 上記 〔1〕 記載の複合体の形 成を阻害または促進する化合物またはその塩のスクリーニング用キット、 [16] (a) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45 or a partial peptide thereof or a salt thereof, and (b) SEQ ID NO: 46; Contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 A kit for screening a compound or a salt thereof that inhibits or promotes the formation of the complex according to the above [1], which comprises a protein or a partial peptide thereof or a salt thereof.
〔1 6 a ] 上記 〔1 3〕 記載のスクリーニング方法または上記 〔1 6〕 記載の スクリーニング用キットを用いて得られる上記 〔1〕 記載の複合体の形成を阻害 する化合物またはその塩、 [16a] a compound or a salt thereof that inhibits the formation of the complex according to [1], obtained by using the screening method according to [13] or the screening kit according to [16],
〔1 6 b〕 上記 〔1 3〕 記載のスクリーニング方法または上記 〔1 6〕 記載の スクリーニング用キットを用いて得られる上記 〔1〕 記載の複合体の形成を促進 する化合物またはその塩、  (16b) a compound or a salt thereof that promotes the formation of the complex according to (1), which is obtained by using the screening method according to (13) or the screening kit according to (16),
〔1 6 c〕 上記 〔1 6 a〕 記載の化合物またはその塩を含有してなる医薬、 〔1 6 d〕 上記 〔1 6 b〕 記載の化合物またはその塩を含有してなる医薬、 〔1 6 e〕 癌細胞のアポトーシス促進剤または癌の予防 ·治療剤である上記 〔1 6 c〕 記載の医薬、 [16c] a drug comprising the compound of the above [16a] or a salt thereof, [16 d] a pharmaceutical containing the compound of the above [16b] or a salt thereof, [1 6 e] The above agent which is a cancer cell apoptosis promoter or cancer prevention and treatment agent (16c) the medicament according to the above,
〔 1 6 f〕 神経細胞のアポトーシス阻害剤または神経変性疾患の予防 ·治療剤 である上記 〔1 6 d〕 記載の医薬、  (16f) the medicament according to the above (16d), which is a neuronal apoptosis inhibitor or a prophylactic / therapeutic agent for a neurodegenerative disease.
〔1 7〕 上記 〔1〕 記載の複合体の形成を阻害する化合物またはその塩を含有 してなる癌細胞のアポトーシス促進剤または癌の予防 ·治療剤、  [17] an agent for promoting apoptosis of cancer cells or an agent for preventing or treating cancer, comprising a compound or a salt thereof that inhibits the formation of the complex according to [1];
〔1 7 a〕 上記 〔1〕 記載の複合体の形成を阻害する化合物またはその塩、 (17a) a compound or a salt thereof that inhibits the formation of the complex according to (1),
〔1 8〕 上記 〔1〕 記載の複合体の形成を促進する化合物またはその塩を含有 してなる神経細胞のアポトーシス阻害剤または神経変性疾患の予防 ·治療剤、[18] a neuronal apoptosis inhibitor or a prophylactic or therapeutic agent for a neurodegenerative disease, comprising the compound or a salt thereof that promotes the formation of the complex according to the above [1];
〔1 8 a〕 上記 〔1〕 記載の複合体の形成を促進する化合物またはその塩、 〔1 9〕 配列番号： 4 6、 配列番号： 4 7、 配列番号： 4 8、 配列番号： 4 9 . 配列番号： 5 0または配列番号： 5 1で表されるアミノ酸配列と同一もしくは実 質的に同一のァミノ酸配列を含有するタンパク質もしくはその部分ペプチドまた はその塩を用いることを特徴とする、 癌または神経変性疾患の予防 ·治療作用を 有する化合物またはその塩のスクリーニング方法、 [18a] a compound or a salt thereof that promotes the formation of the complex according to the above [1], [19] SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 Characterized by using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 50 or SEQ ID NO: 51, a partial peptide thereof, or a salt thereof, A method for screening a compound or a salt thereof having a prophylactic or therapeutic effect on cancer or neurodegenerative disease
〔2 0〕 上記 〔1〕 記載の複合体の形成を阻害することを特徴とする癌細胞の アポトーシス促進法または癌の予防 ·治療法、  [20] a method for promoting apoptosis of cancer cells or a method for preventing or treating cancer, which comprises inhibiting the formation of the complex according to [1].
〔2 1〕 上記 〔1〕 記載の複合体の形成を促進することを特徴とする神経細胞 のアポトーシス阻害法または神経変性疾患の予防 ·治療法、  [21] a method for inhibiting apoptosis of neurons or preventing and treating neurodegenerative diseases, which comprises promoting the formation of the complex according to [1] above;
〔2 2〕 上記 〔4〕 記載の抗体の有効量を、 哺乳動物に対して投与することを 特徴とする癌細胞のアポトーシス促進法または癌の予防 ·治療法、  [22] a method for promoting apoptosis of cancer cells or a method for preventing or treating cancer, which comprises administering to a mammal an effective amount of the antibody according to [4].
〔2 3〕 上記 〔5〕 記載の抗体の有効量を、 哺乳動物に対して投与することを 特徴とする神経細胞のアポトーシス阻害法または神経変性疾患の予防 ·治療法、 (23) a method for inhibiting apoptosis of neurons or preventing and treating neurodegenerative diseases, comprising administering to a mammal an effective amount of the antibody according to (5).
〔2 4〕 癌細胞のアポトーシス促進剤または癌の予防 ·治療剤の製造のための 上記 〔4〕 記載の抗体の使用、 (24) use of the antibody according to (4) for the production of a cancer cell apoptosis promoter or a cancer prevention / treatment agent;
〔2 5〕 神経細胞のアポトーシス阻害剤または神経変性疾患の予防 ·治療剤の 製造のための上記 〔5〕 記載の抗体の使用、  (25) use of the antibody of (5) above for the manufacture of a neuronal apoptosis inhibitor or a prophylactic / therapeutic agent for a neurodegenerative disease,
〔2 6〕 請求項 1記載の複合体の癌または神経変性疾患の診断マーカーとして の使用などを提供する。 図面の簡単な説明 [26] A use of the complex according to claim 1 as a diagnostic marker for cancer or a neurodegenerative disease. Brief Description of Drawings
図 1は、 TACT427- Aの疎水性プロットを表す図である。  FIG. 1 is a diagram showing a hydrophobicity plot of TACT427-A.
図 2は、 TACT427- A2の疎水性プロットを表す図である。  FIG. 2 is a diagram showing a hydrophobicity plot of TACT427-A2.
図 3は、 TACT427- Bの疎水性プロットを表す図である。  FIG. 3 is a diagram showing a hydrophobicity plot of TACT427-B.
図 4は、 TACT427- 2Bの疎水性プロットを表す図である。  FIG. 4 is a diagram showing a hydrophobicity plot of TACT427-2B.
図 5は、 TACT427- Cの疎水性プロットを表す図である。  FIG. 5 is a diagram showing a hydrophobicity plot of TACT427-C.
図 6は、 TACT427- C2の疎水性プロットを表す図である。 発明を実施するための最良の形態  FIG. 6 is a diagram showing a hydrophobicity plot of TACT427-C2. BEST MODE FOR CARRYING OUT THE INVENTION
本明細書では、 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的 に同一のアミノ酸配列を含有するタンパク質を、 TACT427タンパク質と称する。 配列番号： 4 6で表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸 配列を含有するタンパク質を、 FLNAタンパク質と称する。 配列番号： 4 7で表さ れるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含有するタンパ ク質を、 FLNBタンパク質と称する。 配列番号： 4 8で表されるアミノ酸配列と同 一もしくは実質的に同一のアミノ酸配列を含有するタンパク質を、 FLNCタンパク 質と称する。 配列番号： 4 9で表されるアミノ酸配列と同一もしくは実質的に同 一のアミノ酸配列を含有するタンパク質を、 NAV2タンパク質と称する。 配列番 号： 5 0で表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を 含有するタンパク質を、 BTBD2タンパク質と称する。 配列番号： 5 1で表される ァミノ酸配列と同一もしくは実質的に同一のァミノ酸配列を含有するタンパク質 を、 RAB3IL1タンパク質と称する。  In the present specification, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45 is referred to as TACT427 protein. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 46 is referred to as FLNA protein. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 47 is referred to as FLNB protein. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 48 is referred to as FLNC protein. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49 is referred to as NAV2 protein. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 50 is referred to as BTBD2 protein. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 51 is referred to as RAB3IL1 protein.
TACT427タンパク質、 FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質および RAB3IL1タンパク質を、 まとめて、 本発 明のタンパク質または本発明で用いられるタンパク質と、 略称することもある。 本発明のタンパク質は、 ヒトゃ温血動物 （例えば、 モルモット、 ラット、 マウ ス、 ニヮトリ、 ゥサギ、 ブ夕、 ヒッジ、 ゥシ、 サルなど） の細胞 （例えば、 肝細 胞、 脾細胞、 神経細胞、 グリア細胞、 塍臓 jS細胞、 骨髄細胞、 メサンギゥム細胞、 ランゲルハンス細胞、 表皮細胞、 上皮細胞、 杯細胞、 内皮細胞、 平滑筋細胞、 線 維芽細胞、 繊維細胞、 筋細胞、 脂肪細胞、 免疫細胞 （例、 マクロファージ、 τ細 胞、 B細胞、 ナチュラルキラー細胞、 肥満細胞、 好中球、 好塩基球、 好酸球、 単 球） 、 赤血球、 巨核球、 滑膜細胞、 軟骨細胞、 骨細胞、 骨芽細胞、 破骨細胞、 乳 腺細胞、 肝細胞もしくは間質細胞、 またはこれら細胞の前駆細胞、 幹細胞もしく は癌細胞など） もしくはそれらの細胞が存在するあらゆる組織、 例えば、 脳、 脳 の各部位 （例、 嗅球、 扁桃核、 大脳基底球、 海馬、 視床、 視床下部、 大脳皮質、 延髄、 小脳） 、 脊髄、 下垂体、 胃、 塍臓、 腎臓、 肝臓、 生殖腺、 甲状腺、 胆のう、 骨髄、 副腎、 皮膚、 筋肉、 肺、 消化管 （例、 大腸、 小腸） 、 血管、 心臓、 胸腺、 脾臓、 顎下腺、 末梢血、 前立腺、 睾丸、 卵巣、 胎盤、 子宮、 骨、 関節、 骨格筋、 または血球系の細胞もしくはその培養細胞 （例、 MEL, Ml, CTLL-2, HT-2, WEHI- 3, HL-60, JOSK-1, K562, ML-1, MOLT- 3, MOLT- 4， MOLT- 10, CCRF-CEM, TALL- 1, Jurkat, CCRT-HSB-2, KE-37, SKW-3, 匿- 78, HUT- 102， H9, U937, THP-1, HEL, JK-1, CMK, KO-812, MEG- 01など） などに由来するタンパク質であってもよく、 合成タンパク質であってもよい。 The TACT427 protein, FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein and RAB3IL1 protein may be collectively referred to as the protein of the present invention or the protein used in the present invention. The protein of the present invention can be obtained from cells of human warm-blooded animals (eg, guinea pig, rat, mouse, chicken, rabbit, egret, bush, sheep, horsetail, monkey, etc.) (eg, liver cells, spleen cells, nerve cells) , Glial cells, kidney jS cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, lines Fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, τ cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), Red blood cells, megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, breast cells, hepatocytes or stromal cells, or precursors, stem cells or cancer cells of these cells) or All tissues where cells exist, such as the brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, Kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, Testes, ovaries Placental, uterine, bone, joint, skeletal muscle, or cells of the blood cell system or cultured cells thereof (eg, MEL, Ml, CTLL-2, HT-2, WEHI-3, HL-60, JOSK-1, K562, ML -1, MOLT- 3, MOLT-4, MOLT-10, CCRF-CEM, TALL-1, Jurkat, CCRT-HSB-2, KE-37, SKW-3, concealed-78, HUT-102, H9, U937 , THP-1, HEL, JK-1, CMK, KO-812, MEG-01, etc.) or a synthetic protein.
本明細書におけるタンパク質は、 ペプチド標記の慣例に従って左端が N末端 (ァミノ末端) 、 右端が C末端 (カルボキシル末端) である。 本発明のタンパク 質は、 C末端がカルボキシル基 (-C00H) 、 カルボキシレート（-COO—) 、 アミド (-C0NH2) またはエステル （- C00R) の何れであってもよい。 In the protein in the present specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling. Proteins of the present invention, C-terminal, carboxyl group (-C00H), carboxylate (-COO-), amide (-C0NH 2) or ester - may be either (C00R).
ここでエステルにおける Rとしては、 例えば、 メチル、 ェチル、 n—プロピル、 イソプロピル、 n—プチルなどの C HJアルキル基、 例えば、 シクロペンチル、 シ ク口へキシルなどの C 3.8シクロアルキル基、 例えば、 フエニル、 α—ナフチルな どの C 6_12ァリール基、 例えば、 ベンジル、 フエネチルなどのフエニル—C Hァ ルキル基もしくは α—ナフチルメチルなどのひ一ナフチルー C卜2アルキル基など の C7_14ァラルキル基、 ピバロィルォキシメチル基などが用いられる。 Here, as R in the ester, e.g., methyl, Echiru, n- propyl, isopropyl, C HJ alkyl group such as n- heptyl, for example, cyclopentyl, C 3. 8 cycloalkyl groups such as cyclohexyl shea click port, e.g. , phenyl, alpha-naphthyl of which C 6 _ 12 Ariru group, e.g., benzyl, C 7 _ 14 such as flying one Nafuchiru C Bok 2 alkyl groups, such as phenyl -CH § alkyl group or alpha-naphthylmethyl, such as phenethyl Ararukiru And a pivaloyloxymethyl group.
本発明のタンパク質が C末端以外にカルボキシル基 （またはカルポキシレー ト) を有している場合、 カルボキシル基がアミド化またはエステル化されている ものも、 本発明のタンパク質に含まれる。 この場合のエステルとしては、 例えば 上記した C末端のエステルなどが用いられる。  When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the protein of the present invention includes those in which the carboxyl group is amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
さらに、 本発明のタンパク質には、 N末端のアミノ酸残基 （例、 メチォニン残 基） のァミノ基が保護基 （例えば、 ホルミル基、 ァセチル基などの C HJアルカノ ィルなどの C j-sァシル基など） で保護されているもの、 生体内で切断されて生成 する N末端のグルタミン残基がピログルタミン酸化したもの、 分子内のアミノ酸 の側鎖上の置換基 （例えば一〇H、 — S H、 アミノ基、 イミダゾ一ル基、 インド ール基、 グァニジノ基など） が適当な保護基 （例えば、 ホルミル基、 ァセチル基 などの C Η;アルカノィル基などの C HJァシル基など） で保護されているもの、 あ るいは糖鎖が結合したいわゆる糖夕ンパク質などの複合夕ンパク質なども含まれ る。 Furthermore, the protein of the present invention has an N-terminal amino acid residue (eg, a methionine residue). Is protected by a protecting group (for example, a CJsacyl group such as a CHJ alkanol such as a formyl group or an acetyl group), and an N-terminal glutamine formed by cleavage in vivo. Appropriate protection of the residue in which the residue is pyroglutamine-oxidized, or a substituent on the side chain of the amino acid in the molecule (eg, 1H, —SH, amino, imidazole, indole, guanidino, etc.) Protected by a group (for example, CΗ such as formyl group or acetyl group; CHJ acetyl group such as alkanoyl group) or complex protein such as so-called sugar protein to which a sugar chain is bonded. And so on.
以下のアミノ酸配列の相同性は、 相同性計算アルゴリズム NCBI BLAST  The homology of the following amino acid sequences is calculated using the homology calculation algorithm NCBI BLAST.
(Nat ional Center for Biotechnology Informat ion Bas ic Local Al ignment Search Tool) を用い、 以下の条件 （期待値 = 10；ギャップを許す；マトリクス =BL0SUM62；フィルタリング =0FF) にて計算することができる。  It can be calculated using the following conditions (expected value = 10; gap allowed; matrix = BL0SUM62; filtering = 0FF) using the (National Center for Biotechnology Information Basic Basic Alignment Search Tool).
TACT427タンパク質としては、 例えば、 配列番号： 1、 配列番号： 4、 配列番 号： 1 0、 配列番号： 1 5、 配列番号： 1 7、 配列番号： 2 0、 配列番号： 2 2、 配列番号： 2 5または配列番号： 2 7で表されるアミノ酸配列と同一もしくは実 質的に同一のアミノ酸配列を含有するタンパク質などが用いられる。 さらには、 配列番号： 7で表されるァミノ酸配列と同一もしくは実質的に同一のアミノ酸配 列を含有するタンパク質なども、 TACT427タンパク質に含まれる。  Examples of the TACT427 protein include: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: : 25 or a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 is used. Furthermore, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 7 is also included in the TACT427 protein.
配列番号： 1で表されるアミノ酸配列と実質的に同一のアミノ酸配列としては、 配列番号： 1で表されるアミノ酸配列と約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性を有するァ ミノ酸配列などが挙げられる。  As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, about 70% or more, preferably about 80% or more, particularly preferably about 70% or more of the amino acid sequence represented by SEQ ID NO: 1 Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, may be mentioned.
配列番号： 1で表されるァミノ酸配列と実質的に同一のァミノ酸配列を含有す るタンパク質としては、 例えば、 前記の配列番号： 1で表されるアミノ酸配列と 実質的に同一のアミノ酸配列を含有し、 配列番号： 1で表されるアミノ酸配列を 含有するタンパク質と実質的に同質の活性を有するタンパク質などが好ましい。 配列番号： 4で表されるァミノ酸配列と実質的に同一のァミノ酸配列としては、 配列番号： 4で表されるアミノ酸配列と約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性を有するァ ミノ酸配列などが挙げられる。 例えば、 配列番号： 4で表されるアミノ酸配列の 4 7番目から 2 9 6番目のアミノ酸配列に対して約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性 を有するアミノ酸配列なども挙げられる。 Examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by the aforementioned SEQ ID NO: 1 And a protein having substantially the same activity as a protein containing the amino acid sequence represented by SEQ ID NO: 1. The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4 is about 70% or more, preferably about 80% or more, particularly preferably the amino acid sequence represented by SEQ ID NO: 4. Have a homology of about 90% or more, and most preferably about 95% or more. And amino acid sequences. For example, about 70% or more, preferably about 80% or more, particularly preferably about 90% or more, with respect to the amino acid sequence of the 47th to 296th amino acids of the amino acid sequence represented by SEQ ID NO: 4. Most preferably, they include amino acid sequences having about 95% or more homology.
配列番号： 4で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含有す るタンパク質としては、 例えば、 前記の配列番号： 4で表されるアミノ酸配列と 実質的に同一のアミノ酸配列を含有し、 配列番号： 4で表されるアミノ酸配列を 含有するタンパク質と実質的に同質の活性を有するタンパク質などが好ましい。 配列番号： 1 0で表されるアミノ酸配列と実質的に同一のアミノ酸配列として は、 配列番号： 1 0で表されるアミノ酸配列と約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性 を有するアミノ酸配列などが挙げられる。  Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 4 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 4 However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 4 is preferable. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 10 is about 70% or more, preferably about 80% or more, particularly preferably the amino acid sequence represented by SEQ ID NO: 10. Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology.
配列番号： 1 0で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含有 するタンパク質としては、 例えば、 前記の配列番号： 1 0で表されるアミノ酸配 列と実質的に同一のアミノ酸配列を含有し、 配列番号： 1 0で表されるアミノ酸 配列を含有するタンパク質と実質的に同質の活性を有するタンパク質などが好ま しい。  Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 10 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 10 described above. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 10 is preferable.
配列番号： 1 5で表されるアミノ酸配列と実質的に同一のアミノ酸配列として は、 配列番号： 1 5で表されるアミノ酸配列と約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性 を有するアミノ酸配列などが挙げられる。 例えば、 配列番号： 1 5で表されるァ ミノ酸配列の 4 7番目から 2 9 6番目のアミノ酸配列に対して約 7 0 %以上、 好 ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 % 以上の相同性を有するァミノ酸配列なども挙げられる。  The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 15 includes about 70% or more, preferably about 80% or more, particularly preferably, the amino acid sequence represented by SEQ ID NO: 15 Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology. For example, about 70% or more, preferably about 80% or more, particularly preferably about 70% or more with respect to the amino acid sequence at positions 47 to 296 of the amino acid sequence represented by SEQ ID NO: 15. Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, may also be used.
配列番号： 1 5で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含有 するタンパク質としては、 例えば、 前記の配列番号： 1 5で表されるアミノ酸配 列と実質的に同一のアミノ酸配列を含有し、 配列番号： 1 5で表されるアミノ酸 配列を含有するタンパク質と実質的に同質の活性を有するタンパク質などが好ま しい。 配列番号： 1 7で表されるアミノ酸配列と実質的に同一のアミノ酸配列として は、 配列番号： 1 7で表されるアミノ酸配列と約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性 を有するアミノ酸配列などが挙げられる。 例えば、 配列番号： 1 7で表されるァ ミノ酸配列の 4 3番目から 2 9 2番目のアミノ酸配列に対して約 7 0 %以上、 好 ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 % 以上の相同性を有するァミノ酸配列なども挙げられる。 Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 15 include, for example, an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 15 And a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 15 is preferable. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 17 includes about 70% or more, preferably about 80% or more, particularly preferably the amino acid sequence represented by SEQ ID NO: 17 Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology. For example, the amino acid sequence represented by SEQ ID NO: 17 is about 70% or more, preferably about 80% or more, particularly preferably about 70% or more with respect to the 43rd to 292nd amino acid sequence. Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, may also be used.
配列番号： 1 7で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含有 するタンパク質としては、 例えば、 前記の配列番号： 1 7で表されるアミノ酸配 列と実質的に同一のアミノ酸配列を含有し、 配列番号： 1 7で表されるアミノ酸 配列を含有するタンパク質と実質的に同質の活性を有するタンパク質などが好ま しい。  Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 17 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 17 described above. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 17 is preferable.
配列番号： 2 0で表されるアミノ酸配列と実質的に同一のアミノ酸配列として は、 配列番号： 2 0で表されるアミノ酸配列と約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性 を有するアミノ酸配列などが挙げられる。 例えば、 配列番号： 2 0で表されるァ ミノ酸配列の 4 7番目から 2 9 6番目のアミノ酸配列に対して約 7 0 %以上、 好 ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 % 以上の相同性を有するァミノ酸配列なども挙げられる。  The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 20 includes about 70% or more, preferably about 80% or more, particularly preferably, the amino acid sequence represented by SEQ ID NO: 20. Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology. For example, about 70% or more, preferably about 80% or more, particularly preferably about 70% or more of the amino acid sequence from the 47th to the 296th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 20 Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, may also be used.
配列番号： 2 0で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含有 するタンパク質としては、 例えば、 前記の配列番号： 2 0で表されるアミノ酸配 列と実質的に同一のアミノ酸配列を含有し、 配列番号： 2 0で表されるアミノ酸 配列を含有するタンパク質と実質的に同質の活性を有するタンパク質などが好ま しい。  Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 20 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 20 described above. And a protein having substantially the same activity as that of the protein having the amino acid sequence represented by SEQ ID NO: 20 is preferable.
配列番号： 2 2で表されるアミノ酸配列と実質的に同一のアミノ酸配列として は、 配列番号： 2 2で表されるアミノ酸配列と約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性 を有するアミノ酸配列などが挙げられる。 例えば、 配列番号： 2 2で表されるァ ミノ酸配列の 4 3番目から 2 9 2番目のアミノ酸配列に対して約 7 0 %以上、 好 ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 % 以上の相同性を有するァミノ酸配列なども挙げられる。 The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 22 includes about 70% or more, preferably about 80% or more, particularly preferably, the amino acid sequence represented by SEQ ID NO: 22. Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology. For example, about 70% or more of the amino acid sequence represented by SEQ ID NO: 22 from amino acid sequence 43 to 292 in the amino acid sequence represented by SEQ ID NO: 22 is preferable. Preferably, an amino acid sequence having about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology is also included.
配列番号： 2 2で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含有 するタンパク質としては、 例えば、 前記の配列番号： 2 2で表されるアミノ酸配 列と実質的に同一のアミノ酸配列を含有し、 配列番号： 2 2で表されるアミノ酸 配列を含有するタンパク質と実質的に同質の活性を有するタンパク質などが好ま しい。  Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 22 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 22 described above. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 22 is preferable.
配列番号： 2 5で表されるアミノ酸配列と実質的に同一のアミノ酸配列として は、 配列番号： 2 5で表されるアミノ酸配列と約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性 を有するアミノ酸配列などが挙げられる。 例えば、 配列番号： 2 5で表されるァ ミノ酸配列の 4 7番目から 2 9 6番目のアミノ酸配列に対して約 7 0 %以上、 好 ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 % 以上の相同性を有するァミノ酸配列なども挙げられる。  As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25, about 70% or more, preferably about 80% or more, particularly preferably the amino acid sequence represented by SEQ ID NO: 25 Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology. For example, the amino acid sequence represented by SEQ ID NO: 25 is about 70% or more, preferably about 80% or more, particularly preferably about 70% or more of the amino acid sequence from the 47th to the 296th amino acid sequence. Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, may also be used.
配列番号： 2 5で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含有 するタンパク質としては、 例えば、 前記の配列番号： 2 5で表されるアミノ酸配 列と実質的に同一のアミノ酸配列を含有し、 配列番号： 2 5で表されるアミノ酸 配列を含有するタンパク質と実質的に同質の活性を有するタンパク質などが好ま しい。  Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25 described above. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 25 is preferable.
配列番号： 2 7で表されるアミノ酸配列と実質的に同一のアミノ酸配列として は、 配列番号： 2 7で表されるアミノ酸配列と約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性 を有するアミノ酸配列などが挙げられる。 例えば、 配列番号： 2 7で表されるァ ミノ酸配列の 4 3番目から 2 9 2番目のアミノ酸配列に対して約 7 0 %以上、 好 ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 % 以上の相同性を有するアミノ酸配列なども挙げられる。  The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 27 includes about 70% or more, preferably about 80% or more, particularly preferably, the amino acid sequence represented by SEQ ID NO: 27. Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology. For example, the amino acid sequence represented by SEQ ID NO: 27 is at least about 70%, preferably at least about 80%, particularly preferably at least about 80% with respect to the amino acid sequence from the 43rd to 29th amino acid sequence. Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, and the like can also be mentioned.
配列番号： 2 7で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含有 するタンパク質としては、 例えば、 前記の配列番号： 2 7で表されるアミノ酸配 列と実質的に同一のアミノ酸配列を含有し、 配列番号： 2 7で表されるアミノ酸 配列を含有するタンパク質と実質的に同質の活性を有するタンパク質などが好ま しい。 Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 27 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 27 described above. An amino acid represented by SEQ ID NO: 27 Proteins having substantially the same activity as the protein containing the sequence are preferred.
配列番号： 7で表されるアミノ酸配列と実質的に同一のアミノ酸配列としては、 配列番号： 7で表されるアミノ酸配列と約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性を有するァ ミノ酸配列などが挙げられる。 例えば、 配列番号： 7で表されるアミノ酸配列の 5 7 7番目から 5 9 4番目のアミノ酸配列に対して約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同 性を有するァミノ酸配列なども挙げられる。  As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 7, about 70% or more, preferably about 80% or more, particularly preferably about 70% or more of the amino acid sequence represented by SEQ ID NO: 7 Amino acid sequences having a homology of 90% or more, most preferably about 95% or more, may be mentioned. For example, about 70% or more, preferably about 80% or more, particularly preferably about 90% or more with respect to the amino acid sequence from 577th to 594th of the amino acid sequence represented by SEQ ID NO: 7 Also, most preferably, an amino acid sequence having about 95% or more homology is exemplified.
配列番号： 7で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含有す るタンパク質としては、 例えば、 前記の配列番号： 7で表されるアミノ酸配列と 実質的に同一のアミノ酸配列を含有し、 配列番号： 7で表されるアミノ酸配列を 含有するタンパク質と実質的に同質の活性を有するタンパク質などが好ましい。 実質的に同質の活性としては、 例えば、 クロロパ一ォキシダーゼ活性などが挙 げられる。 実質的に同質とは、 それらの性質が性質的に （例、 生理学的に、 また は薬理学的に） 同質であることを示す。 したがって、 クロ口パ一ォキシダーゼ活 性が同等 （例、 約 0 . 0 1〜1 0 0倍、 好ましくは約 0 . 1〜1 0倍、 より好ま しくは 0 . 5〜2倍） であることが好ましいが、 これらの活性の程度、 タンパク 質の分子量などの量的要素は異なっていてもよい。  Examples of the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 7 include, for example, the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 7 However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 7 is preferred. Examples of substantially the same activity include chloropoxidase activity. Substantially identical indicates that the properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, the activity of the black mouth peroxidase is equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). However, quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
クロ口パーォキシダーゼ活性の測定は、 公知の方法に準じて行えばよく、 例え ば、 ジャーナル ォブ バイオロジカル ケミストリー (J. Biol. C em. ) 241 巻、 1763〜1768頁 （1966年） などに記載の方法またはそれに準じる方法に従って 測定することができる。  The measurement of black mouth peroxidase activity may be performed according to a known method, for example, Journal of Biological Chemistry (J. Biol. Cem.) Vol.241, pp.1763-1768 (1966), etc. Can be measured according to the method described in (1) or a method analogous thereto.
また、 TACT427タンパク質としては、 例えば、 （i) 配列番号： 1、 配列番号： 4、 配列番号： 7、 配列番号： 1 0、 配列番号： 1 5、 配列番号： 1 7、 配列番 号： 2 0、 配列番号： 2 2、 配列番号： 2 5または配列番号： 2 7で表されるァ ミノ酸配列中の 1または 2個以上 （例えば 1〜1 0 0個程度、 好ましくは 1〜3 0個程度、 好ましくは 1〜1 0個程度、 さらに好ましくは数 （1〜5 ) 個） のァ ミノ酸が欠失したアミノ酸配列、 （i i) 配列番号： 1、 配列番号： 4、 配列番 号： 7、 配列番号： 10、 配列番号： 15、 配列番号： 17、 配列番号： 20、 配列番号： 22、 配列番号： 25または配列番号： 27で表されるアミノ酸配列 に 1または 2個以上 （例えば 1〜100個程度、 好ましくは 1〜30個程度、 好 ましくは 1〜10個程度、 さらに好ましくは数 （1〜5) 個） のアミノ酸が付加 したアミノ酸配列、 （iii) 配列番号： 1、 配列番号： 4、 配列番号： 7、 配列 番号： 10、 配列番号： 15、 配列番号： 17、 配列番号： 20、 配列番号： 2 2、 配列番号： 25または配列番号： 27で表されるアミノ酸配列に 1または 2 個以上 （例えば 1〜100個程度、 好ましくは 1〜30個程度、 好ましくは 1〜 10個程度、 さらに好ましくは数 （1〜5) 個） のアミノ酸が挿入されたァミノ 酸配列、 （iv) 配列番号： 1、 配列番号： 4、 配列番号： 7、 配列番号： 10、 配列番号： 15、 配列番号： 17、 配列番号： 20、 配列番号： 22、 配列番 号： 25または配列番号： 27で表されるアミノ酸配列中の 1または 2個以上Examples of the TACT427 protein include: (i) SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 2 0, SEQ ID NO: 22, SEQ ID NO: 25 or SEQ ID NO: 27, 1 or 2 or more amino acids in the amino acid sequence (for example, about 1 to 100, preferably 1 to 30) Amino acid sequence in which about 1, preferably about 1 to 10, and more preferably (1 to 5) amino acids have been deleted, (ii) SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: No .: 7, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, 1 or more in the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 27 (For example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably the number (1 to 5) of amino acid sequences; (iii) SEQ ID NO: : 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25 or SEQ ID NO: 27 One or more amino acids (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5)) Amino acid sequence, (iv) SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, sequence No.: 10, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25 or SEQ ID NO: 27 with one or two or more are in the amino acid sequence represented
(例えば 1〜100個程度、 好ましくは 1〜 30個程度、 好ましくは 1〜10個 程度、 さらに好ましくは数 （1〜5) 個） のアミノ酸が他のアミノ酸で置換され たアミノ酸配列、 または （V) それらを組み合わせたアミノ酸配列を含有する夕 ンパク質などのいわゆるムティンも含まれる。 (E.g., an amino acid sequence in which about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) amino acids have been substituted with other amino acids, or V) So-called mucins such as proteins containing amino acid sequences combining them are also included.
上記のようにアミノ酸配列が挿入、 欠失または置換されている場合、 その挿入、 欠失または置換の位置としては、 とくに限定されない。  When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.
TACT427タンパク質の具体例としては、 例えば、 配列番号： 1で表されるアミ ノ酸配列を含有するタンパク質、 配列番号： 4で表されるアミノ酸配列を含有す るタンパク質、 配列番号： 7で表されるアミノ酸配列を含有するタンパク質、 配 列番号： 10で表されるアミノ酸配列を含有するタンパク質、 配列番号： 15で 表されるアミノ酸配列を含有するタンパク質、 配列番号： 17で表されるァミノ 酸配列を含有するタンパク質、 配列番号： 20で表されるアミノ酸配列を含有す るタンパク質、 配列番号： 22で表されるアミノ酸配列を含有するタンパク質、 配列番号： 25で表されるアミノ酸配列を含有するタンパク質、 配列番号： 27 で表されるァミノ酸配列を含有する夕ンパク質などがあげられる。  Specific examples of the TACT427 protein include, for example, a protein containing an amino acid sequence represented by SEQ ID NO: 1, a protein containing an amino acid sequence represented by SEQ ID NO: 4, and a protein represented by SEQ ID NO: 7. A protein containing the amino acid sequence represented by SEQ ID NO: 10; a protein containing the amino acid sequence represented by SEQ ID NO: 15; an amino acid sequence represented by SEQ ID NO: 17 A protein comprising the amino acid sequence represented by SEQ ID NO: 20; a protein comprising the amino acid sequence represented by SEQ ID NO: 22; a protein comprising the amino acid sequence represented by SEQ ID NO: 25 And an amino acid sequence containing the amino acid sequence represented by SEQ ID NO: 27.
TACT427夕ンパク質の部分べプチドとしては、 前記した TACT427夕ンパク質の部 分ペプチドであって、 好ましくは、 前記した TACT427タンパク質と同様の性質を 有するものであればいずれのものでもよい。 The partial peptide of the TACT427 protein is the partial peptide of the TACT427 protein described above, and preferably has the same properties as the TACT427 protein described above. Any of them may be used.
例えば、 TACT427タンパク質の構成アミノ酸配列のうち少なくとも 20個以上、 好ましくは 50個以上、 さらに好ましくは 70個以上、 より好ましくは 100個 以上、 最も好ましくは 200個以上のアミノ酸配列を有するペプチドなどが用い られる。 .  For example, a peptide having at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more amino acid sequences among the constituent amino acid sequences of the TACT427 protein is used. Can be .
具体的には、 配列番号： 45で表されるアミノ酸配列を有するペプチドなどが あげられる。  Specific examples include a peptide having the amino acid sequence represented by SEQ ID NO: 45.
配列番号： 46、 配列番号： 47、 配列番号： 48、 配列番号： 49、 配列番 号： 50または配列番号： 51で表されるアミノ酸配列と実質的に同一のァミノ 酸配列としては、 配列番号： 46、 配列番号： 47、 配列番号： 48、 配列番 号： 49、 配列番号： 50または配列番号： 51で表されるアミノ酸配列と約 7 0%以上、 好ましくは約 80%以上、 特に好ましくは約 90%以上、 最も好まし くは約 95 %以上の相同性を有するアミノ酸配列などが挙げられる。  The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 includes SEQ ID NO: : 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 and about 70% or more, preferably about 80% or more, and particularly preferably about 80% or more Is an amino acid sequence having about 90% or more, most preferably about 95% or more homology.
配列番号： 46、 配列番号： 47、 配列番号： 48、 配列番号： 49、 配列番 号： 50または配列番号： 51で表されるアミノ酸配列と実質的に同一のァミノ 酸配列を含有するタンパク質としては、 例えば、 前記の配列番号： 46、 配列番 号： 47、 配列番号： 48、 配列番号： 49、 配列番号： 50または配列番号： 51で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含有し、 配列番 号： 46、 配列番号： 47、 配列番号： 48、 配列番号： 49、 配列番号： 50 または配列番号： 51で表されるアミノ酸配列を含有するタンパク質と実質的に 同質の活性を有するタンパク質などが好ましい。  As a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 Is, for example, an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or substantially the same as a protein containing the amino acid sequence represented by SEQ ID NO: 51 Proteins having activity are preferred.
実質的に同質とは、 それらの性質が性質的に （例、 生理学的に、 または薬理学 的に） 同質であることを示す。 したがって、 力 る活性が同等 （例、 約 0. 01 〜100倍、 好ましくは約 0. 1〜10倍、 より好ましくは 0. 5〜2倍） であ ることが好ましいが、 これらの活性の程度、 タンパク質の分子量などの量的要素 は異なっていてもよい。  Substantially homogenous indicates that the properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, it is preferable that the potent activities are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). Quantitative factors such as degree and molecular weight of the protein may vary.
FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2 タンパク質または RAB3IL1タンパク質としては、 それぞれ、 例えば、 （i) 配列番 号： 46、 配列番号： 47、 配列番号： 48、 配列番号： 49、 配列番号： 50 または配列番号： 51で表されるアミノ酸配列中の 1または 2個以上 （例えば 1 〜 100個程度、 好ましくは 1〜30個程度、 好ましくは 1〜 10個程度、 さら に好ましくは数 （1〜5) 個） のアミノ酸が欠失したアミノ酸配列、 （ii) 配列 番号： 46、 配列番号： 47、 配列番号： 48、 配列番号： 49、 配列番号： 5 0または配列番号： 51で表されるアミノ酸配列に 1または 2個以上 （例えば 1 〜100個程度、 好ましくは 1〜30個程度、 好ましくは 1〜10個程度、 さら に好ましくは数 （1〜5) 個） のアミノ酸が付加したアミノ酸配列、 （iii) 配 列番号： 46、 配列番号： 47、 配列番号： 48、 配列番号： 49、 配列番号： 50または配列番号： 51で表されるアミノ酸配列に 1または 2個以上 （例えば 1-100個程度、 好ましくは 1〜 30個程度、 好ましくは 1〜 10個程度、 さ らに好ましくは数 （1〜5) 個） のアミノ酸が挿入されたアミノ酸配列、 （iv) 配列番号： 46、 配列番号： 47、 配列番号： 48、 配列番号： 49、 配列番 号： 50または配列番号： 51で表されるアミノ酸配列中の 1または 2個以上 (例えば 1〜 100個程度、 好ましくは 1〜 30個程度、 好ましくは 1〜 10個 程度、 さらに好ましくは数 （1〜5) 個） のアミノ酸が他のアミノ酸で置換され たアミノ酸配列、 または （V) それらを組み合わせたアミノ酸配列を含有する夕 ンパク質などのいわゆるムテインも含まれる。 Examples of the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein include, for example, (i) SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: : 50 Or one or more in the amino acid sequence represented by SEQ ID NO: 51 (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably the number (1 to (Ii) SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 An amino acid having one or more (eg, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5)) amino acids added to the amino acid sequence Sequence, (iii) SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 51 (for example, 1 About -100 pieces, preferably about 1 to 30 pieces, preferably about 1 to 10 pieces, More preferably, an amino acid sequence having a number (1 to 5) of amino acids inserted therein, (iv) SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 Alternatively, one or more or more amino acids in the amino acid sequence represented by SEQ ID NO: 51 (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably about 1 to 5 ) Amino acids are substituted with other amino acids, or (V) so-called muteins such as proteins containing an amino acid sequence combining them.
上記のようにアミノ酸配列が挿入、 欠失または置換されている場合、 その挿入、 欠失または置換の位置としては、 とくに限定されない。  When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.
FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2 タンパク質または RAB3IL1タンパク質の具体例としては、 例えば、 配列番号： 4 6で表されるアミノ酸配列を含有するタンパク質、 配列番号： 47で表されるァ ミノ酸配列を含有するタンパク質、 配列番号： 48で表されるアミノ酸配列を含 有するタンパク質、 配列番号： 49で表されるアミノ酸配列を含有するタンパク 質、 配列番号： 50で表されるアミノ酸配列を含有するタンパク質、 配列番号： 51で表されるアミノ酸配列を含有するタンパク質などが、 それぞれ挙げられる。  Specific examples of the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 46, and a protein represented by SEQ ID NO: 47 A protein having an amino acid sequence represented by SEQ ID NO: 48; a protein having an amino acid sequence represented by SEQ ID NO: 49; a protein having an amino acid sequence represented by SEQ ID NO: 49; A protein containing the amino acid sequence represented by SEQ ID NO: 51, and the like.
FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2 タンパク質または RAB3 IL 1タンパク質の部分べプチドとしては、 前記した FLNA夕 ンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク 質または RAB3IL1タンパク質の部分ペプチドであって、 好ましくは、 前記した FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2夕 ンパク質または RAB3 IL 1夕ンパク質と同様の性質を有するものであればいずれの ものでもよい。 As partial peptides of FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3 IL1 protein, the aforementioned FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein Or a partial peptide of the RAB3IL1 protein, preferably any of the above-mentioned FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein. It may be one of.
例えば、 FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2夕ンパク質または RAB3 IL 1タンパク質の構成アミノ酸配列のうち少なくとも 2 0個以上、 好ましくは 5 0個以上、 さらに好ましくは 7 0個以上、 より好まし くは 1 0 0個以上、 最も好ましくは 2 0 0個以上のアミノ酸配列を有するぺプチ ドなどが用いられる。  For example, FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3 IL1 protein at least 20 or more, preferably 50 or more, more preferably 70 or more of the amino acid sequence, More preferably, peptides having an amino acid sequence of 100 or more, most preferably 200 or more are used.
具体例として、 FLNAタンパク質の部分ペプチドとしては、 例えば、 配列番号： As a specific example, the partial peptide of the FLNA protein includes, for example, SEQ ID NO:
4 6で表されるアミノ酸配列の第 2162番目〜第 2412番目、 第 2206番目〜第 2355番 目または第 2141番目〜第 2351番目のアミノ酸配列を有する部分ペプチドが挙げら れる。 FLNBタンパク質の部分ペプチドとしては、 例えば、 配列番号： 4 7で表さ れるアミノ酸配列の第 1960番目〜第 2299番目、 第 2156番目〜第 2314番目または第 2174番目〜第 2565番目のアミノ酸配列を有する部分ペプチドが挙げられる。 FLNC タンパク質の部分ペプチドとしては、 例えば、 配列番号： 4 8で表されるァミノ 酸配列の第 2295番目〜第 2705番目のアミノ酸配列を有する部分ペプチドが挙げら れる。 NAV2タンパク質の部分ペプチドとしては、 例えば、 配列番号： 4 9で表さ れるアミノ酸配列の第 68番目〜第 286番目のアミノ酸配列を有する部分ペプチド が挙げられる。 BTBD2タンパク質の部分ペプチドとしては、 例えば、 配列番号：And a partial peptide having the amino acid sequence at positions 2162 to 2412, 2206 to 2355, or 2141 to 2351 of the amino acid sequence represented by 46. As a partial peptide of the FLNB protein, for example, it has the amino acid sequence of the 1960th to 2299th, the 2156th to the 2314th or the 2174th to the 2565th of the amino acid sequence represented by SEQ ID NO: 47 And partial peptides. Examples of the partial peptide of the FLNC protein include a partial peptide having the amino acid sequence at positions 2295 to 2705 of the amino acid sequence represented by SEQ ID NO: 48. The partial peptide of the NAV2 protein includes, for example, a partial peptide having the 68th to 286th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 49. As a partial peptide of the BTBD2 protein, for example, SEQ ID NO:
5 0で表されるアミノ酸配列の第 201番目〜第 525番目または第 215番目〜第 506番 目のアミノ酸配列を有する部分べプチドが挙げられる。 RAB3 IL 1夕ンパク質の部 分ペプチドとしては、 例えば、 配列番号： 5 1で表されるアミノ酸配列の第 282 番目〜第 356番目のアミノ酸配列を有する部分ペプチドが挙げられる。 Partial peptides having the 201st to 525th or the 215th to 506th amino acid sequence of the amino acid sequence represented by 50 are exemplified. Examples of the partial peptide of RAB3 IL-1 protein include a partial peptide having the 282nd to 356th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 51.
また、 本発明のタンパク質の部分ペプチド （本発明で用いられる部分べプチ ド） は、 そのアミノ酸配列中の 1または 2個以上 （好ましくは、 1〜1 0個程度、 さらに好ましくは数 （1〜5 ) 個） のアミノ酸が欠失し、 または、 そのアミノ酸 配列に 1または 2個以上 （好ましくは 1〜2 0個程度、 より好ましくは 1〜1 0 個程度、 さらに好ましくは数 （1〜5 ) 個） のアミノ酸が付加し、 または、 その アミノ酸配列に 1または 2個以上 （好ましくは 1〜2 0個程度、 より好ましくは 1〜1 0個程度、 さらに好ましくは数 （1〜5 ) 個） のアミノ酸が挿入され、 ま たは、 そのアミノ酸配列中の 1または 2個以上 （好ましくは 1〜1 0個程度、 よ り好ましくは数個、 さらに好ましくは 1〜5個程度） のアミノ酸が他のアミノ酸 で置換されていてもよい。 The partial peptide of the protein of the present invention (partial peptide used in the present invention) has one or more (preferably about 1 to 10, more preferably 1 to 10) amino acids in the amino acid sequence. 5) amino acids are deleted, or one or more (preferably about 1-20, more preferably about 1-10, and more preferably a number (1-5 ) Amino acids are added or One or two or more amino acids (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably a number (1 to 5)) are inserted into the amino acid sequence. One or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the amino acid sequence may be substituted with another amino acid.
また、 本発明で用いられる部分ペプチドは C末端が力ルポキシル基 （-C00H) 、 カルボキシレート （-C00— ) 、 アミド （-C0NH2) またはエステル （- C00R) の何れ であってもよい。 The partial peptide used in the present invention, the C-terminus force Rupokishiru group (-C00H), carboxylate (-C00-), amide (-C0NH 2) or ester - may be either (C00R).
さらに、 本発明で用いられる部分ペプチドには、 前記した、 本発明のタンパク 質と同様に、 C末端以外に力ルポキシル基 （またはカルポキシレート） を有して いるもの、 N末端のアミノ酸残基 （例、 メチォニン残基） のァミノ基が保護基で 保護されているもの、 N端側が生体内で切断され生成したグルタミン残基がピロ グルタミン酸化したもの、 分子内のアミノ酸の側鎖上の置換基が適当な保護基で 保護されているもの、 あるいは糖鎖が結合したいわゆる糖ペプチドなどの複合べ プチドなども含まれる。  Further, the partial peptide used in the present invention includes, as in the above-mentioned protein of the present invention, those having a carbonyl group (or carboxylate) other than the C-terminal, and the N-terminal amino acid residue (Eg, methionine residue) whose amino group is protected with a protecting group, N-terminal cleavage in vivo, glutamine residue generated by pyroglutamine oxidation, substitution of amino acid in the molecule on the side chain Also included are those in which the group is protected by an appropriate protecting group, and those in which a sugar chain is bonded, such as a complex peptide such as a so-called glycopeptide.
本発明で用いられる部分べプチドは抗体作成のための抗原としても用いること ができる。  The partial peptide used in the present invention can also be used as an antigen for producing an antibody.
本発明のタンパク質またはその部分ペプチドの塩としては、 生理学的に許容さ れる酸 （例、 無機酸、 有機酸） や塩基 （例、 アルカリ金属塩） などとの塩が用い られ、 とりわけ生理学的に許容される酸付加塩が好ましい。 この様な塩としては、 例えば、 無機酸 （例えば、 塩酸、 リン酸、 臭化水素酸、 硫酸） との塩、 あるいは 有機酸 （例えば、 酢酸、 ギ酸、 プロピオン酸、 フマル酸、 マレイン酸、 コハク酸、 酒石酸、 クェン酸、 リンゴ酸、 蓚酸、 安息香酸、 メタンスルホン酸、 ベンゼンス ルホン酸） との塩などが用いられる。  As the salt of the protein of the present invention or its partial peptide, a salt with a physiologically acceptable acid (eg, an inorganic acid, an organic acid) or a base (eg, an alkali metal salt) is used. Acceptable acid addition salts are preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
本発明のタンパク質もしくはその部分ペプチドまたはその塩は、 、 後述の参考 例に従って、 または前述したヒトゃ温血動物の細胞または組織から自体公知の夕 ンパク質の精製方法によって製造することもできるし、 タンパク質をコードする D N Aを含有する形質転換体を培養することによつても製造することができる。 また、 後述のぺプチド合成法に準じて製造することもできる。 ヒトゃ哺乳動物の組織または細胞から製造する場合、 ヒ卜や哺乳動物の組織ま たは細胞をホモジナイズした後、 酸などで抽出を行ない、 該抽出液を逆相クロマ 卜グラフィ一、 イオン交換ク口マトグラフィ一などのクロマトグラフィ一を組み 合わせることにより精製単離することができる。 The protein of the present invention or a partial peptide thereof or a salt thereof can be produced according to the below-mentioned Reference Examples, or from the above-described method for purifying a protein known per se from human or warm-blooded animal cells or tissues, It can also be produced by culturing a transformant containing DNA encoding the protein. It can also be produced according to the peptide synthesis method described below. In the case of production from human or mammalian tissues or cells, human or mammalian tissues or cells are homogenized, and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography, or the like. Purification and isolation can be achieved by combining chromatography such as mouth chromatography.
本発明のタンパク質もしくは部分ペプチドまたはその塩、 またはそのアミド体 の合成には、 通常市販のタンパク質合成用樹脂を用いることができる。 そのよう な樹脂としては、 例えば、 クロロメチル樹脂、 ヒドロキシメチル樹脂、 ベンズヒ ドリルァミン樹脂、 アミノメチル樹脂、 4—ベンジルォキシベンジルアルコール 樹脂、 4一メチルベンズヒドリルァミン樹脂、 P AM樹脂、 4—ヒドロキシメチ ルメチルフエニルァセトアミドメチル樹脂、 ポリアクリルアミド樹脂、 4 _ For the synthesis of the protein or partial peptide of the present invention or a salt thereof, or an amide thereof, a commercially available resin for protein synthesis can be usually used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxy resin. Methyl methylphenylacetamidomethyl resin, polyacrylamide resin, 4 _
( 2 ' , 4， —ジメトキシフエ二ル一ヒドロキシメチル) フエノキシ樹脂、 4 - ( 2， ， 4 ' ージメトキシフエ二ルー Fm o cアミノエチル） フエノキシ樹脂な どを挙げることができる。 このような樹脂を用い、 ひーァミノ基と側鎖官能基を 適当に保護したアミノ酸を、 目的とするタンパク質の配列通りに、 自体公知の各 種縮合方法に従い、 樹脂上で縮合させる。 反応の最後に樹脂からタンパク質また は部分ペプチドを切り出すと同時に各種保護基を除去し、 さらに高希釈溶液中で 分子内ジスルフィド結合形成反応を実施し、 目的のタンパク質もしくは部分ぺプ チドまたはそれらのアミド体を取得する。 (2 ', 4, -dimethoxyphenyl monohydroxymethyl) phenoxy resin, 4- (2,, 4' dimethoxyphenyl Fmoc aminoethyl) phenoxy resin, and the like. Using such a resin, amino acids having appropriately protected amino groups and side chain functional groups are condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, a protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or an amide thereof Get the body.
上記した保護アミノ酸の縮合に関しては、 タンパク質合成に使用できる各種活 性化試薬を用いることができるが、 特に、 カルポジイミド類がよい。 カルポジィ ミド類としては、 D C C、 N, N ' —ジイソプロピルカルポジイミド、 N—ェチ ルー N， ― ( 3—ジメチルァミノプロリル) カルポジイミドなどが用いられる。 これらによる活性化にはラセミ化抑制添加剤 （例えば、 HO B t , HO O B t ) とともに保護アミノ酸を直接樹脂に添加するかまたは、 対称酸無水物または H O B tエステルあるいは HO O B tエステルとしてあらかじめ保護アミノ酸の活性 化を行なった後に樹脂に添加することができる。  Regarding the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carpoimides, DCC, N, N'-diisopropylcarpoimide, N-ethyl N,-(3-dimethylaminoprolyl) carpoimide, and the like are used. For these activations, the protected amino acid may be added directly to the resin with a racemization inhibitor additive (eg, HOBt, HOOBt) or may be pre-protected as a symmetric anhydride or HOBtester or HOOBtester. The amino acid can be added to the resin after activation.
保護アミノ酸の活性化や樹脂との縮合に用いられる溶媒としては、 タンパク質 縮合反応に使用しうることが知られている溶媒から適宜選択されうる。 例えば、 N, N—ジメチルホルムアミド， N, N—ジメチルァセトアミド, N—メチルピ 口リドンなどの酸アミド類、 塩化メチレン， クロ口ホルムなどのハロゲン化炭化 水素類、 トリフルォロエタノールなどのアルコール類、 ジメチルスルホキシドな どのスルホキシド類、 ピリジン， ジォキサン， テトラヒドロフランなどのエーテ ル類、 ァセトニトリル， プロピオ二トリルなどの二トリル類、 酢酸メチル， 酢酸 ェチルなどのエステル類あるいはこれらの適宜の混合物などが用いられる。 反応 温度はタンパク質結合形成反応に使用され得ることが知られている範囲から適宜 選択され、 通常約— 2 0 ° (：〜 5 0 °Cの範囲から適宜選択される。 活性化されたァ ミノ酸誘導体は通常 1 . 5〜 4倍過剰で用いられる。 ニンヒドリン反応を用いた テス卜の結果、 縮合が不十分な場合には保護基の脱離を行なうことなく縮合反応 を繰り返すことにより十分な縮合を行なうことができる。 反応を繰り返しても十 分な縮合が得られないときには、 無水酢酸またはァセチルイミダゾールを用いて 未反応アミノ酸をァセチル化することによって、 後の反応に影響を与えないよう にすることができる。 The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpi Acid amides such as mouth ridone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; sulfoxides such as dimethyl sulfoxide; ethers such as pyridine, dioxane and tetrahydrofuran; and acetonitrile And nitriles such as propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof. The reaction temperature is appropriately selected from the range that can be used for the protein bond formation reaction, and is usually appropriately selected from the range of about −20 ° C. (: to 50 ° C.) Activated amino The acid derivative is usually used in an excess of 1.5 to 4 times.As a result of the test using the ninhydrin reaction, if the condensation is insufficient, it is sufficient to repeat the condensation reaction without removing the protecting group. If sufficient condensation cannot be obtained even after repeating the reaction, the unreacted amino acid is acetylated with acetic anhydride or acetylimidazole so that the subsequent reaction is not affected. Can be
原料のァミノ基の保護基としては、 例えば、 Z、 B o c、 t—ペンチルォキシ カルボニル、 イソポルニルォキシカルボニル、 4ーメトキシベンジルォキシカル ポニル、 C 1 一 Z、 B r— Z、 ァダマンチルォキシカルボニル、 トリフルォロア セチル、 フタロイル、 ホルミル、 2—二トロフエニルスルフエ二ル、 ジフエニル ホスフイノチオイル、 Fm o cなどが用いられる。  Examples of the protecting group for the starting amino group include Z, Boc, t-pentyloxycarbonyl, isopolnyoxycarbonyl, 4-methoxybenzyloxycarponyl, C1-Z, Br-Z, and adaman. Tyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
力ルポキシル基は、 例えば、 アルキルエステル化 (例えば、 メチル、 ェチル、 プロピル、 プチル、 t—ブチル、 シクロペンチル、 シクロへキシル、 シクロヘプ チル、 シクロォクチル、 2—ァダマンチルなどの直鎖状、 分枝状もしくは環状ァ ルキルエステル化) 、 ァラルキルエステル化 （例えば、 ベンジルエステル、 4一 ニトロべンジルエステル、 4ーメトキシベンジルエステル、 4一クロ口べンジル エステル、 ベンズヒドリルエステル化) 、 フエナシルエステル化、 ベンジルォキ シカルポニルヒドラジド化、 t一ブトキシカルポニルヒドラジド化、 トリチルヒ ドラジド化などによつて保護することができる。  The lipoxyl group can be, for example, a linear, branched or cyclic alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Alkyl esterification), aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxy It can be protected by carbonylation, tert-butoxycarbonylhydrazide, tritylhydrazide and the like.
セリンの水酸基は、 例えば、 エステル化またはェ一テル化によって保護するこ とができる。 このエステル化に適する基としては、 例えば、 ァセチル基などの低 級 （C ^ 6) アルカノィル基、 ベンゾィル基などのァロイル基、 ベンジルォキシ カルポニル基、 エトキシカルボニル基などの炭酸から誘導される基などが用いら れる。 また、 エーテル化に適する基としては、 例えば、 ベンジル基、 テトラヒド ロビラニル基、 t一ブチル基などである。 The hydroxyl group of serine can be protected, for example, by esterification or etherification. Suitable groups for this esterification include, for example, lower (C ^ 6 ) alkanol groups such as acetyl group, aroyl groups such as benzoyl group, and benzyloxy group. Groups derived from carbonic acid such as a carbonyl group and an ethoxycarbonyl group are used. Examples of a group suitable for etherification include a benzyl group, a tetrahydrobiranyl group, and a t-butyl group.
チロシンのフエノール性水酸基の保護基としては、 例えば、 Bz l、 C 12- B z l、 2—二トロベンジル、 B r— Z、 t一ブチルなどが用いられる。 The protecting group of the phenolic hydroxyl group of tyrosine, for example, Bz l, C 1 2 - B zl, 2- two Torobenjiru, B r- Z, such as t one-butyl is used.
ヒスチジンのイミダゾールの保護基としては、 例えば、 To s、 4ーメトキシ -2, 3， 6—トリメチルベンゼンスルホニル、 DNP、 ベンジルォキシメチル、 Bum、 Bo c、 Tr t、 Fmo cなどが用いられる。  As a protecting group for imidazole of histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
原料の力ルポキシル基の活性化されたものとしては、 例えば、 対応する酸無水 物、 アジド、 活性エステル 〔アルコール （例えば、 ペンタクロロフエノール、 2， 4， 5_トリクロ口フエノール、 2, 4ージニトロフエノール、 シァノメチルァ ルコール、 パラニトロフエノール、 HONB、 N—ヒドロキシスクシミド、 N— ヒドロキシフタルイミド、 HOB t) とのエステル〕 などが用いられる。 原料の ァミノ基の活性化されたものとしては、 例えば、 対応するリン酸アミドが用いら れる。  Examples of activated raw propyloxyl groups include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5_trichloromouth phenol, 2,4-dinitrophenol) Phenol, cyanomethyl alcohol, para-nitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.
保護基の除去 （脱離） 方法としては、 例えば、 Pd—黒あるいは Pd—炭素な どの触媒の存在下での水素気流中での接触還元や、 また、 無水フッ化水素、 メタ ンスルホン酸、 トリフルォロメタンスルホン酸、 トリフルォロ酢酸あるいはこれ らの混合液などによる酸処理や、 ジイソプロピルェチルァミン、 トリェチルアミ ン、 ピぺリジン、 ピぺラジンなどによる塩基処理、 また液体アンモニア中ナトリ ゥムによる還元なども用いられる。 上記酸処理による脱離反応は、 一般に約一 2 0°C〜40°Cの温度で行なわれるが、 酸処理においては、 例えば、 ァニソ一ル、 フエノール、 チオアニソ一ル、 メタクレゾ一ル、 パラクレゾール、 ジメチルスル フイド、 1, 4—ブタンジチオール、 1， 2一エタンジチオールなどのような力 チオン捕捉剤の添加が有効である。 また、 ヒスチジンのイミダゾ一ル保護基とし て用いられる 2, 4—ジニトロフエニル基はチォフエノール処理により除去され、 トリブトファンのインドール保護基として用いられるホルミル基は上記の 1, 2 —エタンジチオール、 1, 4一ブタンジチオールなどの存在下の酸処理による脱 保護以外に、 希水酸化ナトリウム溶液、 希アンモニアなどによるアルカリ処理に よっても除去される。 Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or trifluoromethane. Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, etc. Is also used. The elimination reaction by the above-mentioned acid treatment is generally performed at a temperature of about 120 ° C to 40 ° C. It is effective to add a force-thione scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol and the like. In addition, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributofan is substituted with 1,2-ethanedithiol, 1,4-dithiol. In addition to deprotection by acid treatment in the presence of butanedithiol, etc., alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc. Therefore, it is also removed.
原料の反応に関与すべきでない官能基の保護ならびに保護基、 およびその保護 基の脱離、 反応に関与する官能基の活性化などは公知の基または公知の手段から 適宜選択しうる。  The protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
タンパク質または部分ペプチドのアミド体を得る別の方法としては、 例えば、 まず、 カルポキシ末端アミノ酸の —力ルポキシル基をアミド化して保護した後、 アミノ基側にペプチド （タンパク質） 鎖を所望の鎖長まで延ばした後、 該ぺプチ ド鎖の N末端のひ—ァミノ基の保護基のみを除いたタンパク質または部分べプチ ドと C末端のカルボキシル基の保護基のみを除去したタンパク質または部分ぺプ チドとを製造し、 これらのタンパク質またはペプチドを上記したような混合溶媒 中で縮合させる。 縮合反応の詳細については上記と同様である。 縮合により得ら れた保護タンパク質またはペプチドを精製した後、 上記方法によりすベての保護 基を除去し、 所望の粗タンパク質またはペプチドを得ることができる。 この粗夕 ンパク質またはペプチドは既知の各種精製手段を駆使して精製し、 主要画分を凍 結乾燥することで所望のタンパク質またはペプチドのアミド体を得ることができ る。  Another method for obtaining an amide form of a protein or partial peptide is, for example, first protecting the carboxy-terminal amino acid with amidation of the amino group, and then adding a peptide (protein) chain on the amino group side to the desired length. After the elongation, a protein or partial peptide from which only the protecting group for the N-terminal amino group of the peptide chain has been removed and a protein or partial peptide from which only the protecting group for the C-terminal carboxyl group has been removed. Is produced and these proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein or peptide. The crude protein or peptide can be purified using various known purification means, and the main fraction can be freeze-dried to obtain the desired protein or peptide amide.
タンパク質またはペプチドのエステル体を得るには、 例えば、 カルポキシ末端 アミノ酸の《—力ルポキシル基を所望のアルコール類と縮合しアミノ酸エステル とした後、 タンパク質またはペプチドのアミド体と同様にして、 所望のタンパク 質またはペプチドのエステル体を得ることができる。  In order to obtain an ester of a protein or peptide, for example, after condensing the carboxy-terminal amino acid with a desired alcohol to form an amino acid ester, the desired protein is synthesized in the same manner as in the amide of a protein or peptide. An ester of a protein or a peptide can be obtained.
本発明で用いられる部分ペプチドまたはそれらの塩は、 自体公知のペプチドの 合成法に従って、 あるいは本発明のタンパク質を適当なぺプチダーゼで切断する ことによって製造することができる。 ペプチドの合成法としては、 例えば、 固相 合成法、 液相合成法のいずれによっても良い。 すなわち、 本発明で用いられる部 分べプチドを構成し得る部分ペプチドもしくはアミノ酸と残余部分とを縮合させ、 生成物が保護基を有する場合は保護基を脱離することにより目的のペプチドを製 造することができる。 公知の縮合方法や保護基の脱離としては、 例えば、 以下の (Ϊ) 〜 （V) に記載された方法が挙げられる。  The partial peptide or a salt thereof used in the present invention can be produced according to a peptide synthesis method known per se or by cleaving the protein of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the target peptide. can do. Examples of the known condensation method and elimination of the protecting group include the methods described in the following (Ϊ) to (V).
(i) M. Bodanszkyおよび M. A. Ondet t i、 ペプチド ·シンセシス（Pept ide Synthes is) , Interscience Publ ishers, New York (1966年) (i) M. Bodanszky and MA Ondet ti, Peptide synthesis Synthes is), Interscience Publ ishers, New York (1966)
(i i) Schroederおよび Luebkeゝ ザ.ペプチド（The Pept ide) , Academic Press, New York (1965年）  (ii) Schroeder and Luebke ゝ The Peptide, Academic Press, New York (1965)
(i i i) 泉屋信夫他、 ペプチド合成の基礎と実験、 丸善 (株） （1975年）  (iii) Nobuo Izumiya et al., Peptide Synthesis Basics and Experiments, Maruzen Co., Ltd. (1975)
(iv) 矢島治明 および榊原俊平、 生化学実験講座 1、 タンパク質の化学 IV、 205、 （1977年）  (iv) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977)
(V) 矢島治明監修、 続医薬品の開発、 第 14巻、 ペプチド合成、 広川書店 また、 反応後は通常の精製法、 例えば、 溶媒抽出 ·蒸留 ·カラムクロマトダラ フィー ·液体クロマトグラフィー ·再結晶などを組み合わせて本発明で用いられ る部分ペプチドを精製単離することができる。 上記方法で得られる部分ペプチド が遊離体である場合は、 公知の方法あるいはそれに準じる方法によって適当な塩 に変換することができるし、 逆に塩で得られた場合は、 公知の方法あるいはそれ に準じる方法によって遊離体または他の塩に変換することができる。  (V) Supervised by Haruaki Yajima, Development of Continuing Pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten Also, after the reaction, conventional purification methods, such as solvent extraction, distillation, column chromatography, liquid chromatography, liquid chromatography, and recrystallization The partial peptides used in the present invention can be purified and isolated by combining these methods. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide is obtained by a salt, a known method or It can be converted into a free form or another salt by an analogous method.
本発明のタンパク質をコ一ドするポリヌクレオチドとしては、 前述した本発明 のタンパク質をコードする塩基配列を含有するものであればいかなるものであつ てもよい。 好ましくは D NAである。 D NAとしては、 ゲノム D NA、 ゲノム D NAライブラリー、 前記した細胞 ·組織由来の c D NA、 前記した細胞 ·組織由 来の c D NAライブラリー、 合成 D NAのいずれでもよい。  The polynucleotide encoding the protein of the present invention may be any polynucleotide as long as it contains the above-described nucleotide sequence encoding the protein of the present invention. Preferably it is DNA. The DNA may be any one of a genomic DNA, a genomic DNA library, the above-described cell and tissue-derived cDNA, the above-described cell and tissue-derived cDNA library, and a synthetic DNA.
ライブラリーに使用するベクターは、 バクテリオファージ、 プラスミド、 コス ミド、 ファージミドなどいずれであってもよい。 また、 前記した細胞，組織より total R N Aまたは mR N A画分を調製したものを用いて直接 Reverse  The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, using a preparation of a total RNA or mRNA fraction from the cells and tissues described above, direct reverse
Transcriptase Polymerase Chain React ion (以下、 R T— P C R法と略称す る） によって増幅することもできる。 It can also be amplified by transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method).
TACT427タンパク質をコードする D NAとしては、 例えば、  Examples of DNA encoding the TACT427 protein include:
(i) 配列番号： 2で表される塩基配列を含有する D NA、 または配列番号： 2 で表される塩基配列とハイストリンジェントな条件下でハイプリダイズする塩基 配列を含有し、 配列番号： 1で表されるアミノ酸配列を含有するタンパク質と実 質的に同質の性質を有するタンパク質をコードする D NA、  (i) a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions; A DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by 1,
(i i) 配列番号： 5で表される塩基配列を含有する D NA、 または配列番号： 5 で表される塩基配列とハイストリンジェントな条件下でハイブリダィズする塩基 配列を含有し、 配列番号： 4で表されるアミノ酸配列を含有するタンパク質と実 質的に同質の性質を有するタンパク質をコードする D NA、 (ii) DNA containing the nucleotide sequence represented by SEQ ID NO: 5, or SEQ ID NO: 5 Encodes a protein having a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 4, and having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 4. DNA,
(i i i) 配列番号： 8で表される塩基配列を含有する D NA、 または配列番号： 8で表される塩基配列とハイストリンジェン卜な条件下でハイプリダイズする塩 基配列を含有し、 配列番号： 7で表されるアミノ酸配列を含有するタンパク質と 実質的に同質の性質を有するタンパク質をコードする DNA、  (iii) a sequence containing a DNA containing the base sequence represented by SEQ ID NO: 8 or a base sequence that hybridizes with the base sequence represented by SEQ ID NO: 8 under high stringent conditions; DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by No. 7;
(iv) 配列番号： 1 1で表される塩基配列を含有する D NA、 または配列番号： 1 1で表される塩基配列とハイストリンジェントな条件下でハイプリダイズする 塩基配列を含有し、 配列番号： 1 0で表されるアミノ酸配列を含有するタンパク 質と実質的に同質の性質を有するタンパク質をコードする D NA、  (iv) a DNA containing the nucleotide sequence represented by SEQ ID NO: 11 or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 11 under high stringency conditions; No .: DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by 10,
(v) 配列番号： i 6で表される塩基配列を含有する D NA、 または配列番号： (v) SEQ ID NO: DNA containing the nucleotide sequence represented by i6, or SEQ ID NO:
1 6で表される塩基配列とハイストリンジェントな条件下でハイブリダィズする 塩基配列を含有し、 配列番号： 1 5で表されるアミノ酸配列を含有するタンパク 質と実質的に同質の性質を有するタンパク質をコードする D NA、 A protein comprising a nucleotide sequence which hybridizes under high stringent conditions to the nucleotide sequence represented by 16 and having substantially the same properties as the protein comprising the amino acid sequence represented by SEQ ID NO: 15 D NA, which codes
(vi) 配列番号： 1 8で表される塩基配列を含有する D NA、 または配列番号： 1 8で表される塩基配列とハイストリンジェントな条件下でハイプリダイズする 塩基配列を含有し、 配列番号： 1 7で表されるアミノ酸配列を含有するタンパク 質と実質的に同質の性質を有するタンパク質をコードする D NA、  (vi) a DNA containing the nucleotide sequence represented by SEQ ID NO: 18 or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 18 under high stringent conditions; DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by No. 17;
(vi i) 配列番号： 2 1で表される塩基配列を含有する D NA、 または配列番 号： 2 1で表される塩基配列とハイストリンジェン卜な条件下でハイブリダィズ する塩基配列を含有し、 配列番号： 2 0で表されるアミノ酸配列を含有するタン パク質と実質的に同質の性質を有するタンパク質をコードする D NA、  (vii) DNA containing the nucleotide sequence represented by SEQ ID NO: 21 or a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 21 A DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 20;
(vi i i) 配列番号： 2 3で表される塩基配列を含有する D NA、 または配列番 号： 2 3で表される塩基配列とハイストリンジェン卜な条件下でハイブリダィズ する塩基配列を含有し、 配列番号： 2 2で表されるアミノ酸配列を含有するタン パク質と実質的に同質の性質を有するタンパク質をコードする D NA、  (vi ii) DNA containing the nucleotide sequence represented by SEQ ID NO: 23 or a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 23 A DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 22;
(ix) 配列番号： 2 6で表される塩基配列を含有する D NA、 または配列番号： 2 6で表される塩基配列とハイストリンジェントな条件下でハイプリダイズする 塩基配列を含有し、 配列番号： 25で表されるアミノ酸配列を含有するタンパク 質と実質的に同質の性質を有するタンパク質をコードする DNA、 (ix) DNA containing the nucleotide sequence represented by SEQ ID NO: 26, or hybridizing with the nucleotide sequence represented by SEQ ID NO: 26 under high stringent conditions DNA encoding a protein having a base sequence and having substantially the same properties as a protein having an amino acid sequence represented by SEQ ID NO: 25;
(x) 配列番号： 28で表される塩基配列を含有する DNA、 または配列番号： 28で表される塩基配列とハイストリンジェントな条件下でハイプリダイズする 塩基配列を含有し、 配列番号： 27で表されるアミノ酸配列を含有するタンパク 質と実質的に同質の性質を有するタンパク質をコードする DN Aであれば何れの ものでもよい。  (x) a DNA containing the nucleotide sequence represented by SEQ ID NO: 28, or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 28 under high stringent conditions, SEQ ID NO: 27 Any DNA may be used as long as it encodes a protein having substantially the same properties as the protein having the amino acid sequence represented by
配列番号： 2で表される塩基配列とハイストリンジェントな条件下でハイプリ ダイズできる DNAとしては、 例えば、 配列番号： 2で表される塩基配列と約 7 0%以上、 好ましくは約 80%以上、 特に好ましくは約 90%以上、 最も好まし くは約 95 %以上の相同性を有する塩基配列を含有する D N Aなどが用いられる, 配列番号： 5で表される塩基配列とハイストリンジェン卜な条件下でハイプリ ダイズできる DNAとしては、 例えば、 配列番号： 5で表される塩基配列と約 7 0%以上、 好ましくは約 80%以上、 特に好ましくは約 90%以上、 最も好まし くは約 95%以上の相同性を有する塩基配列を含有する DNAなどが用いられる, 例えば、 配列番号： 5で表される塩基配列の 139番目から 888番目の塩基配 列に対して約 70 %以上、 好ましくは約 80 %以上、 特に好ましくは約 90 %以 上、 最も好ましくは約 95%以上の相同性を有する塩基配列を含有する DN Aな ども用いられる。  Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 under high stringency conditions include, for example, about 70% or more, preferably about 80% or more of the nucleotide sequence represented by SEQ ID NO: 2. Particularly preferably, DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used. It is highly stringent with the nucleotide sequence represented by SEQ ID NO: 5. Examples of the DNA that can hybridize under the conditions include, for example, about 70% or more, preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 90% or more of the nucleotide sequence represented by SEQ ID NO: 5. DNA containing a nucleotide sequence having a homology of 95% or more is used, for example, about 70% or more, preferably, about 139 to 888 of the nucleotide sequence represented by SEQ ID NO: 5. Is about 80% or more, particularly preferred Is used, such as DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more.
配列番号： 8で表される塩基配列とハイストリンジェントな条件下でハイプリ ダイズできる DNAとしては、 例えば、 配列番号： 8で表される塩基配列と約 7 0%以上、 好ましくは約 80%以上、 特に好ましくは約 90%以上、 最も好まし くは約 95 %以上の相同性を有する塩基配列を含有する D N Aなどが用いられる t 例えば、 配列番号： 8で表される塩基配列の 1728番目から 1782番目の塩 基配列に対して約 70%以上、 好ましくは約 80%以上、 特に好ましくは約 9 0%以上、 最も好ましくは約 95%以上の相同性を有する塩基配列を含有する D NAなども用いられる。 Examples of a DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 8 under high stringency conditions include, for example, about 70% or more, preferably about 80% or more of the nucleotide sequence represented by SEQ ID NO: 8 , particularly preferably about 90%, most preferably rather t is a DNA containing the base sequence having about 95% homology, for example, is used, SEQ ID NO: from 1728 th nucleotide sequence represented by 8 About 70% or more, preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more of a base sequence having a homology of about 1782 with the nucleotide sequence, etc. Is also used.
配列番号： 1 1で表される塩基配列とハイストリンジェントな条件下でハイブ リダィズできる DNAとしては、 例えば、 配列番号： 11で表される塩基配列と 約 70%以上、 好ましくは約 80%以上、 特に好ましくは約 90%以上、 最も好 ましくは約 95 %以上の相同性を有する塩基配列を含有する DN Aなどが用いら れる。 Examples of DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 11 under high stringency conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 11 A DNA containing a nucleotide sequence having a homology of about 70% or more, preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more is used.
配列番号： 16で表される塩基配列とハイストリンジェントな条件下でハイブ リダィズできる DNAとしては、 例えば、 配列番号： 16で表される塩基配列と 約 70%以上、 好ましくは約 80%以上、 特に好ましくは約 90%以上、 最も好 ましくは約 95 %以上の相同性を有する塩基配列を含有する D N Aなどが用いら れる。  Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 16 under high stringency conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 16 and about 70% or more, preferably about 80% or more. Particularly preferably, a DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used.
配列番号： 18で表される塩基配列とハイストリンジェントな条件下でハイブ リダィズできる DNAとしては、 例えば、 配列番号： 18で表される塩基配列と 約 70%以上、 好ましくは約 80%以上、 特に好ましくは約 90%以上、 最も好 ましくは約 95 %以上の相同性を有する塩基配列を含有する DNAなどが用いら れる。  Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 18 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 18 and about 70% or more, preferably about 80% or more. Particularly preferably, a DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used.
配列番号： 21で表される塩基配列とハイストリンジェントな条件下でハイブ リダィズできる DNAとしては、 例えば、 配列番号： 21で表される塩基配列と 約 70%以上、 好ましくは約 80%以上、 特に好ましくは約 90%以上、 最も好 ましくは約 95 %以上の相同性を有する塩基配列を含有する D N Aなどが用いら れる。  Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 21 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 21 and about 70% or more, preferably about 80% or more. Particularly preferably, a DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used.
配列番号： 23で表される塩基配列とハイストリンジェン卜な条件下でハイブ リダィズできる DNAとしては、 例えば、 配列番号： 23で表される塩基配列と 約 70%以上、 好ましくは約 80%以上、 特に好ましくは約 90%以上、 最も好 ましくは約 95 %以上の相同性を有する塩基配列を含有する DN Aなどが用いら れる。  Examples of the DNA which can hybridize with the nucleotide sequence represented by SEQ ID NO: 23 under high stringent conditions include, for example, about 70% or more, preferably about 80% or more of the nucleotide sequence represented by SEQ ID NO: 23 Particularly preferably, a DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used.
配列番号： 26で表される塩基配列とハイストリンジェン卜な条件下でハイブ リダィズできる DNAとしては、 例えば、 配列番号： 26で表される塩基配列と 約 70%以上、 好ましくは約 80%以上、 特に好ましくは約 90%以上、 最も好 ましくは約 95%以上の相同性'を有する塩基配列を含有する DN Aなどが用いら れる。  Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 26 under high stringent conditions include, for example, about 70% or more, preferably about 80% or more of the nucleotide sequence represented by SEQ ID NO: 26 Particularly preferably, a DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is used.
配列番号： 28で表される塩基配列とハイストリンジェントな条件下でハイブ リダィズできる DNAとしては、 例えば、 配列番号： 28で表される塩基配列と 約 70%以上、 好ましくは約 80%以上、 特に好ましくは約 90 %以上、 最も好 ましくは約 95 %以上の相同性を有する塩基配列を含有する D N Aなどが用いら れる。 Hive under high stringent conditions with the base sequence represented by SEQ ID NO: 28 Examples of the DNA that can be resized include a homology of about 70% or more, preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more with the nucleotide sequence represented by SEQ ID NO: 28. For example, DNA containing a base sequence having a property is used.
塩基配列の相同性は、 相同性計算アルゴリズム NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) を用レ , 以下の条件 （期待値 =10；ギャップを許す；フィルタリング =0N；マッチスコア =1；ミスマッチスコア =-3) にて計算することができる。  The homology of the nucleotide sequences was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gap allowed; filtering = 0N; match score = 1). The mismatch score = -3).
ハイブリダィゼーシヨンは、 自体公知の方法あるいはそれに準じる方法、 例え ば、 Molecular Cloning 2nd (J. Sambrook et al. , Cold Spring Harbor Lab. Press, 1989) に記載の方法などに従って行なうことができる。 また、 市販のラ ィブラリ一を使用する場合、 添付の使用説明書に記載の方法に従つて行なうこと ができる。 より好ましくは、 ハイストリンジェントな条件に従って行なうことが できる。  Hybridization can be carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be performed under high stringency conditions.
ハイストリンジェントな条件とは、 例えば、 ナトリウム濃度が約 19〜40m High stringency conditions are, for example, when the sodium concentration is about 19 to 40 m
M、 好ましくは約 19〜 20 mMで、 温度が約 50〜 70 ° (、 好ましくは約 60M, preferably about 19-20 mM and a temperature of about 50-70 ° (preferably about 60
〜651：の条件を示す。 特に、 ナトリウム濃度が約 19 mMで温度が約 65 の 場合が最も好ましい。 ~ 651: Indicates the condition. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.
より具体的には、 （i) 配列番号： 1で表されるアミノ酸配列を含有するタン パク質をコードする DNAとしては、 配列番号： 2で表される塩基配列を含有す る DNAまたは配列番号： 3で表される塩基配列を含有する DNAなどが、 (ii) 配列番号： 4で表されるアミノ酸配列を含有するタンパク質をコードする More specifically, (i) the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 includes a DNA containing the base sequence represented by SEQ ID NO: 2 or a DNA containing the amino acid sequence represented by SEQ ID NO: 2 And (ii) a protein containing the amino acid sequence represented by SEQ ID NO: 4.
DNAとしては、 配列番号： 5で表される塩基配列を含有する DNAまたは配列 番号： 6で表される塩基配列を含有する DNAなどが、 Examples of the DNA include a DNA containing the nucleotide sequence represented by SEQ ID NO: 5 or a DNA containing the nucleotide sequence represented by SEQ ID NO: 6;
(iii) 配列番号： 7で表されるアミノ酸配列を含有するタンパク質をコードす る DNAとしては、 配列番号： 8で表される塩基配列を含有する DNAまたは配 列番号： 9で表される塩基配列を含有する DNAなどが、  (iii) The DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 7 is a DNA containing the base sequence represented by SEQ ID NO: 8 or the base represented by SEQ ID NO: 9 DNA containing the sequence,
(iv) 配列番号： 10で表されるアミノ酸配列を含有するタンパク質をコ一ドす る DNAとしては、 配列番号： 11で表される塩基配列を含有する DNAまたは 配列番号： 12で表される塩基配列を含有する DNAなどが、(iv) The DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 10 may be a DNA containing the base sequence represented by SEQ ID NO: 11 or DNA containing the base sequence represented by SEQ ID NO: 12, etc.
(v) 配列番号： 15で表されるアミノ酸配列を含有するタンパク質をコードす る DNAとしては、 配列番号： 16で表される塩基配列を含有する DNAまたは 配列番号： 19で表される塩基配列を含有する DNAなどが、 (v) As the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 15, DNA containing the nucleotide sequence represented by SEQ ID NO: 16 or the nucleotide sequence represented by SEQ ID NO: 19 DNA containing
(vi) 配列番号： 17で表されるアミノ酸配列を含有するタンパク質をコードす る DNAとしては、 配列番号： 18で表される塩基配列を含有する DNAまたは 配列番号： 19で表される塩基配列を含有する DNAなどが、  (vi) The DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 17 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 18 or the nucleotide sequence represented by SEQ ID NO: 19 DNA containing
(vii) 配列番号： 20で表されるアミノ酸配列を含有するタンパク質をコード する DNAとしては、 配列番号： 21で表される塩基配列を含有する DNAまた は配列番号： 24で表される塩基配列を含有する DNAなどが、  (vii) The DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 20 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 21 or the nucleotide sequence represented by SEQ ID NO: 24 DNA containing
(viii) 配列番号： 22で表されるアミノ酸配列を含有するタンパク質をコード する DNAとしては、 配列番号： 23で表される塩基配列を含有する DNAまた は配列番号： 24で表される塩基配列を含有する DNAなどが、  (viii) The DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 22 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 23 or the nucleotide sequence represented by SEQ ID NO: 24 DNA containing
(ix) 配列番号： 25で表されるアミノ酸配列を含有するタンパク質をコードす る DNAとしては、 配列番号： 26で表される塩基配列を含有する DNAまたは 配列番号： 29で表される塩基配列を含有する DN Aなどが、  (ix) As the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 25, DNA containing the nucleotide sequence represented by SEQ ID NO: 26 or the nucleotide sequence represented by SEQ ID NO: 29 Such as DNA containing
(X) 配列番号： 27で表されるアミノ酸配列を含有するタンパク質をコードす る DNAとしては、 配列番号： 28で表される塩基配列を含有する DNAまたは 配列番号： 29で表される塩基配列を含有する DNAなどが用いられる。  (X) The DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 27 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 28 or the nucleotide sequence represented by SEQ ID NO: 29 And the like containing DNA.
FLNAタンパク質をコードする DNAとしては、 例えば、 配列番号： 52で表さ れる塩基配列を含有する DNA、 または配列番号： 52で表される塩基配列とハ イストリンジェントな条件下でハイプリダイズする塩基配列を含有し、 配列番 号： 46で表されるアミノ酸配列を含有するタンパク質と実質的に同質の性質を 有するタンパク質をコードする D N Aなどが挙げられる。  Examples of the DNA encoding the FLNA protein include a DNA containing the nucleotide sequence represented by SEQ ID NO: 52, or a base that hybridizes with the nucleotide sequence represented by SEQ ID NO: 52 under high stringent conditions. DNA encoding a protein having a sequence and having substantially the same properties as a protein having the amino acid sequence represented by SEQ ID NO: 46.
FLNBタンパク質をコードする DNAとしては、 例えば、 配列番号： 53で表さ れる塩基配列を含有する DNA、 または配列番号： 53で表される塩基配列とハ イストリンジ； ントな条件下でハイプリダイズする塩基配列を含有し、 配列番 号： 47で表されるアミノ酸配列を含有するタンパク質と実質的に同質の性質を 有するタンパク質をコードする D N Aなどが挙げられる。 FLNCタンパク質をコードする D NAとしては、 例えば、 配列番号： 5 4で表さ れる塩基配列を含有する D NA、 または配列番号： 5 4で表される塩基配列とハ イストリンジェントな条件下でハイブリダィズする塩基配列を含有し、 配列番 号： 4 8で表されるアミノ酸配列を含有するタンパク質と実質的に同質の性質を 有するタンパク質をコードする D N Aなどが挙げられる。 Examples of the DNA encoding the FLNB protein include a DNA containing the base sequence represented by SEQ ID NO: 53, or a base sequence represented by SEQ ID NO: 53 and a string that hybridizes under simple conditions. DNA encoding a protein having a sequence and having substantially the same properties as a protein having the amino acid sequence represented by SEQ ID NO: 47. Examples of the DNA encoding the FLNC protein include, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 54 or the nucleotide sequence represented by SEQ ID NO: 54 under stringent conditions. Examples include DNA encoding a protein containing a hybridizing base sequence and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 48.
NAV2タンパク質をコードする D NAとしては、 例えば、 配列番号： 5 5で表さ れる塩基配列を含有する D NA、 または配列番号： 5 5で表される塩基配列とハ イストリンジェン卜な条件下でハイプリダイズする塩基配列を含有し、 配列番 号： 4 9で表されるアミノ酸配列を含有するタンパク質と実質的に同質の性質を 有するタンパク質をコードする D N Aなどが挙げられる。  Examples of the DNA encoding the NAV2 protein include, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 55 or the nucleotide sequence represented by SEQ ID NO: 55 under highly stringent conditions. And a DNA encoding a protein having a property substantially the same as that of the protein having the amino acid sequence represented by SEQ ID NO: 49.
BTBD2タンパク質をコードする D NAとしては、 例えば、 配列番号： 5 6で表 される塩基配列を含有する D NA、 または配列番号： 5 6で表される塩基配列と ハイストリンジェントな条件下でハイプリダイズする塩基配列を含有し、 配列番 号： 5 0で表されるアミノ酸配列を含有するタンパク質と実質的に同質の性質を 有するタンパク質をコードする D N Aなどが挙げられる。  Examples of the DNA encoding the BTBD2 protein include, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 56 or a DNA comprising the nucleotide sequence represented by SEQ ID NO: 56 in a highly stringent condition. Examples include a DNA encoding a protein containing a soybean base sequence and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 50.
MB3IL1タンパク質をコードする D NAとしては、 例えば、 配列番号： 5 7で 表される塩基配列を含有する D NA、 または配列番号： 5 7で表される塩基配列 とハイストリンジェントな条件下でハイプリダイズする塩基配列を含有し、 配列 番号： 5 1で表されるアミノ酸配列を含有するタンパク質と実質的に同質の性質 を有するタンパク質をコードする D N Aなどが挙げられる。  Examples of the DNA encoding the MB3IL1 protein include, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 57 or a DNA which hybridizes with the nucleotide sequence represented by SEQ ID NO: 57 under high stringent conditions. Examples include a DNA encoding a protein containing a soybean base sequence and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 51.
配列番号： 5 2、 配列番号： 5 3、 配列番号： 5 4、 配列番号： 5 5、 配列番 号： 5 6または配列番号： 5 7で表される塩基配列とハイストリンジェントな条 件下でハイブリダイズできる D N Aとしては、 例えば、 配列番号： 5 2、 配列番 号： 5 3、 配列番号： 5 4、 配列番号： 5 5、 配列番号： 5 6または配列番号： 5 7で表される塩基配列と約 7 0 %以上、 好ましくは約 8 0 %以上、 特に好まし くは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性を有する塩基配列を含 有する D N Aなどが用いられる。  The nucleotide sequence represented by SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57 and under high stringent conditions The DNA that can be hybridized with, for example, is represented by SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57 DNA having a nucleotide sequence having a homology of about 70% or more, preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more with the nucleotide sequence is used. Can be
塩基配列の相同性は、 相同性計算アルゴリズム NCBI BLAST (Nat ional Center for Biotechnology Informat ion Bas ic Local Al ignment Search Tool) を用い、 以下の条件 （期待値 = 10；ギャップを許す；フィルタリング =0N；マッチスコア = 1；ミスマッチスコア =-3) にて計算することができる。 The homology of the nucleotide sequences was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Basic Local Search Tool). It can be calculated under the following conditions (expected value = 10; gap allowed; filtering = 0N; match score = 1; mismatch score = -3).
ハイブリダィゼーシヨンは、 自体公知の方法あるいはそれに準じる方法、 例え ば、 Molecular Cloning 2nd (J. Sambrook et al. , Cold Spring Harbor Lab. Press, 1989) に記載の方法などに従って行なうことができる。 また、 市販のラ ィブラリ一を使用する場合、 添付の使用説明書に記載の方法に従つて行なうこと ができる。 より好ましくは、 ハイストリンジェントな条件に従って行なうことが できる。  Hybridization can be carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be performed under high stringency conditions.
ハイストリンジェントな条件とは、 例えば、 ナトリゥム濃度が約 1 9〜 4 0 m M、 好ましくは約 1 9〜 2 0 mMで、 温度が約 5 0〜 7 0 ° (：、 好ましくは約 6 0 〜6 5 °Cの条件を示す。 特に、 ナトリウム濃度が約 1 9 mMで温度が約 6 5 °Cの 場合が最も好ましい。  The high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° (:, preferably, about 60 ° C.). The condition is about 65 ° C. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
具体的には、 （i) 配列番号： 4 6で表されるアミノ酸配列を含有するタンパ ク質をコードする D NAとしては、 配列番号： 5 2で表される塩基配列を含有す る D NAなどが、 （i i) 配列番号： 4 7で表されるアミノ酸配列を含有するタン パク質をコードする D NAとしては、 配列番号： 5 3で表される塩基配列を含有 する D NAなどが、 （i i i) 配列番号： 4 8で表されるアミノ酸配列を含有する タンパク質をコードする D NAとしては、 配列番号： 5 4で表される塩基配列を 含有する D NAなどが、 （iv) 配列番号： 4 9で表されるアミノ酸配列を含有す るタンパク質をコードする D NAとしては、 配列番号： 5 5で表される塩基配列 を含有する D NAなどが、 （V) 配列番号： 5 0で表されるアミノ酸配列を含有 するタンパク質をコードする D NAとしては、 配列番号： 5 6で表される塩基配 列を含有する D NAなどが、 （vi) 配列番号： 5 0で表されるアミノ酸配列を含 有するタンパク質をコードする D NAとしては、 配列番号： 5 6で表される塩基 配列を含有する D N Aなどが用いられる。  Specifically, (i) the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 46 includes the DNA containing the base sequence represented by SEQ ID NO: 52 (Ii) Examples of the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 47 include a DNA having the nucleotide sequence represented by SEQ ID NO: 53. (Iii) DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 48 includes, for example, DNA having the nucleotide sequence represented by SEQ ID NO: 54, and (iv) SEQ ID NO: The DNA encoding the protein having the amino acid sequence represented by 49 is, for example, the DNA having the nucleotide sequence represented by SEQ ID NO: 55. The DNA encoding the protein containing the represented amino acid sequence includes SEQ ID NO: The DNA containing the base sequence represented by 56 is, for example, (vi) the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 50 is represented by SEQ ID NO: 56. DNA containing the base sequence to be used is used.
本発明で用いられる部分ペプチドをコードするポリヌクレオチド '(例、 D N A) としては、 前述した本発明で用いられる部分ペプチドをコードする塩基配列 を含有するものであればいかなるものであってもよい。 また、 ゲノム D NA、 ゲ 一、 前記した細胞 '組織由来の c D NA、 前記した細胞 . 組織由来の c DNAライブラリー、 合成 DN Aのいずれでもよい。 The polynucleotide (eg, DNA) encoding the partial peptide used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the partial peptide used in the present invention. In addition, genomic DNA, cDNA, the above-mentioned cell-derived tissue-derived cDNA, and the above-mentioned cells. It may be either a tissue-derived cDNA library or a synthetic DNA.
TACT427タンパク質の部分ペプチドをコードする DNAとしては、 例えば、 配 列番号： 2、 配列番号： 5、 配列番号： 8、 配列番号： 11、 配列番号： 16、 配列番号： 18、 配列番号： 21、 配列番号： 23、 配列番号： 26または配列 番号： 28で表される塩基配列を含有する DNAの一部分を有する DNA、 また は配列番号： 2、 配列番号： 5、 配列番号： 8、 配列番号： 11、 配列番号： 1 6、 配列番号： 18、 配列番号： 21、 配列番号： 23、 配列番号： 26または 配列番号： 28で表される塩基配列とハイストリンジェン卜な条件下でハイプリ ダイズする塩基配列を含有し、 TACT427タンパク質と実質的に同質の活性を有す るタンパク質をコードする D N Aの一部分を含有する D N Aなどが用いられる。 配列番号： 2、 配列番号： 5、 配列番号： 8、 配列番号： 11、 配列番号： 1 6、 配列番号： 18、 配列番号： 21、 配列番号： 23、 配列番号： 26または 配列番号： 28で表される塩基配列とハイブリダィズできる DNAは、 前記と同 意義を示す。  DNAs encoding partial peptides of the TACT427 protein include, for example, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21, DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 23, SEQ ID NO: 26 or SEQ ID NO: 28, or SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11. SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 26 or hybridizes with the nucleotide sequence represented by SEQ ID NO: 28 under high stringent conditions. A DNA containing a base sequence and a part of a DNA encoding a protein having substantially the same activity as the TACT427 protein may be used. SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 26 or SEQ ID NO: 28 DNA that can hybridize with the base sequence represented by has the same significance as described above.
ハイブリダィゼ一シヨンの方法およびハイストリンジェントな条件は前記と同 様のものが用いられる。  Hybridization methods and high stringency conditions are the same as described above.
FLNAタンパク質の部分ペプチドをコードする DNAとしては、 例えば、 配列番 号： 52で表される塩基配列を含有する DN Aの一部分を有する DNA、 または 配列番号： 52で表される塩基配列とハイストリンジェン卜な条件下でハイプリ ダイズする塩基配列を含有し、 配列番号： 46で表されるアミノ酸配列を含有す るタンパク質と実質的に同質の活性を有するタンパク質をコードする DNAの一 部分を含有する D N Aなどが用いられる。  Examples of the DNA encoding the partial peptide of the FLNA protein include, for example, DNA having a portion of DNA containing the nucleotide sequence represented by SEQ ID NO: 52, or the nucleotide sequence represented by SEQ ID NO: 52 and high string Contains a nucleotide sequence that hybridizes under mild conditions and contains a portion of DNA encoding a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 46. DNA or the like is used.
FLNBタンパク質の部分ペプチドをコードする DNAとしては、 例えば、 配列番 号： 53で表される塩基配列を含有する DNAの一部分を有する DNA、 または 配列番号： 53で表される塩基配列とハイストリンジェン卜な条件下でハイプリ ダイズする塩基配列を含有し、 配列番号： 47で表されるアミノ酸配列を含有す るタンパク質と実質的に同質の性質を有するタンパク質をコードする DN Aの一 部分を含有する D N Aなどが用いられる。  Examples of the DNA encoding the partial peptide of the FLNB protein include, for example, a DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 53, or the base sequence represented by SEQ ID NO: 53 and high stringency. Contains a nucleotide sequence that hybridizes under simple conditions, and contains a portion of DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 47. DNA or the like is used.
FLNCタンパク質の部分ペプチドをコードする DNAとしては、 例えば、 配列番 号： 54で表される塩基配列を含有する DNAの一部分を有する DNA、 または 配列番号： 54で表される塩基配列とハイストリンジェントな条件下でハイプリ ダイズする塩基配列を含有し、 配列番号： 48で表されるアミノ酸配列を含有す るタンパク質と実質的に同質の性質を有するタンパク質をコードする DN Aの一 部分を含有する DN Aなどが用いられる。 DNA encoding a partial peptide of FLNC protein includes, for example, SEQ ID NO: No .: a DNA having a portion of the DNA containing the base sequence represented by 54, or a base sequence which hybridizes with the base sequence represented by SEQ ID NO: 54 under high stringent conditions; For example, a DNA containing a part of a DNA encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by 48 may be used.
NAV2タンパク質の部分ペプチドをコードする DNAとしては、 例えば、 配列番 号： 55で表される塩基配列を含有する DNAの一部分を有する DNA、 または 配列番号： 55で表される塩基配列とハイストリンジェントな条件下でハイプリ ダイズする塩基配列を含有し、 配列番号： 49で表されるアミノ酸配列を含有す るタンパク質と実質的に同質の性質を有するタンパク質をコードする DNAの一 部分を含有する DN Aなどが用いられる。  Examples of the DNA encoding the partial peptide of the NAV2 protein include, for example, a DNA having a part of the DNA containing the nucleotide sequence represented by SEQ ID NO: 55, or the nucleotide sequence represented by SEQ ID NO: 55 and high stringency Containing a portion of DNA encoding a protein having a nucleotide sequence which hybridizes under simple conditions and having substantially the same properties as a protein having an amino acid sequence represented by SEQ ID NO: 49 Are used.
BTBD2タンパク質の部分ペプチドをコードする DNAとしては、 例えば、 配列 番号： 56で表される塩基配列を含有する DNAの一部分を有する DNA、 また は配列番号： 56で表される塩基配列とハイストリンジェン卜な条件下でハイブ リダィズする塩基配列を含有し、 配列番号： 50で表されるアミノ酸配列を含有 するタンパク質と実質的に同質の性質を有するタンパク質をコードする DNAの 一部分を含有する DN Aなどが用いられる。  Examples of the DNA encoding the partial peptide of the BTBD2 protein include, for example, a DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 56, or the base sequence represented by SEQ ID NO: 56 and high stringency. DNA containing a portion of DNA encoding a protein having a nucleotide sequence that hybridizes under simple conditions and having substantially the same properties as a protein having an amino acid sequence represented by SEQ ID NO: 50, etc. Is used.
RAB3IL1タンパク質の部分ペプチドをコードする DNAとしては、 例えば、 配 列番号： 57で表される塩基配列を含有する DNAの一部分を有する DNA、 ま たは配列番号： 57で表される塩基配列とハイストリンジェントな条件下でハイ ブリダィズする塩基配列を含有し、 配列番号： 51で表されるアミノ酸配列を含 有するタンパク質と実質的に同質の性質を有するタンパグ質をコードする DN A の一部分を含有する DNAなどが用いられる。  Examples of the DNA encoding the partial peptide of the RAB3IL1 protein include a DNA having a portion of the DNA containing the nucleotide sequence represented by SEQ ID NO: 57, or a DNA having a nucleotide sequence represented by SEQ ID NO: 57. Contains a nucleotide sequence that hybridizes under stringent conditions and contains a portion of DNA encoding a protein having substantially the same properties as a protein having the amino acid sequence represented by SEQ ID NO: 51 DNA or the like is used.
配列番号： 52、 配列番号： 53、 配列番号： 54、 配列番号： 55、 配列番 号： 56または配列番号： 57で表される塩基配列とハイプリダイズできる DN Aは、 前記と同意義を示す。  SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57 .
ハイプリダイゼーションの方法およびハイストリンジェントな条件は前記と同 様のものが用いられる。  The same hybridization method and high stringency conditions as described above are used.
本発明のタンパク質およびその部分ペプチド （以下、 これらをコードする DN Aのクローニングおよび発現の説明においては、 これらを単に本発明のタンパク 質と略記する場合がある） を完全にコードする DN Aのクローニングの手段とし ては、 本発明のタンパク質をコードする塩基配列の一部分を有する合成 DNAプ ライマ一を用いて PC R法によって増幅するか、 ま は適当なベクターに組み込 んだ DNAを本発明のタンパク質の一部あるいは全領域をコードする DNA断片 もしくは合成 DN Aを用いて標識したものとのハイブリダィゼ一シヨンによって 選別することができる。 ハイブリダィゼ一シヨンの方法は、 例えば、 モレキユラ 一-クローニング (Molecular Cloning) 2nd (J. Sambrook et al. , Cold The protein of the present invention and its partial peptide (hereinafter, DNs encoding them) In the description of the cloning and expression of A, these may be simply abbreviated as the protein of the present invention.) As a means for cloning DNA that completely encodes, the nucleotide sequence encoding the protein of the present invention Amplification by the PCR method using a synthetic DNA primer having a portion, or DNA integration into an appropriate vector, DNA fragment encoding a part or all of the protein of the present invention, or synthetic DNA Can be selected by hybridization with those labeled using Hybridization methods are described, for example, in Molecular Cloning 2nd (J. Sambrook et al., Cold
Spring Harbor Lab. Press, 1989) に記載の方法などに従って行なうことができ る。 また、 市販のライブラリーを使用する場合、 添付の使用説明書に記載の方法 に従って行なうことができる。 Spring Harbor Lab. Press, 1989). When a commercially available library is used, the procedure can be performed according to the method described in the attached instruction manual.
DNAの塩基配列の変換は、 PCR、 公知のキット、 例えば、 Mutan™- super Express Km (宝酒造 （株） ） 、 Mutan™- K (宝酒造 （株） ） 等を用いて、 0M-LA PCR法、 Gapped duplex法、 Kunkel法等の自体公知の方法あるいはそれらに準じる 方法に従って行なうことができる。  The DNA base sequence can be converted using PCR, a known kit, for example, Mutan ™ -Super Express Km (Takara Shuzo Co., Ltd.), Mutan ™ -K (Takara Shuzo Co., Ltd.), etc., using the 0M-LA PCR method. The method can be carried out according to a method known per se, such as the gapped duplex method or the Kunkel method, or a method analogous thereto.
クローン化されたタンパク質をコードする DNAは目的によりそのまま、 また は所望により制限酵素で消化したり、 リンカーを付加したりして使用することが できる。 該 DNAはその 5' 末端側に翻訳開始コドンとしての ATGを有し、 ま た 3' 末端側には翻訳終止コドンとしての TAA、 TGAまたは TAGを有して いてもよい。 これらの翻訳開始コドンや翻訳終止コドンは、 適当な合成 DNAァ ダブ夕一を用いて付加することもできる。  The DNA encoding the cloned protein can be used as it is depending on the purpose, or it can be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
本発明のタンパク質の発現べクタ一は、 例えば、 （ィ） 本発明のタンパク質を コードする DNAから目的とする DNA断片を切り出し、 （口） 該 DNA断片を 適当な発現ベクター中のプロモー夕一の下流に連結することにより製造すること ができる。  The expression vector for the protein of the present invention can be prepared, for example, by (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) converting the DNA fragment into a promoter in an appropriate expression vector. It can be manufactured by connecting downstream.
ベクターとしては、 大腸菌由来のプラスミド （例、 pBR322， pBR32 5, pUC 12, pUC13) 、 枯草菌由来のプラスミド （例、 pUB 110， pTP 5, pC194) 、 酵母由来プラスミド （例、 p SH19, p SH15) . λファージなどのバクテリオファージ、 レトロウイルス， ワクシニアウィルス， バキュロウィルスなどの動物ウィルスなどの他、 pAl— 11、 pXTl、 pR c/CMV、 pRcZRSV、 p cDNA IZNe oなどが用いられる。 Examples of vectors include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15) bacteriophage such as λ phage, retrovirus, vaccinia virus, In addition to animal viruses such as baculovirus, pAl-11, pXTl, pRc / CMV, pRcZRSV, pcDNA IZNeo and the like are used.
本発明で用いられるプロモータ一としては、 遺伝子の発現に用いる宿主に対応 して適切なプロモーターであればいかなるものでもよい。 例えば、 動物細胞を宿 主として用いる場合は、 SRaプロモーター、 SV40プロモータ一、 LTRプ 口モータ—、 CMVプロモーター、 HSV-TKプロモーターなどが挙げられる c これらのうち、 CMV (サイトメガロウィルス） プロモーター、 SRo!プロモ 一夕一などを用いるのが好ましい。 宿主がェシエリヒア属菌である場合は、 t r ρプロモ一夕一、 l a cプロモーター、 r e cAプロモーター、 APLプロモ一 夕一、 1 p pプロモーター、 T 7プロモータ一などが、 宿主がバチルス属菌であ る場合は、 SP〇1プロモ一夕一、 SP02プロモータ一、 p e nPプロモ一夕 —など、 宿主が酵母である場合は、 PH05プロモ一ター、 PGKプロモータ一、 GAPプロモーター、 ADHプロモータ一などが好ましい。 宿主が昆虫細胞であ る場合は、 ポリヘドリンプロモーター、 P 10プロモーターなどが好ましい。 発現ベクターには、 以上の他に、 所望によりェンハンサー、 スプライシングシ ダナル、 ポリ A付加シグナル、 選択マ一カー、 S V40複製オリジン (以下、 S V40 o r iと略称する場合がある） などを含有しているものを用いることがで きる。 選択マーカ一としては、 例えば、 ジヒドロ葉酸還元酵素 （以下、 dh f r と略称する場合がある） 遺伝子 〔メソトレキセート （MTX) 耐性〕 、 アンピシ リン耐性遺伝子 （以下、 Amp fと略称する場合がある） 、 ネオマイシン耐性遺 伝子 （以下、 Ne orと略称する場合がある、 G418耐性） 等が挙げられる。 特に、 dh f r遺伝子欠損チャイニーズハムスター細胞を用いて dh f r遺伝子 を選択マーカーとして使用する場合、 目的遺伝子をチミジンを含まない培地によ つても選択できる。 The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when animal cells are used as host, SRa promoter, SV40 promoter, LTR motor, CMV promoter, HSV-TK promoter, etc. are included.c Among them, CMV (cytomegalovirus) promoter, SRo! It is preferable to use a promoter overnight. When the host is Eshierihia genus bacteria, tr [rho promoter Isseki one, lac promoter, re cA promoter, AP L promoter one evening one, 1 pp promoter, T 7 promoter one is Ru host Bacillus der When the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. For example, SP〇1 promoter, SP02 promoter, penP promoter, etc. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. The expression vector may further contain, if desired, an enhancer, a splicing signal, a poly-A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like. Can be used. Examples of the selectable marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance], an ampicillin resistance gene (hereinafter sometimes abbreviated as Amp f), neomycin resistance gene (hereinafter sometimes abbreviated as Ne o r, G418 resistance). In particular, when the dh fr gene is used as a selection marker using dh fr gene-deficient Chinese hamster cells, the target gene can be selected using a thymidine-free medium.
また、 必要に応じて、 宿主に合ったシグナル配列を、 本発明のタンパク質の N 端末側に付加する。 宿主がェシエリヒア属菌である場合は、 PhoA 'シグナル配 列、 0即 A ·シグナル配列などが、 宿主がバチルス属菌である場合は、 α—アミ ラーゼ ·シグナル配列、 サブチリシン ·シグナル配列などが、 宿主が酵母である 場合は、 MFo; ·シグナル配列、 SUC2 ·シグナル配列など、 宿主が動物細胞 である場合には、 インシュリン .シグナル配列、 ひ一インターフェロン ·シグナ ル配列、 抗体分子 ·シグナル配列などがそれぞれ利用できる。 If necessary, a signal sequence suitable for the host is added to the N-terminal of the protein of the present invention. When the host is a bacterium belonging to the genus Escherichia, the PhoA ′ signal sequence and the 0-A signal sequence are included. When the host is a bacterium belonging to the genus Bacillus, the α-amylase signal sequence and the subtilisin signal sequence are included. If the host is yeast, MFo; signal sequence, SUC2, signal sequence, etc. In this case, an insulin signal sequence, a single interferon / signal sequence, an antibody molecule / signal sequence, etc. can be used.
このようにして構築された本発明のタンパク質をコードする DN Aを含有する ベクターを用いて、 形質転換体を製造することができる。  Using the vector containing the DNA encoding the protein of the present invention thus constructed, a transformant can be produced.
宿主としては、 例えば、 ェシエリヒア属菌、 バチルス属菌、 酵母、 昆虫細胞、 昆虫、 動物細胞などが用いられる。  As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
ェシエリヒア属菌の具体例としては、 例えば、 ェシエリヒア .コリ  Specific examples of the genus Escherichia include, for example, Escherichia coli.
(Escherichia coli) K 12 · DH 1 [Proc. Natl. Acad. Sci. USA, 60 巻， 160 (1968)〕 ， J M 103 [Nucleic Acids Research, 9巻， 309 (1981)〕 ， J A 221 [Journal of Molecular Biology, 120巻， 517 (1978)〕 , HB 101  (Escherichia coli) K 12 · DH 1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM 103 [Nucleic Acids Research, 9, 309 (1981)], JA 221 [Journal of Molecular Biology, 120, 517 (1978)], HB 101
[Journal of Molecular Biology, 41巻， 459(1969)〕 ， C 600 [Genetics, 39 巻， 440 (1954)〕 などが用いられる。  [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like are used.
バチルス属菌としては、 例えば、 バチルス ·サブチルス (Bacillus  Examples of Bacillus bacteria include, for example, Bacillus subtilis (Bacillus
subtilis) M I 1 14 〔Gene， 24巻， 255 (1983)〕 ， 207 - 21 [Journal of Biochemistry, 95 , 87(1984)] などが用いられる。 subtilis) M I 1 14 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)] and the like are used.
酵母としては、 例えば、 サッカロマイセス セレピシェ （Saccharomyces cerevisiae) AH22, AH22R—， NA 87 - 1 1 A, DKD— 5D， 20 B— 12、 シゾサッカロマイセス ボンべ （Scliizosaccharomyces pombe) NC YC 1913, NCYC 2036, ピキア パストリス （Pichia pastoris) K M71などが用いられる。  Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R—, NA87-11A, DKD—5D, 20B—12, and Scliizosaccharomyces pombe NC YC 1913, NCYC 2036, Pichia Pastoris (Pichia pastoris) K M71 or the like is used.
昆虫細胞としては、 例えば、 ウィルスが AcNPVの場合は、 夜盗蛾の幼虫由 来株化細胞 (Spodoptera frugiperda cell； S f細胞) 、 Tric oplusia niの中 . 腸由来の MG1細胞、 Trichoplusia niの卵由来の High Five™細胞、 Mamestra brassicae由来の細胞または Estigmena acrea由来の細胞などが用いられる。 ウイ ルスが BmNPVの場合は、 蚕由来株化細胞 （Bombyx mori N細胞； BmN細 胞） などが用いられる。 該 S f細胞としては、 例えば、 S f 9細胞 （ATCC CRL1711) 、 S f 21細胞 （以上、 Vaughn, J. L.ら、 In Vivo, 13, 213- 217, (1977)) などが用いられる。  Insect cells include, for example, when the virus is AcNPV, a cell line derived from a larva of night roth moth (Spodoptera frugiperda cell; S f cell), in Tricoplusia ni. High Five ™ cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a cell line derived from a silkworm (Bombyx mori N cell; BmN cell) is used. As the Sf cells, for example, Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, J.L., et al., In Vivo, 13, 213-217, (1977)) and the like are used.
昆虫としては、 例えば、 カイコの幼虫などが用いられる 〔前田ら、 Nature, 315 巻， 592 (1985)〕 。 Examples of insects include silkworm larvae [Maeda et al., Nature, 315 Vol., 592 (1985)].
動物細胞としては、 例えば、 サル細胞 COS— 7， Ve r o， チャイニーズハ ムス夕一細胞 CHO (以下、 CHO細胞と略記） ， dh f r遺伝子欠損チヤィニ —ズハムスター細胞 CHO (以下、 CHO (dh f r~) 細胞と略記） ， マウス L細胞， マウス At T— 20, マウスミエ口一マ細胞， マウス ATDC5細胞， ラット GH3, ヒト FL細胞などが用いられる。  Examples of animal cells include monkey cells COS-7, Vero, Chinese Hams Yuichi cell CHO (hereinafter abbreviated as CHO cells), dh fr gene-deficient chicks, and Chinese hamster cell CHO (hereinafter CHO (dh fr ~ ) Cells), mouse L cells, mouse At T-20, mouse myeoma cells, mouse ATDC5 cells, rat GH3, human FL cells, etc. are used.
ェシエリヒア属菌を形質転換するには、 例えば、 Proc. Natl. Acad. Sci.  To transform Escherichia sp., For example, Proc. Natl. Acad. Sci.
USA, 69巻, 2110 (1972)や Gene, 17巻， 107 (1982)などに記載の方法に従って行なうこ とができる。 USA, 69, 2110 (1972) and Gene, 17, 107 (1982).
バチルス属菌を形質転換するには、 例えば、 Molecular & General  To transform Bacillus, for example, use Molecular & General
Genetics, 168巻， 111 (1979)などに記載の方法に従って行なうことができる。  Genetics, Vol. 168, 111 (1979) can be used.
酵母を形質転換するには、 例えば、 Methods in Enzymology, 194巻， 182- 187 (1991)、 Proc. Natl. Acad. Sci. USA, 75巻, 1929 (1978) などに記載の方法 に従って行なうことができる。  Transformation of yeast can be performed, for example, according to the method described in Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). it can.
昆虫細胞または昆虫を形質転換するには、 例えば、 Bio/Technology, 6, Ai- SS (1988)などに記載の方法に従って行なうことができる。  Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, Ai-SS (1988).
動物細胞を形質転換するには、 例えば、 細胞工学別冊 8 新細胞工学実験プロ トコール. 263-267 (1995) (秀潤社発行) 、 Virology, 52巻, 456 (1973)に記載の方 法に従って行なうことができる。  Transformation of animal cells can be performed, for example, according to the method described in Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). Can do it.
このようにして、 タンパク質をコ一ドする DNAを含有する発現ベクターで形 質転換された形質転換体を得ることができる。  Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.
宿主がェシエリヒア属菌、 バチルス属菌である形質転換体を培養する際、 培養 に使用される培地としては液体培地が適当であり、 その中には該形質転換体の生 育に必要な炭素源、 窒素源、 無機物その他が含有せしめられる。 炭素源としては、 例えば、 グルコース、 デキストリン、 可溶性澱粉、 ショ糖など、 窒素源としては、 例えば、 アンモニゥム塩類、 硝酸塩類、 コーンスチープ ·リカー、 ペプトン、 力 ゼイン、 肉エキス、 大豆粕、 バレイショ抽出液などの無機または有機物質、 無機 物としては、 例えば、 塩化カルシウム、 リン酸二水素ナトリウム、 塩化マグネシ ゥムなどが挙げられる。 また、 酵母エキス、 ビタミン類、 生長促進因子などを添 加してもよい。 培地の pHは約 5〜8が望ましい。 When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein. , Nitrogen sources, inorganic substances and others. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, potato zein, meat extract, soybean meal, and potato extract. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. Also, add yeast extract, vitamins, growth promoting factors, etc. May be added. The pH of the medium is preferably about 5-8.
ェシエリヒア属菌を培養する際の培地としては、 例えば、 グルコース、 カザミ ノ酸を含む M 9培地 〔ミラ一 （Miller) , Journal of Experiments in  As a medium for cultivating a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in
Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York
1972〕 が好ましい。 ここに必要によりプロモータ一を効率よく働かせるために、 例えば、 3 /3—インドリルァクリル酸のような薬剤を加えることができる。 1972] is preferred. Here, for example, a drug such as 3 / 3-indolylacrylic acid can be added to make the promoter work efficiently if necessary.
宿主がェシエリヒア属菌の場合、 培養は通常約 15〜43°Cで約 3〜24時間 行ない、 必要により、 通気や撹拌を加えることもできる。  When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.
宿主がバチルス属菌の場合、 培養は通常約 30〜 40 °Cで約 6〜 24時間行な レ 必要により通気や撹拌を加えることもできる。  When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours. If necessary, aeration and stirring may be applied.
宿主が酵母である形質転換体を培養する際、 培地としては、 例えば、 バークホ 一ルダー (Burkholder) 最小培地 〔Bostian, K. L. ら、 Proc. Natl. Acad. Sci. USA, 77巻， 4505 (1980)〕 や 0. 5 %カザミノ酸を含有する S D培地 〔Bitter, G. A. ら、 Proc. Natl. Acad. Sci. USA, 81巻， 5330 (1984)〕 が挙げられる。 培地の pH ：は約 5〜8に調整するのが好ましい。 培養は通常約 20°C〜35°Cで約 24〜7 2時間行ない、 必要に応じて通気や撹拌を加える。  When culturing a transformant in which the host is yeast, for example, Burkholder's minimal medium [Bostian, KL et al., Proc. Natl. Acad. Sci. USA, 77, 4505 (1980) And SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. The cultivation is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added as necessary.
宿主が昆虫細胞または昆虫である形質転換体を培養する際、 培地としては、 Grace's Insect Medium (Grace, T. C. C. , Nature, 195, 788 (1962)) に非動化した 10 %ゥシ血清等の添加物を適宜加えたものなどが用いられる。 培地の pHは約 6. 2-6. 4に調整するのが好ましい。 培養は通常約 27 °Cで約 3〜 5日間行 ない、 必要に応じて通気や撹拌を加える。  When culturing an insect cell or a transformant whose host is an insect, add 10% serum inactivated to Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). What added the thing suitably is used. Preferably, the pH of the medium is adjusted to about 6.2-6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
宿主が動物細胞である形質転換体を培養する際、 培地としては、 例えば、 約 5 〜20%の胎児牛血清を含む MEM培地 [Science, 122巻， 501 (1952)〕 ， DME M培地 [Virology, 8巻, 396 (1959)] , RPM I 1640培地 [The Journal of the American Medical Association 199巻, 519 (1967)3 , 199培地  When culturing a transformant in which the host is an animal cell, the culture medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology , Volume 8, 396 (1959)], RPM I 1640 medium [The Journal of the American Medical Association Volume 199, 519 (1967) 3, 199 medium
[Proceeding of the Society for the Biological Medicine, 73巻, 1 (1950)〕 などが用いられる。 pHは約 6〜8であるのが好ましい。 培養は通常約 30° (：〜 40でで約15〜60時間行ない、 必要に応じて通気や撹拌を加える。  [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)]. Preferably, the pH is about 6-8. Culture is usually performed at about 30 ° (: ~ 40 for about 15 to 60 hours, and aeration and agitation are added as necessary.
以上のようにして、 形質転換体の細胞内、 細胞膜または細胞外に本発明のタン パク質を生成せしめることができる。 As described above, the protein of the present invention is placed inside the cell, in the cell membrane, or outside the cell of the transformant. It can produce protein.
上記培養物から本発明のタンパク質を分離精製するには、 例えば、 下記の方法 により行なうことができる。  The protein of the present invention can be separated and purified from the above culture by, for example, the following method.
本発明のタンパク質を培養菌体あるいは細胞から抽出するに際しては、 培養後、 公知の方法で菌体あるいは細胞を集め、 これを適当な緩衝液に懸濁し、 超音波、 リゾチームおよび/または凍結融解などによって菌体あるいは細胞を破壊したの ち、 遠心分離やろ過によりタンパク質の粗抽出液を得る方法などが適宜用いられ る。 緩衝液の中に尿素や塩酸グァニジンなどのタンパク質変性剤や、 トリトン X 一 1 0 0 TMなどの界面活性剤が含まれていてもよい。 培養液中にタンパク質が 分泌される場合には、 培養終了後、 それ自体公知の方法で菌体あるいは細胞と上 清とを分離し、 上清を集める。 When extracting the protein of the present invention from the cultured cells or cells, after the culture, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme, and / or freeze-thawing. After the cells or cells are destroyed by the method described above, a method of obtaining a crude protein extract by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X100 ™ . When the protein is secreted into the culture solution, after completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.
このようにして得られた培養上清、 あるいは抽出液中に含まれるタンパク質の 精製は、 自体公知の分離 '精製法を適切に組み合わせて行なうことができる。 こ れらの公知の分離、 精製法としては、 塩析ゃ溶媒沈澱法などの溶解度を利用する 方法、 透析法、 限外ろ過法、 ゲルろ過法、 および S D S—ポリアクリルアミドゲ ル電気泳動法などの主として分子量の差を利用する方法、 イオン交換クロマ卜グ ラフィーなどの荷電の差を利用する方法、 ァフィ二ティークロマトグラフィーな どの特異的親和性を利用する方法、 逆相高速液体クロマトグラフィーなどの疎水 性の差を利用する方法、 等電点電気泳動法などの等電点の差を利用する方法など が用いられる。  Purification of the protein contained in the culture supernatant or extract thus obtained can be carried out by appropriately combining known separation and purification methods. These known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Methods that mainly use differences in molecular weight, methods that use differences in charges such as ion-exchange chromatography, methods that use specific affinity such as affinity chromatography, and methods that use reversed-phase high-performance liquid chromatography. A method utilizing a difference in hydrophobicity, a method utilizing a difference in isoelectric point such as isoelectric focusing, and the like are used.
かくして得られるタンパク質が遊離体で得られた場合には、 自体公知の方法あ るいはそれに準じる方法によって塩に変換することができ、 逆に塩で得られた場 合には自体公知の方法あるいはそれに準じる方法により、 遊離体または他の塩に 変換することができる。  When the protein thus obtained is obtained as a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when the protein is obtained as a salt, a method known per se or The compound can be converted into a free form or another salt by an analogous method.
なお、 組換え体が産生するタンパク質を、 精製前または精製後に適当な蛋白修 飾酵素を作用させることにより、 任意に修飾を加えたり、 ポリペプチドを部分的 に除去することもできる。 蛋白修飾酵素としては、 例えば、 トリプシン、 キモト リブシン、 アルギニルェンドぺプチダーゼ、 プロテインキナーゼ、 グリコシダ一 ゼなどが用いられる。 かくして生成する本発明のタンパク質の存在は、 特異抗体を用いたェンザィム ィムノアツセィゃウエスタンブロッテイングなどにより測定することができる。 以下、 TACT427タンパク質もしくはその部分ペプチドまたはその塩を、 単に TACT427タンパク質と、 FLNAタンパク質もしくはその部分ペプチドまたはその塩 を、 単に FLNAタンパク質と、 FLNBタンパク質もしくはその部分ペプチドまたはそ の塩を、 単に FLNBタンパク質と、 FLNCタンパク質もしくはその部分ペプチドまた はその塩を、 単に FLNCタンパク質と、 NAV2タンパク質もしくはその部分ペプチド またはその塩を、 単に NAV2タンパク質と、 BTBD2タンパク質もしくはその部分べ プチドまたはその塩を、 単に BTBD2タンパク質と、 RAB3IL1タンパク質もしくはそ の部分ペプチドまたはその塩を、 単に RAB3IL1タンパク質とそれぞれ略記する。 The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein-modifying enzyme before or after purification. As the protein-modifying enzyme, for example, trypsin, chymotoribsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. The presence of the protein of the present invention thus produced can be measured by, for example, enzymatic immunoassay western blotting using a specific antibody. Hereinafter, the TACT427 protein or its partial peptide or its salt, simply referred to as the TACT427 protein, the FLNA protein or its partial peptide or its salt, simply referred to as the FLNA protein, the FLNB protein or its partial peptide or its salt, and simply referred to as the FLNB protein FLNC protein or its partial peptide or its salt, simply FLNC protein, NAV2 protein or its partial peptide or its salt, simply NAV2 protein, BTBD2 protein or its partial peptide or its salt, and simply BTBD2 protein. RAB3IL1 protein or its partial peptide or a salt thereof is simply abbreviated as RAB3IL1 protein, respectively.
(a) TACT427タンパク質、 および （b) FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質および RAB3IL1タンパク質か ら選ばれる一または二種以上を含有する複合体を、 本発明の複合体と略称するこ ともある。  A complex containing one or more selected from (a) TACT427 protein and (b) FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein and RAB3IL1 protein is abbreviated as the complex of the present invention. Sometimes.
本発明の複合体は、 TACT427タンパク質、 FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質または （および） RAB3IL1夕 ンパク質に加え、 上記以外のタンパク質、 ペプチド、 R NA、 核酸、 脂質、 糖類、 アミド基、 リン酸基などを含んでいてもよい。 例えば、 TACT427タンパク質、 FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タ ンパク質および RAB3IL1タンパク質は、 アミド化されていてもよく、 リン酸化さ れたアミノ酸を含んでいてもよく、 脂質 （例、 ミリスチン酸など） 結合アミノ酸 を含んでいてもよく、 いかなる翻訳後修飾を受けていても限定されない。 翻訳後 修飾を受ける領域として、 例えば、 配列番号： 4 5で表わされるアミノ酸配列に 相当する領域などが挙げられる。  The complex of the present invention comprises, in addition to TACT427 protein, FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or (and) RAB3IL1 protein, proteins, peptides, RNAs, nucleic acids, lipids, and saccharides other than those described above. , An amide group, a phosphate group and the like. For example, TACT427 protein, FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein and RAB3IL1 protein may be amidated, may contain phosphorylated amino acids, and may contain lipids (eg, , Myristic acid, etc.), and may be bound by any post-translational modification. Examples of the region subjected to post-translational modification include a region corresponding to the amino acid sequence represented by SEQ ID NO: 45, and the like.
本発明の複合体としては、 例えば、 （a) TACT427タンパク質の細胞質側ドメイ ン （例、 配列番号： 4 5で表されるアミノ酸配列を有するペプチドなど） と (b) FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質および RAB3IL1タンパク質から選ばれる一または二種以上を含有 している複合体、 例えば、 （a) TACT427タンパク質の細胞質側ドメイン （例、 配 列番号： 4 5で表されるアミノ酸配列を有するペプチドなど） と （b) FLNAタン パク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質 および RAB3IL1タンパク質から選ばれる一または二種以上とが結合している複合 体などが挙げられる。 具体例としては、 （1) TACT427タンパク質の細胞質側ドメ イン （例、 配列番号： 4 5で表されるアミノ酸配列を有するペプチドなど） と FLNAタンパク質とが結合している複合体、 （2) TACT427タンパク質の細胞質側ド メイン （例、 配列番号： 4 5で表されるアミノ酸配列を有するペプチドなど） と FLNBタンパク質とが結合している複合体、 （3) TACT427タンパク質の細胞質側ド メイン （例、 配列番号： 4 5で表されるアミノ酸配列を有するペプチドなど） と FLNCタンパク質とが結合している複合体、 （4) TACT427タンパク質の細胞質側ド メイン （例、 配列番号： 4 5で表されるアミノ酸配列を有するペプチドなど） と NAV2タンパク質とが結合している複合体、 （5) TACT427タンパク質の細胞質側ド メイン （例、 配列番号： 4 5で表されるアミノ酸配列を有するペプチドなど） と BTBD2タンパク質とが結合している複合体、 （6) TACT427タンパク質の細胞質側 ドメイン （例、 配列番号： 4 5で表されるアミノ酸配列を有するペプチドなど） と RAB3IL1タンパク質とが結合している複合体などが挙げられる。 Examples of the complex of the present invention include (a) a cytoplasmic domain of TACT427 protein (eg, a peptide having the amino acid sequence represented by SEQ ID NO: 45) and (b) FLNA protein, FLNB protein, FLNC Complex containing one or more selected from proteins, NAV2 protein, BTBD2 protein and RAB3IL1 protein, for example, (a) cytoplasmic domain of TACT427 protein (eg, Such as a peptide having an amino acid sequence represented by SEQ ID NO: 45) and (b) one or more selected from FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein and RAB3IL1 protein Complex. Specific examples include (1) a complex in which the cytoplasmic domain of TACT427 protein (eg, a peptide having the amino acid sequence represented by SEQ ID NO: 45) and FLNA protein are bound; A complex in which the cytoplasmic domain of the protein (eg, a peptide having the amino acid sequence represented by SEQ ID NO: 45) binds to the FLNB protein; (3) a cytoplasmic domain of the TACT427 protein (eg, A complex in which FLNC protein is bound to a peptide having the amino acid sequence represented by SEQ ID NO: 45) and (4) a cytoplasmic domain of TACT427 protein (eg, represented by SEQ ID NO: 45) A complex in which an NAV2 protein is bound to a peptide having an amino acid sequence, etc .; (5) a cytoplasmic domain of TACT427 protein (eg, the amino acid sequence represented by SEQ ID NO: 45 (6) Cytoplasmic domain of TACT427 protein (eg, peptide having the amino acid sequence represented by SEQ ID NO: 45) and RAB3IL1 protein And the like.
FLNAタンパク質の、 TACT427タンパク質との結合活性を有する領域としては、 例えば、 配列番号： 4 6で表されるアミノ酸配列の第 2162番目〜第 2412番目、 第 2206番目〜第 2355番目または第 2141番目〜第 2351番目が挙げられる。  Examples of the region of the FLNA protein having a binding activity to the TACT427 protein include, for example, the 2162nd to 2412th, the 2206th to the 2355th or the 2141th to the amino acid sequence represented by SEQ ID NO: 46. No. 2351 is mentioned.
FLNBタンパク質の、 TACT427タンパク質との結合活性を有する領域としては、 例えば、 配列番号： 4 7で表されるアミノ酸配列の第 1960番目〜第 2299番目、 第 2156番目〜第 2314番目または第 2174番目〜第 2565番目が挙げられる。  Examples of a region of the FLNB protein having a binding activity with the TACT427 protein include, for example, the 1960th to 2299th, the 2156th to the 2314th or the 2174th to the amino acid sequence represented by SEQ ID NO: 47. The 2565th is mentioned.
FLNCタンパク質の、 TACT427タンパク質との結合活性を有する領域としては、 例えば、 配列番号： 4 8で表されるアミノ酸配列の第 2295番目〜第 2705番目が挙 げられる。  Examples of a region of the FLNC protein having a binding activity with the TACT427 protein include the 2295th to 2705th amino acids of the amino acid sequence represented by SEQ ID NO: 48.
NAV2タンパク質の、 TACT427タンパク質との結合活性を有する領域としては、 例えば、 配列番号： 4 9で表されるアミノ酸配列の第 68番目〜第 286番目が挙げ られる。  Examples of the region having binding activity to the TACT427 protein of the NAV2 protein include the 68th to 286th amino acids of the amino acid sequence represented by SEQ ID NO: 49.
BTBD2タンパク質の、 TACT427タンパク質との結合活性を有する領域としては、 例えば、 配列番号： 5 0で表されるアミノ酸配列の第 201番目〜第 525番目または 第 215番目〜第 506番目が挙げられる。 The region of the BTBD2 protein having a binding activity with the TACT427 protein includes: Examples include the 201st to 525th or the 215th to 506th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50.
RAB3IL1タンパク質の、 TACT427タンパク質との結合活性を有する領域としては、 例えば、 配列番号： 5 1で表されるアミノ酸配列の第 282番目〜第 356番目が挙げ られる。  Examples of the region of the RAB3IL1 protein having a binding activity to the TACT427 protein include the 282nd to 356th amino acids of the amino acid sequence represented by SEQ ID NO: 51.
本発明の複合体の形成を阻害または促進する活性を有する TACT427タンパク質 に対する抗体 （以下、 本発明の抗体と略称することもある） は、 本発明の複合体 の形成を阻害または促進する活性を有し、 TACT427タンパク質を認識し得る抗体 であれば、 ポリクローナル抗体、 モノクローナル抗体の何れであってもよい。 本発明の抗体は、 TACT427タンパク質を抗原 （以下、 抗原タンパク質と略する こともある） として用い、 自体公知の抗体または抗血清の製造法に従って製造す ることができる。  An antibody against the TACT427 protein having the activity of inhibiting or promoting the formation of the complex of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) has the activity of inhibiting or promoting the formation of the complex of the present invention. However, any antibody that can recognize the TACT427 protein may be a polyclonal antibody or a monoclonal antibody. The antibody of the present invention can be produced by using the TACT427 protein as an antigen (hereinafter sometimes abbreviated as antigen protein) according to a method for producing an antibody or antiserum known per se.
さらに、 本発明の複合体を認識する抗体も本発明のタンパク質を抗原として用 レ 自体公知の抗体または抗血清の製造法に従って製造することができる。  Furthermore, an antibody that recognizes the complex of the present invention can also be produced using the protein of the present invention as an antigen according to a method for producing an antibody or antiserum known per se.
〔モノクローナル抗体の作製〕 [Preparation of monoclonal antibody]
( a ) モノクローナル抗体産生細胞の作製  (a) Preparation of monoclonal antibody-producing cells
抗原夕ンパク質は、 温血動物に対して投与により抗体産生が可能な部位にそれ 自体あるいは担体、 希釈剤とともに投与される。 投与に際して抗体産生能を高め るため、 完全フロイン卜アジュバントや不完全フロイントアジュバントを投与し てもよい。 投与は通常 2〜 6週毎に 1回ずつ、 計 2〜 1 0回程度行われる。 用い られる温血動物としては、 例えば、 サル、 ゥサギ、 ィヌ、 モルモット、 マウス、 ラット、 ヒッジ、 ャギ、 ニヮトリが挙げられるが、 マウスおよびラットが好まし く用いられる。  The antigen protein is administered to a warm-blooded animal by itself or together with a carrier or a diluent at a site capable of producing an antibody upon administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. The warm-blooded animals used include, for example, monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
モノクローナル抗体産生細胞の作製に際しては、 抗原で免疫された温血動物、 例えばマウスから抗体価の認められた個体を選択し最終免疫の 2〜 5日後に脾臓 またはリンパ節を採取し、 それらに含まれる抗体産生細胞を同種または異種動物 の骨髄腫細胞と融合させることにより、 モノクローナル抗体産生ハイプリドーマ を調製することができる。 抗血清中の抗体価の測定は、 例えば、 後記の標識化夕 ンパク質と抗血清とを反応させたのち、 抗体に結合した標識剤の活性を測定する ことにより行なうことができる。 融合操作は既知の方法、 例えば、 ケーラ一とミ ルスタインの方法 〔Nature、 256、 495 (1975)〕 に従い実施することができる。 融合促進剤としては、 例えば、 ポリエチレングリコール （PEG) やセンダイゥ ィルスなどが挙げられるが、 好ましくは P E Gが用いられる。 When preparing monoclonal antibody-producing cells, select a warm-blooded animal immunized with the antigen, for example, an individual with an antibody titer from a mouse, collect the spleen or lymph nodes 2 to 5 days after the final immunization, and include them in By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The measurement of the antibody titer in the antiserum can be performed, for example, by the following labeling method. After reacting the protein with the antiserum, the activity can be measured by measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
骨髄腫細胞としては、 例えば、 NS— 1、 P3U1、 S P 2/0、 AP— 1な どの温血動物の骨髄腫細胞が挙げられるが、 P 3U1が好ましく用いられる。 用 いられる抗体産生細胞 （脾臓細胞） 数と骨髄腫細胞数との好ましい比率は 1 ： 1 〜20 ： 1程度であり、 PEG (好ましくは PEG 1000〜PEG6000) 力 S 10〜 80 %程度の濃度で添加され、 20〜 40 °C、 好ましくは 30〜 37 °C で 1〜 10分間ィンキュベー卜することにより効率よく細胞融合を実施できる。 モノクローナル抗体産生ハイプリドーマのスクリ一二ングには種々の方法が使 用できるが、 例えば、 タンパク質抗原を直接あるいは担体とともに吸着させた固 相 （例、 マイクロプレート） にハイブリド一マ培養上清を添加し、 次に放射性物 質や酵素などで標識した抗免疫グロプリン抗体 （細胞融合に用いられる細胞がマ ウスの場合、 抗マウス免疫グロブリン抗体が用いられる） またはプロテイン Aを 加え、 固相に結合したモノクローナル抗体を検出する方法、 抗免疫グロブリン抗 体またはプロテイン Aを吸着させた固相にハイプリドーマ培養上清を添加し、 放 射性物質や酵素などで標識したタンパク質を加え、 固相に結合したモノクローナ ル抗体を検出する方法などが挙げられる。  Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and the concentration of PEG (preferably PEG 1000 to PEG6000) S is about 10 to 80%. By incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes, cell fusion can be carried out efficiently. Various methods can be used for screening monoclonal antibody-producing hybridomas.For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which protein antigens are directly or adsorbed together with a carrier. Then, an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is mouse) or protein A labeled with a radioactive substance or an enzyme is added, and the mixture is bound to the solid phase. Monoclonal antibody detection method, hybridoma supernatant was added to a solid phase to which anti-immunoglobulin antibody or protein A was adsorbed, proteins labeled with radioactive substances, enzymes, etc. were added and bound to the solid phase. Examples include a method for detecting a monoclonal antibody.
モノクローナル抗体の選別は、 自体公知あるいはそれに準じる方法に従って行 なうことができる。 通常 HAT (ヒポキサンチン、 アミノプテリン、 チミジン） を添加した動物細胞用培地で行なうことができる。 選別および育種用培地として は、 ハイプリドーマが生育できるものならばどのような培地を用いても良い。 例 えば、 1〜20%、 好ましくは 10〜20%の牛胎児血清を含む RPMI 164 0培地、 1〜10%の牛胎児血清を含む G I T培地 （和光純薬工業 （株） ） ある いはハイブリド一マ培養用無血清培地 （SFM— 101、 日水製薬 （株） ) など を用いることができる。 培養温度は、 通常 20〜40°C、 好ましくは約 37°Cで ある。 培養時間は、 通常 5日〜 3週間、 好ましくは 1週間〜 2週間である。 培養 は、 通常 5 %炭酸ガス下で行なうことができる。 ハイプリドーマ培養上清の抗体 価は、 上記の抗血清中の抗体価の測定と同様にして測定できる。 The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as it can grow a hybridoma. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or hybrid A serum-free culture medium for culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. culture Can be carried out usually under 5% carbon dioxide gas. The antibody titer of the culture supernatant of the hybridoma can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
( b ) モノクローナル抗体の精製  (b) Purification of monoclonal antibodies
モノクローナル抗体の分離精製は、 自体公知の方法、 例えば、 免疫グロブリン の分離精製法 〔例、 塩析法、 アルコール沈殿法、 等電点沈殿法、 電気泳動法、 ィ オン交換体 （例、 D E A E ) による吸脱着法、 超遠心法、 ゲルろ過法、 抗原結合 固相あるいはプロテイン Aあるいはプロテイン Gなどの活性吸着剤により抗体の みを採取し、 結合を解離させて抗体を得る特異的精製法〕 に従って行なうことが できる。  Monoclonal antibodies can be separated and purified by methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)) Absorption / desorption method, ultracentrifugation method, gel filtration method, antigen binding Solid phase or specific purification method of collecting antibody only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody) You can do it.
〔ポリクローナル抗体の作製〕  (Preparation of polyclonal antibody)
本発明のポリクローナル抗体は、 それ自体公知あるいはそれに準じる方法に従 つて製造することができる。 例えば、 免疫抗原 （タンパク質抗原） 自体、 あるい はそれとキャリアータンパク質との複合体をつくり、 上記のモノクローナル抗体 の製造法と同様に温血動物に免疫を行ない、 該免疫動キ匆から抗原タンパク質に対 する抗体含有物を採取して、 抗体の分離精製を行なうことにより製造することが できる。  The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, an immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody. The antibody can be produced by collecting the antibody-containing substance and separating and purifying the antibody.
温血動物を免疫するために用いられる免疫抗原とキャリアータンパク質との複 合体に関し、 キヤリァータンパク質の種類およびキヤリァ一とハプテンとの混合 比は、 キャリアーに架橋させて免疫したハプテンに対して抗体が効率良くできれ ば、 どの様なものをどの様な比率で架橋させてもよいが、 例えば、 ゥシ血清アル ブミンゃゥシサイログロブリン、 へモシァニン等を重量比でハプテン 1に対し、 約 0 . 1〜2 0、 好ましくは約 1〜5の割合でカプルさせる方法が用いられる。 また、 ハプテンとキャリア一の力プリングには、 種々の縮合剤を用いることが できるが、 ダルタルアルデヒドゃカルポジィミド、 マレイミド活性エステル、 チ オール基、 ジチオビリジル基を含有する活性エステル試薬等が用いられる。 縮合生成物は、 温血動物に対して、 抗体產生が可能な部位にそれ自体あるいは 担体、 希釈剤とともに投与される。 投与に際して抗体産生能を高めるため、 完全 フロイントアジュバントゃ不完全フロイントアジュバントを投与してもよい。 投 与は、 通常約 2〜 6週毎に 1回ずつ、 計約 3〜1 0回程度行なわれる。 ポリクローナル抗体は、 上記の方法で免疫された温血動物の血液、 腹水など、 好ましくは血液から採取することができる。 Regarding the complex of the immunizing antigen and the carrier protein used to immunize a warm-blooded animal, the type of carrier protein and the mixing ratio of the carrier and the hapten depend on the antibody against the hapten immunized by cross-linking with the carrier. If it can be efficiently performed, any kind may be crosslinked at any ratio.For example, serum serum albumin, thyroglobulin, hemocyanin, etc. may be used in a weight ratio of about 0 to 1 for hapten. A method of coupling at a rate of 1 to 20, preferably about 1 to 5 is used. In addition, various condensing agents can be used for force coupling between the hapten and the carrier. For example, an active ester reagent containing a daltaraldehyde-carpoimide, a maleimide active ester, a thiol group, or a dithioviridyl group is used. The condensation product is administered to a warm-blooded animal at a site where antibody production is possible, by itself or together with a carrier or diluent. Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The dose is usually given about every 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.
抗血清中のポリクローナル抗体価の測定は、 上記の抗血清中の抗体価の測定と 同様にして測定できる。 ポリクローナル抗体の分離精製は、 上記のモノクロ一ナ ル抗体の分離精製と同様の免疫グロプリンの分離精製法に従って行なうことがで きる。  The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same method for separation and purification of immunoglobulin as in the above-described separation and purification of the monoclonal antibody.
以下に、 本発明のタンパク質もしくは部分ペプチドまたはその塩 （以下、 本発 明のタンパク質と略記する場合がある） 、 本発明のタンパク質または部分べプチ ドをコードする D NA (以下、 本発明の D NAと略記する場合がある） 、 本発明 の抗体、 および本発明の複合体などの用途を説明する。  Hereinafter, the protein or partial peptide of the present invention or a salt thereof (hereinafter, sometimes abbreviated as the protein of the present invention), the DNA encoding the protein or partial peptide of the present invention (hereinafter referred to as D The use of the antibody of the present invention and the complex of the present invention will be described.
〔1〕 疾病に対する医薬候補化合物のスクリーニング  [1] Screening of drug candidate compounds for diseases
TACT427タンパク質は癌組織で発現が亢進しており、 さらに、 TACT427タンパク 質の機能 （活性、 発現など） を阻害すると癌細胞がアポトーシスを起こす。  The expression of TACT427 protein is increased in cancer tissues, and when the function (activity, expression, etc.) of TACT427 protein is inhibited, cancer cells undergo apoptosis.
また、 FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質または RAB3IL1タンパク質は、 TACT427タンパク質の細胞質側ド メインに結合し、 TACT427タンパク質の機能を担っていると考えられる。  Further, it is considered that the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein binds to the cytoplasmic domain of TACT427 protein and plays a role of TACT427 protein.
従って、 本発明の複合体の形成を阻害する化合物 （例、 ペプチド、 タンパク質、 抗体、 非ペプチド性化合物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物組織抽出液、 血漿など） 、 例えば、 （a) TACT427タンパク質と （b) FLNA夕 ンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク 質および RAB3IL1タンパク質から選ばれる一または二種以上による複合体の形成 を阻害する化合物、 好ましくは、 （a) TACT427タンパク質と （b) FLNAタンパク . 質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質およ び RAB3IL1タンパク質から選ばれる一または二種以上との結合 〔例、 （1)  Therefore, compounds that inhibit the formation of the complex of the present invention (eg, peptides, proteins, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc.) For example, a compound that inhibits the formation of a complex by (a) TACT427 protein and (b) one or more selected from FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein, and RAB3IL1 protein; Preferably, the binding between (a) the TACT427 protein and (b) one or more selected from the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein and RAB3IL1 protein [eg, (1)
TACT427タンパク質と FLNAタンパク質との結合、 （2) TACT427タンパク質と FLNB タンパク質との結合、 （3) TACT427タンパク質と FLNCタンパク質との結合、Binding between TACT427 protein and FLNA protein, (2) binding between TACT427 protein and FLNB protein, (3) binding between TACT427 protein and FLNC protein,
(4) TACT427タンパク質と NAV2タンパク質との結合、 （5) TACT427タンパク質と BTBD2タンパク質との結合、 （6) TACT427タンパク質と RAB3IL1タンパク質との結 合など〕 を阻害する化合物またはその塩は、 癌細胞のアポトーシス誘導作用を有 し、 例えば、 癌 （例、 大腸癌、 乳癌、 肺癌、 前立腺癌、 食道癌、 胃癌、 肝臓癌、 胆道癌、 脾臓癌、 腎癌、 膀胱癌、 子宮癌、 精巣癌、 卵巣癌、 甲状腺癌、 塍臓癌、 脳腫瘍、 血液腫瘍など） などの予防 ·治療剤、 癌細胞のアポトーシス促進剤など として使用することができる。 (4) binding of TACT427 protein to NAV2 protein, (5) binding of TACT427 protein to BTBD2 protein, (6) binding of TACT427 protein to RAB3IL1 protein, etc.) Has apoptosis-inducing effect And, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, ovarian cancer, thyroid cancer, It can be used as a prophylactic / therapeutic agent for kidney cancer, brain tumor, blood tumor, etc., and as an apoptosis promoter for cancer cells.
本発明の複合体の形成を促進する化合物 （例、 ペプチド、 タンパク質、 抗体、 非ペプチド性化合物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物 組織抽出液、 血漿など） 、 例えば、 （a) TACT427タンパク質と （b) FLNAタンパ ク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質お よび RAB3IL1タンパク質から選ばれる一または二種以上による複合体の形成を促 進する化合物、 好ましくは、 （a) TACT427タンパク質と （b) FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質および RAB3IL1タンパク質から選ばれる一または二種以上との結合 〔例、 （1) TACT427 タンパク質と FLNAタンパク質との結合、 （2) TACT427タンパク質と FLNBタンパク 質との結合、 （3) TACT427タンパク質と FLNCタンパク質との結合、 （4) TACT427 タンパク質と NAV2タンパク質との結合、 （5) TACT427タンパク質と BTBD2タンパ ク質との結合、 （6) TACT427タンパク質と RAB3IL1タンパク質との結合など〕 を 促進する化合物またはその塩は、 神経細胞のアポトーシス抑制作用を有し、 例え ば、 神経変性疾患 （例、 アルツハイマー病 （家族性アルツハイマー病、 若年性ァ ルツハイマー病、 孤発性アルツハイマー病など） などの予防 ·治療剤、 神経細胞 のアポトーシス阻害 （抑制） 剤などとして使用することができる。  Compounds that promote the formation of the complex of the present invention (eg, peptides, proteins, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc.), for example A compound that promotes the formation of a complex by one or more selected from (a) TACT427 protein and (b) FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein, and RAB3IL1 protein, preferably Is the binding between (a) TACT427 protein and (b) one or more selected from FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein and RAB3IL1 protein [eg, (1) TACT427 protein and FLNA protein (3) TACT427 protein and FLNB protein, (3) TACT427 protein and FLNC protein Compound that promotes (4) binding of TACT427 protein to NAV2 protein, (5) binding of TACT427 protein to BTBD2 protein, (6) binding of TACT427 protein to RAB3IL1 protein, etc.) The salt has an inhibitory effect on apoptosis of nerve cells. For example, it is a preventive and therapeutic agent for neurodegenerative diseases (eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.)) It can be used as an agent for inhibiting (suppressing) apoptosis of nerve cells.
したがって、 本発明のタンパク質は、 本発明の複合体の形成を阻害または促進 する化合物またはその塩のスクリーニングのための試薬として有用である。  Therefore, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that inhibits or promotes the formation of the complex of the present invention.
すなわち、 本発明は、 本発明のタンパク質を用いることを特徴とする本発明の 複合体の形成 〔例、 上記 （1) 〜 （6) の結合〕 を阻害または促進する化合物また はその塩のスクリーニング方法を提供する。  That is, the present invention provides a method for screening a compound or a salt thereof that inhibits or promotes the formation of the complex of the present invention (eg, the binding of the above (1) to (6)), which comprises using the protein of the present invention. Provide a method.
本発明のスクリーニング方法には、 本発明のタンパク質が用いられ、 さらには、 TACT427タンパク質の細胞質側ドメインに相当するペプチドが用いられてもよい また、 本発明のスクリーニング方法は、 本発明のタンパク質を産生する能力を有 する細胞 （好ましくは、 本発明のタンパク質をコードする D NAで形質転換され た形質転換体 （例、 酵母、 動物細胞などの細胞など） ） が用いられてもよい。 該 細胞としては、 例えば、 （a) TACT427タンパク質をコードする D NA (例、 TACT427タンパク質の細胞質側ドメインに相当するペプチドをコードする D N A) および （b) FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タン パク質、 BTBD2タンパク質または RAB3IL1タンパク質をコードする D NAで形質転 換された形質転換体が用いられる。 The protein of the present invention may be used in the screening method of the present invention, and a peptide corresponding to the cytoplasmic domain of TACT427 protein may be used. (Preferably, transformed with a DNA encoding the protein of the present invention). Transformants (eg, cells such as yeast and animal cells) may be used. Examples of such cells include (a) DNA encoding TACT427 protein (eg, DNA encoding a peptide corresponding to the cytoplasmic domain of TACT427 protein) and (b) FLNA protein, FLNB protein, FLNC protein, NAV2 protein. A transformant transformed with DNA encoding the protein, BTBD2 protein or RAB3IL1 protein is used.
〔l a〕 In vitro結合試験によるスクリーニング [La] Screening by in vitro binding test
FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2 タンパク質または RAB3IL1タンパク質を、 FLNAタンパク質、 FLNBタンパク質、 FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein, FLNA protein, FLNB protein,
FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質または RAB3IL1タンパク質に 対する抗体を用いて、 固相 （例、 EIAプレート） に固定化するか、 または、 FLNA タンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパ ク質または RAB3IL1タンパク質を、 Tagタンパク質 （例、 His- Tag、 GST (ダル夕チ オン- S-トランスフェラーゼ） 等） と融合させた後、 固相に固定化する。 Immobilize on a solid phase (eg, EIA plate) using antibodies against FLNC, NAV2, BTBD2, or RAB3IL1 protein, or use FLNA, FLNB, FLNC, NAV2, BTBD2 proteins Alternatively, the RAB3IL1 protein is fused to a Tag protein (eg, His-Tag, GST (Dalphinthione-S-transferase), etc.) and then immobilized on a solid phase.
FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2 タンパク質または RAB3IL1タンパク質として、 該タンパク質の部分ペプチドを用 いる場合には、 好ましくは TACT427タンパク質との結合活性を有する部分べプチ ド （例、 配列番号： 4 6で表されるアミノ酸配列の第 2162番目〜第 2412番目、 第 2206番目〜第 2355番目または第 2141番目〜第 2351番目のアミノ酸配列を有する部 分ペプチド、 配列番号： 4 7で表されるアミノ酸配列の第 1960番目〜第 2299番目. 第 2156番目〜第 2314番目または第 2174番目〜第 2565番目のアミノ酸配列を有する 部分ペプチド、 配列番号： 4 8で表されるアミノ酸配列の第 2295番目〜第 2705番 目のアミノ酸配列を有する部分ペプチド、 配列番号： 4 9で表されるアミノ酸配 列の第 68番目〜第 286番目のアミノ酸配列を有する部分ペプチド、 配列番号： 5 0で表されるアミノ酸配列の第 201番目〜第 525番目または第 215番目〜第 506番目 のアミノ酸配列を有する部分ペプチド、 配列番号： 5 1で表されるアミノ酸配列 の第 282番目〜第 356番目のアミノ酸配列を有する部分ペプチドなど） などが用い られる。 Tag付きタンパク質の固相への固定に際しては、 His- Tagであればニッケル、 GSTであればダル夕チォンが用いられる。 When a partial peptide of the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein is used, a partial peptide having a binding activity to TACT427 protein (eg, SEQ ID NO: : A partial peptide having the amino acid sequence at positions 2162 to 2412, 2206 to 2355 or 2141 to 2351 of the amino acid sequence represented by 46, represented by SEQ ID NO: 47 1960th to 2299th amino acid sequence of the amino acid sequence. Partial peptide having the 2156th to 2314th or 2174th to 2565th amino acid sequence, the 2295th amino acid sequence represented by SEQ ID NO: To a partial peptide having the 2705th amino acid sequence, and the 68th to 286th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 49. Partial peptide having a sequence, Partial peptide having the 201st to 525th or 215th to 506th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 50, represented by SEQ ID NO: 51 A partial peptide having the 282nd to 356th amino acid sequence of the amino acid sequence) and the like. When immobilizing a protein with a tag to a solid phase, nickel is used for His-Tag, and Dalcon is used for GST.
(i) 標識剤 （例、 ピオチンなど） で標識した TACT427タンパク質 （例、 細胞質 側ドメインに相当する部分ペプチド （例、 配列番号： 4 5で表されるアミノ酸配 列を有する部分ペプチドなど） ） を、 固相化した FLNAタンパク質、 FLNBタンパク 質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質または RAB3IL1タンパク 質に接触させる場合の結合量と、 （i i) 標識剤 （例、 ピオチンなど） で標識した TACT427タンパク質 （例、 細胞質側ドメインに相当する部分ペプチド （例、 配列 番号： 4 5で表されるアミノ酸配列を有する部分ペプチドなど） ） および試験化 合物を、 固相化した FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2夕 ンパク質、 BTBD2タンパク質または RAB3IL1タンパク質に同時に接触させる場合の 結合量とを測定し、 比較する。  (i) TACT427 protein (eg, a partial peptide corresponding to the cytoplasmic domain (eg, a partial peptide having the amino acid sequence represented by SEQ ID NO: 45) labeled with a labeling agent (eg, biotin) The amount of binding when contacting with immobilized FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein, and (ii) TACT427 protein labeled with a labeling agent (eg, biotin) For example, FLNA protein, FLNB protein, FLNC protein in which a partial peptide corresponding to the cytoplasmic domain (eg, a partial peptide having the amino acid sequence represented by SEQ ID NO: 45) and a test compound are immobilized. , NAV2 protein, BTBD2 protein, or RAB3IL1 protein. To.
結合量は、 公知の方法、 例えば、 固相化された TACT427タンパク質またはその 部分ペプチドを、 標識剤 （例、 ピオチンなど） を検出する市販のキットまたは TACT427タンパク質に対する抗体を用いて、 測定する。  The binding amount is measured by a known method, for example, using a solid-phased TACT427 protein or a partial peptide thereof using a commercially available kit for detecting a labeling agent (eg, biotin or the like) or an antibody against the TACT427 protein.
試験化合物としては、 例えば、 ペプチド、 タンパク質、 抗体、 非ペプチド性化 合物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物組織抽出液、 血 漿などがあげられる。  Test compounds include, for example, peptides, proteins, antibodies, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.
例えば、 上記 （i i) の場合における結合量が上記 （i) の場合に比べて、 約 2 0 %以上、 好ましくは 3 0 %以上、 より好ましくは約 5 0 %以上減少させる試験 化合物を、 本発明の複合体の形成を阻害する化合物 （以下、 結合阻害剤と略記す る場合がある） として、 上記 （i i) の場合における活性が上記 （i) の場合に比 ベて、 約 2 0 %以上、 好ましくは 3 0 %以上、 より好ましくは約 5 0 %以上増加 させる試験化合物を、 本発明の複合体の形成を促進する化合物 （以下、 結合促進 剤と略記する場合がある） として選択する。  For example, a test compound that reduces the amount of binding in the case (ii) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case (i), As a compound that inhibits the formation of the complex of the invention (hereinafter sometimes abbreviated as a binding inhibitor), the activity in the case of the above (ii) is about 20% as compared with the case of the above (i). As described above, a test compound that increases the concentration by preferably 30% or more, more preferably about 50% or more, is selected as a compound that promotes the formation of the complex of the present invention (hereinafter sometimes abbreviated as a binding promoter). .
また、 TACT427タンパク質 （例、 TACT427タンパク質の細胞質側ドメインに相当 する部分ペプチドなど） を固相に固定化し、 FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質または RAB3IL1タンパク質を 接触させ、 同様に結合量を測定してもよい。 TACT427タンパク質を固相へ固定化 する際には、 標識剤 （例、 ピオチンなど） で標識された TACT427タンパク質また はその部分ペプチド、 およびアビジンで標識された固相 （例、 プレート） が好ま しく用いられる。 TACT427 protein (eg, a partial peptide corresponding to the cytoplasmic domain of TACT427 protein) is immobilized on a solid phase, and FLNA, FLNB, FLNC, NAV2, BTBD2, or RAB3IL1 protein is immobilized on a solid phase. After contact, the amount of binding may be measured in the same manner. When the TACT427 protein is immobilized on a solid phase, a TACT427 protein or a partial peptide thereof labeled with a labeling agent (eg, biotin) or a solid phase (eg, a plate) labeled with avidin is preferably used. Can be
(i i i) 固相化した TACT427タンパク質に、 FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質または RAB3IL1タンパク質を 接触させる場合の結合量と、 （iv) 固相化した TACT427タンパク質またはその部 分ペプチドに、 FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパ ク質、 BTBD2タンパク質または RAB3IL1タンパク質および試験化合物を同時に接触 させる場合の結合量とを、 測定し、 比較する。  (iii) the amount of binding when the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein is brought into contact with the immobilized TACT427 protein, and (iv) the immobilized TACT427 protein or a part thereof. The amount of binding when the peptide is simultaneously contacted with the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein and the test compound is measured and compared.
結合量は、 公知の方法、 例えば、 固相化された FLNAタンパク質、 FLNBタンパク 質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質または RAB3IL1タンパク 質を、 該タンパク質に対する抗体を用いて、 測定する。  The binding amount is measured by a known method, for example, by immobilizing FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein using an antibody against the protein.
試験化合物としては、 例えばペプチド、 タンパク質、 抗体、 非ペプチド性化合 物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物組織抽出液、 血漿 などがあげられる。  Test compounds include, for example, peptides, proteins, antibodies, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.
この方法において、 FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2 タンパク質、 BTBD2タンパク質または RAB3IL1タンパク質は、 Tagタンパク質と融 合させたタンパク質を用いてもよく、 その際には、 FLNAタンパク質、 FLNBタンパ ク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質または RAB3IL1タンパ ク質を、 該タンパク質に対する抗体で検出、 定量してもよく、 また Tagタンパク 質に対する抗体で検出、 定量してもよい。  In this method, the FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein may be a protein fused with a Tag protein, in which case the FLNA protein, FLNB protein, The FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein may be detected and quantified using an antibody against the protein, or may be detected and quantified using an antibody against the Tag protein.
例えば、 上記 （iv) の場合における結合量が上記 （i i i) の場合に比べて、 約 2 0 %  For example, the binding amount in the case of the above (iv) is about 20% as compared with the case of the above (iii).
以上、 好ましくは 3 0 %以上、 より好ましくは約 5 0 %以上減少させる試験化合 物を本発明の複合体の形成を阻害する化合物 （以下、 結合阻害剤と略記する場合 がある） として、 上記 （iv) の場合における活性が上記 （i i i) の場合に比べて、 約 2 0 %以上、 好ましくは 3 0 %以上、 より好ましくは約 5 0 %以上増加させる 試験化合物を結合促進剤として選択する。 As described above, a test compound that preferably reduces by 30% or more, more preferably about 50% or more, is referred to as a compound that inhibits the formation of the complex of the present invention (hereinafter, may be abbreviated as a binding inhibitor). The activity in the case of (iv) is increased by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii). A test compound is selected as the binding promoter.
〔1 b〕 ツーハイプリッド法によるスクリーニング [1 b] Screening by two-hybrid method
〔1 b _ 1〕 酵母ツーハイプリッド法によるスクリーニング  [1 b _ 1] Screening by yeast two-hybrid method
(a) TACT427タンパク質の細胞質側ドメインに相当する部分ペプチド （例えば、 配列番号： 4 5で表わされるアミノ酸配列を含有する部分ペプチド） にレポ一夕 —遺伝子結合ドメインを融合させたキメラタンパク質をコードする D NA、 およ び （b) FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2夕ンパク質または RAB3 IL 1タンパク質にレポ一夕一遺伝子転写活性化ドメイ ンを融合させたキメラタンパク質をコードする D NAを酵母 （例、  (a) encodes a chimeric protein in which a partial peptide corresponding to the cytoplasmic domain of TACT427 protein (for example, a partial peptide containing the amino acid sequence represented by SEQ ID NO: 45) and a repo overnight-gene binding domain are fused DNA, and (b) encode a chimeric protein obtained by fusing a repo overnight gene transcription activation domain to FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein, or RAB3 IL1 protein. DNA is converted to yeast (eg,
Saccharomyces cerevis iae、 より好ましくは S. cerevis iae Y190株） において同 時に発現させると、 ツーハイブリッドの作用により、 レポーター遺伝子である β 一ガラクトシダーゼ遺伝子およびヒスチジン合成系遺伝子 HIS3の表現型が発現す る。 この酵母株を試験化合物の存在下、 一定時間培養し、 酵母株の β—ガラクト シダーゼ活性を減少させるか、 または酵母株をヒスチジン要求性に転換させる試 験化合物を、 結合阻害剤として選択する。  When expressed simultaneously in Saccharomyces cerevis iae (more preferably, S. cerevis iae strain Y190), the phenotype of the reporter gene β-galactosidase gene and the histidine synthesis gene HIS3 is expressed by the action of the two hybrids. The yeast strain is cultured in the presence of the test compound for a certain period of time, and a test compound that reduces the β-galactosidase activity of the yeast strain or converts the yeast strain to histidine requirement is selected as a binding inhibitor.
該酵母株は、 上記した宿主が酵母である形質転換体の培養と同様に培養できる。 β_ガラクトシダーゼの活性は、 X- Gal (5-bromo-4-chloro-3-indolyl-p-D- galactopyranos ide) 、 ONPG (o-ni t rophenyl β-D-galactopyranos ide) または CPRG (c lorop enyl red-p-D-galactopyranos ide) を基質として公知の方法に 従って測定することができる。 HIS3表現型の発現は、 ヒスチジン欠損の最小培地 で酵母を培養することにより測定することができる。  The yeast strain can be cultured in the same manner as in the culture of a transformant whose host is yeast as described above. β-galactosidase activity can be measured using X-Gal (5-bromo-4-chloro-3-indolyl-pD-galactopyranoside), ONPG (o-nitrophenyl β-D-galactopyranoside) or CPRG (clorop enyl red). -pD-galactopyranoside) can be measured according to a known method as a substrate. Expression of the HIS3 phenotype can be measured by culturing the yeast in a minimal medium lacking histidine.
選択された化合物のうち、 細胞毒性のある化合物、 およびレポーター遺伝子産 物との相互作用などでレポーター遺伝子産物自身の活性を阻害する化合物などは、 擬陽性化合物として除外する。  Among the selected compounds, cytotoxic compounds and compounds that inhibit the activity of the reporter gene product itself due to interaction with the reporter gene product are excluded as false positive compounds.
〔1 b— 2〕 動物細胞ッ一ハイブリツド法によるスクリーニング [1 b-2] Screening by animal cell hybrid method
動物細胞 （例、 CH0細胞など） にレポーター遺伝子 （例、 クロラムフエニコー ルァセチルトランスフェラーゼ （CAT) 遺伝子、 ホ夕ルルシフェラ一ゼ遺伝子な ど） を導入する。 レポーター遺伝子の転写調節領域は、 例えば動物細胞で機能す るプロモーター （例、 アデノウイルス Elb由来の最小プロモーター （TATA box) など） の下流に、 例えば GAL1の転写活性化配列 （UAS) を結合したものを用い、 酵母ッ一ハイプリッドシステムの GAL4- GAL1の転写調節系を動物細胞内に導入し、 動物細胞内でレポーター遺伝子の発現が誘導されるようにする。 得られる細胞に 対し、 （a) TACT427タンパク質の細胞質側ドメインに相当する部分ペプチド （例 えば、 配列番号： 4 5で表わされるアミノ酸配列を含有する部分ペプチド） に GAL4- DNA結合ドメインを融合させたキメラタンパク質をコードする D NA、 およ び （b) FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質または MB3IL1タンパク質に単純へルぺスウィルス由来 VP16タン パク質を融合させたキメラタンパク質をコードする D N Aを同時に発現させると、 ッ一ハイプリッドの作用により、 レポ一夕一遺伝子が発現する動物細胞株が得ら れる。 この細胞株を試験化合物の存在下、 一定時間培養し、 レポ一夕一遺伝子産 物の活性を測定し、 活性を減少させる化合物を、 結合阻害剤として選択する。 該動物細胞株は、 上記した宿主が動物細胞である形質転換体の培養と同様に培 養できる。 レポーター遺伝子産物 （例、 CAT、 ルシフェラーゼなど） の活性は、 公知の方法に従って、 市販のキットを用いて測定できる。 Reporter genes (eg, chloramphenicol acetylacetyltransferase (CAT) gene, chromium luciferase gene) in animal cells (eg, CH0 cells) ) Is introduced. The transcriptional regulatory region of the reporter gene is, for example, a promoter that functions in animal cells (eg, a minimal promoter (TATA box) derived from the adenovirus Elb, etc.) downstream of which a transcriptional activation sequence (UAS) of GAL1 is linked. The transcriptional control system of GAL4-GAL1 of the yeast hybrid system is introduced into animal cells to induce the expression of a reporter gene in animal cells. In the obtained cells, (a) a GAL4-DNA binding domain was fused to a partial peptide corresponding to the cytoplasmic domain of TACT427 protein (for example, a partial peptide containing the amino acid sequence represented by SEQ ID NO: 45). DNA encoding the chimeric protein, and (b) a chimeric protein obtained by fusing a simple virus-derived VP16 protein to the FLNA, FLNB, FLNC, NAV2, BTBD2, or MB3IL1 protein. By co-expressing the encoding DNA, an animal cell line expressing the repo-all-one gene is obtained by the action of the single hybrid. This cell line is cultured for a certain period of time in the presence of the test compound, the activity of the repo overnight gene product is measured, and the compound that reduces the activity is selected as a binding inhibitor. The animal cell strain can be cultured in the same manner as the culture of the above-mentioned transformant whose host is an animal cell. The activity of a reporter gene product (eg, CAT, luciferase, etc.) can be measured using a commercially available kit according to a known method.
選択された化合物のうち、 細胞毒性のある化合物、 およびレポ一夕一遺伝子産 物との相互作用などでレポ一夕一遺伝子産物自身の活性を阻害する化合物などは、 擬陽性化合物として除外する。  Among the selected compounds, cytotoxic compounds and compounds that inhibit the activity of the repo overnight gene product itself due to interaction with the repo overnight gene product are excluded as false positive compounds.
試験化合物としては、 例えばペプチド、 タンパク質、 抗体、 非ペプチド性化合 物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物組織抽出液、 血漿 などがあげられる。  Test compounds include, for example, peptides, proteins, antibodies, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.
本発明のスクリーニング用キットは、 FLNAタンパク質、 FLNBタンパク質、 FLNC タンパク質、 NAV2タンパク質、 BTBD2タンパク質または RAB3IL1タンパク質を含有 し、 さらに TACT427タンパク質またはその部分ペプチド （例、 細胞質側ドメイン に相当する部分ペプチドなど） を含有していてもよい。 また、 本発明のスクリー ニング用キットは、 本発明のタンパク質を産生する能力を有する細胞 （好ましく は、 本発明のタンパク質をコードする D NAで形質転換された形質転換体 （例、 酵母、 動物細胞などの細胞） ） を含有する。 該細胞は、 FLNAタンパク質、 FLNBタ ンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2タンパク質または RAB3IL1夕 ンパク質をコードする D NA、 および TACT427タンパク質 （例、 細胞質側ドメイ ンに相当する部分ペプチドなど） をコードする D NAで形質転換された形質転換 体であってもよい。 The screening kit of the present invention contains FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein, and further contains TACT427 protein or a partial peptide thereof (eg, a partial peptide corresponding to a cytoplasmic domain). It may be contained. In addition, the screening kit of the present invention includes cells capable of producing the protein of the present invention (preferably, a transformant transformed with a DNA encoding the protein of the present invention (eg, Cells such as yeast and animal cells)). The cells encode FLNA protein, FLNB protein, FLNC protein, DNA encoding NAV2 protein, BTBD2 protein or RAB3IL1 protein, and TACT427 protein (eg, a partial peptide corresponding to the cytoplasmic domain). The transformant may be a transformant transformed with DNA.
本発明のスクリ一ニング方法またはスクリーニング用キットを用いて得られる 化合物またはその塩は、 上記試験化合物 （例、 ペプチド、 タンパク質、 抗体、 非 ペプチド性化合物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物組 織抽出液、 血漿など） から選ばれた化合物であり、 例えば、 TACT427タンパク質 と、 FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention may be a test compound (eg, peptide, protein, antibody, non-peptide compound, synthetic compound, fermentation product, cell extract, Plant extract, animal tissue extract, plasma, etc.). For example, TACT427 protein, FLNA protein, FLNB protein, FLNC protein, NAV2 protein,
BTBD2タンパク質または RAB3IL1タンパク質との結合を阻害または促進する化合物 である。 A compound that inhibits or promotes binding to BTBD2 protein or RAB3IL1 protein.
該化合物の塩としては、 前記した本発明のタンパク質の塩と同様のものが用い られる。  As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.
本発明の複合体の形成を阻害する化合物は、 例えば、 癌 （例、 大腸癌、 乳癌、 肺癌、 前立腺癌、 食道癌、 胃癌、 肝臓癌、 胆道癌、 脾臓癌、 腎癌、 膀胱癌、 子宮 癌、 精巣癌、 卵巣癌、 甲状腺癌、 膝臓癌、 脳腫瘍、 血液腫瘍など） などの予防 - 治療剤として、 または癌細胞のアポトーシス促進剤として有用である。  Compounds that inhibit the formation of the complex of the present invention include, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterus) It is useful as a preventive-therapeutic agent for cancer, testicular cancer, ovarian cancer, thyroid cancer, knee cancer, brain tumor, blood tumor, etc.) or as an apoptosis promoter for cancer cells.
本発明の複合体の形成を促進する化合物は、 例えば、 神経変性疾患 （例、 アル ッハイマー病 (家族性アルツハイマー病、 若年性アルツハイマー病、 孤発性アル ッハイマー病など） などの予防 ·治療剤として、 または神経細胞のアポトーシス 阻害 （抑制） 剤として有用である。  The compound that promotes the formation of the complex of the present invention may be used as a prophylactic / therapeutic agent for neurodegenerative diseases (eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.)) Or as an agent for inhibiting (suppressing) apoptosis of nerve cells.
本発明のスクリーニング方法またはスクリーニング用キットを用いて得られる 化合物またはその塩を上述の予防 ·治療剤として使用する場合、 常套手段に従つ て製剤化することができる。  When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method.
例えば、 経口投与のための組成物としては、 固体または液体の剤形、 具体的に は錠剤 （糖衣錠、 フィルムコーティング錠を含む) 、 丸剤、 顆粒剤、 散剤、 カブ セル剤 （ソフトカプセル剤を含む） 、 シロップ剤、 乳剤、 懸濁剤などがあげられ る。 かかる組成物は自体公知の方法によって製造され、 製剤分野において通常用 いられる担体、 希釈剤もしくは賦形剤を含有するものである。 例えば、 錠剤用の 担体、 賦形剤としては、 乳糖、 でんぷん、 蔗糖、 ステアリン酸マグネシウムなど が用いられる。 For example, compositions for oral administration include solid or liquid dosage forms, such as tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). ), Syrups, emulsions, suspensions and the like. Such a composition is produced by a method known per se, and is commonly used in the pharmaceutical field. It contains a carrier, diluent or excipient. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
非経口投与のための組成物としては、 例えば、 注射剤、 坐剤などが用いられ、 注射剤は静脈注射剤、 皮下注射剤、 皮内注射剤、 筋肉注射剤、 点滴注射剤、 関節 内注射剤などの剤形を包含する。 かかる注射剤は、 自体公知の方法に従って、 例 えば、 上記化合物またはその塩を通常注射剤に用いられる無菌の水性もしくは油 性液に溶解、 懸濁または乳化することによって調製する。 注射用の水性液として は、 例えば、 生理食塩水、 ブドウ糖やその他の補助薬を含む等張液などが用いら れ、 適当な溶解補助剤、 例えば、 アルコール （例、 エタノール） 、 ポリアルコー ル （例、 プロピレングリコ一ル、 ポリエチレングリコール） 、 非イオン界面活性 剤 〔例、 ポリソルべ一ト 8 0、 H C O - 5 0 (polyoxyet ylene (50mol) adduct of hydrogenated cas tor oi l) 〕 などと併用してもよい。 油性液としては、 例 えば、 ゴマ油、 大豆油などが用いられ、 溶解補助剤として安息香酸ベンジル、 ベ ンジルアルコールなどを併用してもよい。 調製された注射液は、 通常、 適当なァ ンプルに充填される。 直腸投与に用いられる坐剤は、 上記化合物またはその塩を 通常の坐薬用基剤に混合することによって調製される。  Examples of compositions for parenteral administration include injections, suppositories, and the like. Injections are intravenous, subcutaneous, intradermal, intramuscular, intravenous, intraarticular. Dosage forms such as agents. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above compound or a salt thereof in a sterile aqueous or oily liquid usually used for injections. As an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants is used, and a suitable solubilizing agent, for example, alcohol (eg, ethanol), polyalcohol (eg, , Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyetylene (50mol) adduct of hydrogenated catalyst)], etc. Good. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in an appropriate sample. Suppositories used for rectal administration are prepared by mixing the above compound or a salt thereof with a usual suppository base.
上記の経口用または非経口用医薬組成物は、 活性成分の投与量に適合するよう な投薬単位の剤形に調製されることが好都合である。 かかる投薬単位の剤形とし ては、 錠剤、 丸剤、 カプセル剤、 注射剤 （アンプル） 、 坐剤などが例示され、 そ れぞれの投薬単位剤形当たり通常 5〜5 0 0 m g、 とりわけ注射剤では 5〜 1 0 0 m g、 その他の剤形では 1 0〜2 5 O m gの上記化合物が含有されていること が好ましい。  The above-mentioned oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit forms to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually 5 to 500 mg, especially It is preferable that the injection contains 5 to 100 mg of the above compound, and other dosage forms contain 10 to 25 mg of the above compound.
なお前記した各組成物は、 上記化合物との配合により好ましくない相互作用を 生じない限り他の活性成分を含有してもよい。  Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above compound.
このようにして得られる製剤は安全で低毒性であるので、 例えば、'ヒトまたは 温血動物 （例えば、 マウス、 ラット、 ゥサギ、 ヒッジ、 ブ夕、 ゥシ、 ゥマ、 トリ. ネコ、 ィヌ、 サル、 チンパンジーなど) に対して経口的にまたは非経口的に投与 することができる。 該化合物 （好ましくは結合阻害剤） またはその塩の投与量は、 その作用、 対象 疾患、 投与対象、 投与ルートなどにより差異はあるが、 例えば、 乳癌の治療の目 的で該化合物を経口投与する場合、 一般的に成人 （体重 60 kgとして） におい ては、 一日につき該化合物またはその塩を約 0. l〜100mg、 好ましくは約 1. 0〜50mg、 より好ましくは約 1. 0〜2 Omg投与する。 非経口的に投 与する場合は、 該化合物またはその塩の 1回投与量は投与対象.、 対象疾患などに よっても異なるが、 例えば、 乳癌の治療の目的で該化合物を注射剤の形で通常成 人 （体重 6 O kgとして） に投与する場合、 一日につき該化合物またはその塩を 約 0. 01〜3 Omg、 好ましくは約 0. 1〜2 Omg、 より好ましくは約 0. 1〜1 Omgを癌病変部に注射により投与するのが好都合である。 他の動物の場 合も、 体重 6 O kg当たりに換算した量を投与することができる。 The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or in warm-blooded animals (for example, mice, rats, puppies, higgs, bushus, puppies, pumas, birds, cats, dogs). , Monkeys, chimpanzees, etc.) orally or parenterally. The dose of the compound (preferably a binding inhibitor) or a salt thereof varies depending on its action, target disease, subject to be administered, administration route and the like. For example, the compound is orally administered for the purpose of treating breast cancer. In general, for an adult (assuming a body weight of 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 mg of the compound or a salt thereof per day is used. Omg is administered. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the subject to be administered, the target disease, and the like, but, for example, the compound is in the form of an injection for the purpose of treating breast cancer. Usually, when administered to an adult (assuming a body weight of 6 O kg), the compound or a salt thereof is administered in an amount of about 0.01 to 3 Omg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 0 Omg per day. It is convenient to administer 1 Omg to the cancerous lesion by injection. In the case of other animals, the dose can be administered in terms of the weight per 6 O kg of body weight.
〔2〕 本発明の複合体の定量 [2] Quantification of the complex of the present invention
本発明の複合体を特異的に認識する抗体は、 被検液中の本発明の複合体の定量. 特にサンドイッチ免疫測定法による定量などに使用することができる。  An antibody that specifically recognizes the complex of the present invention can be used for quantification of the complex of the present invention in a test solution, particularly for quantification by a sandwich immunoassay.
すなわち、 本発明は、  That is, the present invention
(i) 本発明の抗体と、 被検液および標識化された本発明の複合体とを競合的に 反応させ、 該抗体に結合した標識化された本発明の複合体の割合を測定すること を特徴とする被検液中の本発明の複合体の定量法、 および  (i) reacting the antibody of the present invention with a test solution and the labeled complex of the present invention competitively, and measuring the ratio of the labeled complex of the present invention bound to the antibody; A method for quantifying the complex of the present invention in a test solution, which comprises:
(ii) 被検液と担体上に不溶化した本発明の抗体および標識化された本発明の別 の抗体とを同時あるいは連続的に反応させたのち、 不溶化担体上の標識剤の活性 を測定することを特徴とする被検液中の本発明の複合体の定量法を提供する。 上記 （ii) の定量法においては、 2種類の抗体が本発明の複合体の異なる部位 を認識する抗体であることが望ましい。  (ii) After reacting the test solution with the antibody of the present invention insolubilized on the carrier and another labeled antibody of the present invention simultaneously or continuously, the activity of the labeling agent on the insolubilized carrier is measured. A method for quantifying the complex of the present invention in a test solution is provided. In the quantitative method (ii), it is desirable that the two kinds of antibodies are antibodies that recognize different sites of the complex of the present invention.
また、 本発明の複合体に対するモノクローナル抗体 （以下、 本発明のモノクロ —ナル抗体と称する場合がある） を用い本発明の複合体の定量を行なえるほか、 組織染色等による検出を行なうこともできる。 これらの目的には、 抗体分子その ものを用いてもよく、 また、 抗体分子の F(ab')2 、 Fab'、 あるいは Fab画分を用 いてもよい。 本発明の抗体を用いる本発明の複合体の定量法は、 特に制限されるべきもので はなく、 被測定液中の抗原量 （例えば、 タンパク質量） に対応した抗体、 抗原も しくは抗体一抗原複合体の量を化学的または物理的手段により検出し、 これを既 知量の抗原を含む標準液を用いて作製した標準曲線より算出する測定法であれば、 いずれの測定法を用いてもよい。 例えば、 ネフロメトリー、 競合法、 ィムノメト リック法およびサンドイッチ法が好適に用いられるが、 感度、 特異性の点で、 後 述するサンドイッチ法を用いるのが特に好ましい。 In addition, the complex of the present invention can be quantified using a monoclonal antibody against the complex of the present invention (hereinafter, sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. . For these purposes, the antibody molecule itself may be used, or F (ab ′) 2 , Fab ′, or Fab fraction of the antibody molecule may be used. The method for quantifying the complex of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any method that detects the amount of the antigen complex by chemical or physical means and calculates this from a standard curve prepared using a standard solution containing a known amount of antigen can be used. Is also good. For example, nephrometry, a competition method, an immunometric method and a sandwich method are suitably used, but it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity.
標識物質を用いる測定法に用いられる標識剤としては、 例えば、 放射性同位元 素、 酵素、 蛍光物質、 発光物質などが用いられる。 放射性同位元素としては、 例 えば、 〔1251〕 、 〔1311〕 、 〔¾〕 、 〔14c〕 などが用いられる。 上記酵素としては、 安定で比活性の大きなものが好ましく、 例えば、 /3—ガラクトシダーゼ、 ]3—グ ルコシダ一ゼ、 アルカリフォスファターゼ、 パーォキシダーゼ、 リンゴ酸脱水素 酵素などが用いられる。 蛍光物質としては、 例えば、 フルォレスカミン、 フルォ レツセンイソチオシァネートなどが用いられる。 発光物質としては、 例えば、 ル ミノ一ル、 ルミノール誘導体、 リレシフェリン、 ルシゲニンなどが用いられる。 さ らに、 抗体あるいは抗原と標識剤との結合にピオチン—アビジン系を用いること もできる。 As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, [125 1], [131 1], [¾], and the like are used [14 c]. As the above-mentioned enzyme, a stable enzyme having a large specific activity is preferable. For example, / 3-galactosidase,] 3-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiosinate and the like are used. As the luminescent substance, for example, luminol, luminol derivative, reluciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
抗原あるいは抗体の不溶化に当っては、 物理吸着を用いてもよく、 また通常夕 ンパク質あるいは酵素等を不溶化、 固定化するのに用いられる化学結合を用いる 方法でもよい。 担体としては、 ァガロース、 デキストラン、 セルロースなどの不 溶性多糖類、 ポリスチレン、 ポリアクリルアミド、 シリコン等の合成樹脂、 ある いはガラス等が挙げられる。  For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
サンドイッチ法においては不溶化した本発明のモノクローナル抗体に被検液を 反応させ （1次反応） 、 さらに標識化した別の本発明のモノクローナル抗体を反 応させ （2次反応） たのち、 不溶化担体上の標識剤の活性を測定することにより 被検液中の本発明の複合体量を定量することができる。 1次反応と 2次反応は逆 の順序に行っても、 また、 同時に行なってもよいし時間をずらして行なってもよ レ^ 標識化剤および不溶化の方法は前記のそれらに準じることができる。 また、  In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the complex of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or staggered. The labeling agent and the method of insolubilization may be the same as those described above. . Also,
-法による免疫測定法において、 固相用抗体あるいは標識用抗体に用 いられる抗体は必ずしも 1種類である必要はなく、 測定感度を向上させる等の目 的で 2種類以上の抗体の混合物を用いてもよい。 -For immunoassay by solid-phase or labeling antibodies The type of antibody used is not necessarily one type, and a mixture of two or more types of antibodies may be used for the purpose of improving measurement sensitivity and the like.
本発明のサンドィツチ法による本発明の複合体の測定法においては、 1次反応 と 2次反応に用いられる本発明のモノクローナル抗体は、 本発明の複合体の結合 する部位が相異なる抗体が好ましく用いられる。 すなわち、 1次反応および 2次 反応に用いられる抗体は、 例えば、 2次反応で用いられる抗体が、 TACT427の C 端部によって形成されている複合体部分を認識する場合、 1次反応で用いられる 抗体は、 好ましくはその部位以外、 例えば TACT427の N端部によって形成されて レ る複合体部分を認識する抗体が用いられる。  In the method for measuring the complex of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site of the complex of the present invention. Can be That is, the antibody used in the primary reaction and the secondary reaction is used in the primary reaction, for example, when the antibody used in the secondary reaction recognizes the complex portion formed by the C-terminal of TACT427. As the antibody, an antibody that recognizes a site other than the site, for example, a complex formed by the N-terminal of TACT427 is used.
本発明のモノクローナル抗体をサンドイッチ法以外の測定システム、 例えば、 競合法、 ィムノメトリック法あるいはネフロメトリーなどに用いることができる c 競合法では、 被検液中の抗原と標識抗原とを抗体に対して競合的に反応させた のち、 未反応の標識抗原（F ) と、 抗体と結合した標識抗原 （B ) とを分離し (B / F分離） 、 B， Fいずれかの標識量を測定し、 被検液中の抗原量を定量す る。 本反応法には、 抗体として可溶性抗体を用い、 B /F分離をポリエチレング リコール、 前記抗体に対する第 2抗体などを用いる液相法、 および、 第 1抗体と して固相化抗体を用いるか、 あるいは、 第 1抗体は可溶性のものを用い第 2抗体 として固相化抗体を用いる固相化法とが用いられる。 In a measurement system other than the sandwich method using the monoclonal antibody of the present invention, for example, a competition method, an immunometric method, or a c- competition method that can be used in nephrometry, an antigen in a test solution and a labeled antigen are used for the antibody. After reacting competitively, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), and the amount of labeled B or F is measured. Then, the amount of the antigen in the test solution is determined. In this reaction method, a soluble antibody is used as an antibody, polyethylene glycol is used for B / F separation, a liquid phase method using a second antibody against the antibody, and a solid phase antibody is used as the first antibody. Alternatively, an immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used.
ィムノメトリック法では、 被検液中の抗原と固相化抗原とを一定量の標識化抗 体に対して競合反応させた後固相と液相を分離するか、 あるいは、 被検液中の抗 原と過剰量の標識化抗体とを反応させ、 次に固相化抗原を加え未反応の標識化抗 体を固相に結合させたのち、 固相と液相を分離する。 次に、 いずれかの相の標識 量を測定し被検液中の抗原量を定量する。  In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of the label in either phase is measured to determine the amount of the antigen in the test solution.
また、 ネフロメトリーでは、 ゲル内あるいは溶液中で抗原抗体反応の結果生じ た不溶性の沈降物の量を測定する。 被検液中の抗原量が僅かであり、 少量の沈降 物しか得られない場合にもレーザーの散乱を利用するレーザーネフロメトリーな どが好適に用いられる。  In nephelometry, the amount of insoluble sediment resulting from the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
これら個々の免疫学的測定法を本発明の定量方法に適用するにあたっては、 特 別の条件、 操作等の設定は必要とされない。 それぞれの方法における通常の条件、 2004/014527 In applying these individual immunoassays to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The usual conditions for each method, 2004/014527
59 操作法に当業者の通常の技術的配慮を加えて本発明の複合体の測定系を構築すれ ばよい。 これらの一般的な技術手段の詳細については、 総説、 成書などを参照す ることができる。 59 A measurement system for the complex of the present invention may be constructed by adding ordinary technical considerations to those skilled in the art to the operation method. For details of these general technical means, reference can be made to reviews and written documents.
例えば、 入江 寛編 「ラジオィムノアツセィ」 （講談社、 昭和 4 9年発行） 、 入江 寛編 「続ラジオィムノアツセィ」 （講談社、 昭和 5 4年発行） 、 石川栄治 ら編 「酵素免疫測定法」 （医学書院、 昭和 5 3年発行） 、 石川栄治ら編 「酵素免 疫測定法」 （第 2版） （医学書院、 昭和 5 7年発行） 、 石川栄治ら編 「酵素免疫 測定法」 （第 3版） （医学書院、 昭和 6 2年発行） 、 「Methods in  For example, edited by Hiro Irie, "Radio Nonotsusei" (Kodansha, published in Showa 49), edited by Hiroshi Irie, "Radio Imunoatsushi" (Kodansha, published in 1954), Eiji Ishikawa et al. "Measurement Method" (Medical Shoin, published in 1958), edited by Eiji Ishikawa et al., "Enzyme Immunoassay" (Second Edition) (Medical School, published in 1977), Eiji Ishikawa, et al., "Enzyme Immunoassay" (3rd edition) (Issue Shoin, published in 1962), "Methods in
ENZYM0L0GY」 Vol. 70 (Imunochemical Techni ues (Part A) ) , 同書 Vol. ENZYM0L0GY ”Vol. 70 (Imunochemical Technologies (Part A)), Ibid.
73 (I匪謹 chemical Techniaues (Part B) )、 同書 Vol. 74 (I画 nochemical Techniaues (Part 0 ) ^ 同書 Vol. 84 (Inununocheiiiical Techniques (Part 73 (I married chemical techniaues (Part B)), ibid.
D: Selected Immunoassays) ) ^ 同書 Vol. 92 (Inmunoc emical Techniques (Part E: Monoclonal Ant ibodies and General Immunoassay Methods) ) , 同書 Vol. 121 (I匪 nochemical Techniaues (Part I :Hybridoma Technology and Monoclonal Ant ibodies) ) (以上、 アカデミックプレス社発行）などを参照することができる。 以上のようにして、 本発明の抗体を用いることによって、 本発明の複合体を感 度良く定量することができる。 D: Selected Immunoassays)) ^ Ibid.Vol. 92 (Inmunocemical Techniques (Part E: Monoclonal Ant ibodies and General Immunoassay Methods)), Ibid.Vol. 121 (I married nochemical Techniaues (Part I: Hybridoma Technology and Monoclonal Ant ibodies)) (Above, published by Academic Press). As described above, the complex of the present invention can be quantified with good sensitivity by using the antibody of the present invention.
さらには、 本発明の抗体を用いて本発明の複合体の濃度を定量することによつ て、 本発明の複合体の濃度の増加が検出された場合、 例えば癌 （例、 大腸癌、 乳 癌、 肺癌、 前立腺癌、 食道癌、 胃癌、 肝臓癌、 胆道癌、 脾臓癌、 腎癌、 膀胱癌、， 子宮癌、 精巣癌、 卵巣癌、 甲状腺癌、 膝臓癌、 脳腫瘍、 血液腫瘍など） などであ る、 または将来罹患する可能性が高いと診断することができる。 一方、 本発明の 抗体を用いて本発明の複合体の濃度を定量することによって、 本発明の複合体の 濃度の低下が検出された場合、 例えば神経変性疾患 （例、 アルツハイマー病 （家 族性アルツハイマー病、 若年性アルツハイマー病、 孤発性アルツハイマー病な ど） などである、 または将来罹患する可能性が高いと診断すること力できる。 また、 本発明の抗体は、 体液や組織などの被検体中に存在する本発明の複合体 を検出するために使用することができる。 また、 本発明の複合体を精製するため に使用する抗体カラムの作製、 精製時の各分画中の本発明の複合体の検出、 被検 細胞内における本発明の複合体の挙動の分析などのために使用することができる。 〔3〕 本発明の抗体を含有する医薬 Furthermore, when an increase in the concentration of the complex of the present invention is detected by quantifying the concentration of the complex of the present invention using the antibody of the present invention, for example, cancer (eg, colon cancer, breast cancer) Cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, ovarian cancer, thyroid cancer, knee cancer, brain tumor, blood tumor, etc.) Etc. or can be diagnosed as likely to be affected in the future. On the other hand, when a decrease in the concentration of the complex of the present invention is detected by quantifying the concentration of the complex of the present invention using the antibody of the present invention, for example, a neurodegenerative disease (eg, Alzheimer's disease (family (Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.) or the likelihood of future disease is high. In addition, it can be used to detect the complex of the present invention present in the antibody, and the production of an antibody column used for purifying the complex of the present invention, and the present invention in each fraction at the time of purification. Complex detection, test It can be used for analysis of the behavior of the complex of the present invention in cells. [3] A drug containing the antibody of the present invention
本発明の複合体の形成を阻害する活性を有する TACT427タンパク質に対する抗 体は、 アポトーシス促進作用を有し、 例えば癌 （例、 大腸癌、 乳癌、 肺癌、 前立 腺癌、 食道癌、 胃癌、 肝臓癌、 胆道癌、 脾臓癌、 腎癌、 膀胱癌、 子宮癌、 精巣癌、 卵巣癌、 甲状腺癌、 膝臓癌、 脳腫瘍、 血液腫瘍など） などの予防 ·治療剤として、 また、 癌細胞のアポト一シス促進剤として使用することもできる。  An antibody against the TACT427 protein having an activity of inhibiting the formation of the complex of the present invention has an apoptosis-promoting effect and includes, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver). Cancer, biliary tract, spleen, kidney, bladder, uterus, testes, ovary, thyroid, knee, brain, blood, etc.) It can also be used as a monocis accelerator.
一方、 本発明の複合体の形成を促進する活性を有する TACT427タンパク質に対 する抗体は、 アポトーシス抑制作用を有し、 例えば神経変性疾患 （例、 アルッハ イマ一病 (家族性アルツハイマー病、 若年性アルツハイマー病、 孤発性アルッハ イマ一病など） などの予防 ·治療剤として、 また、 神経細胞のアポト一シス阻害 (抑制） 剤として使用することもできる。  On the other hand, an antibody against the TACT427 protein having an activity of promoting the formation of the complex of the present invention has an apoptosis-suppressing effect, and is used for, for example, neurodegenerative diseases (eg, Alhaima disease (familial Alzheimer disease, juvenile Alzheimer disease, It can also be used as a prophylactic / therapeutic agent for diseases such as Alzheimer's disease and sporadic Alhaima disease, and as an inhibitor of neuronal apoptosis (suppression).
上記の本発明の抗体を含有する上記疾患の予防 ·治療剤、 促進剤、 阻害剤など は低毒性であり、 そのまま液剤として、 または適当な剤型の医薬組成物として、 ヒトまたは哺乳動物 （例、 ラット、 ゥサギ、 ヒッジ、 ブタ、 ゥシ、 ネコ、 ィヌ、 サルなど） に対して経口的または非経口的 （例、 血管内投与、 皮下投与など） に 投与することができる。 ワクチンとして定法に従って投与することもできる。 本発明の抗体は、 それ自体を投与しても良いし、 または適当な医薬組成物とし て投与しても良い。 投与に用いられる医薬組成物としては、 本発明の抗体および その塩と薬理学的に許容され得る担体、 希釈剤もしくは賦形剤とを含むものであ つても良い。 このような医薬組成物は、 経口または非経口投与に適する剤形とし て提供される。  The above-mentioned prophylactic / therapeutic agents, accelerators, inhibitors and the like for the above-mentioned diseases containing the antibody of the present invention have low toxicity, and are used as they are as liquids or as pharmaceutical compositions in appropriate dosage forms. It can be administered orally or parenterally (eg, intravascular, subcutaneous, etc.) to rats, rabbits, rabbits, sheep, pigs, mice, cats, dogs, monkeys, etc.). It can also be administered as a vaccine according to the usual methods. The antibody of the present invention may be administered as it is, or may be administered as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the antibody of the present invention or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.
非経口投与のための組成物としては、 例えば、 注射剤、 坐剤、 ワクチン等が用 いられ、 注射剤は静脈注射剤、 皮下注射剤、 皮内注射剤、 筋肉注射剤、 点滴注射 剤等の剤形を包含しても良い。 このような注射剤は、 公知の方法に従って調製で きる。 注射剤の調製方法としては、 例えば、 上記本発明の抗体またはその塩を通 常注射剤に用いられる無菌の水性液、 または油性液に溶解、 懸濁または乳化する ことによって調製できる。 注射用の水性液としては、 例えば、 生理食塩水、 ブド ゥ糖やその他の補助薬を含む等張液等が用いられ、 適当な溶解補助剤、 例えば、 アルコール (例、 エタノール） 、 ポリアルコール （例、 プロピレングリコール、 ポリエチレングリコ一ル） 、 非イオン界面活性剤 〔例、 ポリソルべ一ト 8 0、 H C O— 5 0 (.polyoxyethylene (50mol) adduct of hydrogenated castor oil) j 等と併用してもよい。 油性液としては、 例えば、 ゴマ油、 大豆油等が用いられ、 溶解補助剤として安息香酸ベンジル、 ベンジルアルコール等を併用してもよい。 調製された注射液は、 適当なアンプルに充填されることが好ましい。 直腸投与に 用いられる坐剤は、 上記抗体またはその塩を通常の坐薬用基剤に混合することに よって調製されても良い。 Compositions for parenteral administration include, for example, injections, suppositories, vaccines, and the like. May be included. Such an injection can be prepared according to a known method. Injection preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody of the present invention or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. Aqueous liquids for injection include, for example, saline, bud Isotonic solutions containing sugar and other adjuvants are used, and suitable solubilizers, for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants Agents [eg, polysorbate 80, HCO-50 (.polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. A suppository for rectal administration may be prepared by mixing the antibody or a salt thereof with a conventional suppository base.
経口投与のための組成物としては、 固体または液体の剤形、 具体的には錠剤 Compositions for oral administration include solid or liquid dosage forms, specifically tablets
(糖衣錠、 フィルムコーティング錠を含む） 、 丸剤、 顆粒剤、 散剤、 カプセル剤 (ソフトカプセル剤を含む） 、 シロップ剤、 乳剤、 懸濁剤等が挙げられる。 この ような組成物は公知の方法によって製造され、 製剤分野において通常用いられる 担体、 希釈剤もしくは賦形剤を含有していても良い。 錠剤用の担体、 賦形剤とし ては、 例えば、 乳糖、 でんぷん、 蔗糖、 ステアリン酸マグネシウムが用いられる。 上記の非経口用または経口用医薬組成物は、 活性成分の投与量に適合するよう な投薬単位の剤形に調製されることが好都合である。 このような投薬単位の剤形 としては、 例えば、 錠剤、 丸剤、 カプセル剤、 注射剤 （アンプル） 、 坐剤が挙げ られる。 抗体の含有量としては、 投薬単位剤形当たり通常 5〜5 0 O m g程度、 とりわけ注射剤では 5〜1 0 O m g程度、 その他の剤形では 1 0〜2 5 0 m g程 度の上記抗体が含有されていることが好ましい。 (Including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions and the like. Such compositions are prepared by known methods and may contain carriers, diluents or excipients commonly used in the field of formulation. As carriers and excipients for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used. The above-mentioned parenteral or oral pharmaceutical composition is conveniently prepared in the form of a dosage unit so as to match the dosage of the active ingredient. Such dosage unit forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The antibody content is usually about 5 to 50 mg per dosage unit dosage form, especially about 5 to 100 mg for injections, and about 10 to 250 mg for other dosage forms. Is preferably contained.
本発明の抗体を含有する上記予防 ·治療剤、 促進剤、 阻害剤の投与量は、 投与. 対象、 対象疾患、 症状、 投与ルートなどによっても異なるが、 例えば、 成人の乳 癌の治療 ·予防のために使用する場合には、 本発明の抗体を 1回量として、 通常 0. 0 1 - 2 O m g / k g体重程度、 好ましくは 0. 1〜1 0 m g / k g体重程度、 さらに好ましくは 0. l〜5 m g /k g体重程度を、 1日 1〜5回程度、 好まし くは 1日 1〜3回程度、 静脈注射により投与するのが好都合である。 他の非経口 投与および経口投与の場合もこれに準ずる量を投与することができる。 症状が特 に重い場合には、 その症状に応じて増量してもよい。 本発明の抗体は、 それ自体または適当な医薬組成物として投与することができ る。 上記投与に用いられる医薬組成物は、 上記抗体またはその塩と薬理学的に許 容され得る担体、 希釈剤もしくは賦形剤とを含むものである。 かかる組成物は、 経口または非経口投与 （例、 血管内注射、 皮下注射など） に適する剤形として提 供される。 The dosage of the above-mentioned prophylactic / therapeutic agent, promoter, or inhibitor containing the antibody of the present invention is administered. The amount varies depending on the subject, target disease, symptoms, administration route, and the like. When the antibody of the present invention is used for a single dose, it is usually about 0.01 to 2 Omg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 10 mg / kg body weight. It is convenient to administer 0.1-5 mg / kg body weight by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent dose can be administered. If the symptoms are particularly severe, the dose may be increased accordingly. The antibody of the present invention can be administered by itself or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a composition is provided as a dosage form suitable for oral or parenteral administration (eg, intravascular injection, subcutaneous injection, etc.).
なお前記した各組成物は、 上記抗体との配合により好ましくない相互作用を生 じない限り他の活性成分を含有してもよい。  Each of the above-mentioned compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
〔4〕 本発明の複合体の用途 [4] Uses of the composite of the present invention
TACT427タンパク質は癌組織で発現が亢進しており、 さらに、 TACT427タンパク 質の機能 （活性、 発現など） を阻害すると癌細胞がアポトーシスを起こす。 また、 FLNAタンパク質、 FLNBタンパク質、 FLNCタンパク質、 NAV2タンパク質、 BTBD2夕 ンパク質または RAB3IL1タンパク質は、 TACT427タンパク質の細胞質側ドメインに 結合し、 TACT427夕ンパク質の機能を担っていると考えられる。  The expression of TACT427 protein is increased in cancer tissues, and when the function (activity, expression, etc.) of TACT427 protein is inhibited, cancer cells undergo apoptosis. Further, it is considered that FLNA protein, FLNB protein, FLNC protein, NAV2 protein, BTBD2 protein or RAB3IL1 protein binds to the cytoplasmic domain of TACT427 protein and plays a role of TACT427 protein.
従って、 本発明の複合体は、 例えば癌組織での発現亢進など、 癌や神経変性疾 患などでの発現変動が予想されるため、 癌 （例、 大腸癌、 乳癌、 肺癌、 前立腺癌、 食道癌、 胃癌、 肝臓癌、 胆道癌、 脾臓癌、 腎癌、 膀胱癌、 子宮癌、 精巣癌、 卵巣 癌、 甲状腺癌、 滕臓癌、 脳腫瘍、 血液腫瘍など） 、 神経変性疾患 （例、 アルッハ イマ一病 (家族性アルツハイマー病、 若年性アルツハイマー病、 孤発性アルッハ ィマ一病など） の診断マーカ一として利用することが出来る。  Therefore, since the complex of the present invention is expected to fluctuate in expression in cancer and neurodegenerative diseases such as enhanced expression in cancer tissues, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus) Cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testis cancer, ovarian cancer, thyroid cancer, thyroid cancer, brain cancer, blood tumor, etc., neurodegenerative diseases (eg, Alhaima) It can be used as a diagnostic marker for a single disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.).
本明細書において、 塩基やアミノ酸などを略号で表示する場合、 IUPAC- IUB Commiss ion on Biochemical Nomenclature による略号あるいは当該分野におけ る慣用略号に基づくものであり、 その例を下記する。 またアミノ酸に関し光学異 性体があり得る場合は、 特に明示しなければ L体を示すものとする。  In the present specification, bases, amino acids and the like are indicated by abbreviations based on the abbreviations by IUPAC-IUB Communication on Biochemical Nomenclature or commonly used abbreviations in the field, and examples thereof are described below. When there is an optical isomer for an amino acid, the L-form is indicated unless otherwise specified.
D NA デォキシリポ核酸  D NA Deoxylipo nucleic acid
c D NA 相補的デォキシリポ核酸  c DNA Complementary deoxyliponucleic acid
A アデニン  A adenine
T チミン  T thymine
G グァニン C G Guanin C
RNA リポ核酸  RNA liponucleic acid
mRNA メッセンジャーリポ核酸  mRNA messenger liponucleic acid
dATP デォキシアデノシン三リン酸  dATP Deoxyadenosine triphosphate
dTTP デォキシチミジン三リン酸  dTTP Deoxythymidine triphosphate
dGTP デォキシグアノシン三リン酸  dGTP Deoxyguanosine triphosphate
dCTP デォキシシチジン三リン酸  dCTP Deoxycytidine triphosphate
ATP アデノシン三リン酸  ATP adenosine triphosphate
EDTA エチレンジァミン四酢酸  EDTA ethylenediaminetetraacetic acid
EGTA エチレングリコール-ビス- （ベー夕アミノエチルェ一テ ル） 四酢酸  EGTA Ethylene glycol-bis- (behino aminoethyl ether) tetraacetic acid
SDS ドデシル硫酸ナトリウム  SDS sodium dodecyl sulfate
G 1 y グリシン  G 1 y Glycine
A 1 a ァラニン  A 1 a Alanin
Va 1 バリン  Va 1 Valine
Leu ロイシン  Leu Leucine
I 1 e  I 1 e
S e r セリン  S e r serine
Th r スレオニン  Th r threonine
Cy s  Cy s
Me t メチォニン  Me t Methionin
G 1 u グルタミン酸  G 1 u Glutamic acid
As p  As p
L y s リジン  Lys lysine
A r g アルギニン  A r g Arginine
H i s ヒスチジン  H is histidine
P h e フエ二ルァラニン  P h e feniralanin
Ty r チロシン  Ty r tyrosine
T r p トリブトファン P r o プロリン T rp Tribute fan Pro proline
A s n ァスパラギン  A s n asparagine
G 1 n グルタミン  G 1 n Glutamine
p G 1 u ピログルタミン酸  p G 1 u pyroglutamic acid
S e c セレノシスティン （selenocysteine)  S e c selenocysteine
また、 本明細 :中で繁用される置換基、 保護基および試薬を下記の記号で表記 する。 Further, this specification: referred substituents frequently used medium, the protecting groups and reagents the following symbols.
Me メチル基  Me methyl group
E t ェチル基  Etethyl group
B u ブチル基  B u butyl group
P h フエニル基  P h phenyl group
TC チアゾリジン— 4 (R) —力ルポキサミド基  TC thiazolidine—4 (R) —caprolupamide group
T o s P—トルエンスルフォニル  T os P—Toluenesulfonyl
CHO ホルミル  CHO Holmill
B z 1  B z 1
Cl2-Bzl 2, 6—ジクロロべンジル Cl 2 -Bzl 2, 6-dichlorobenzyl
Bom ベンジルォキシメチル  Bom benzyloxymethyl
Z ベンジルォキシカルポニル  Z benzyloxycarponyl
C 1— Z 2—クロ口べンジルォキシカルボニル  C 1— Z 2—Black benzyloxycarbonyl
B r -Z 2一ブロモベンジルォキシカルポニル  B r -Z 2 -Bromobenzyloxycarponyl
B o c t一ブトキシカルポニル  B oc t-butoxycarponyl
DNP ジニトロフエニル  DNP dinitrophenyl
T r t 卜 Uチル  T r t u U chill
B um t一ブトキシメチル  Bum t-butoxymethyl
Fmo c N— 9一フルォレニルメトキシカルボニル  Fmo c N—9-Fluorenylmethoxycarbonyl
HOB t  HOB t
HOOB t 3, 4ージヒドロー 3—ヒドロキシ一 4—ォキソ一  HOOB t 3,4 dihydro-3-hydroxy-1 4-oxo-1
1, 2, 3—べンゾ卜リアジン  1, 2, 3—Benzotriazine
HONB 1 -ヒドロキシ -5-ノルポルネン- 2, 3 -ジカルポキシィ ドヽ 本願明細書の配列表の配列番号は、 以下の配列を示す。 HONB 1-Hydroxy-5-norporene-2,3-dicarboxide The sequence numbers in the sequence listing in the present specification indicate the following sequences.
〔配列番号： 1〕  [SEQ ID NO: 1]
FLJ20539のアミノ酸配列を示す。  2 shows the amino acid sequence of FLJ20539.
〔配列番号： 2〕  [SEQ ID NO: 2]
配列番号： 1で表されるアミノ酸配列を有する FLJ20539をコードする DNAの塩 基配列を示す。  This shows the base sequence of DNA encoding FLJ20539 having the amino acid sequence represented by SEQ ID NO: 1.
〔配列番号： 3〕  [SEQ ID NO: 3]
FLJ20539をコードする全長遺伝子を含む DNAの塩基配列を示す。  The nucleotide sequence of a DNA containing the full length gene encoding FLJ20539 is shown.
〔配列番号： 4〕  [SEQ ID NO: 4]
MP50177のアミノ酸配列を示す。  2 shows the amino acid sequence of MP50177.
〔配列番号： 5〕  [SEQ ID NO: 5]
配列番号： 4で表されるアミノ酸配列を有する hCP50177をコードする DNAの塩 基配列を示す。  This shows the base sequence of DNA encoding hCP50177 having the amino acid sequence represented by SEQ ID NO: 4.
〔配列番号： 6〕  [SEQ ID NO: 6]
hCP50177をコードする全長遺伝子を含む DNAの塩基配列を示す。  The nucleotide sequence of a DNA containing the full length gene encoding hCP50177 is shown.
〔配列番号： 7〕  [SEQ ID NO: 7]
MP1762319のアミノ酸配列を示す。  2 shows the amino acid sequence of MP1762319.
〔配列番号： 8〕  [SEQ ID NO: 8]
配列番号： 7で表されるアミノ酸配列を有する MP1762319をコードする DNAの 塩基配列を示す。  This shows the base sequence of DNA encoding MP1762319 having the amino acid sequence represented by SEQ ID NO: 7.
〔配列番号： 9〕  [SEQ ID NO: 9]
MP1762319をコードする全長遺伝子を含む DNAの塩基配列を示す。  The nucleotide sequence of the DNA containing the full length gene encoding MP1762319 is shown.
〔配列番号： 1 0〕  [SEQ ID NO: 10]
FLJ 13515のアミノ酸配列を示す。  3 shows the amino acid sequence of FLJ 13515.
〔配列番号： 1 1〕  [SEQ ID NO: 11]
配列番号： 1 0で表されるアミノ酸配列を有する FLJ 13515をコードする DNAの 塩基配列を示す。  This shows the base sequence of DNA encoding FLJ13515 having the amino acid sequence represented by SEQ ID NO: 10.
〔配列番号： 1 2〕 FU13515をコードする全長遺伝子を含む DNAの塩基配列を示す。 [SEQ ID NO: 1 2] Shows the nucleotide sequence of DNA containing the full-length gene encoding FU13515.
〔配列番号： 13〕  [SEQ ID NO: 13]
参考例 2、 参考例 19、 参考例 20および参考例 21で用いられたァンチセン スオリゴヌクレオチドの塩基配列を示す。  The base sequences of the antisense oligonucleotides used in Reference Examples 2, 19, 20, and 21 are shown.
〔配列番号： 14〕  [SEQ ID NO: 14]
参考例 2、 参考例 19、 参考例 20および参考例 21で用いられたァンチセン スオリゴヌクレオチドの塩基配列を示す。  The base sequences of the antisense oligonucleotides used in Reference Examples 2, 19, 20, and 21 are shown.
〔配列番号： 15〕  [SEQ ID NO: 15]
TACT427 - Aのアミノ酸配列を示す。  2 shows the amino acid sequence of TACT427-A.
〔配列番号： 16〕  [SEQ ID NO: 16]
配列番号： 15で表されるアミノ酸配列を有する TACT427- Aをコードする DN Aの塩基配列を示す。  This shows the base sequence of DNA encoding TACT427-A having the amino acid sequence represented by SEQ ID NO: 15.
〔配列番号： 17〕  [SEQ ID NO: 17]
TACT427-A2のアミノ酸配列を示す。  2 shows the amino acid sequence of TACT427-A2.
〔配列番号： 18〕  [SEQ ID NO: 18]
配列番号： 17で表されるアミノ酸配列を有する TACT427- A2をコードする DN Aの塩基配列を示す。  This shows the nucleotide sequence of DNA encoding TACT427-A2 having the amino acid sequence represented by SEQ ID NO: 17.
〔配列番号： 19〕  [SEQ ID NO: 19]
TACT427- Aおよび TACT427- A2をコードする全長遺伝子を含む DNAの塩基配列を示 す。  Shows the nucleotide sequence of DNA containing the full-length gene encoding TACT427-A and TACT427-A2.
〔配列番号： 20〕  [SEQ ID NO: 20]
TACT427- Bのアミノ酸配列を示す。  2 shows the amino acid sequence of TACT427-B.
〔配列番号： 21〕  [SEQ ID NO: 21]
配列番号： 20で表されるアミノ酸配列を有する TACT427-Bをコードする DNAの 塩基配列を示す。  This shows the base sequence of DNA encoding TACT427-B having the amino acid sequence represented by SEQ ID NO: 20.
〔配列番号： 22〕 —  [SEQ ID NO: 22] —
TACT427 - B2のアミノ酸配列を示す。  2 shows the amino acid sequence of TACT427-B2.
〔配列番号： 23〕  [SEQ ID NO: 23]
配列番号： 22で表されるアミノ酸配列を有する TACT427- B2をコードする DNA の塩基配列を示す。 DNA encoding TACT427-B2 having the amino acid sequence represented by SEQ ID NO: 22 Shows the base sequence.
〔配列番号： 24〕  [SEQ ID NO: 24]
TACT427- Bおよび TACT427- B2をコードする全長遺伝子を含む DNAの塩基配列を示 す。  The nucleotide sequence of a DNA containing the full-length gene encoding TACT427-B and TACT427-B2 is shown.
〔配列番号： 2 5〕  [SEQ ID NO: 25]
TACT427-Cのアミノ酸配列を示す。  2 shows the amino acid sequence of TACT427-C.
〔配列番号： 2 6〕  [SEQ ID NO: 26]
配列番号： 2 5で表されるアミノ酸配列を有する TACT427- Cをコードする DNAの 塩基配列を示す。  This shows the base sequence of DNA encoding TACT427-C having the amino acid sequence represented by SEQ ID NO: 25.
〔配列番号： 2 7〕  [SEQ ID NO: 27]
TACT427- C2のアミノ酸配列を示す。  2 shows the amino acid sequence of TACT427-C2.
〔配列番号： 2 8〕  [SEQ ID NO: 28]
配列番号： 2 7で表されるアミノ酸配列を有する TACT427- C2をコードする DNA の塩基配列を示す。  This shows the base sequence of DNA encoding TACT427-C2 having the amino acid sequence represented by SEQ ID NO: 27.
〔配列番号： 2 9〕  [SEQ ID NO: 29]
TACT427-Cおよび TACT427- C2をコードする全長遺伝子を含む DNAの塩基配列を示 す。  The nucleotide sequence of the DNA containing the full-length gene encoding TACT427-C and TACT427-C2 is shown.
〔配列番号： 3 0〕  [SEQ ID NO: 30]
参考例 3で用いられたプライマー 1の塩基配列を示す。  3 shows the base sequence of primer 1 used in Reference Example 3.
〔配列番号： 3 1〕  [SEQ ID NO: 31]
参考例 3で用いられたプライマー 2の塩基配列を示す。  3 shows the base sequence of primer 2 used in Reference Example 3.
〔配列番号： 3 2〕  [SEQ ID NO: 32]
参考例 4で用いられたプライマー 3の塩基配列を示す。  7 shows the base sequence of primer 3 used in Reference Example 4.
〔配列番号： 3 3〕  [SEQ ID NO: 33]
参考例 4で用いられたプライマー 4の塩基配列を示す。  7 shows the base sequence of primer 4 used in Reference Example 4.
〔配列番号： 34〕  [SEQ ID NO: 34]
参考例 5で用いられたプライマ一 5の塩基配列を示す。  7 shows the nucleotide sequence of Primer 15 used in Reference Example 5.
〔配列番号： 3 5〕  [SEQ ID NO: 35]
参考例 5で用いられたプライマ一 6の塩基配列を示す。 〔配列番号： 36〕 7 shows the nucleotide sequence of primer 16 used in Reference Example 5. [SEQ ID NO: 36]
参考例 6、 参考例 7、 参考例 8、 および参考例 20で用いられたプライマ一 7 の塩基配列を示す。  The base sequence of primer 17 used in Reference Example 6, Reference Example 7, Reference Example 8, and Reference Example 20 is shown.
〔配列番号： 37〕  [SEQ ID NO: 37]
参考例 6、 参考例 7、 参考例 8、 および参考例 20で用いられたプライマー 8 の塩基配列を示す。  The base sequence of the primer 8 used in Reference Examples 6, 7, 8, and 20 is shown.
〔配列番号： 38〕  [SEQ ID NO: 38]
参考例 6、 参考例 7、 参考例 8、 および参考例 20で用いられた TanManプロ一 ブ 1の塩基配列を示す。  The base sequence of TanMan probe 1 used in Reference Example 6, Reference Example 7, Reference Example 8, and Reference Example 20 is shown.
〔配列番号： 39〕  [SEQ ID NO: 39]
参考例 10で用いられたプライマー 9の塩基配列を示す。  13 shows the base sequence of primer 9 used in Reference Example 10.
〔配列番号： 40〕  [SEQ ID NO: 40]
参考例 10で用いられたプライマ一 10の塩基配列を示す。  13 shows the nucleotide sequence of Primer-10 used in Reference Example 10.
〔配列番号： 41〕  [SEQ ID NO: 41]
参考例 11で用いられたペプチド 1のアミノ酸配列を示す。  14 shows the amino acid sequence of peptide 1 used in Reference Example 11.
〔配列番号： 42〕  [SEQ ID NO: 42]
参考例 1 1で用いられたペプチド 2のァミノ酸配列を示す。  2 shows the amino acid sequence of peptide 2 used in Reference Example 11;
〔配列番号： 43〕  [SEQ ID NO: 43]
参考例 1 1で用いられたペプチド 3のアミノ酸配列を示す。  7 shows the amino acid sequence of peptide 3 used in Reference Example 11.
〔配列番号： 44〕  [SEQ ID NO: 44]
参考例 19、 参考例 20および参考例 21で用いられたセンスオリゴヌクレオ チドの塩基配列を示す。  The nucleotide sequences of the sense oligonucleotides used in Reference Examples 19, 20, and 21 are shown.
〔配列番号： 45〕  [SEQ ID NO: 45]
TACT427-Aのアミノ酸配列中、 第 956番目〜第 1024番目のアミノ酸配列を示す。 〔配列番号： 46〕  The amino acid sequence at positions 956 to 1024 in the amino acid sequence of TACT427-A is shown. [SEQ ID NO: 46]
ヒト FLNAのアミノ酸配列を示す。  2 shows the amino acid sequence of human FLNA.
〔配列番号： 47〕  [SEQ ID NO: 47]
ヒト FLNBのアミノ酸配列を示す。  2 shows the amino acid sequence of human FLNB.
〔配列番号： 48〕 ヒト FLNCのアミノ酸配列を示す。 [SEQ ID NO: 48] 2 shows the amino acid sequence of human FLNC.
〔配列番号： 49〕  [SEQ ID NO: 49]
ヒ卜 NAV2のアミノ酸配列を示す。  2 shows the amino acid sequence of human NAV2.
〔配列番号： 50〕  [SEQ ID NO: 50]
ヒト BTBD2のアミノ酸配列を示す。  2 shows the amino acid sequence of human BTBD2.
〔配列番号： 51〕  [SEQ ID NO: 51]
ヒト RAB3IL1のアミノ酸配列を示す。  2 shows the amino acid sequence of human RAB3IL1.
〔配列番号： 52〕  [SEQ ID NO: 52]
配列番号： 46で表されるアミノ酸配列を有する FLNAをコードする DNAの塩基 配列を示す。  This shows the base sequence of DNA encoding FLNA having the amino acid sequence represented by SEQ ID NO: 46.
〔配列番号： 53〕  [SEQ ID NO: 53]
配列番号： 47で表されるアミノ酸配列を有する FLNBをコードする DNAの塩基 配列を示す。  This shows the base sequence of DNA encoding FLNB having the amino acid sequence represented by SEQ ID NO: 47.
〔配列番号： 54〕  [SEQ ID NO: 54]
配列番号： 48で表されるアミノ酸配列を有する FLNCをコードする DNAの塩基 配列を示す。  This shows the base sequence of DNA encoding FLNC having the amino acid sequence represented by SEQ ID NO: 48.
〔配列番号： 55〕  [SEQ ID NO: 55]
配列番号： 49で表されるアミノ酸配列を有する NAV2をコードする DNAの塩基 配列を示す。  This shows the base sequence of DNA encoding NAV2 having the amino acid sequence represented by SEQ ID NO: 49.
〔配列番号： 56〕  [SEQ ID NO: 56]
配列番号： 50で表されるアミノ酸配列を有する BTBD2をコードする DNAの塩基 配列を示す。  This shows the base sequence of DNA encoding BTBD2 having the amino acid sequence represented by SEQ ID NO: 50.
〔配列番号： 57〕  [SEQ ID NO: 57]
配列番号： 51で表されるアミノ酸配列を有する RAB3IL1をコードする DNAの塩 基配列を示す。  This shows the base sequence of DNA encoding RAB3IL1 having the amino acid sequence represented by SEQ ID NO: 51.
〔配列番号： 58〕  [SEQ ID NO: 58]
参考例 26で用いられたプライマ一 1 1の塩基配列を示す。  26 shows the nucleotide sequence of primer 111 used in Reference Example 26.
〔配列番号： 59〕  [SEQ ID NO: 59]
参考例 26で用いられたプライマー 12の塩基配列を示す。 〔配列番号： 6 0〕 26 shows the base sequence of primer 12 used in Reference Example 26. [SEQ ID NO: 60]
参考例 2 6で用いられたプライマー 1 3の塩基配列を示す。  The base sequence of primer 13 used in Reference Example 26 is shown.
〔配列番号： 6 1〕  [SEQ ID NO: 61]
参考例 2 6で用いられたプライマ一 1 4の塩基配列を示す。  The base sequence of the primer 14 used in Reference Example 26 is shown.
後述の参考例 4で得られた形質転換体 Escherichia col i T0P10/47427A/pCR- The transformant Escherichia col i T0P10 / 47427A / pCR- obtained in Reference Example 4 described later
Bluntll- T0P0は、 2002年 12月 3日から茨城県つくば市東 1丁目 1番地 1 中央第 6 (郵 便番号 305- 8566) の独立行政法人産業技術総合研究所 特許生物寄託センタ一に 寄託番号 FERM BP- 8253として寄託されている。 Bluntll-T0P0 has been deposited with the National Institute of Advanced Industrial Science and Technology (AIST) Patent Depositary No. 1 at 1-1, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture since December 3, 2002 (postal number 305-8566). Deposited as BP-8253.
後述の参考例 4で得られた形質転換体 Escherichia col i TOP10/47427B/pCR- Blunt l l- T0P0は、 2002年 12月 3日から茨城県つくば市東 1丁目 1番地 1 中央第 6 (郵 便番号 305- 8566) の独立行政法人産業技術総合研究所 特許生物寄託セン夕一に 寄託番号 FERM ΒΡ-8254として寄託されている。  The transformant Escherichia col i TOP10 / 47427B / pCR-Blunt l-T0P0 obtained in Reference Example 4 described below has been used since December 3, 2002, 1-1 1-1 Higashi, Tsukuba-shi, Ibaraki Prefecture 1 Chuo No. 6 (mail service No. 305-8566) has been deposited with the National Institute of Advanced Industrial Science and Technology (AIST) under the deposit number FERM ΒΡ-8254.
後述の参考例 5で得られた形質転換体 Escherichia col i TOP10/47427C/pC -Bl unt l l-TOPOは、 2002年 12月 3日から茨城県つくば巿東 1丁目 1番地 1 中央第 6 (郵便 番号 305- 8566) の独立行政法人産業技術総合研究所 特許生物寄託センターに寄 託番号 FERM BP- 8255として寄託されている。 実 施 例  The transformant Escherichia col i TOP10 / 47427C / pC -Bl unt l l-TOPO obtained in Reference Example 5 described below has been used since December 3, 2002, 1-1 1-1 Tsukuba East, Ibaraki Prefecture 1 Central No. 6 ( It has been deposited with the National Institute of Advanced Industrial Science and Technology, Japan Patent Organism Depositary with the postal code 305-8566) under the deposit number FERM BP-8255. Example
以下に参考例および実施例を挙げて本発明を更に具体的に説明するが、 本発明 はそれに限定されるものではない。 なお、 大腸菌を用いての遺伝子操作法は、 Molecular cloning, 2nd, Cold Spring Harbor Lab. Press, 1989年に記載の方 法に準じて行った。 参考例 1 Hereinafter, the present invention will be described more specifically with reference to Reference Examples and Examples, but the present invention is not limited thereto. The gene manipulation using Escherichia coli, Molecular Cloning, 2 nd, was carried out in accordance with Cold Spring Harbor Lab. Press, how the 1989. Reference example 1
遺伝子発現解析  Gene expression analysis
乳癌組織および肺癌組織で特異的に発現亢進している遺伝子群を明らかにする ため、 乳癌組織 8例、 正常乳房組織 4例から抽出された total RNA (表 1 ) 、 およ び肺癌組織 4例、 正常肺組織 5例から抽出された total RNA (表 3 ) を材料とし、 ol igonucleot ide microarray (Human Genome U95A, U95B, U95C, U95D, U95E;Affymetrix¾) を用いて遺伝子発現解析を行った。 Total RNA extracted from eight cases of breast cancer tissue, four cases of normal breast tissue (Table 1), and four cases of lung cancer tissue to identify genes that are specifically up-regulated in breast and lung cancer tissues Using total RNA (Table 3) extracted from 5 normal lung tissues as a material, ol igonucleotide ide microarray (Human Genome U95A, U95B, U95C, U95D, U95E; Affymetrix;) was used to analyze gene expression.
実験方法は、 Aifymetrix社の実験手引き書 （Expression analysis technical manual) に従った。 その結果、 乳癌組織 （lot.0009- 192- 00101、 lot.0009-192- 00120、 lot.0009— 192— 00153、 lot.0009-192-00178) および肺癌組織 (lot.0009- 192-00122, lot.0011-192-01293, lot.0011-192-01297) においてそれぞれ正常 乳房組織、 正常肺組織に比べ、 （1) FU20539遺伝子 （配列番号： 2) 、  The experiment method was in accordance with the Aifymetrix experiment manual (Expression analysis technical manual). As a result, breast cancer tissue (lot.0009-192-00101, lot.0009-192-00120, lot.0009—192—00153, lot.0009-192-00178) and lung cancer tissue (lot.0009-192-00122, lot.0011-192-01293, lot.0011-192-01297), compared to normal breast tissue and normal lung tissue, respectively. (1) FU20539 gene (SEQ ID NO: 2),
FLJ20539関連遺伝子である hCP50177遺伝子 (配列番号 : 5) 、 F 20539関連遺伝 子である MP1762319遺伝子 （配列番号： 8) ならびに FLJ13515遺伝子 （配列番 号： 1 1) 、 および （2) 後述の参考例 4または参考例 5で得られた TACT427-A遺 伝子 （配列番号： 1 6) 、 TACT427- A2遺伝子 （配列番号： 1 8) 、 TACT427- B遺 伝子 （配列番号： 2 1) 、 TACT427- B2遺伝子 （配列番号： 2 3) 、 TACT427-C遺 伝子 （配列番号： 26) ならびに TACT427-C2遺伝子 （配列番号： 2 8) の発現亢 進が検出された （表 2および表 4) 。 表 The hCP50177 gene (SEQ ID NO: 5), which is a FLJ20539-related gene, the MP1762319 gene (SEQ ID NO: 8), which is a F20539-related gene, and the FLJ13515 gene (SEQ ID NO: 11), and (2) Reference Example 4 described below Alternatively, TACT427-A gene (SEQ ID NO: 16), TACT427-A2 gene (SEQ ID NO: 18), TACT427-B gene (SEQ ID NO: 21), TACT427- Enhanced expression of B2 gene (SEQ ID NO: 23), TACT427-C gene (SEQ ID NO: 26) and TACT427-C2 gene (SEQ ID NO: 28) were detected (Tables 2 and 4). table
RNAを抽出した組織 販売兀  Tissue from which RNA was extracted
乳がん組織 (lot.0009-192-00101) BioClinical Partners 社 Breast cancer tissue (lot.0009-192-00101) BioClinical Partners
乳がん組織 (lot.0009-192-00120) BioClinical Partners 社 Breast cancer tissue (lot.0009-192-00120) BioClinical Partners
乳がん組織 (lot.0009-192-00153) BioClinical Partners 社 Breast cancer tissue (lot.0009-192-00153) BioClinical Partners
乳がん組織 (lot.0009-192-00155) BioClinical Partners 社 Breast cancer tissue (lot.0009-192-00155) BioClinical Partners
乳がん組織 (lot.0009-192-00157) BioClinical Partners 社 Breast cancer tissue (lot.0009-192-00157) BioClinical Partners
乳がん組織 (lot.0009-192-00178) BioClinical Partners 社 Breast cancer tissue (lot.0009-192-00178) BioClinical Partners
乳がん組織 (lot.0011-192-01284) BioClinical Partners 社 Breast cancer tissue (lot.0011-192-01284) BioClinical Partners
乳がん組織 (lot.0011-192-01287) BioClinical Partners 社 Breast cancer tissue (lot.0011-192-01287) BioClinical Partners
正常乳房組織 (lot.0008-192-00404) BioClinical Partners 社 Normal breast tissue (lot.0008-192-00404) BioClinical Partners
正常乳房組織 (lot.0008-192-00422) BioClinical Partners 社 Normal breast tissue (lot.0008-192-00422) BioClinical Partners
正常乳房組織 (lot.0009-192-00153) BioClinical Partners 社 Normal breast tissue (lot.0009-192-00153) BioClinical Partners
正常乳房組織 (lot.0011-192-01286) BioClinical Partners 社 表 2 Normal breast tissue (lot.0011-192-01286) BioClinical Partners Table 2
組織 遺伝子発現 Tissue Gene expression
乳がん組織 (lot. 0009-192-00101) 3. 7 Breast cancer tissue (lot.0009-192-00101) 3.7
乳がん組織 (lot. 0009-192-00120) 9. 0 Breast cancer tissue (lot.0009-192-00120) 9.0
乳がん組織 (lot. 0009-192-00153) 2. 1 Breast cancer tissue (lot.0009-192-00153) 2.1
乳がん組織 (lot. 0009-192-00155) ND Breast cancer tissue (lot.0009-192-00155) ND
乳がん組織 (lot. 0009-192-00157) 0. 54 Breast cancer tissue (lot.0009-192-00157) 0.54
乳がん組織 (lot. 0009-192-00178) 2. 1 Breast cancer tissue (lot.0009-192-00178) 2.1
乳がん組織 (lot. 0011-192-01284) ND Breast cancer tissue (lot.0011-192-01284) ND
乳がん組織 (lot. 0011-192-01287) ND Breast cancer tissue (lot.0011-192-01287) ND
正常乳房組織 (lot. 0008-192-00404) ND Normal breast tissue (lot.0008-192-00404) ND
正常乳房組織 (lot. 0008-192-00422) ND Normal breast tissue (lot.0008-192-00422) ND
正常乳房組織 (lot. 0009-192-00153) 1. 6 Normal breast tissue (lot.0009-192-00153) 1.6
正常乳房組織 (lot. 0011-192-01286) 1. 2 Normal breast tissue (lot.0011-192-01286) 1.2
遺伝子発現量は、 ol igonucleot ide microarrayで発現が 検出された全遺伝子の発現量の中央値を 1として標準化した ND; not detected 表 3  The gene expression level was normalized using the median expression level of all genes whose expression was detected by the oligonucleotide microarray as 1, ND; not detected.Table 3
RNAを抽出した組織 販売兀  Tissue from which RNA was extracted
肺がん組織 (lot. 0009· -192-00122) BioCl inical Partners 社 肺がん組織 (lot. 0011· -192-01285) BioCl inical Partners 社 肺がん組織 (lot. 0011· -192-01293) BioCl inical Partners 社 肺がん組織 (lot. 0011· -192-01297) BioCl inical Partners 社 正常肺組織 (lot. 0009· -192-00150) BioCl inical Partners 社 正常肺組織 (lot. 0009- -192-00168) BioCl inical Partners 社 正常肺組織 (lot. 0011. -192-01283) BioCl inical Partners 社 正常肺組織 (lot. 0011· -192-01285) BioCl inical Partners 社 正常肺組織 (lot. 0011· -192-01297) BioCl inical Partners—社 表 4 Lung cancer tissue (lot. 0009-192-00122) BioCl inical Partners Lung cancer tissue (lot. 0011 (lot. 0011 -192-01297) Normal lung tissue from BioCl inical Partners (lot.0009 -192-00150) Normal lung tissue from BioCl inical Partners (lot. Tissue (lot.0011.-192-01283) BioCl inical Partners Normal lung tissue (lot.0011 · -192-01285) BioCl inical Partners Normal lung tissue (lot.0011 Table 4
組織— 遺伝子発現 Tissue-gene expression
肺がん組織 (lot. 0009- 192-00122) 2. 8 Lung cancer tissue (lot.0009-192-00122) 2.8
肺がん組織 (lot. 0011- 192-01285) 0. 67 Lung cancer tissue (lot.0011-192-01285) 0.67
肺がん組織 (lot. 0011- 192-01293) 1. 3 Lung cancer tissue (lot.0011-192-01293) 1.3
肺がん組織 (lot. 0011- 192-01297) 1. 5 Lung cancer tissue (lot.0011-192-01297) 1.5
正常肺組織 (lot. 0009- 192-00150) ND Normal lung tissue (lot.0009-192-00150) ND
正常肺組織 (lot. 0009- 192-00168) 0. 38 Normal lung tissue (lot.0009-192-00168) 0.38
正常肺組織 (lot. 0011- 192-01283) ND Normal lung tissue (lot.0011-192-01283) ND
正常肺組織 (lot. 0011- 192-01285) ND Normal lung tissue (lot.0011- 192-01285) ND
正常肺組織 (lot. 0011- 192-01297) ND Normal lung tissue (lot.0011-192-01297) ND
遺伝子発現量は、 ol igonucleot ide microarrayで発現が  The gene expression level was determined by ol igonucleotide microarray.
検出された全遺伝子の発現量の中央値を 1として標準化した  The median expression level of all detected genes was standardized as 1.
ND; not detected 参考例 2  ND; not detected Reference Example 2
ヒト肺癌細胞株のアポトーシス誘発  Apoptosis induction of human lung cancer cell line
TACT427タンパク質遺伝子の発現を抑制することにより、 ヒ卜肺癌細胞株のァ ポト一シスが誘発されるか否かを調べた。  It was investigated whether suppression of the expression of the TACT427 protein gene would induce apoptosis in a human lung cancer cell line.
まず、 アメリカンタイプカルチャーコレクション （ATCC) より購入したヒト非 小細胞肺癌細胞株 NCI- H460を、 RPMI- 1640培地 （25mM HEPES含有） （Invitrogen 社） に牛胎仔血清 （ATCC) を 10%加えた培地で懸濁し、 1ゥエル当たり 4000個の 細胞密度で 96穴平底組織培養プレート （BDファルコン社） に播種した。 5%炭酸 ガス気流中、 37°Cで一晩培養した後、 アンチセンスオリゴヌクレオチドをトラン スフエクシヨンした。  First, NCI-H460, a human non-small cell lung cancer cell line purchased from the American Type Culture Collection (ATCC), was added to RPMI-1640 medium (containing 25 mM HEPES) (Invitrogen) and supplemented with 10% bovine fetal serum (ATCC). And seeded on a 96-well flat bottom tissue culture plate (BD Falcon) at a cell density of 4000 cells / well. After culturing overnight at 37 ° C. in a 5% carbon dioxide gas stream, antisense oligonucleotides were transfected.
具体的には、 (1) FU20539遺伝子 (配列番号 : 2 ) 、 FLJ20539関連遺伝子で ある hCP50177遺伝子 (配列番号 : 5 ) 、 FLJ20539関連遺伝子である MP1762319遺 伝子 （配列番号： 8 ) ならびに FLJ13515遺伝子 （配列番号： 1 1 ) 、 および (2) 後述の参考例 4または参考例 5で得られた TACT427- A遺伝子 （配列番号： 1 6 ) 、 TACT427- A2遺伝子 （配列番号： 18) 、 TACT427-B遺伝子 （配列番号： 2 1 ) 、 TACT427- B2遺伝子 （配列番号： 23) 、 TACT427- C遺伝子 （配列番号： 2 6) ならびに TACT427-C2遺伝子 （配列番号： 28) のタンパク質コード領域配列 または 3 ' 非翻訳領域にハイプリダイズするアンチセンスオリゴヌクレオチド配 列 （配列番号： 1 3) を設計後、 phosphorothioate化オリゴヌクレオチドを合成 し、 HP LC精製して導入実験に用いた （以下、 アンチセンスオリゴヌクレオチ ドと略する） 。 コントロールとしては、 配列番号： 13で示される塩基配列のリ バース配列 (配列番号： 14) を同様に phosphorothioate化し、 HPLC精製して用 いた （以下、 コントロールオリゴヌクレオチドと略する） 。 Specifically, (1) the FU20539 gene (SEQ ID NO: 2), the hCP50177 gene (SEQ ID NO: 5), which is a FLJ20539-related gene, the MP1762319 gene (SEQ ID NO: 8), which is a FLJ20539-related gene, and the FLJ13515 gene ( SEQ ID NOs: 1 1), and (2) TACT427-A gene (SEQ ID NO: 16), TACT427-A2 gene (SEQ ID NO: 18), TACT427-B gene (SEQ ID NO: 21) obtained in Reference Example 4 or Reference Example 5 described below Antibodies that hybridize to the protein coding region sequence or 3 'untranslated region of the TACT427-B2 gene (SEQ ID NO: 23), TACT427-C gene (SEQ ID NO: 26) and TACT427-C2 gene (SEQ ID NO: 28) After designing a sense oligonucleotide sequence (SEQ ID NO: 13), a phosphorothioated oligonucleotide was synthesized, purified by HP LC, and used for introduction experiments (hereinafter abbreviated as antisense oligonucleotide). As a control, the reverse sequence (SEQ ID NO: 14) of the nucleotide sequence represented by SEQ ID NO: 13 was similarly phosphorothioated and purified by HPLC (hereinafter abbreviated as control oligonucleotide).
ァンチセンスオリゴヌクレオチドまたはコントロールォリゴヌクレオチドを、 Opt i -MEM (Invitrogen社） で希釈し、 さらに FuGENE6試薬 (Roche Diagnostics 社） を 4倍量 （4/2L/ zgオリゴヌクレオチド） の割合で加えた混合液を室温で 30 分間放置した。 このオリゴヌクレオチド溶液を、 1ゥエル当たり の割合で プレートに添加した。 オリゴヌクレオチドの終濃度は 140nMとなるよう調整した 上記の条件で更に 3日間培養した後、 Cell Death Detection ELISAPLUSキットAntisense oligonucleotides or control oligonucleotides were diluted with Opti-MEM (Invitrogen), and mixed with FuGENE6 reagent (Roche Diagnostics) at a ratio of 4 times (4 / 2L / zg oligonucleotide). The solution was left at room temperature for 30 minutes. This oligonucleotide solution was added to the plate at a rate of 1 μl. The final concentration of the oligonucleotide was adjusted to 140 nM. After culturing for another 3 days under the above conditions, Cell Death Detection ELISA PLUS kit
(Roche Diagnostics社） を用いて、 添付プロトコールに従い上記オリゴヌクレ ォチドのアポト一シス誘導活性を測定した。 The apoptosis-inducing activity of the above oligonucleotides was measured using (Roche Diagnostics) according to the attached protocol.
その結果、 ァンチセンスォリゴヌクレオチドは陰性対照として用いたコント口 ールオリゴヌクレオチドに比べ、 約 2.8倍のアポ卜一シス誘導活性を示し、 統計 学的に有意な差 （P=0.0005) を示した （表 5) 。 表 5 アポ卜一シス誘導活性 （A4。5— A49 平均値 標準偏差 As a result, antisense oligonucleotide showed about 2.8 times the apoptosis-inducing activity as compared to the control oligonucleotide used as a negative control, showing a statistically significant difference (P = 0.0005). (Table 5). Table 5 apo Bok one cis-inducing activity (A 4 5 -. A 49 mean standard deviation
ブランク 0. 297 0. 053 Blank 0.297 0.053
コント口ール才リゴヌクレ才チド 0. 764 0. 096 Cont's Mouth Talent Rigonucle Taide 0.7764 0.096
(配列番号： 1 4)  (SEQ ID NO: 14)
ァンチセンス才リゴヌクレ才チド 1. 57 0. 093 (配列番号： 1 3) 参考例 3 Antisense Gengo 1.57 0.093 (SEQ ID NO: 13) Reference Example 3
アンチセンスオリゴヌクレオチド導入による NCI-H460のアポトーシス誘導 アンチセンスオリゴヌクレオチド投与により、 （1) FLJ20539遺伝子 （配列番 号 ·· 2) 、 FU20539関連遺伝子である MP50177遺伝子 （配列番号： 5) 、  Induction of apoptosis of NCI-H460 by introduction of antisense oligonucleotide By administration of antisense oligonucleotide, (1) FLJ20539 gene (SEQ ID NO: 2), FU20539-related gene MP50177 gene (SEQ ID NO: 5),
FLJ20539関連遺伝子である MP1762319遺伝子 (配列番号： 8) ならびに FU13515 遺伝子 （配列番号： 11) 、 および （2) 後述の参考例 4または参考例 5で得ら れた TACT427- A遺伝子 （配列番号： 16) 、 TACT427- A2遺伝子 （配列番号： 1 8 ) 、 TACT427- B遺伝子 （配列番号： 21) 、 TACT427- B2遺伝子 （配列番号： 2 3) 、 TACT427-C遺伝子 （配列番号： 26) ならびに TACT427-C2遺伝子 （配列番 号： 28) の発現量が低下するか否か調べた。  The MP1762319 gene (SEQ ID NO: 8) and the FU13515 gene (SEQ ID NO: 11), which are FLJ20539-related genes, and (2) the TACT427-A gene (SEQ ID NO: 16) obtained in Reference Example 4 or Reference Example 5 described below. ), TACT427-A2 gene (SEQ ID NO: 18), TACT427-B gene (SEQ ID NO: 21), TACT427-B2 gene (SEQ ID NO: 23), TACT427-C gene (SEQ ID NO: 26) and TACT427- It was examined whether the expression level of the C2 gene (SEQ ID NO: 28) was reduced.
参考例 2で用いたヒト非小細胞肺癌細胞株 NCI-H460を参考例 2と同じ培地に懸 濁し、 1ゥエル当たり 24， 000個の細胞密度で 24穴平底組織培養プレート （BDファ ルコン社） に播種した。 5%炭酸ガス気流中、 37°Cで一晩培養した後、 アンチセ ンスォリゴヌクレオチドとコント口ールォリゴヌクレオチドをトランスフエクシ ヨンした。 オリゴヌクレオチド溶液の添加量は 1ゥエル当たり 390 xLとし、 オリ ゴヌクレオチドの終濃度は 200nMとなるよう調整した。 トランスフエクション後、 5%炭酸ガス気流中、 37 で 24時間培養を継続した後に RNeasy Mini Total RNA Kit (QIAGEN社） を用いてトータル RNAを抽出した。 約 400 ngのトータル RNAを踌 型として、 TaaMan Reverse Transcription Reagents (Applied Biosystems社） を用いて添付プロトコールに従い逆転写反応を行った。 トータル RNAにして 10 ng に相当する cDNAを鐯型とし、 2種類のプライマー 〔プライマー 1 (配列番号： 3 0) およびプライマ一 2 (配列番号： 31) 〕 と SYBR Green PCR Master Mix (Applied Biosystems社) を用いて、 (1) FLJ20539遺伝子 (配列番号 : 2) 、 FU20539関連遺伝子である hCP50177遺伝子 （配列番号： 5) 、 FLJ20539関連遺伝 子である P1762319遺伝子 （配列番号： 8) ならびに FU13515遺伝子 （配列番 号： 11) 、 および （2) 後述の参考例 4で得られた TACT427-A遺伝子 （配列番 号： 16) 、 TACT427- A2遺伝子 （配列番号： 18) 、 TACT427-B遺伝子 （配列番 号： 21 ) 、 TACT427- B2遺伝子 （配列番号： 23) 、 TACT427- C遺伝子 （配列番 号： 26) ならびに TACT427-C2遺伝子 （配列番号： 28) の発現コピー数を測定 した。 The human non-small cell lung cancer cell line NCI-H460 used in Reference Example 2 was suspended in the same medium as in Reference Example 2, and a 24-well flat bottom tissue culture plate (BD Falcon) was used at a cell density of 24,000 cells / well. Seeded. After culturing overnight at 37 ° C. in a 5% carbon dioxide gas stream, antisense oligonucleotide and control oligonucleotide were transfected. The addition amount of the oligonucleotide solution was adjusted to 390 xL / well, and the final concentration of the oligonucleotide was adjusted to 200 nM. After transfection, culturing was continued for 24 hours at 37 in a 5% carbon dioxide gas stream, and then total RNA was extracted using RNeasy Mini Total RNA Kit (QIAGEN). Using about 400 ng of total RNA as a template, a reverse transcription reaction was performed using TaaMan Reverse Transcription Reagents (Applied Biosystems) according to the attached protocol. A cDNA corresponding to 10 ng of total RNA was designated as type II, and two types of primers (primer 1 (SEQ ID NO: 30) and primer 1 (SEQ ID NO: 31)) and SYBR Green PCR Master Mix (Applied Biosystems) ), The FLJ20539 gene (SEQ ID NO: 2), the hCP50177 gene (SEQ ID NO: 5), which is a FU20539-related gene, the P1762319 gene (SEQ ID NO: 8), which is a FLJ20539-related gene, and the FU13515 gene (SEQ ID NO: 8) No .: 11), and (2) TACT427-A gene (SEQ ID NO: 16), TACT427-A2 gene (SEQ ID NO: 18), TACT427-B gene (SEQ ID NO: 18) obtained in Reference Example 4 described below. No .: 21), TACT427-B2 gene (SEQ ID NO: 23), TACT427-C gene (SEQ ID NO: 26), and expression copy number of TACT427-C2 gene (SEQ ID NO: 28) were measured.
同量の铸型 cDNA中に含まれる β -ァクチン遺伝子発現量を TaaMan β -act in Control Reagents (Applied Biosystems社) を用いて測定し内部標準とした。 7 ンチセンスオリゴヌクレオチドをトランスフエクシヨンしない場合には、 上記 10 遺伝子の発現量は i3-ァクチン遺伝子の発現量の 0.10%であったのに対し、 配列 番号： 13で示されるアンチセンスオリゴヌクレオチド投与群では 0.046%であ り統計学的に有意 （P≤0.05) な発現量低下が認められた。 一方、 コントロール オリゴヌクレオチド （配列番号： 14) 投与群では 0.088%であり非トランスフ ェクション群と比べて統計学的に有意な発現量低下は認められなかった。 この結 果より、 上記 10遺伝子の発現量の低下によりヒト肺癌細胞株のアポトーシスが誘 発されたことが示された。 参考例 4  The expression amount of β-actin gene contained in the same amount of type III cDNA was measured using TaaMan β-act in Control Reagents (Applied Biosystems) and used as an internal standard. 7 When the antisense oligonucleotide was not transfected, the expression level of the 10 genes was 0.10% of the expression level of the i3-actin gene, whereas the antisense oligonucleotide represented by SEQ ID NO: 13 was administered. In the group, the expression level was 0.046%, indicating a statistically significant (P≤0.05) decrease in the expression level. On the other hand, the control oligonucleotide (SEQ ID NO: 14) administration group was 0.088%, and no statistically significant decrease in the expression level was observed as compared with the non-transfection group. The results showed that a decrease in the expression level of the above 10 genes induced apoptosis of the human lung cancer cell line. Reference example 4
ヒト脳由来タンパク質 TACT427-A、 TACT427-A2 TACT427- Bおよび TACT427- B2を コードする cDNAのクローニングと塩基配列の決定  Cloning and sequencing of cDNAs encoding human brain-derived proteins TACT427-A, TACT427-A2, TACT427-B and TACT427-B2
ヒト脳 Marathon- Ready cDNA (CL0NTECH社） を銬型とし、 2種のプライマ一 〔プ ライマ一 3 (配列番号： 32) およびプライマー 4 (配列番号： 33) 〕 を用い て PCR反応を行った。 該反応における反応液の組成は、 上記 cDNAl xlを錶型とし て使用し、 PfuTurbo DNA Polymerase (STRATAGENE社） 6· 25U、 プライマー 3 (配 列番号： 32) およびプライマー 4 (配列番号： 33) を各 1.0 xM、 dNTPsを 200 M、 および Pfu Buffer (STRATAGENE社） を 5 1加え、 50 ilの液量とした。 PCR反応は、 95°C · 1分の後、 95 · 10秒、 55°C · 30秒、 72°C · 6分のサイクルを 40回繰り返した。 該 PCR反応産物を PCR Purification Kit (QIAGEN社） を用いて 精製した。 これを Zero Blunt T0P0 PCRクロ一ニングキット （Invitrogen社） の 処方に従いプラスミドベクター pCR-BluntII-T0P0 (Invitrogen社) へサブクロー エングした。 これを大腸菌 T0P10に導入し、 cDNAを持つクローンをカナマイシン を含む LB寒天培地中で選択した。 個々のクローンの配列を解析した結果、 配列 番号： 1 6および配列番号： 2 1で表される cDNAの塩基配列をそれぞれ得た。 配列番号： 3で表される FU20539全長遺伝子塩基配列の 1〜160番目および 2483 〜2755番目の塩基配列を、 配列番号： 1 6で表される塩基配列の 5 ' 端および 3 ' 端にそれぞれ付加した塩基配列を、 配列番号： -1 9に示す。 Using human brain Marathon-Ready cDNA (CL0NTECH) as type III, PCR was carried out using two types of primers (primer 3 (SEQ ID NO: 32) and primer 4 (SEQ ID NO: 33)). The composition of the reaction solution used in the reaction was as follows. Using cDNAl xl as type I, 6 · 25 U of PfuTurbo DNA Polymerase (STRATAGENE), primer 3 (SEQ ID NO: 32) and primer 4 (SEQ ID NO: 33) 1.0 xM of each, 200 M of dNTPs, and 51 of Pfu Buffer (STRATAGENE) were added to make a liquid volume of 50 il. In the PCR reaction, a cycle of 95 ° C for 1 minute, 95 ° C for 10 seconds, 55 ° C for 30 seconds, and 72 ° C for 6 minutes was repeated 40 times. The PCR reaction product was purified using a PCR Purification Kit (QIAGEN). This was subcloned into a plasmid vector pCR-BluntII-T0P0 (Invitrogen) according to the prescription of Zero Blunt T0P0 PCR Cloning Kit (Invitrogen). This was introduced into E. coli T0P10, and a clone having cDNA was selected on an LB agar medium containing kanamycin. As a result of analyzing the sequence of each clone, the sequence The nucleotide sequences of the cDNAs represented by No. 16 and SEQ ID No. 21 were obtained. The nucleotide sequence at positions 1 to 160 and 2483 to 2755 of the full length gene sequence of FU20539 represented by SEQ ID NO: 3 is added to the 5 'end and 3' end of the nucleotide sequence represented by SEQ ID NO: 16, respectively. The base sequence thus obtained is shown in SEQ ID NO: -19.
配列番号： 3で表される FLJ20539全長遺伝子塩基配列の 1〜160番目および 2483 〜2755番目の塩基配列を、 配列番号： 2 1で表される塩基配列の 5 ' 端および 3 ' 端にそれぞれ付加した塩基配列を、 配列番号： 2 4に示す。  The nucleotide sequence at positions 1 to 160 and 2483 to 2755 of the FLJ20539 full-length gene represented by SEQ ID NO: 3 is added to the 5 'end and 3' end of the nucleotide sequence represented by SEQ ID NO: 21, respectively. The base sequence thus obtained is shown in SEQ ID NO: 24.
配列番号： 1 6で表される塩基配列を有する DNA断片を有するプラスミドを TACT427_A/pCR_Blimt I I- T0P0と、 配列番号： 2 1で表される塩基配列を有する DNA断片を有するプラスミドを TACT427-B/PCR- Blunt l l- T0P0と名付けた。  A plasmid having a DNA fragment having the nucleotide sequence represented by SEQ ID NO: 16 is called TACT427_A / pCR_Blimt II-TOP0, and a plasmid having a DNA fragment having the nucleotide sequence represented by SEQ ID NO: 21 is called TACT427-B / PCR-Blunt l-T0P0.
配列番号： 1 6で表される塩基配列がコードするアミノ酸配列 （配列番号： 1 5 ) を含有するタンパク質を TACT427- Aと命名した。  A protein containing the amino acid sequence (SEQ ID NO: 15) encoded by the nucleotide sequence represented by SEQ ID NO: 16 was designated as TACT427-A.
配列番号： 2 1で表される塩基配列がコードするアミノ酸配列 （配列番号： 2 0 ) を含有するタンパク質を TACT427- Bと命名した。  A protein containing the amino acid sequence (SEQ ID NO: 20) encoded by the nucleotide sequence represented by SEQ ID NO: 21 was designated as TACT427-B.
さらに、 プラスミド TACT427-A/pCR-Blunt I I- T0P0が導入された形質転換体を Escherichia col i T0P10/47427A/pCR- Blunt ll- T0P0と、 プラスミド TACT427- B/pCR- Blunt l l- T0P0が導入された形質転換体を Escherichia col i  Furthermore, the transformant into which the plasmid TACT427-A / pCR-Blunt II-T0P0 was introduced was transformed into Escherichia col i T0P10 / 47427A / pCR-Bluntll-T0P0 and plasmid TACT427-B / pCR-BluntIl-T0P0. The transformed transformant is transformed into Escherichia coli
TOP 10/47427B/pCR-B luntl I-TOPOとそれぞれ命名した。 TOP 10 / 47427B / pCR-Bluntl I-TOPO.
TACT427- Βのアミノ酸配列 （配列番号： 2 0 ) では、 TACT427-Aのアミノ酸配列 (配列番号： 1 5 ) の 278番目の Argが Ginに、 825番目の Gluが Lysに、 826番目の Alaが Proに、 970番目の Valが Alaにそれぞれ置換され、 340番目の Serが欠失して いる。  In the amino acid sequence of TACT427-Β (SEQ ID NO: 20), the 278th Arg of the TACT427-A amino acid sequence (SEQ ID NO: 15) is Gin, the 825th Glu is Lys, and the 826th Ala is In Pro, the 970th Val has been replaced with Ala, respectively, and the 340th Ser has been deleted.
TACT427- Bをコードする DNAの塩基配列 （配列番号： 2 1 ) では、 TACT427-Aを コードする DNAの塩基配列 （配列番号： 1 6 ) の 833番目の gが aに、 1482番目の g が cに、 1590番目の aが に、 2473番目の gが aに、 2476番目の gが cに、 2909番目の t が cにそれぞれ置換され、 1015〜1017番目の ageが欠失している。  In the nucleotide sequence of the DNA encoding TACT427-B (SEQ ID NO: 21), the 833th g of the DNA encoding TACT427-A (SEQ ID NO: 16) is a and the 1482th g is In c, the 1590th a is replaced with a, the 2473th g is replaced with a, the 2476th g is replaced with c, and the 2909th t is replaced with c, and the 1015-1017 age is deleted.
TACT427-Aのアミノ酸配列の 1〜4番目のアミノ酸配列が欠失している配列を配 列番号： 1 7に示す。 配列番号： 1 7で示されるアミノ酸配列を有するタンパク 質を TACT427- と命名する。 TACT427- A2をコ一ドする DNAの塩基配列を配列番 号： 1 8に示す。 SEQ ID NO: 17 shows a sequence in which the first to fourth amino acid sequences of the amino acid sequence of TACT427-A are deleted. The protein having the amino acid sequence represented by SEQ ID NO: 17 is designated as TACT427-. TACT427- The nucleotide sequence of the DNA encoding A2 No .: See 18
TACT427- Bのアミノ酸配列の 1〜4番目のアミノ酸配列が欠失している配列を配 列番号： 2 2に示す。 配列番号： 2 2で示されるアミノ酸配列を有するタンパク 質を TACT427- B2と命名する。 TACT427- B2をコードする DNAの塩基配列を配列番 号： 2 3に示す。  SEQ ID NO: 22 shows a sequence in which the first to fourth amino acid sequences of TACT427-B are deleted. The protein having the amino acid sequence represented by SEQ ID NO: 22 is designated as TACT427-B2. The nucleotide sequence of the DNA encoding TACT427-B2 is shown in SEQ ID NO: 23.
TACT427- Aをコードする DNAの塩基配列 （配列番号： 1 6 ) は、 ヒトでは FU20539をコードする DNAの塩基配列 （配列番号： 2 ) に最も高い相同性を示し た。 配列番号： 1 6で表される塩基配列の 1〜138番目および 889〜3072番目の塩 基配列に対しては、 配列番号： 2で表される塩基配列の 1〜138番目および 139〜 2322番目の塩基配列が対応し、 各部分配列において、 99. 3%および 100%の相同 性をそれぞれ示す。 FU20539をコードする DNAの塩基配列 （配列番号： 2 ) は、 TACT427- Aをコードする DNAの塩基配列 (配列番号： 1 6 ) の 139〜888番目に相当 する塩基配列を欠いていることより、 この配列は TACT427- Aに特異的な配列であ る。  The nucleotide sequence of the DNA encoding TACT427-A (SEQ ID NO: 16) showed the highest homology to the nucleotide sequence of the DNA encoding FU20539 (SEQ ID NO: 2) in humans. With respect to the nucleotide sequence at positions 1 to 138 and 889 to 3072 of the nucleotide sequence represented by SEQ ID NO: 16, Correspond to each other, and show 99.3% and 100% homology in each partial sequence, respectively. The nucleotide sequence of the DNA encoding FU20539 (SEQ ID NO: 2) lacks the nucleotide sequence corresponding to positions 139 to 888 of the nucleotide sequence of the DNA encoding TACT427-A (SEQ ID NO: 16). This sequence is specific to TACT427-A.
TACT427- A2、 TACT427-Bおよび TACT427-B2についても、 TACT427- Aと同様に、 FU20539に最も高い相同性を示し、 かつ、 同様な塩基置換および塩基配列の欠失 を有する。  Similarly to TACT427-A, TACT427-A2, TACT427-B and TACT427-B2 show the highest homology to FU20539, and have similar base substitutions and deletions of base sequences.
TACT427-Aのアミノ酸配列 （配列番号： 1 5 ) の 735〜792番目までのアミノ酸 配列、 TACT427- A2のアミノ酸配列 （配列番号： 1 7 ) の 731〜788番目までのアミ ノ酸配列、 TACT427 - Bのアミノ酸配列 （配列番号：. 2 0 ) の 734〜791番目までの アミノ酸配列、 および TACT427- B2のアミノ酸配列 （配列番号： 2 2 ) の 730〜787 番目までのアミノ酸配列は、 ともにアミノ酸ドメインモチーフ検索サイト SMART (ht tp ://smart. embl-heidel erg. de/) での検索で、 クロ口パーォキシダーゼモ チーフを有することからクロロパ一ォキシダーゼ活性を持つと考えられる。  TACT427-A amino acid sequence (SEQ ID NO: 15) at amino acids 735 to 792; TACT427-A2 amino acid sequence (SEQ ID NO: 17) at amino acids 731 to 788; The amino acid sequence of amino acids 734 to 791 of amino acid sequence B (SEQ ID NO: 20) and the amino acid sequence of amino acids 730 to 787 of amino acid sequence of TACT427-B2 (SEQ ID NO: 22) are both amino acid domains. A search on the motif search site SMART (ht tp: // smart. Embl-heidel erg. De /) indicates that it has chloropa-oxidase activity because it has a black mouth peroxidase chief.
TACT427- Aの疎水性プロット図を 〔図 1〕 に、 TACT427- A2の疎水性プロット図 を 〔図 2〕 に、 TACT427-Bの疎水性プロット図を 〔図 3〕 に、 TACT427- B2の疎水 性プロット図を 〔図 4〕 にそれぞれ示す。 参考例 5 ヒト肺癌細胞株 NCI- H522由来タンパク質 TACT427- Cおよび TACT427- C2をコード する cDNAのクローニングと塩基配列の決定 Fig. 1 shows the hydrophobicity plot of TACT427-A, Fig. 2 shows the hydrophobicity plot of TACT427-A2, and Fig. 3 shows the hydrophobicity plot of TACT427-B2. The sex plots are shown in Figure 4 respectively. Reference example 5 Cloning and nucleotide sequencing of cDNAs encoding TACT427-C and TACT427-C2 from human lung cancer cell line NCI-H522
ヒト非小細胞肺癌細胞株 NCI- H522 (ATCCより購入） を RPMI- 1640培地  Human non-small cell lung cancer cell line NCI-H522 (purchased from ATCC) in RPMI-1640 medium
(Invitrogen社） に牛胎仔血清を 10%加えた培地で培養し、 R easy Mini Total RNA Kit (QIAGEN社） を用いてトータル RNAを抽出した。 トータル RNAを鍀型とし て、 Ta Man Reverse Transcription Reagents (Applied Biosystems社) を用レ て添付プロトコールに従い逆転写反応し cDNAを得た。 ここで得られた cDNAを錶型 とし、 2種のプライマ一 〔プライマー 5 (配列番号： 34) およびプライマ一 6 (配列番号： 35) 〕 を用いて PCR反応を行った。 該反応における反応液の組成 は上記 cDNAを铸型として使用し、 PiuTurbo Hotstart DNA Polymerase  (Invitrogen) and 10% fetal bovine serum were added to the medium, and the total RNA was extracted using Reasy Mini Total RNA Kit (QIAGEN). Using total RNA as type III, reverse transcription was performed using Ta Man Reverse Transcription Reagents (Applied Biosystems) according to the attached protocol to obtain cDNA. The cDNA obtained here was designated as type III, and a PCR reaction was carried out using two types of primers [Primer 5 (SEQ ID NO: 34) and Primer 6 (SEQ ID NO: 35)]. The composition of the reaction solution for this reaction was prepared using the above cDNA as type II, PiuTurbo Hotstart DNA Polymerase.
(STRATAGENE社） 2.5 ϋ、 プライマ一 5 (配列番号： 34) およびプライマー 6 (配列番号： 35) を各 1. O M、 dNTPsを 200 Μ、 および GC Bufferl (TaKaRa 社） を 10 zl加え、 20 1の液量とした。 PCR反応は、 95°C · 1分の後、 95°C · 30秒、 60°C ' 20秒、 72°C · 3分のサイクルを 35回繰り返した。 該 PCR反応産物をァガロー スゲルにて電気泳動後、 MinElute Gel Extraction Kit (QIAGEN社） を用いて精 製した。 これを Zero Blunt T0P0 PCRクローニングキット （Invitrogen社） の処 方に従いプラスミドベクター pCR-Bluntll-TOPO (Invitrogen社） へサブクロー二 ングした。 これを大腸菌 TOP10に導入し、 cDNAを持つクローンをカナマイシンを 含む LB寒天培地中で選択した。 個々のクローンの配列を解析した結果、 配列番 号： 26で表される cDNAの塩基配列を得た。  (STRATAGENE) 2.5 l, Primer 1 (SEQ ID NO: 34) and Primer 6 (SEQ ID NO: 35) each 1. Add 200 l of OM, dNTPs, and 10 zl of GC Bufferl (TaKaRa), and add 20 1 Of liquid. In the PCR reaction, a cycle of 95 ° C for 1 minute, 95 ° C for 30 seconds, 60 ° C for 20 seconds, and 72 ° C for 3 minutes was repeated 35 times. The PCR reaction product was electrophoresed on an agarose gel, and then purified using MinElute Gel Extraction Kit (QIAGEN). This was subcloned into a plasmid vector pCR-Bluntll-TOPO (Invitrogen) according to the procedure of Zero Blunt T0P0 PCR Cloning Kit (Invitrogen). This was introduced into Escherichia coli TOP10, and clones having cDNA were selected in LB agar medium containing kanamycin. As a result of analyzing the sequence of each clone, the nucleotide sequence of cDNA represented by SEQ ID NO: 26 was obtained.
配列番号： 3で表される FLJ20539全長遺伝子塩基配列の 1〜160番目および 2483 〜2755番目の塩基配列を、 配列番号： 26で表される塩基配列の 5' 端および 3' . 端にそれぞれ付加した塩基配列を、 配列番号 29 ：に示す。  The nucleotide sequences at positions 1 to 160 and 2483 to 2755 of the FLJ20539 full length gene sequence represented by SEQ ID NO: 3 are added to the 5 'end and 3'. End of the nucleotide sequence represented by SEQ ID NO: 26, respectively. The base sequence thus obtained is shown in SEQ ID NO: 29.
配列番号： 26で表される塩基配列を有する DNA断片を有するプラスミドを TACT427- C/pCR-BluntII- T0P0と名付けた。  A plasmid having a DNA fragment having the base sequence of SEQ ID NO: 26 was designated as TACT427-C / pCR-BluntII-TOP0.
配列番号： 26で表される DNAの塩基配列がコードするアミノ酸配列 （配列番 号： 25) を含有するタンパク質を TACT427- Cと命名した。  A protein containing the amino acid sequence (SEQ ID NO: 25) encoded by the nucleotide sequence of DNA represented by SEQ ID NO: 26 was designated as TACT427-C.
プラスミド TACT427- C/pCR- BlunUI- T0P0が導入された形質転換体を、  The transformant into which the plasmid TACT427-C / pCR-BlunUI-T0P0 has been introduced,
Escherichia coli T0P10/47427C/pCR- Blimtll- T0P0と命名した。 TACT427- Cのアミノ酸配列 （配列番号： 2 5 ) では、 TACT427-Aのアミノ酸配列 (配列番号： 1 5 ) の 491番目の Valが Metに、 825番目の Gluが Lysに、 826番目の Alaが Proに、 970番目の Valが Alaにそれぞれ置換され、 340番目の Serが欠失して いる。 It was named Escherichia coli T0P10 / 47427C / pCR-Blimtll-T0P0. In the amino acid sequence of TACT427-C (SEQ ID NO: 25), Val of 491 in the amino acid sequence of TACT427-A (SEQ ID NO: 15) is Met, Glu of 825 is Lys, and Ala of 826 is In Pro, the 970th Val has been replaced with Ala, respectively, and the 340th Ser has been deleted.
TACT427- Cをコードする DNAの塩基配列 （配列番号： 2 6 ) では、 TACT427- Aを コードする DNAの塩基配列 （配列番号： 1 6 ) の 504番目の aがじに、 939番目の aが gに、 1471番目の gが aに、 1482番目の gが cに、 1590番目の aが gに、 2473番目の が aに、 2476番目の gが cに、 2909番目の tが cにそれぞれ置換され、 1015〜1017番目 の ageが欠失している。  In the nucleotide sequence of the DNA encoding TACT427-C (SEQ ID NO: 26), the base sequence of the DNA encoding TACT427-A (SEQ ID NO: 16) has the 504th a and the 939th a g, 1471th g to a, 1482th g to c, 1590th a to g, 2473th to a, 2476th g to c, 2909th t to c It has been replaced and age 1015-1017 has been deleted.
TACT427-Cのアミノ酸配列の 1〜4番目のアミノ酸配列が欠失している配列を配 列番号： 2 7に示す。 配列番号： 2 7で示されるアミノ酸配列を有するタンパク 質を TACT427- C2と命名する。 TACT427- C2をコ一.ドする DNAの塩基配列を配列番 号： 2 8に示す。  SEQ ID NO: 27 shows a sequence in which the first to fourth amino acid sequences of TACT427-C are deleted. The protein having the amino acid sequence represented by SEQ ID NO: 27 is named TACT427-C2. The nucleotide sequence of the DNA encoding TACT427-C2 is shown in SEQ ID NO: 28.
TACT427- Cをコードする DNAの塩基配列 （配列番号： 2 6 ) は、 ヒトでは FU20539をコードする DNAの塩基配列 （配列番号： 2 ) に最も高い相同性を示し た。 配列番号： 2 6で表される塩基配列の 1〜138番目および 886〜3069番目の DNA 塩基配列に対しては、 配列番号： 2で表される塩基配列の 1〜138番目および 139 番目〜 2322番目の塩基配列が対応し、 各部分配列において、 99. 3%および 99. 5% の相同性をそれぞれ示す。 FU20539コードする DNAの塩基配列 （配列番号： 2 ) は、 TACT427- C (配列番号： 2 6 ) で表される 139〜885番目に相当する DNA塩基配 列を欠いていることより、 この配列は、 TACT427-Cに特異的な配列である。  The nucleotide sequence of the DNA encoding TACT427-C (SEQ ID NO: 26) showed the highest homology to the nucleotide sequence of the DNA encoding FU20539 (SEQ ID NO: 2) in humans. For the DNA base sequence at positions 1 to 138 and 886 to 3069 of the base sequence represented by SEQ ID NO: 26, the base sequence represented by SEQ ID NO: 2 is represented by positions 1 to 138 and 139 to 2322. The third base sequence corresponds, and shows 99.3% and 99.5% homology in each partial sequence, respectively. The nucleotide sequence of the DNA encoding FU20539 (SEQ ID NO: 2) lacks the DNA base sequence corresponding to positions 139 to 885 represented by TACT427-C (SEQ ID NO: 26). , A sequence specific to TACT427-C.
TACT427-C2についても、 TACT427- Cと同様に FLJ20539に最も高い相同性を示し、 かつ同様な塩基配列の欠失を有する。 Similarly to TACT427-C, TACT427-C2 shows the highest homology to FLJ20539 and has a similar nucleotide sequence deletion.
TACT427- Cのアミノ酸配列 （配列番号： 2 5 ) の 734〜791番目までの配列およ び TACT427-C2のアミノ酸配列 （配列番号： 2 7 ) の 730〜787番目までの配列は、 ともにアミノ酸ドメインモチーフ検索サイト SMART (ht tp ://smart. einbl- hei del berg, de/) での検索でク口口パーォキシダーゼモチーフを有することから クロ口パーォキシダーゼ活性を持つと考えられる。  The amino acid sequence of amino acids 734-791 of TACT427-C (SEQ ID NO: 25) and the amino acids 730-787 of amino acid sequence of TACT427-C2 (SEQ ID NO: 27) are both amino acid domains. A motif search site, SMART (ht tp: // smart. Einbl-hei del berg, de /), indicates that it has a black mouth peroxidase activity because it has a mouth mouth peroxidase motif.
TACT427- Cの疎水性プロット図を 〔図 5〕 に、 TACT427- Cの疎水性プロット図を 〔図 6〕 に示す。 参考例 6 The hydrophobicity plot of TACT427-C is shown in Fig. 5, and the hydrophobicity plot of TACT427-C is shown. [Figure 6] shows. Reference example 6
以下の参考例において、 TACT427-A遺伝子 （配列番号： 16) 、 TACT427- A2遺 伝子 （配列番号： 18) 、 TACT427-B遺伝子 （配列番号： 21) 、 TACT427- B2遺 伝子 （配列番号： 23) 、 TACT427-C遺伝子 （配列番号： 26) および TACT427 - C2遺伝子 （配列番号： 28) をまとめて TACT427遺伝子と略記することもある。 以下の参考例において、 TACT427- Aタンパク質 （配列番号： 15) 、 TACT427- A2タンパク質 （配列番号： 17) 、 TACT427-Bタンパク質 （配列番号： 20) 、 TACT427-B2タンパク質 （配列番号： 22) 、 TACT427-Cタンパク質 （配列番号： 25) および TACT427-C2タンパク質 （配列番号： 27) をまとめて TACT427タン パク質と略記することもある。  In the following Reference Examples, the TACT427-A gene (SEQ ID NO: 16), the TACT427-A2 gene (SEQ ID NO: 18), the TACT427-B gene (SEQ ID NO: 21), the TACT427-B2 gene (SEQ ID NO: : 23), TACT427-C gene (SEQ ID NO: 26) and TACT427-C2 gene (SEQ ID NO: 28) may be collectively abbreviated as TACT427 gene. In the following Reference Examples, TACT427-A protein (SEQ ID NO: 15), TACT427-A2 protein (SEQ ID NO: 17), TACT427-B protein (SEQ ID NO: 20), TACT427-B2 protein (SEQ ID NO: 22), The TACT427-C protein (SEQ ID NO: 25) and TACT427-C2 protein (SEQ ID NO: 27) may be collectively abbreviated as TACT427 protein.
ヒ卜癌組織における遺伝子発現量の検討 （その 1 )  Examination of gene expression level in human cancer tissue (Part 1)
ヒト癌患者組織 （乳癌、 肺癌、 大腸癌、 直腸癌、 卵巣癌） 由来の Matched Tumor/Normal cDNA Pair (CLONTECH社） を鍀型として、 FAM標識した TaQManプロ ーブを用いた定量的 PCR反応を行うことにより、 癌組織と正常組織での FLJ20539 遺伝子 （配列番号： 2) 、 P5G177遺伝子 （配列番号： 5) 、 MP1762319遺伝子 (配列番号： 8) ならびに FU13515遺伝子 （配列番号： 11) 、 および参考例 4 または参考例 5で得られた TACT427遺伝子の発現量を測定した。  Using matched Tumor / Normal cDNA Pair (CLONTECH) derived from human cancer patient tissues (breast, lung, colorectal, rectal, and ovarian cancer) as a type III, quantitative PCR reaction using FAM-labeled TaQMan probe was performed. By doing so, FLJ20539 gene (SEQ ID NO: 2), P5G177 gene (SEQ ID NO: 5), MP1762319 gene (SEQ ID NO: 8), FU13515 gene (SEQ ID NO: 11), and Reference Examples in cancer tissues and normal tissues 4 or the expression level of the TACT427 gene obtained in Reference Example 5 was measured.
該反応における反応液の組成は、 上記 cDNA l ilを铸型として使用し、  The composition of the reaction solution in the reaction was determined using the cDNA
TaaMan™ Universal PCR Master Mix (Ap lied Biosystems社) を 7.5/^1、 プラ イマ一 7 (配列番号： 36) およびプライマー 8 (配列番号： 37) を各 0.5 M、 TaaManプローブ 1 (配列番号： 38) を ΙΟΟηΜとなるよう加え、 15 1の液量とし た。 PCR反応は、 50で · 2分、 95°C · 10分の後、 95°C■ 15秒、 60°C · 1分のサイク ルを 40回繰り返した。  7.5 / ^ 1 of TaaMan ™ Universal PCR Master Mix (Applied Biosystems), 0.5 M each of primer 7 (SEQ ID NO: 36) and primer 8 (SEQ ID NO: 37), TaaMan probe 1 (SEQ ID NO: 38) ) Was added to obtain ΙΟΟηΜ, and the liquid volume was set to 151. The PCR reaction was repeated 40 times at 50 ° C for 2 minutes and at 95 ° C for 10 minutes, then at 95 ° C for 15 seconds and at 60 ° C for 1 minute.
その結果、 上記遺伝子の総量は、 ヒト乳癌組織 4例中 2例で周辺正常組織に比べ それぞれ約 7倍、 約 16倍の、 ヒト肺癌組織 3例中 2例で周辺正常組織に比べそれぞ れ約 3倍、 約 2倍の、 ヒト大腸癌組織 5例中 4例で周辺正常組織に比べそれぞれ約 2 倍、 約 8倍、 約 3倍、 約 4倍の、 ヒト直腸癌組織 3例中 2例で周辺正常組織に比べ約 10倍、 約 5倍の、 ヒト卵巣癌組織 5例中 2例で周辺正常組織に比べそれぞれ約 3倍、 約 20倍の発現量を示した。 As a result, the total amount of the above genes was about 7 times and about 16 times that of the surrounding normal tissues in 2 out of 4 human breast cancer tissues, respectively, and in 2 out of 3 human lung cancer tissues compared to the surrounding normal tissues, respectively. About 3 times, about 2 times, human colorectal cancer tissue about 4 times out of 5 cases, about 2 times, about 8 times, about 3 times, about 4 times higher than surrounding normal tissues, respectively, 2 out of 3 human rectal cancer tissues In some cases, compared to surrounding normal tissue Two out of five human ovarian cancer tissues, 10-fold and about 5-fold, showed about 3-fold and about 20-fold expression levels, respectively, compared to surrounding normal tissues.
これより、 上記遺伝子の総量は、 癌組織において顕著に発現上昇していること がわかる。 参考例 7  This indicates that the total amount of the above genes is significantly increased in cancer tissues. Reference Example 7
ヒト癌組織における遺伝子発現量の検討 （その 2 )  Examination of gene expression level in human cancer tissues (Part 2)
ヒト肺癌組織 （Direct Cl inical Access社、 および BioCl inical Partners社よ り購入） より調製した cDNAを銬型として参考例 6と同様の方法により、 癌組織、 および正常組織における参考例 6で用いた遺伝子の発現量の比較を行った。 該反応における反応液の組成は上記 cDNA 1 / ^を鎢型として使用し、 参考例 6 と同一条件で PCR反応を行った。 並行して TadMan™ Human i3 -act in Control Reagents (Appl ied Biosystems社) を用いて上記 cDNA 1 1に含まれる J3 -ァクチ ン遺伝子のコピー数を算出し内部標準とした。 β -ァクチン遺伝子発現量で標準 化した場合、 上記遺伝子の総量は、 DirectCl inical Access社のサンプルでは、 正常肺組織に比べてヒト肺癌組織 10例中 8例で約 144倍、 約 3倍、 約 5倍、 約 4倍、 約 4倍、 約 13倍、 約 3倍、 約 19倍に増加しており、 ヒト肺癌組織における顕著な発 現上昇がみられた。  A cDNA prepared from human lung cancer tissue (purchased from Direct Clinical Access and BioClinical Partners) was used as a type II cDNA in the same manner as in Reference Example 6, and the genes used in Reference Example 6 in cancer tissues and normal tissues were used. Was compared. The composition of the reaction solution in this reaction was the same as that in Reference Example 6, except that the above cDNA 1 / ^ was used as type III, and a PCR reaction was performed. In parallel, the copy number of the J3-actin gene contained in the cDNA 11 was calculated using TadMan ™ Human i3-act in Control Reagents (Applied Biosystems) and used as an internal standard. When normalized by the expression level of β-actin gene, the total amount of the above genes was about 144 times, about 3 times, and about 3 times in 8 out of 10 human lung cancer tissues compared to normal lung tissues in the sample of Direct Clinical Access Co. It increased 5 times, about 4 times, about 4 times, about 13 times, about 3 times, and about 19 times, and markedly increased expression in human lung cancer tissues.
また、 BioCl inical Partners社のサンプルでは、 上記遺伝子の総量が j3 -ァク チン遺伝子発現量の 1 %を超えるものが、 正常肺組織では 7例中 1例であつたのに 対し、 ヒト肺癌組織では 11例中 5例と高頻度に認められた。  In addition, in the case of BioClinical Partners, the total amount of the above genes exceeded 1% of the expression level of the j3-actin gene in 1 of 7 normal lung tissues, whereas in human lung cancer tissues Of the 11 cases, 5 cases were frequently observed.
これより、 上記遺伝子がヒト肺癌組織において顕著に発現上昇していることが 示された。 参考例 8  This indicated that the expression of the above gene was significantly increased in human lung cancer tissues. Reference Example 8
ヒト培養細胞株における遺伝子発現量の検討  Examination of gene expression level in human cultured cell line
以下で使用される脳腫瘍細胞株 SK- N- M SK- N-AS、 SK-N-BE, SK- N- DZ、 SK-N- FI、 SK-N- SH、 D341Med、 Daoy、 DBTRG_05MG、 U-118 MG、 U-87 MG、 CCF- STTGlおよ び SW 1088；ヒト乳癌細胞株 HCC1937、 ZR- 75- 1、 AU565, MCF-7および DA- MB - 231；ヒ卜大腸癌細胞株 Caco - 2、 COL020K COLO 205、 COLO 3画、 HCT- 8、 HT - 29、 LoVo、 LS123、 SNU-CK SK-CO-K SW 403、 SW 48、 SW480, SW 620、 SW 837および SW 948；ヒト胎児腎臓細胞株 HEK293；ヒト小細胞肺癌細胞株 NCI- H187、 NCI-H378, NCI-H526, NCI-H889, NCI-H1672, NCI-H1836, NCI-H2227, NCI- N417および SHP - 77；ヒ卜非小細胞肺癌細胞株 A549、 NCI-H23, NCI-H226, NCI-H358, NCI- H460、 NCI- H522、 NCI-H66 L NCI-H810, NCI- HI 155、 NCI-H1299, NCI-H1395, NCI- H1417、 NCI-H1435, NCI-H158K NCI-H165K NCI-H1703, NCI-H1793, NCI- H1963、 NCI - 顧 3、 NCト薩 5、 NCI- H2106、 NCI-H2228, NCI- H2342および NCI- H2347；ヒト卵 巣癌細胞株 ES - 2、 Caov- 3、 MDAH2774, NIH: 0VCAR3、 OV- 90、 SK普 3、 丽- 112Dお よび丽- 21G ;ヒ卜塍臓癌細胞株 PANC-L MIA-PaCa-2, AsPC- 1、 BxPC- 3、 Capan-1 および Capan-2；ヒト前立腺癌細胞株 DU145；ヒト網膜芽腫細胞株 WERI- Rb- 1およ び Y79； ヒト精巣癌細胞株 Cates- 1Bの 8 6株は、 ATCCより購入した。 ヒト正常気 道上皮細胞 SAECおよびヒト正常前立腺上皮細胞 HPrECは、 Clonet ics社より購入し た。 ヒ卜大腸癌細胞株 C0CM1、 ヒト非小細胞肺癌細胞株 VMRC-LCDおよびヒ卜前立 腺癌細胞株 PC3は、 JCRBより購入した。 これら細胞株は参考例 9以降の参考例で も用いることがある。 Brain tumor cell lines used in SK-N-M SK-N-AS, SK-N-BE, SK-N-DZ, SK-N-FI, SK-N-SH, D341Med, Daoy, DBTRG_05MG, U -118 MG, U-87 MG, CCF-STTGl and SW 1088; human breast cancer cell lines HCC1937, ZR-75-1, AU565, MCF-7 and DA-MB- 231; human colon cancer cell line Caco-2, COL020K COLO 205, COLO 3 strokes, HCT-8, HT-29, LoVo, LS123, SNU-CK SK-CO-K SW403, SW48, SW480, SW620 SW837 and SW948; human fetal kidney cell line HEK293; human small cell lung cancer cell line NCI-H187, NCI-H378, NCI-H526, NCI-H889, NCI-H1672, NCI-H1836, NCI-H2227, NCI- N417 and SHP-77; human non-small cell lung cancer cell lines A549, NCI-H23, NCI-H226, NCI-H358, NCI-H460, NCI-H522, NCI-H66L NCI-H810, NCI-HI155, NCI -H1299, NCI-H1395, NCI-H1417, NCI-H1435, NCI-H158K NCI-H165K NCI-H1703, NCI-H1793, NCI-H1963, NCI-Customer 3, NC Tosatsu 5, NCI-H2106, NCI-H2228 , NCI-H2342 and NCI-H2347; human ovarian cancer cell lines ES-2, Caov-3, MDAH2774, NIH: 0VCAR3, OV-90, SK-P3, 丽 -112D and 21-21G; Cancer cell lines PANC-L MIA-PaCa-2, AsPC-1, BxPC-3, Capan-1 and Capan-2; human prostate cancer cell line DU145; human retinoblastoma cell lines WERI-Rb-1 and Y79 ; 8 6 strains of testicular cancer cell lines Cates- 1B were purchased from ATCC. Human normal airway epithelial cells SAEC and human normal prostate epithelial cells HPrEC were purchased from Clonetics. Human colon cancer cell line C0CM1, human non-small cell lung cancer cell line VMRC-LCD, and human prostate cancer cell line PC3 were purchased from JCRB. These cell lines may also be used in Reference Examples 9 and thereafter.
上記記載の細胞株 91株より RNeasy Mini Total RNA Ki t (QIAGEN社） を用いて I タル RNAを調製した。 この! タル RNAを铸型として TaQMan Reverse  I total RNA was prepared from 91 cell lines described above using RNeasy Mini Total RNA Kit (QIAGEN). TaQMan Reverse
Transcript ion Reagents (Appl ied Biosys tems社） の添付プロトコ一ルに従い、 ランダムプライマーを用いた逆転写反応で cDNAを調製した。 この cDNAを铸型とし て定量的 PCR反応を行うことにより、 遺伝子 FLJ20539遺伝子 （配列番号： 2 ) 、 MP50177遺伝子 (配列番号 : 5 ) 、 MP1762319遺伝子 (配列番号 : 8 ) ならびに FLJ 13515遺伝子 （配列番号： 1 1 ) 、 および TACT427遺伝子の発現量の検討を行 つた。 According to the protocol attached to Transcript ion Reagents (Applied Biosystems), cDNA was prepared by a reverse transcription reaction using random primers. By performing a quantitative PCR reaction using this cDNA as type III, the genes FLJ20539 (SEQ ID NO: 2), MP50177 (SEQ ID NO: 5), MP1762319 (SEQ ID NO: 8) and FLJ13515 (SEQ ID NO: 8) were obtained. : 1 1) and the expression level of the TACT427 gene were examined.
該反応は上記トータル RNA 5 ngより得られた cDNAを鎢型として使用し、 参考例 6と同様の方法により行った。 並行して TadMan™ Human i3 - act in Control Reagents (Appl ied Biosystems社） を用いて上記トータル RNA 1 ngに含まれる i8 -ァクチン遺伝子のコピー数を算出し内部標準とした。  The reaction was carried out in the same manner as in Reference Example 6, using the cDNA obtained from 5 ng of the above total RNA as Form III. In parallel, the copy number of the i8-actin gene contained in 1 ng of the total RNA was calculated using TadMan ™ Human i3-act in Control Reagents (Applied Biosystems) and used as an internal standard.
上記遺伝子全体の発現量を、 β -ァゥチン遺伝子発現量で標準化した相対的発 現率を 〔表 6〕 および 〔表 7〕 に示す。 Relative expression normalized to the expression level of the β-patin gene The current rates are shown in [Table 6] and [Table 7].
上記遺伝子発現量の総量が β -ァクチン遺伝子発現量の 1 %を超える癌細胞株が 13株見出され、 上記遺伝子の癌細胞株における高発現が認められた。  Thirteen cancer cell lines in which the total amount of the above gene expression exceeded 1% of the β-actin gene expression amount were found, and high expression of the above gene in the cancer cell lines was confirmed.
表 6 Table 6
Figure imgf000085_0002
表 7
Figure imgf000085_0002
Table 7
Figure imgf000085_0001
NCI-H1963 1. 25 IH:0VCAR3 1. 04 HPrEC 0. 55
Figure imgf000085_0001
NCI-H1963 1.25 IH: 0VCAR3 1.04 HPrEC 0.55
NC I-H2073 0. 08 0V-90 0. 40 DU 145 0. 84  NC I-H2073 0.08 0V-90 0.40 DU 145 0.84
NCI-H2085 0. 22 SK-0V-3 0. 26 PC3 0. 23  NCI-H2085 0.22 SK-0V-3 0.26 PC3 0.23
NC I-H2106 0. 82 T0V-112D 0. 88 ER I-Rb-1 0. 89  NC I-H2106 0.82 T0V-112D 0.88 ER I-Rb-1 0.89
NCI -H2228 0. 20 T0V-21G 0. 53 Y79 0. 81  NCI -H2228 0.20 T0V-21G 0.53 Y79 0.81
NC I-H2342 0. 48 PANC-1 0. 34 Cates - I B 0. 31  NC I-H2342 0.48 PANC-1 0.34 Cates-I B 0.31
NC I-H2347 0. 21 MIA-PaCa-2 0. 03  NC I-H2347 0.21 MIA-PaCa-2 0.03
VMRC-LCD 0. 74 AsPC-1 0. 06 参考例 9  VMRC-LCD 0.74 AsPC-1 0.06 Reference example 9
組換え型完全長タンパク質の動物細胞用発現ベクターの構築 （その 1 ) , TACT427- Aタンパク質および TACT427- Bタンパク質の C末端に 3xFLAGタグを融合 したタンパク質を発現する動物細胞用発現ベクターを構築した。  Construction of Expression Vector for Recombinant Full-Length Protein for Animal Cells (Part 1), Expression vector for animal cells expressing a protein in which a 3xFLAG tag was fused to the C-terminal of TACT427-A and TACT427-B proteins was constructed.
参考例 4で得た TACT427- A/pCR- Bluntll- T0P0、 TACT427-B/pC -B 1 un 111 -T0P0, および p3xFLAG- CMV- 14 (Sigma社） を制限酵素 EcoRIおよび Xbalにて処理し、 ァガ ロースゲル電気泳動で分離後、 TACT427- A、 TACT427- Bおよび p3xFLAG-CMV- 14に相 当する DNA断片を回収し、 Gel Extract ion Ki t (QIAGEN社）を用いて精製した。 そ れぞれの DNA断片を DNA Ligat ion Ki t ver. 2 (Takara Bio社） を用いてライゲー シヨン反応を行った後、 大腸菌 TOP10に導入し、 アンピシリンを含む LB寒天培地 中で選択した。 個々のクローンの配列を解析した結果、 TACT427- Aタンパク質 (配列番号： 1 5 ) 、 および TACT427-Bタンパク質 （配列番号： 2 0 ) をコード する cDNA配列を有する動物細胞用発現ベクター p3xFLAG- TACT427-A、 および p3xFLAG- TACT427- Bを得た。 参考例 1 0  TACT427-A / pCR-Bluntll-T0P0, TACT427-B / pC-B1un111-T0P0, and p3xFLAG-CMV-14 (Sigma) obtained in Reference Example 4 were treated with restriction enzymes EcoRI and Xbal, After separation by agarose gel electrophoresis, DNA fragments corresponding to TACT427-A, TACT427-B and p3xFLAG-CMV-14 were recovered and purified using Gel Extraction Kit (QIAGEN). Each DNA fragment was subjected to a ligation reaction using DNA Ligation Kit ver. 2 (Takara Bio), then introduced into E. coli TOP10, and selected in LB agar medium containing ampicillin. As a result of analyzing the sequence of each clone, an expression vector p3xFLAG-TACT427- for animal cells having a cDNA sequence encoding the TACT427-A protein (SEQ ID NO: 15) and the TACT427-B protein (SEQ ID NO: 20) was obtained. A and p3xFLAG-TACT427-B were obtained. Reference example 10
組換え型完全長夕ンパク質の動物細胞用発現べクタ一の構築 （その 2 )  Construction of expression vector of recombinant full-length protein for animal cells (Part 2)
TACT427- Aタンパク質 （配列番号： 1 5 ) を発現する動物細胞用発現ベクター を構築した。  An expression vector for animal cells expressing the TACT427-A protein (SEQ ID NO: 15) was constructed.
参考例 9で得られた p3xFLAG- TACT427- Aを鍀型とし、 プライマ一 9 (配列番 号： 3 9 ) およびプライマー 1 0 (配列番号： 4 0 ) を用いて PCR反応を行った - 該反応における反応液の組成は p3xFLAG- TACT427- A200 ngを錶型として使用し、 Pfu Turbo Hotstart DNA Polymerase (STRATAGENE社） を 2. 5 U、 プライマー 9Using p3xFLAG-TACT427-A obtained in Reference Example 9 as type I, PCR was carried out using Primer 9 (SEQ ID NO: 39) and Primer 10 (SEQ ID NO: 40)-this reaction The composition of the reaction mixture in p3xFLAG-TACT427-A 200 ng 2.5 U of Pfu Turbo Hotstart DNA Polymerase (STRATAGENE), primer 9
(配列番号： 3 9 ) およびプライマー 1 0 (配列番号： 4 0 ) を各 1 M、 dNTPs を 200 M、 および GC Buf fer I (Takara Bio社） を 25 1加え、 50 1の液量とし た。 PCR反応は、 95°C · 1分の後、 95°C · 15秒、 60°C · 15秒、 72°C · 3分のサイク ルを 25回繰り返し行った。 次に PCR Purif icat ion Ki t (QIAGEN社) にて該 PCR反 応産物を精製した後、 制限酵素 Xbalおよび EcoRIにて処理した。 pcDNA3. 1 (+)(SEQ ID NO: 39) and primer 10 (SEQ ID NO: 40) were added at 1 M each, dNTPs at 200 M, and GC Buf fer I (Takara Bio) at 25 1 to give a liquid volume of 501. . The PCR reaction was repeated 25 times at 95 ° C for 1 minute, followed by 95 ° C for 15 seconds, 60 ° C for 15 seconds, and 72 ° C for 3 minutes. Next, the PCR reaction product was purified using PCR Purification Kit (QIAGEN), and then treated with restriction enzymes Xbal and EcoRI. pcDNA3.1 (+)
(Invi trogen社） も制限酵素 Xbalおよび EcoRIにて処理した。 これらをァガ口一 スゲル電気泳動で分離後、 TACT427- Aタンパク質をコードする塩基配列を含む DNA 断片と PCDNA3. 1 (+)に相当する DNA断片をそれぞれ回収し、 Gel Extract ion Ki t (QIAGEN社）を用いて精製した。 それぞれの DNA断片を DNA Ligat ion Ki t ver. 2 (Takara Bio社） を用いてライゲーシヨン反応を行った後、 大腸菌 T0P10に導入 し、 アンピシリンを含む LB寒天培地中で選択した。 個々のクロ一ンの配列を解析 した結果、 TACT427- A夕ンパク質をコードする cDNA配列を有する動物細胞用発現 ベクター PCDNA3. 1 (+) - TACT427- Aを得た。 (Invitrogen) was also treated with restriction enzymes Xbal and EcoRI. After separating these by agarose gel electrophoresis, a DNA fragment containing the nucleotide sequence encoding the TACT427-A protein and a DNA fragment corresponding to PCDNA3.1 (+) were recovered, respectively, and extracted using Gel Extraction Kit (QIAGEN Was used for purification. After each DNA fragment was subjected to a ligation reaction using DNA Ligation Kit ver. 2 (Takara Bio), it was introduced into E. coli T0P10 and selected in LB agar medium containing ampicillin. As a result of analyzing the sequence of each clone, an expression vector PCDNA3.1 (+)-TACT427-A for animal cells having a cDNA sequence encoding TACT427-A protein was obtained.
ペプチド抗体の作製と精製 Preparation and purification of peptide antibodies
TACT427タンパク質のアミノ酸配列に基づき、 15アミノ酸からなる以下の 3種の ペプチド （ペプチド 1〜3 ) を Fmoc固相合成法を用いて合成した。  Based on the amino acid sequence of the TACT427 protein, the following three peptides (peptides 1 to 3) consisting of 15 amino acids were synthesized by Fmoc solid phase synthesis.
ぺプチド 1のアミノ酸配列 〔Gly-Ser-Gly-Glu-Glu-Asn- Asp-Pro- Gly_Glu- Gin - Ala- Leu- Pro- Cys (配列番号: 4 1 ) 〕 は、 TACT427- Aタンパク質 (配列番号： 1 5 ) の 220番目から 233番目までのアミノ酸配列の C末端に、 Cysを付加した配列 である。  The amino acid sequence of peptide 1 [Gly-Ser-Gly-Glu-Glu-Glu-Asn-Asp-Pro-Gly_Glu-Gin-Ala-Leu-Pro-Cys (SEQ ID NO: 41)] is the TACT427-A protein (sequence). This sequence has Cys added to the C-terminal of the amino acid sequence from position 220 to position 233 of No. 15).
ぺプチド 2 CGly-Pro-Ala-Glu-Gly-Pro-Ala-Glu-Pro-Ala-Ala-Glu-Ala-Ser- Cys (配列番号： 4 2 ) 〕 のアミノ酸配列は、 TACT427- Aタンパク質 （配列番号： 1 5 ) の 517番目から 530番目までのアミノ酸配列の C末端に、 Cysを付加した配 列である。  The peptide 2 CGly-Pro-Ala-Glu-Gly-Pro-Ala-Glu-Pro-Ala-Ala-Glu-Ala-Ser-Cys (SEQ ID NO: 42)] has an amino acid sequence of TACT427-A protein ( This is a sequence in which Cys has been added to the C-terminal of the amino acid sequence from position 517 to position 530 of SEQ ID NO: 15).
ぺプチド 3のアミノ酸配列 〔Gly- Ser- Va卜 Gly-Gly - Asn- Thr-Gly- Va卜 Arg- Gly - Lys-Phe-Glu-Cys (配列番号： 4 3 ) 〕 は、 TACT427- Aタンパク質 (配列番号 : 1 5 ) の 800番目から 813番目までのアミノ酸配列の C末端に、 Cysを付加した配列 である。 ホールリンペットへモシァニン （KLH) をキャリアータンパク質として結合させ 抗原とし、 以下のように、 ゥサギポリクローナル抗体を作製した。 The amino acid sequence of peptide 3 [Gly-Ser-Vat Gly-Gly-Asn-Thr-Gly-Vat Arg-Gly-Lys-Phe-Glu-Cys (SEQ ID NO: 43)] is the TACT427-A protein (SEQ ID NO: 1 This is a sequence in which Cys has been added to the C-terminal of the amino acid sequence from position 800 to position 813 in 5). Mossinin (KLH) was bound to whole limpet as a carrier protein and used as an antigen to prepare a Perian polyclonal antibody as follows.
免疫動物は雄性ゥサギ K B L : J W ( 1 1週齢、 オリエンタル酵母） 一羽を用 レ 初回感作は完全フロインドアジュバンド （Di ico社） 懸濁液、 2回目以降は不 完全フロインドアジュバンド （Diico社） 懸濁液を用いた。 感作は背部皮下注射 により行い、 1回の感作には各抗原 0. 5mgを用い、 初回感作後 14日毎に 3回繰り返 した。 初回感作後 52日目に麻酔下頸動脈採血を行い、 血清約 50mlを得た。 このよ うにして得られた血清を硫酸アンモニゥム塩析法により濃縮し、 得られた粗 IgG 画分全量をプロテイン Aァフィ二ティーカラム （Amersham- Bioscience社） により 精製し、 ペプチド 1、 ペプチド 2またはペプチド 3を免疫したゥサギから、 それ ぞれ約 223mg、 約 495 mgおよび約 390 mgの精製 IgGを得た。 さらにペプチド 1に対 する精製 IgG l l lmg、 ペプチド 2に対する精製 IgG 248mgおよびペプチド 3に対す る精製 IgG195mgを材料として、 各々の免疫源ペプチドを固定化したカラムに結合 する IgG画分を取得した。 固定化には各ペプチドの C末端の Cysを利用し、 ホウ酸 緩衝液を用いてセファロ一スカラム （Amersham- Bioscience社） にカップリング した。 カラムからの溶出には 8M尿素/リン酸緩衝化生理食塩水 （PBS) を用いた < 溶出液を PBSに対して透析して尿素を除いた後、 限外濃縮、 フィルターろ過滅菌 することにより、 ペプチド 1、 ペプチド 2およびペプチド 3に対するァフィニテ ィー精製抗体 AS-2480、 AS - 2481および AS - 2482を、 約 3. 7mg、 約 0. 69 mgおよび約 17 mgずつ取得した。 参考例 1 2  Male immunized male male egret KBL: JW (1 week old, Oriental yeast) Use one bird Les Initial sensitization is complete Freund's adjuvant (Di ico) suspension, second and subsequent times are incomplete Freund's adjuvant (Diico) The suspension was used. The sensitization was performed by subcutaneous injection into the back, and 0.5 mg of each antigen was used for one sensitization, and the sensitization was repeated three times every 14 days after the first sensitization. On the 52nd day after the first sensitization, blood was collected from the carotid artery under anesthesia to obtain about 50 ml of serum. The serum thus obtained was concentrated by the ammonium sulfate salting-out method, and the entire amount of the obtained crude IgG fraction was purified using a protein A affinity column (Amersham-Bioscience) to obtain peptide 1, peptide 2 or About 223 mg, about 495 mg and about 390 mg of purified IgG were obtained from the egret immunized with peptide 3. Furthermore, IgG fractions that bind to the column on which each immunogenic peptide was immobilized were obtained using purified IgG 11 mg for peptide 1, 248 mg of purified IgG for peptide 2 and 195 mg of purified IgG for peptide 3 as materials. For immobilization, Cys at the C-terminal of each peptide was used and coupled to a Sepharose column (Amersham-Bioscience) using a borate buffer. The column was eluted with 8M urea / phosphate-buffered saline (PBS). <The eluate was dialyzed against PBS to remove urea, then ultra-concentrated and sterilized by filtration. Affinity purified antibodies AS-2480, AS-2481 and AS-2482 against Peptide 1, Peptide 2 and Peptide 3 were obtained at about 3.7 mg, about 0.69 mg and about 17 mg, respectively. Reference example 1 2
ゥサギぺプチド抗体を用いたウェスタンブロッティング  Western blotting using rabbit antibodies
TACT427- Aタンパク質 （配列番号： 1 5 ) の検出は、 参考例 1 1で作製したぺ プチド抗体を含むゥサギ血清を用いて行った。 ヒト胎児腎臓由来 HEK293細胞 1. 0 X 106個を 10%牛胎仔血清 （JRH社） を含むダルベッコ改変型イーグル最少培 地 （Invi trogen社） 9 mlに懸濁し、 直径 10cmのペトリディッシュに播種した。 5%炭酸ガス気流下、 37°Cでー晚培養した後、 予め FuGene6トランスフエクシヨン 試薬 18 1 (Roche Diagnost ics社） および OPTI- MEM I (Invi trogen社） と混合し 室温で 15分間放置しておいた p3xFLAG-TACT427- A 6 / gを添加し同条件で培養を継 続した。 2日後に細胞を PBSで洗浄後、 氷冷した RIPA緩衝液 〔50mMトリス，塩酸緩 衝液、 pH 7. 5、 150 mM塩化ナトリウム、 l %Tri ton X- 100、 0. 1 %SDS、 1 %デォ キシコール酸、 Complete™タブレツト (Roche Diagnost ics社) 、 Phosphatase Inhibi tor Cocktai l-2 (Sigma社） 〕 800 1を添加し 4°Cで 15分間放置した。 この RIPA緩衝液を回収し、 15， OOOrpmで 20分間遠心分離した上澄液を無細胞抽出液と し、 20 1を 7. 5%アクリルアミドゲルでの SDS-PAGEに供した。 泳動分離したタン パク質は、 常法に従いクリアブロット P膜 （ATT0社） に転写した後、 ブロッキン グ溶液 （トリス緩衝化生理食塩水、 0. l %Tween- 20、 5%スキムミルク） 中に 1時 間室温放置した。 次に参考例 1 1で作製したペプチド抗体 AS- 2480、 AS-2481また は AS- 2482を含む 3種類のゥサギ血清を、 ブロッキング溶液中で各々 100倍に希釈 して加え、 4でにて一晩インキュベートし、 続いて HRP標識抗ゥサギ IgG抗体The detection of the TACT427-A protein (SEQ ID NO: 15) was performed using the rabbit serum containing the peptide antibody prepared in Reference Example 11. Dulbecco's modified Eagle minimum culture containing 1.0 x 10 6 HEK293 cells from human fetal kidney containing 10% fetal bovine serum (JRH) The suspension was suspended in 9 ml of ground (Invitrogen) and seeded on a 10 cm diameter petri dish. After culturing at 37 ° C in a 5% carbon dioxide gas stream, mix with FuGene6 Transfection Reagent 181 (Roche Diagnostics) and OPTI-MEM I (Invitrogen) and leave at room temperature for 15 minutes. The previously added p3xFLAG-TACT427-A6 / g was added, and the culture was continued under the same conditions. After 2 days, the cells are washed with PBS and then ice-cold RIPA buffer [50 mM Tris, hydrochloric acid buffer, pH 7.5, 150 mM sodium chloride, l% Triton X-100, 0.1% SDS, 1% Deoxycholic acid, Complete ™ tablets (Roche Diagnostics), Phosphatase Inhibitor Cocktail 1-2 (Sigma)] 8001 were added, and the mixture was allowed to stand at 4 ° C for 15 minutes. The RIPA buffer was collected, and the supernatant obtained by centrifugation at 15, OOO rpm for 20 minutes was used as a cell-free extract, and 201 was subjected to SDS-PAGE on a 7.5% acrylamide gel. The proteins separated by electrophoresis were transferred to a clear blot P membrane (ATT0) according to a conventional method, and then transferred to a blocking solution (Tris buffered saline, 0.1% Tween-20, 5% skim milk). It was left at room temperature for a time. Next, three kinds of egret serum containing the peptide antibodies AS-2480, AS-2481 or AS-2482 prepared in Reference Example 1 were diluted 100-fold in a blocking solution, and added at 4 Incubate overnight, followed by HRP-labeled anti-Peagle IgG antibody
(Amersham- Bioscience社） をブロッキング溶液で 10万倍に希釈した 2次抗体溶液 中で 1時間室温放置した。 検出は ECL plus (Amersham- Bioscience社） の添付プロ トコ一ルに従い行った。 (Amersham-Bioscience) was left at room temperature for 1 hour in a secondary antibody solution diluted 100,000 times with a blocking solution. Detection was performed according to the protocol attached to ECL plus (Amersham-Bioscience).
ペプチド抗体 AS- 2480、 AS- 2481または AS- 2482を含む 3種類のゥサギ血清のいず れを用いても、 分子量 150kD近傍の位置に TACT427-Aタンパク質に由来する特異的 なバンドが認められた。 参考例 1 3  A specific band derived from the TACT427-A protein was observed at a molecular weight of around 150 kD using any of the three kinds of egret serum containing the peptide antibodies AS-2480, AS-2481, or AS-2482. . Reference Example 1 3
ペプチド抗体を用いた免疫沈降  Immunoprecipitation using peptide antibodies
参考例 1 1で作製したペプチド抗体を用いて、 TACT427- Aタンパク質に対する 免疫沈降を非変性状態にて行った。  Using the peptide antibody prepared in Reference Example 11, immunoprecipitation against the TACT427-A protein was performed in a nondenaturing state.
参考例 9で得た p3xFLAG- TACT427- Aを用いて参考例 1 2と同様の操作で調製し た無細胞抽出液 lmlに、 Protein G-Sepharose 4FF (Amersliam-Bioscience社) 懸 濁液 （等容量の RIPA緩衝液で懸濁したもの） 50 1とゥサギ血清 3 1とを加え、 4°Cにて一晩撹拌を行った。 ゥサギ血清としては、 参考例 1 1で作製したぺプチ ド抗体 AS- 2480、 AS- 2481または AS- 2482を含む 3種類のゥサギ血清のいずれかを用 いた。 ProteinG- Sepharose 4FF共沈殿画分を RIPA緩衝液にて洗浄後、 1 % 2-メル カプトエタノールを含む SDS- PAGE用サンプルバッファー （Bio Rad社） に懸 濁し 95 で 5分間加熱した後、 20 1を 7. 5%ァクリルアミド'ゲルでの SDS- PAGEに 供した。 検出は参考例 1 2に記載の方法に準じた。 但し一次抗体としてマウス抗 FLAGM2抗体 （Sigma社） をブロッキング溶液で 10 g/mlとなるよう希釈したもの、 二次抗体として HRP標識抗マウス IgG抗体 （Amersham- Bioscience社） をブロッキ ング溶液で 5万倍に希釈したものを用いた。 ペプチド抗体 AS- 2480、 AS-2481また は AS- 2482を含む 3種類のゥサギ血清のいずれを用いて免疫沈降を行った場合にも、 分子量 150kD近傍に TACT427- Aタンパク質に由来する特異的なバンドが認められた これより、 ペプチド抗体 AS-2480、 AS- 2481および AS- 2482は、 未変性の Protein G-Sepharose 4FF (Amersliam-Bioscience) suspension (equal volume) was added to 1 ml of the cell-free extract prepared by the same procedure as in Reference Example 12 using p3xFLAG-TACT427-A obtained in Reference Example 9. Suspended with RIPA buffer of 50) Stirring was performed overnight at 4 ° C. As the egret serum, any one of three kinds of egos serum including the peptide antibodies AS-2480, AS-2481 or AS-2482 prepared in Reference Example 11 was used. After washing the ProteinG-Sepharose 4FF coprecipitate fraction with RIPA buffer, suspend it in SDS-PAGE sample buffer (Bio Rad) containing 1% 2-mercaptoethanol and heat at 95 for 5 minutes. Was subjected to SDS-PAGE on 7.5% acrylamide gel. Detection was performed according to the method described in Reference Example 12. However, mouse anti-FLAGM2 antibody (Sigma) as the primary antibody was diluted to 10 g / ml with a blocking solution, and HRP-labeled anti-mouse IgG antibody (Amersham-Bioscience) as the secondary antibody was used in a blocking solution of 50,000. A one-fold dilution was used. When immunoprecipitation was performed using any of the three types of egret serum containing the peptide antibodies AS-2480, AS-2481 or AS-2482, a specific band derived from the TACT427-A protein with a molecular weight of around 150 kD. Thus, peptide antibodies AS-2480, AS-2481 and AS-2482
TACT427- Aタンパク質と結合することが明らかとなった。 参考例 1 4 It was found to bind to TACT427-A protein. Reference example 1 4
癌細胞株における TACT427タンパク質の発現検討  Examination of TACT427 protein expression in cancer cell lines
直径 10cmのべトリディッシュに播種した肺癌細胞株 A549、 NCI- H226ならびに NCI-H522および乳癌細胞株 ZR- 75-1を PBSで洗浄後、 参考例 1 2に記載の方法に従 い無細胞抽出液を作製した。 A549、 NCI-H226, NCI-H522および ZR- 75- 1の無細胞 抽出液各々 lmlに、 ProteinG-Sepharose 4FF (Amersham- Bioscience社) 懸濁液 (等容量の RIPA緩衝液で懸濁したもの） と、 参考例 1 1で作製したぺプチ ド抗体 AS- 2482を 3 ^g加え、 4°Cにて一晩撹拌を行った。 ProteinG-Sepharose 4FF 共沈殿画分を RIPA緩衝液にて洗浄後、 1% 2-メルカプトエタノールを含む SDS - PAGE用サンプルバッファー （Bio Rad社） 1に懸濁し 95°Cで 5分間加熱した後、 20 1を 7· 5%アクリルアミド、ゲルでの SDS- PAGEに供した。 ペプチド抗体 AS- 2482 を用いて参考例 1 2に記載の方法に準じて検出を行った。  After washing the lung cancer cell lines A549, NCI-H226 and NCI-H522 and breast cancer cell line ZR-75-1 seeded in a 10 cm diameter dish with PBS, cell-free extraction according to the method described in Reference Example 12 A liquid was prepared. A549, NCI-H226, NCI-H522 and ZR-75-1 cell-free extracts each in 1 ml, ProteinG-Sepharose 4FF (Amersham-Bioscience) suspension (suspended in an equal volume of RIPA buffer) And 3 ^ g of the peptide antibody AS-2482 prepared in Reference Example 11 was added thereto, followed by stirring at 4 ° C overnight. After washing the ProteinG-Sepharose 4FF coprecipitate fraction with RIPA buffer, suspend it in SDS-PAGE sample buffer (Bio Rad) 1 containing 1% 2-mercaptoethanol and heat at 95 ° C for 5 minutes. 201 was subjected to SDS-PAGE on 7.5% acrylamide gel. Detection was performed using the peptide antibody AS-2482 according to the method described in Reference Example 12.
A549、 NCI-H226, NCI- H522および ZR- 75-1のいずれにおいても、 分子量 150kD近 傍に、 TACT427タンパク質に由来する特異的なバンドが認められた。  In each of A549, NCI-H226, NCI-H522, and ZR-75-1, a specific band derived from the TACT427 protein was observed near a molecular weight of 150 kD.
これより、 上記夕ンパク質が上記 5種類の癌細胞株で発現していることが明ら かとなつた。 参考例 15 It is clear from the above that the protein is expressed in the above five types of cancer cell lines. I got it. Reference Example 15
TACT427- Aタンパク質の局在性検討 （細胞染色）  Localization of TACT427-A protein (cell staining)
ヒト胎児腎臓由来 HEK293細胞 1X105個を 10%牛胎仔血清 （JRH社） を含むダル べッコ改変型イーグル最少培地 (Invitrogen社） に懸濁し、 2ゥエルポリ- D-リ ジンコートカルチャースライド （BDファルコン社） に播種した。 同様に 5X104個 のヒト非小細胞肺癌細胞株 NCI- H460を 10%牛胎仔血清 （JRH社） を含む RPMI1640 培地 （Invitrogen社） に懸濁し 2ゥエルポリ- D-リジンコートカルチャースライ ド （BDファルコン社） に播種した。 5%炭酸ガス気流下、 37°Cで一晩培養した後、 予め FuGene6トランスフエクシヨン試薬 5.3 xl (Roche Diagnostics社） および OPTI-MEM I (Invitrogen社） と混合し室温で 15分間放置しておいた p3xFLAG - TACT427-A 1. を添加し同条件で培養を継続した。 2日後に細胞を PBSで洗浄 後、 10%中性ホルマリン緩衝液を加え室温で 30分間固定した。 その後 PBSで 0.1% に希釈した TritonX- 100を加え室温で 5分放置した後、 再び PBSで洗浄し、 更に 1% BSAを含む PBSを加え 4°Cで 24時間放置し、 抗体の非特異的結合サイトをブロック した。 次に 1% BSAを含む PBSで 10 g/mlとなるよう希釈したマウス抗 FLAG M2抗 体 （Sigma社） を加え、 室温で 45分間反応させてから PBSで洗浄し、 続いて 1% BSAを含む PBSで lO ig/mlとなるよう希釈した Alexa488標識抗マウス IgG抗体 Human embryonic kidney-derived HEK293 cells (1 × 10 5 cells) were suspended in Dulbecco's modified Eagle's minimal medium (Invitrogen) containing 10% fetal bovine serum (JRH), and 2 ゥ L-poly-D-lysine-coated culture slides (BD (Falcon). Similarly, 5 × 10 4 human non-small cell lung cancer cell lines NCI-H460 were suspended in RPMI1640 medium (Invitrogen) containing 10% fetal bovine serum (JRH) and 2 ゥ L-poly-D-lysine-coated culture slide (BD Falcon). Company). After culturing overnight at 37 ° C in a 5% carbon dioxide gas stream, mix with FuGene6 transfection reagent 5.3 xl (Roche Diagnostics) and OPTI-MEM I (Invitrogen) in advance and leave at room temperature for 15 minutes. The added p3xFLAG-TACT427-A1 was added, and the culture was continued under the same conditions. Two days later, the cells were washed with PBS, 10% neutral formalin buffer was added, and the cells were fixed at room temperature for 30 minutes. Then, add TritonX-100 diluted to 0.1% with PBS, leave at room temperature for 5 minutes, wash again with PBS, further add PBS containing 1% BSA, leave at 4 ° C for 24 hours, Binding sites were blocked. Next, mouse anti-FLAG M2 antibody (Sigma) diluted to 10 g / ml with PBS containing 1% BSA was added, reacted at room temperature for 45 minutes, washed with PBS, and then 1% BSA was added. Alexa488-labeled anti-mouse IgG antibody diluted to 10 ig / ml with PBS
(Molecular Probes社） を加えた。 再び室温で 45分間反応させてから PBSで洗浄 した後、 蛍光顕微鏡により観察した。  (Molecular Probes) was added. After reacting again at room temperature for 45 minutes, the plate was washed with PBS, and observed with a fluorescence microscope.
その結果、 TACT427- Aタンパク質は、 HEK293, NCI-H460のいずれにおいても細 胞質膜上に発現していることが明らかとなった。  As a result, it was revealed that the TACT427-A protein was expressed on the cell membrane in both HEK293 and NCI-H460.
また同様の方法で HEK293細胞に、 pcDNA3.1 (+) -TACT427- Aプラスミドを導入し、 lO ig/mlの AS- 2480、 AS- 2481および AS- 2482を一次抗体として、 10/zg/mlの  Also, pcDNA3.1 (+)-TACT427-A plasmid was introduced into HEK293 cells in the same manner, and 10 oz / ml of lOig / ml AS-2480, AS-2481 and AS-2482 as primary antibodies. of
Alexa488標識抗ゥサギ IgG抗体 （MolecularProbes社） を二次抗体として用いて、 TACT427- Aタンパク質の局在性を調べたところ、 同じく細胞質膜上に発現してい ることが明らかとなった。 参考例 1 6 When the localization of the TACT427-A protein was examined using Alexa488-labeled anti-Peacock IgG antibody (Molecular Probes) as a secondary antibody, it was revealed that the protein was also expressed on the cytoplasmic membrane. Reference Example 1 6
TACT427- A夕ンパク質の局在性検討 （ピオチン標識）  Localization of TACT427-A protein (Piotin labeling)
参考例 1 2と同様の方法で、 HEK293細胞に p3xFLAG_TACT427- Aプラスミドを導 入し、 48時間後に細胞表層上に露出したタンパク質を Cel lular Label ing and Immunoprecipi tat ion Ki t (Roche Diagnos t ics社） を用いてビォチン標 ΐ戠を行つ た。 さらに参考例 1 2の方法に従い調製した無細胞抽出液 lmlとマウス抗 FLAG Μ2 抗体 （Sigma社） 3 /i gとを用いて参考例 1 3の方法に従って免疫沈降し SDS- PAGE を行った。 HRP標識したストレプトアビジン （Amersham- Bioscience社） を用いて 検出したところ、 分子量 150kD近傍に TACT427- Aタンパク質に由来するバンドが認 められ、 TACT427- A夕ンパク質が細胞質膜上に発現することが明らかとなった。 参考例 1 7  In the same manner as in Reference Example 12, the p3xFLAG_TACT427-A plasmid was introduced into HEK293 cells, and the protein exposed on the cell surface 48 hours later was subjected to Cellular Labeling and Immunoprecipitation tation Kit (Roche Diagnostics). A biotin marker was performed by using. Furthermore, SDS-PAGE was performed by immunoprecipitation using lml of the cell-free extract prepared according to the method of Reference Example 12 and mouse anti-FLAG 社 2 antibody (Sigma) 3 / ig according to the method of Reference Example 13. Detection using HRP-labeled streptavidin (Amersham-Bioscience) revealed a band derived from TACT427-A protein at a molecular weight of around 150 kD, indicating that TACT427-A protein was expressed on the cytoplasmic membrane. It became clear. Reference Example 1 7
TACT427タンパク質の局在性検討 （ピオチン標識）  Localization of TACT427 protein (Piotin labeling)
直径 10cmのペトリディッシュに播種したヒト非小細胞肺癌細胞株 A549、 NCI- H226および NCI- H522の細胞表層上に露出しているタンパク質を Cel lular  Cellular proteins exposed on the cell surface of human non-small cell lung cancer cell lines A549, NCI-H226 and NCI-H522 seeded on 10 cm diameter Petri dishes
Label ing and Immunoprec ipi tat ion Ki t (Roche Diagnost ics社) を用レてビォ チン標識を行った。 さらに参考例 1 2の方法に従い調製した無細胞抽出液 lmlと ゥサギペプチド抗体 AS- 2482 3 とを用いて参考例 1 3の方法に従って免疫沈降 し SDS- PAGEを行った。 HRP標識したストレプトアビジン （Amersham- Biosc ience 社） を用いて検出したところ、 A549、 NCI-H226および NCI-H522のいずれにおいて も、 分子量 150kD近傍に TACT427- Aタンパク質、 TACT427-A2タンパク質、 TACT427 - Bタンパク質、 TACT427-B2タンパク質、 TACT427- Cタンパク質および TACT427- C2夕 ンパク質に由来するバンドが認められた。  Biotin labeling was performed using Labeling and Immunoprecipitation Kit (Roche Diagnostics). Further, immunoprecipitation was performed using lml of the cell-free extract prepared according to the method of Reference Example 12 and the egret peptide antibody AS-24823 according to the method of Reference Example 13, and SDS-PAGE was performed. HRP-labeled streptavidin (Amersham-Bioscience) was used to detect TACT427-A, TACT427-A2, and TACT427-B proteins with a molecular weight of around 150 kD in all of A549, NCI-H226 and NCI-H522. Bands derived from protein, TACT427-B2 protein, TACT427-C protein and TACT427-C2 protein were observed.
これより、 TACT427タンパク質が細胞質膜上に発現していることが明らかとな つた。 参考例 1 8  This revealed that the TACT427 protein was expressed on the cytoplasmic membrane. Reference Example 1 8
TACT427夕ンパク質の局在性検討 （FACS解析）  TACT427 Localization of protein (FACS analysis)
直径 10cmのべトリディッシュに播種しサブコンフルェントにまで培養したヒト 非小細胞肺癌細胞株 A549を PBSで洗浄後、 3%BSAおよび 5niM EDTAを含む PBSを加え 室温で 15分間放置し A549細胞を分散した。 次に緩衝液 A 〔2%牛胎仔血清 （JRH 社） および 0. 1 %アジ化ナトリウムを含む HBSS (Hanks ' Balanced Sal t Human seeded in a 10 cm diameter dish and cultured to subconfluent After washing the non-small cell lung cancer cell line A549 with PBS, PBS containing 3% BSA and 5niM EDTA was added, and the mixture was left at room temperature for 15 minutes to disperse the A549 cells. Next, buffer A [HBSS (Hanks' Balanced Salt) containing 2% fetal calf serum (JRH) and 0.1% sodium azide
Solut ions, Invi trogen社) 〕 で l x 106個 /mlの濃度になるように A549細胞を懸濁 し、 終濃度 5 /i g/mlとなるように AS- 2482または非免疫ゥサギ IgG (Jackson社） を 加え、 氷上に 5時間放置した。 続いて緩衝液 Aで細胞を洗浄した後、 10 g/mlの Alexa488標識抗ゥサギ IgG抗体 （Molecular Probes社） を含む緩衝液 Aで懸濁し、 氷上にて 1. 5時間放置した。 緩衝液 Aで再び洗浄後、 FACScan (BDバイオサイェン ス社） にて解析した。 その結果、 ゥサギペプチド抗体 AS- 2482特異的に A549細胞 が染色され、 TACT427- Aタンパク質、 TACT427-A2タンパク質、 TACT427-Bタンパク 質、 TACT427-B2タンパク質、 TACT427-Cタンパク質および TACT427-C2タンパク質 が細胞質膜上に発現していることが明らかとなった。 参考例 1 9 A549 cells were suspended at a concentration of lx 10 6 cells / ml with AS-2482 or non-immune rabbits (Jackson, Inc.) to a final concentration of 5 / ig / ml. ) And left on ice for 5 hours. Subsequently, the cells were washed with buffer A, suspended in buffer A containing 10 g / ml of Alexa488-labeled anti-Peacock IgG antibody (Molecular Probes), and allowed to stand on ice for 1.5 hours. After washing again with buffer A, analysis was performed with FACScan (BD Biosciences). As a result, A549 cells were stained specifically for the egret peptide antibody AS-2482. It was revealed that it was expressed above. Reference Example 1 9
アンチセンスオリゴヌクレオチド導入によるヒト非小細胞肺癌細胞株 A549およ び NCI- H226のアポ 1 シス誘導  Apoptosis induction of human non-small cell lung cancer cell lines A549 and NCI-H226 by introduction of antisense oligonucleotide
参考例 2に記載の NCI- H460以外のヒト非小細胞肺癌細胞株においてもアンチセ ンスオリゴヌクレオチド導入によりアポトーシスが誘発されるか否かを検討した c ヒト非小細胞肺癌細胞株 A549および NCI- H226 (いずれも ATCCより購入） を、 そ れぞれ Kaighn' s改変 F - 12 Nutrient Mixture (Invi trogen社） および 25mM HEPES 含有 RPMI- 1640培地 （Invi t rogen社） に牛胎仔血清 (層社) を 10%加えた培地で 懸濁し、 1ゥエル当たり I X 104個の細胞密度で 96穴平底組織培養プレート （BDフ . アルコン社） に播種した。 5%炭酸ガス気流中、 37°Cで一晩培養した後、 オリゴ ヌクレオチドを導入した。 In human non-small cell lung cancer cell lines other than NCI-H460 described in Reference Example 2, c human non-small cell lung cancer cell lines A549 and NCI-H226 were examined to determine whether apoptosis was induced by the introduction of antisense oligonucleotides. (Both purchased from ATCC), and fetal calf serum (Layer) in Kaighn's modified F-12 Nutrient Mixture (Invitrogen) and RPMI-1640 medium (Invitrogen) containing 25 mM HEPES, respectively. It was suspended in 10% added medium, and seeded into 1 Ueru per IX 10 4 cells density in 96-well flat-bottom tissue culture plates (BD off. Alcon). After culturing overnight at 37 ° C. in a 5% carbon dioxide gas stream, oligonucleotides were introduced.
以下に述べる参考例 1 9、 2 0および 2 1で用いたセンスオリゴヌクレオチド (配列番号： 4 4 ) は、 参考例 2に記載のアンチセンスオリゴヌクレオチド （配 列番号： 1 3 ) に相補的な配列を有するように設計し、 phosphorothioate化した 後、 HPLC精製して用いた （以下、 センスオリゴヌクレオチドと略する） 。  The sense oligonucleotide (SEQ ID NO: 44) used in Reference Examples 19, 20, and 21 described below was complementary to the antisense oligonucleotide (SEQ ID NO: 13) described in Reference Example 2. It was designed to have a sequence, phosphorothioated, purified by HPLC, and used (hereinafter abbreviated as sense oligonucleotide).
具体的には、 A549細胞の場合には、 参考例 2で得られたアンチセンスオリゴヌ クレオチド （配列番号： 13) 、 コントロールォリゴヌクレオチド (配列番号： 14) 、 およびセンスオリゴヌクレオチド （配列番号： 44) 、 それぞれ Specifically, in the case of A549 cells, the antisense oligonucleotide obtained in Reference Example 2 was used. Nucleotide (SEQ ID NO: 13), control oligonucleotide (SEQ ID NO: 14), and sense oligonucleotide (SEQ ID NO: 44), respectively
0. をリポフエクトァミン 2000 (Invitrogen社） 0.8 Uと共に、 OPTI-MEM I (Invitrogen社） 50 1と混合し、 室温で 20分間放置した。 予め OPTI- MEM I (Invitrogen社） 50 1に培地交換しておいた A549細胞に該混合液中を全量添加 し、 更に 3時間培養を継続した後、 10%牛胎仔血清 （JRH社） を含む Kaighn' s改変 F-12Nutrient Mixture (Invitrogen社） 100 1に培地交換した。 Was mixed with OPTI-MEM I (Invitrogen) 501 together with 0.8 U of Lipofectamine 2000 (Invitrogen) and left at room temperature for 20 minutes. Add the entire amount of the mixture to A549 cells whose medium has been changed to OPTI-MEM I (Invitrogen) 501 beforehand, continue culturing for 3 hours, and contain 10% fetal bovine serum (JRH) The medium was changed to Kaighn's modified F-12 Nutrient Mixture (Invitrogen) 1001.
NCI- H226細胞の場合では、 アンチセンスオリゴヌクレオチド （配列番号： 1 3) 、 コントロールオリゴヌクレオチド （配列番号： 14) 、 およびセンスオリ ゴヌクレオチド （配列番号： 44) のそれぞれ 0.13/1 gを、 ォリゴフェクトアミ ン （Invitrogen社） 0.8 1と共に、 OPT I -MEM I (Invitrogen社) 50 1と混合し， 室温で 20分間放置した。 予め OPTI- MEM I (Invitrogen社） 50 1に培地交換して おいた NCI- H226細胞に該混合液を全量添加し、 更に 3時間培養を継続した後、 30%牛胎仔血清 （JRH社） を含む 25mM HEPES含有 RPMI- 1640培地 （Invitrogen社） 50 1を添加した。  In the case of NCI-H226 cells, 0.13 / 1 g of each of the antisense oligonucleotide (SEQ ID NO: 13), the control oligonucleotide (SEQ ID NO: 14), and the sense oligonucleotide (SEQ ID NO: 44) was The solution was mixed with OPTI-MEMI (Invitrogen) 501 together with 0.81 of Feptamine (Invitrogen) and left at room temperature for 20 minutes. The whole volume of the mixture was added to NCI-H226 cells whose medium had been changed to OPTI-MEM I (Invitrogen) 501 in advance, and the culture was further continued for 3 hours. Then, 30% fetal bovine serum (JRH) was added. RPMI-1640 medium (Invitrogen) containing 25 mM HEPES was added.
オリゴヌクレオチドを導入して更に 2日間培養した後、 Cell Death Detection ELISAPLUS (Roche Diagnostics社)の添付プロトコールに従い、 上記オリゴヌクレ ォチドのアポト一シス誘導活性を測定した。 After the introduction of the oligonucleotide and culturing for another 2 days, the apoptosis-inducing activity of the above oligonucleotide was measured according to the protocol attached to Cell Death Detection ELISA PLUS (Roche Diagnostics).
その結果、 両細胞株においてアンチセンスオリゴヌクレオチドは陰性対象とし て用いたコントロールオリゴヌクレオチドおよびセンスオリゴヌクレオチドに比 ベ、 それぞれ 1.65倍および 3.03倍のアポトーシス誘導活性を示し、 統計学的に有 意な差 （P≤0.01) を示した。 参考例 20  As a result, in both cell lines, the antisense oligonucleotide showed 1.65-fold and 3.03-fold apoptosis-inducing activities, respectively, as compared to the control oligonucleotide and the sense oligonucleotide used as negative controls, and a statistically significant difference (P≤0.01). Reference Example 20
アンチセンスオリゴヌクレオチド導入によるヒト非小細胞肺癌細胞株 A549およ び NCI- H226における FLJ20539遺伝子 (配列番号 : 2) 、 hCP50177遺伝子 （配列番 号： 5) 、 hCP1762319遺伝子 (配列番号 : 8) ならびに FLJ13515遺伝子 (配列番 号： 11) 、 および TACT427遺伝子の mRNA発現量低下  FLJ20539 gene (SEQ ID NO: 2), hCP50177 gene (SEQ ID NO: 5), hCP1762319 gene (SEQ ID NO: 8) and FLJ13515 in human non-small cell lung cancer cell lines A549 and NCI-H226 by introducing antisense oligonucleotides Gene (SEQ ID NO: 11) and mRNA expression of TACT427 gene decreased
参考例 3に記載の NCI- H460以外のヒ卜非小細胞肺癌細胞株においてもアンチセ ンスオリゴヌクレオチド投与により、 上記遺伝子の mRNA発現量が低下するか否か 調べた。 Anti-serum was also detected in human non-small cell lung cancer cell lines other than NCI-H460 described in Reference Example 3. It was examined whether or not the administration of the oligonucleotide reduced the mRNA expression level of the above gene.
ヒト非小細胞肺癌細胞株 A549および NCI-H226をそれぞれ参考例 19と同じ培地 に懸濁し、 1ゥエル当たり A549細胞株では 7.5X104個、 NCI- H226細胞株では Human non-small cell lung cancer cell lines A549 and NCI-H226 respectively suspended in the same medium as Reference Example 19, 7.5 × 10 4 pieces in the 1 Ueru per A549 cell lines, with NCI H226 cell lines
5 X104個の細胞密度で 24穴平底組織培養プレート （BDファルコン社） に播種した c 5%炭酸ガス気流中、 37°Cで一晩培養した後、 オリゴヌクレオチドをトランスフ ェクションした。 After culturing overnight at 37 ° C in a 5% carbon dioxide gas stream seeded on a 24-well flat bottom tissue culture plate (BD Falcon) at a cell density of 5 x 10 4 cells, the oligonucleotide was transfected.
具体的に、 A549細胞株では、 アンチセンスオリゴヌクレオチド （配列番号： 1 3) 、 コントロールオリゴヌクレオチド （配列番号： 14) 、 およびセンスオリ ゴヌクレオチド （配列番号： 44) のそれぞれ 0· 84 をリポフエクトァミン 2000 (Invitrogen社） 3.2 1と共に、 Opti-MEMI (Invitrogen社) 200 lと混合 し、 室温で 20分間放置した。 予め 0PTI- MEM I (Invitrogen社） 200 1に培地交 換しておいた Α549細胞に該混合液を全量添加し、 更に 3時間培養を継続した後、 10%牛胎仔血清 （JRH社） を含む Kaiglm's改変 F- 12Nutrient Mixture  Specifically, in the A549 cell line, 0 · 84 of each of the antisense oligonucleotide (SEQ ID NO: 13), the control oligonucleotide (SEQ ID NO: 14), and the sense oligonucleotide (SEQ ID NO: 44) is lipofector. The mixture was mixed with 200 l of Opti-MEMI (Invitrogen) together with Min 2000 (Invitrogen) 3.21 and left at room temperature for 20 minutes. Add the entire volume of the mixture to Α549 cells that had been medium-exchanged to 0PTI-MEM I (Invitrogen) 2001 before continuing cultivation for 3 hours, then containing 10% fetal bovine serum (JRH). Kaiglm's Modified F-12 Nutrient Mixture
(Invitrogen社） 1に培地交換した。  (Invitrogen) The medium was changed to 1.
NCI- H226細胞の場合では、 アンチセンスオリゴヌクレオチド （配列番号： 1 3) 、 コントロールオリゴヌクレオチド （配列番号： 14) 、 およびセンスオリ ゴヌクレオチド （配列番号： 44) のそれぞれ 0· 13 xgを、 ォリゴフェクトアミ ン （Invitrogen社） 2 1と共に Opt i- MEM I (invitrogen社） 125/xlと混合し、 室 温で 20分間放置した。 予め 0PTI- MEM I (Invitrogen社） 125 1に培地交換して おいた NCI- H226細胞に該混合液を全量添加し、 更に 4時間培養を継続した後、 30%牛胎仔血清 （JRH社） を含む 25mM HEPES含有 RPMI- 1640培地 （Invitrogen社） 125/11を加えた。  In the case of NCI-H226 cells, 0.13 xg each of the antisense oligonucleotide (SEQ ID NO: 13), the control oligonucleotide (SEQ ID NO: 14), and the sense oligonucleotide (SEQ ID NO: 44) was The mixture was mixed with Opti-MEMI (Invitrogen) 125 / xl together with Ffectamine (Invitrogen) 21 and allowed to stand at room temperature for 20 minutes. The whole volume of the mixture was added to NCI-H226 cells whose medium had been changed to 0PTI-MEM I (Invitrogen) 1251 in advance, and cultivation was further continued for 4 hours. Then, 30% fetal bovine serum (JRH) was added. RPMI-1640 medium (Invitrogen) containing 25 mM HEPES was added.
オリゴヌクレオチドをトランスフエクシヨンし、 更に 16時間培養を継続した後 に、 RNeasyMini Total RNA Kit (QIAGEN社) を用いて A549細胞および NCI- H226細 胞から 1 ^一タル R Aを抽出した。 TaaMan™ Reverse Transcription Reagents  After transfection of the oligonucleotide and culturing for further 16 hours, 1 ^ -tal RA was extracted from A549 cells and NCI-H226 cells using the RNeasyMini Total RNA Kit (QIAGEN). TaaMan ™ Reverse Transcription Reagents
(Applied Biosystems社） の添付プロトコ一ルに従い、 ランダムプライマーを用 いた逆転写反応によってトータル RNAから cDNAを調製した。 トータル R A5 ngから 調製した cDNAを錶型とし、 参考例 6と同様の方法により FLJ20539遺伝子 （配列番 号： 2) 、 hCP50177遺伝子 (配列番号： 5) 、 hCP1762319遺伝子 (配列番号： 8) ならびに FLJ13515遺伝子 （配列番号： 1 1) 、 および TACT427遺伝子の発現 量を測定した。 同量の鎢型 cDNA中に含まれる ァクチン遺伝子発現量を According to the attached protocol of (Applied Biosystems), cDNA was prepared from total RNA by reverse transcription using random primers. The cDNA prepared from 5 ng of total RA was designated as type I, and the FLJ20539 gene (sequence number No .: 2), hCP50177 gene (SEQ ID NO: 5), hCP1762319 gene (SEQ ID NO: 8), FLJ13515 gene (SEQ ID NO: 11), and expression level of TACT427 gene were measured. The amount of actin gene expression in the same amount of type III cDNA
Ta Man™ /3-actin Control Reagents (Applied Biosystems社） を用いて測定し 内部標準とした。  It was measured using Ta Man ™ / 3-actin Control Reagents (Applied Biosystems) and used as an internal standard.
i3 -ァクチン遺伝子発現量に対する上記遺伝子の相対的発現量比率 (%) は、 陰性対照として用いたコントロールォリゴヌクレオチド （配列番号： 14) また はセンスオリゴヌクレオチド （配列番号： 44) を導入した場合に比べて、 アン チセンスオリゴヌクレオチド （配列番号： 13) を導入した場合に顕著に低下し ており、 統計学的に有意 （P≤0.01) な発現量低下が認められた （表 8) 。  The relative expression ratio (%) of the above gene to the i3-actin gene expression is based on the introduction of the control oligonucleotide (SEQ ID NO: 14) or the sense oligonucleotide (SEQ ID NO: 44) used as a negative control. In contrast, the expression level was significantly reduced when the antisense oligonucleotide (SEQ ID NO: 13) was introduced, and a statistically significant (P≤0.01) decrease in the expression level was observed (Table 8).
この結果より、 ヒト非小細胞肺癌細胞株 A549および NCI-H226においても、 FU20539遺伝子 （配列番号： 2) 、 hCP50177遺伝子 （配列番号： 5) 、  From these results, the human non-small cell lung cancer cell lines A549 and NCI-H226 also showed the FU20539 gene (SEQ ID NO: 2), hCP50177 gene (SEQ ID NO: 5),
MP1762319遺伝子 （配列番号： 8) ならびに FU13515遺伝子 （配列番号： 11) . および TACT427遺伝子の mRNA発現量低下により、 アポト一シスが誘発されたこと が示された。  It was shown that apoptosis was induced by the decreased mRNA expression level of the MP1762319 gene (SEQ ID NO: 8), the FU13515 gene (SEQ ID NO: 11) and the TACT427 gene.
表 8  Table 8
Figure imgf000096_0001
参考例 21
Figure imgf000096_0001
Reference Example 21
アンチセンスオリゴヌクレオチド導入による A549および NCI- H226における  In A549 and NCI-H226 by introduction of antisense oligonucleotide
TACT427タンパク質の発現量低下 ヒト非小細胞肺癌細胞株 A549および NCI-H226をそれぞれ参考例 1 9と同じ培地 に懸濁し、 A549細胞株では 2. 25 X 106個、 NCI- H226細胞株では 1. 45X 106個の細胞 を直径 10cmのペトリディッシュ （BDファルコン社） に播種した。 5%炭酸ガス気 流中、 37°Cで一晚培養した後、 オリゴヌクレオチドをトランスフエクシヨンした t 具体的には、 A549細胞株では、 アンチセンスオリゴヌクレオチド （配列番号： 1 3 ) 、 コントロールオリゴヌクレオチド （配列番号： 1 4 ) およびセンスオリ ゴヌクレオチド （配列番号： 4 4 ) のそれぞれ 5. 8 i gを、 リポフエクトァミン 2000 (Invitrogen社) 96 1と共に、 Opt i -MEM I (Invi trogen社) 6 mlと混合し， 室温で 20分間放置した。 予め 0PTI- MEM I (Invi trogen社） 6 mlに培地交換して おいた A549細胞に該混合液を全量添加し、 更に 3時間培養を継続した後、 10%牛 胎仔血清 (週社) を含む Kaighn' s改変 F - 12 Nutrient Mixture (Invitrogen社） 15 mlに培地交換した。 Decreased expression level of TACT427 protein Human non-small cell lung cancer cell lines A549 and NCI-H226 were suspended in the same medium as each Example 1 9, the A549 cell line 2. 25 X 10 6 cells, in NCI H226 cell lines 1. 45X 10 6 pieces of Cells were seeded on a 10 cm diameter Petri dish (BD Falcon). 5% carbon dioxide gas flow, after one晚incubated at 37 ° C, the t specifically that transflector Ekushi Yung oligonucleotides, the A549 cell line, the antisense oligonucleotide (SEQ ID NO: 1 3), control oligo 5.8 ig of each of the nucleotide (SEQ ID NO: 14) and the sense oligonucleotide (SEQ ID NO: 44) together with Lipofectamine 2000 (Invitrogen) 961 together with Opti-MEM I (Invitrogen) The mixture was mixed with 6 ml and left at room temperature for 20 minutes. Add the entire volume of the mixture to A549 cells whose medium has been exchanged to 6 ml of 0PTI-MEM I (Invitrogen) in advance, and continue culturing for an additional 3 hours, then containing 10% fetal bovine serum (Weekly) The medium was changed to 15 ml of Kaighn's modified F-12 Nutrient Mixture (Invitrogen).
NCI- H226細胞株では、 アンチセンスオリゴヌクレオチド （配列番号： 1 3 ) 、 コントロールオリゴヌクレオチド （配列番号： 1 4 ) およびセンスオリゴヌクレ ォチド （配列番号： 4 4 ) のそれぞれ 9. 7 gを、 オリゴフエクトァミン  In the NCI-H226 cell line, 9.7 g each of the antisense oligonucleotide (SEQ ID NO: 13), the control oligonucleotide (SEQ ID NO: 14) and the sense oligonucleotide (SEQ ID NO: 44) were Fectamine
(Invitrogen社） 60 /z 1と共に 0pt i_MEM I (Invi trogen社) 3. 75 mlと混合し、 室 温で 20分間放置した。 予め 0PTI- MEM I (Invi trogen社） 3. 75 mlに培地交換して おいた NCI- H226細胞に該混合液を全量添加し、 更に 4時間培養を継続した後、 30%牛胎仔血清 （JRH社） を含む 25mM HEPES含有 RPMI-1640培地 （Invitrogen社） 3. 75 mlを加えた。  The mixture was mixed with 3.75 ml of 0pt i_MEM I (Invitrogen) together with (Invitrogen) 60 / z1, and left at room temperature for 20 minutes. The total volume of the mixture was added to NCI-H226 cells whose medium had been changed to 0 PTI-MEM I (Invitrogen) 3.75 ml in advance, and cultivation was continued for another 4 hours, followed by 30% fetal bovine serum (JRH). 3.75 ml of RPMI-1640 medium (Invitrogen) containing 25 mM HEPES containing the following.
トランスフエクシヨン後、 更に 24時間、 48時間、 72時間培養を継続した後に、 参考例 1 2の方法に従い無細胞抽出液を調製した。 得られた無細胞抽出液のタン パク質濃度を BCAProtein Assay Ki t (Pierce社） にて測定し、 各無細胞抽出液中 のタンパク質濃度をそろえた。 A549細胞株の無細胞抽出液 100 g、 および NCI- H226細胞株の無細胞抽出液 140 gを、 参考例 1 2の方法に準じて SDS-PAGEおよび ウエスタンプロッティングをそれぞれ行った。 一次抗体として参考例 1 1で作製 した AS- 2482を 3 g/mlの濃度で、 二次抗体として HRP標識抗ゥサギ IgG抗体  After the transfection, the culture was further continued for 24 hours, 48 hours, and 72 hours, and then a cell-free extract was prepared according to the method of Reference Example 12. The protein concentration of the obtained cell-free extract was measured using a BCAProtein Assay Kit (Pierce), and the protein concentration in each cell-free extract was adjusted. 100 g of the cell-free extract of the A549 cell line and 140 g of the cell-free extract of the NCI-H226 cell line were subjected to SDS-PAGE and Western plotting according to the method of Reference Example 12 respectively. AS-2482 prepared in Reference Example 11 was used as the primary antibody at a concentration of 3 g / ml, and HRP-labeled anti-Peagle IgG antibody was used as the secondary antibody.
(Amersliam- Bioscience社） を用いた。 検出は SuperSignal™ West Femto  (Amersliam-Bioscience) was used. Detection is SuperSignal ™ West Femto
Maximum Sensit ivity Substrate (Pierce社） を用いて添付のマニュアルに従つ た。 Follow the attached manual using Maximum Sensitivity Substrate (Pierce) It was.
その結果、 両細胞株ともァンチセンスォリゴヌクレオチドを導入した場合にの み、 TACT427タンパク質がほぼ消失しているのが確認され、 かつ、 該タンパク質 の消失は 24時間で認められた。 また、 同時にサイトケラチン 8タンパク質の発現 量を抗ヒトサイトケラチン 8抗体 （Oncogene社） を用いたウエスタンプロッティ ング法で調べたが、 いずれのオリゴヌクレオチド処理でも発現量減少は認められ なかった。 これより、 TACT427タンパク質発現量の特異的低下により、 ヒト肺癌 細胞株のアポトーシスが誘発されたことが示された。 参考例 2 2  As a result, it was confirmed that the TACT427 protein almost disappeared only when the antisense oligonucleotide was introduced into both cell lines, and the disappearance of the protein was observed in 24 hours. At the same time, the expression level of the cytokeratin 8 protein was examined by Western blotting using an anti-human cytokeratin 8 antibody (Oncogene), but no decrease in the expression level was observed with any of the oligonucleotide treatments. This indicated that a specific decrease in the expression level of the TACT427 protein induced apoptosis of a human lung cancer cell line. Reference Example 2 2
組換え型完全長夕ンパク質の安定発現細胞株の樹立  Establishment of a cell line that stably expresses recombinant full-length protein
TACT427- Aタンパク質 （配列番号： 1 5 ) の C末端に 3xFLAGタグを融合した夕 ンパク質を構成的に発現する細胞株の樹立をマウス胎仔由来線維芽細胞株 Balb3T3-A31 (以後、 A31細胞と略記する) を用いて行った。 A31細胞 1. 25 X 105個 を 10%牛胎仔血清 （JRH社） および 50 g/mlゲンタマイシン （Invi trogen社） を 含むダルベッコ改変型イーグル最少培地 （Invi trogen社） 2· 5 mlに懸濁し、 6穴 プレートの 1ゥエルに播種した後、 5%炭酸ガス気流下、 37でで一晩培養した。 さ らに同じ培地 2. 5mlに培地交換し 4時間培養を継続した後、 予め FUGENE6トランス フエクシヨン試薬 3. 8 1 (Roche Diagnos t ics社) および OPTI- MEM I The establishment of a cell line that constitutively expresses a protein with a 3xFLAG tag fused to the C-terminus of the TACT427-A protein (SEQ ID NO: 15) (Abbreviated). A31 cells 1. 25 X 10 5 cells with 10% fetal bovine serum (JRH Co.) and 50 g / ml gentamicin (Invi trogen Inc.) Dulbecco's modified Eagle's medium containing (Invi trogen Inc.) were suspended in 2 · 5 ml After seeding in a 1-well of a 6-well plate, the cells were cultured overnight at 37 in a 5% carbon dioxide gas stream. Further, after changing the medium to 2.5 ml and continuing the culture for 4 hours, FUGENE6 transfection reagent 3.81 (Roche Diagnostics) and OPTI-MEM I
(Invi trogen社） 93. 3 x 1と混合し室温で 15分間放置しておいたプラスミド p3xFLAG-TACT427-Al. を添加し培養を継続した。 翌日にトリプシン · EDTA (Invitrogen) 93.3. The plasmid p3xFLAG-TACT427-Al., Which had been mixed with 1 × 3 and left at room temperature for 15 minutes, was added thereto, and the culture was continued. Next day trypsinEDTA
(Invi trogen社) を用いて細胞を回収し、 0. 5 mg/mlの G418 (Promega社) を含む 上記培地 （G418選択培地） 10mlに懸濁し、 直径 10 cmのペトリディッシュに播種 した。 G418選択培地で培養を継続し 2回継代培養した後、 1ゥエルあたり細胞 100 個 （培地容量 0. 1 ml) の割合から始まる 2倍希釈系列を 12系列作成し 96穴プレー トにそれぞれ播種し、 3日ごとに G418選択培地を交換しながら培養を継続した。 ゥエルあたり 0. 8個〜 3. 2個の細胞が増殖しコロニーを形成したゥエルから 11日後 に細胞を回収'し、 24穴プレートの 2ゥエルに等しく播種した。 G418選択培地でコ ：なるまで培養を継続した後、 1ゥエル分の細胞をスクレイパーで 回収し、 1 % 2-メルカプトエタノールを含む SDS- PAGE用サンプルバッファー (Bio Rad社） に懸濁した。 95°Cで 5分間加熱処理した後、 12 1を 10%ァク リルアミドゲルでの SDS-PAGEに供した。 参考例 1 3で記載した方法に準じ、 マウ ス抗 FLAGM2抗体 （Sigma社、 1000倍希釈） を用いてウエスタンブロッテイングを 行い、 TACT427-Aタンパク質 （配列番号： 1 5 ) の C末端に 3xFLAGタグが付加した タンパク質を構成的に発現する細胞株 TACT- 1を得た。 参考例 2 3 Cells were collected using (Invitrogen), suspended in 10 ml of the above-mentioned medium (G418 selective medium) containing 0.5 mg / ml of G418 (Promega), and seeded on a Petri dish having a diameter of 10 cm. Continue culturing with G418 selection medium and subculture twice, then prepare 12 series of 2-fold dilutions starting from 100 cells / well (medium volume: 0.1 ml) and inoculate them into 96-well plates respectively. The culture was continued while replacing the G418 selection medium every three days. Cells were harvested 11 days after the 0.8-3.2 cells grew and formed a colony per well, and seeded equally in 2 wells of a 24-well plate. After continuing the culture in the G418 selection medium until the cells reach 1%, the cells of 1 μl are scraped with a scraper. The cells were collected and suspended in a sample buffer for SDS-PAGE (Bio Rad) containing 1% 2-mercaptoethanol. After heat treatment at 95 ° C for 5 minutes, 121 was subjected to SDS-PAGE on a 10% acrylamide gel. According to the method described in Reference Example 13 and 3, Western blotting was performed using a mouse anti-FLAGM2 antibody (Sigma, diluted 1000-fold). A cell line TACT-1 that constitutively expresses the protein added with was obtained. Reference Example 2 3
TACT- 1のアポトーシス耐性能の評価  Evaluation of anti-apoptotic performance of TACT-1
参考例 2 2で得られた TACT- 1およびその親株 A31細胞を 10%牛胎仔血清 （JRH 社） および SO ^ g/mlゲンタマイシン （Invi trogen社） を含むダルベッコ改変型ィ 一ダル最少培地 （Invi trogen社） 0. lmlに懸濁し 1ゥエルあたり 6 x 103個になるよ うそれぞれ組織培養用 96穴プレートに播種した。 5%炭酸ガス気流下、 37°Cで一 晚培養した後、 トポイソメラーゼ I阻害剤カンプトテシン （WakoPure Chemical 社） あるいはタンパク質合成阻害剤ァニソマイシン （Wako Pure Chemical社） を 種々の濃度になるよう添加した上記培地に交換し培養を継続した。 24時間後に Cel l Death Detect ion ELISA腿 (Roche Diagnost ics社) を用いてアポトーシ スを検出した。 終濃度 1 /x g/mlのカンプトテシン添加により TACT- 1に誘発された アポトーシスは、 同じ条件で A31に認められたアポト一シスの 56 %にとどまった。 また終濃度 1 ^ g/mlのァニソマイシン添加により TACT- 1に誘発されたアポトーシ スは、 同じ条件で A31に認められたアポトーシスの 69%にとどまった。 以上の結 果は、 カンプトテシンやァニソマイシンによって誘発されるアポトーシスに対し、 TACT-1細胞が耐性になっていることを示しており、 TACT427- A強制発現によりァ ポトーシス耐性能を獲得することが明らかとなった。 参考例 2 4 The TACT-1 and its parental A31 cells obtained in Reference Example 22 were transformed with Dulbecco's modified idal minimal medium (Invitrogen) containing 10% fetal bovine serum (JRH) and SO ^ g / ml gentamicin (Invitrogen). The suspension was suspended in 0.1 ml and seeded on a 96-well plate for tissue culture at 6 x 10 3 cells / well. After culturing at 37 ° C in a 5% carbon dioxide gas stream, the above medium to which various concentrations of topoisomerase I inhibitor camptothecin (WakoPure Chemical) or protein synthesis inhibitor anisomycin (Wako Pure Chemical) is added. And the culture was continued. After 24 hours, apoptosis was detected using Cell Death Detection ion ELISA thigh (Roche Diagnostics). Apoptosis induced by TACT-1 by adding camptothecin at a final concentration of 1 / xg / ml was only 56% of the apoptosis observed in A31 under the same conditions. Under the same conditions, apoptosis induced by TACT-1 with the addition of anisomycin at a final concentration of 1 ^ g / ml was 69% of the apoptosis observed in A31 under the same conditions. The above results indicate that TACT-1 cells are resistant to apoptosis induced by camptothecin and anisomycin, and that TACT427-A is forced to express apoptosis resistance by forced expression. became. Reference example 2 4
TACT- 1における ERK1/2リン酸化増強  Enhanced phosphorylation of ERK1 / 2 in TACT-1
参考例 2 3で記載した TACT- 1細胞株のアポトーシス耐性現象のメカニズムを調 ベる一環として、 抗アポトーシス作用に関与する MAPキナーゼ、 即ち ERK1/2タン パク質のリン酸化を A31細胞株と比較した。 A31細胞には 10%牛胎仔血清 （JRH 社） および 50 g/mlゲン夕マイシン （Invi trogen社） を含むダルベッコ改変型ィ —ダル最少培地（Invi trogen社）を用い、 TACT- 1細胞には同培地に更に 0. 6 mg/mlG418 (Promega社） を添加したものを用いて、 直径 10 cmのペトリディッシ ュ（BD ファルコン社）で 5%炭酸ガス気流中、 37°Cで培養した。 細胞密度が約 80% コンフルェントになった時点で、 l i g/mlァニソマイシン （Wako Pure Chemical 社） を含む上記培地に交換し、 37°Cで 1時間、 4時間および 8時間培養を続けた。 - ァニソマイシン処理前の細胞も含めて、 これらそれぞれの細胞を氷冷した PBS (Ca、 Mg不含） 10mlで 2回洗浄し、 0. 5 mlの細胞溶解用緩衝液 [1 % Tri ton X- 100、 1 % デォキシコール酸、 0. 05% SDS、 5. 25mM EGTA, EDTA不含 Co即 lete™タブレット (Roche Diagnost ics社） 、 Phosphatase inhibi tor cocktai l-2 (Sigmafi) 、 150mM塩化ナトリゥムを含む 50 トリス ·塩酸緩衝液、 pH7. 5〕 を加えて 4°Cで 20 分間放置した。 スクレイパーを用いて細胞破砕液を回収し、 4°C、 15000回転で 20 分間遠心分離して沈殿物を除去した。 この細胞破砕液 80 1に 10% 2-メルカプト エタノールを含む 5倍濃縮 SDS- PAGE用サンプルバッファー （Bio Rad社） を 20 1 加え、 95°Cで 5分間加熱した。 なお、 5 1細胞破砕液を蒸留水で 10倍希釈し、 Micro BCA protein assay reagent (Pierce社）の処方に準じてタンパク質濃度を 測定し、 2% 2-メルカプトエタノールを含む SDS- PAGE用サンプルバッファー As a part of investigating the mechanism of the apoptosis resistance phenomenon of the TACT-1 cell line described in Reference Example 23, MAP kinase involved in the anti-apoptotic action, that is, ERK1 / 2 protein Protein phosphorylation was compared to the A31 cell line. Dulbecco's modified minimal medium (Invitrogen) containing 10% fetal calf serum (JRH) and 50 g / ml Genymycin (Invitrogen) was used for A31 cells, and TACT-1 cells were used for TACT-1 cells. Using the same medium supplemented with 0.6 mg / ml G418 (Promega), the cells were cultured at 37 ° C. in a 10% diameter petri dish (BD Falcon) in a stream of 5% carbon dioxide gas. When the cell density reached about 80% confluence, the medium was replaced with the above medium containing lig / ml anisomicin (Wako Pure Chemical), and culturing was continued at 37 ° C for 1, 4 and 8 hours. -Wash each of these cells, including cells before anisomycin treatment, twice with 10 ml of ice-cold PBS (without Ca and Mg), and add 0.5 ml of cell lysis buffer [1% Triton X- 100, 1% Deoxycholic acid, 0.05% SDS, 5.25mM EGTA, EDTA-free Co Immediate lete ™ Tablets (Roche Diagnostics), Phosphatase inhibi tor cocktai l-2 (Sigmafi), 150mM sodium chloride 50 Tris-HCl buffer, pH 7.5] was added and left at 4 ° C for 20 minutes. The cell lysate was recovered using a scraper, and centrifuged at 15,000 rpm for 20 minutes at 4 ° C to remove the precipitate. To the cell lysate 801, 20 1 of a 5-fold concentrated SDS-PAGE sample buffer (Bio Rad) containing 10% 2-mercaptoethanol was added, and the mixture was heated at 95 ° C for 5 minutes. The 50-cell lysate was diluted 10-fold with distilled water, and the protein concentration was measured according to the prescription of Micro BCA protein assay reagent (Pierce). Sample buffer for SDS-PAGE containing 2% 2-mercaptoethanol
(BioRad社） で適宜希釈してタンパク質濃度を揃えた。 総タンパク質 24 ^ gを 7. 5% ポリアクリルアミドゲルでの SDS- PAGEに供した後、 PVDF膜に転写した。 転 写した膜は 5%スキムミルクを含む TTBS (0. l %Tween 20を含む TBS) で室温、 1時 間ブロッキングした後、 10分間の TTBS洗浄を 2回行った。 その後、 抗 ERK1/2抗体 (Cel l s ignal ing社) または抗リン酸化 ERK1/2抗体 (Cel l s ignal ing社） を 5% BSA (Sigma社） を含む TTBSでそれぞれ 5000倍または 1000倍に希釈した一次抗体液 を用いて 4 で一晩反応させた後、 10分間の TTBS洗浄を 4回行った。 次に、 5 %ス キムミルクを含む TTBSで 10000倍に希釈した HRP標識抗ゥサギ IgG抗体 (Amersham Bioscience社）を添加し室温で 1時間保温した後、 10分間の TTBS洗浄を 4回行った。 ECLpIus試薬 (Amersham Bioscience社) を用いて ERK1/2タンパク質およびリン酸 化 ERK1/2タンパク質に相当するバンドを検出した結果、 TACT-1細胞株では A31細 胞株と比べ、 ァニソマイシン添加で誘導される ERK1/2タンパク質のリン酸化、 即 ち活性化が増強されていた。 TACT- 1細胞における ERK1/2活性化亢進程度を求める ため、 現像したフィルムをルミノイメージアナライザー LAS- lOOOplus (FUJIFILM 社） で画像として読み取り、 付属のイメージゲージソフトウェアを用いてリン酸 化 ERK1とリン酸化 ERK2のバンド強度をそれぞれ数値化した。 ァニソマイシン処理 時間 （2時間、 4時間および 8時間） 毎に、 A31細胞のバンド強度を 100%として TACT- 1細胞のリン酸化亢進率を算出したところ、 TACT- 1細胞における ERK1のリン 酸化亢進率は 164%、 150%および 158%、 ER 2のリン酸化亢進率は 130%、 137% および 172%であった。 (BioRad) to make the protein concentration uniform. 24 ^ g of the total protein was subjected to SDS-PAGE on a 7.5% polyacrylamide gel, and then transferred to a PVDF membrane. The transferred membrane was blocked with TTBS containing 5% skim milk (TBS containing 0.1% Tween 20) for 1 hour at room temperature, and then washed twice with TTBS for 10 minutes. Thereafter, the anti-ERK1 / 2 antibody (Cel lsignaling) or anti-phosphorylated ERK1 / 2 antibody (Cel lsignaling) was diluted 5000-fold or 1000-fold with TTBS containing 5% BSA (Sigma), respectively. After overnight reaction at 4 using the primary antibody solution, TTBS washing for 10 minutes was performed four times. Next, HRP-labeled anti-Egret IgG antibody (Amersham Bioscience) diluted 10000-fold with TTBS containing 5% skim milk was added, and the mixture was incubated at room temperature for 1 hour, and then washed 10 times with TTBS for 10 minutes. Bands corresponding to ERK1 / 2 protein and phosphorylated ERK1 / 2 protein were detected using ECLpIus reagent (Amersham Bioscience). Phosphorylation of ERK1 / 2 protein induced by the addition of anisomycin, ie, activation, was enhanced as compared to the vesicle strain. To determine the degree of ERK1 / 2 activation enhancement in TACT-1 cells, read the developed film as an image with a lumino image analyzer LAS-LOOOOplus (FUJIFILM), and use the attached image gauge software to phosphorylate ERK1 and phosphorylation. The band intensity of ERK2 was quantified. The rate of phosphorylation enhancement of TACT-1 cells was calculated for each of the anisomicin treatment times (2 hours, 4 hours, and 8 hours), with the band intensity of A31 cells as 100%. Were 164%, 150% and 158%, and the phosphorylation rate of ER2 was 130%, 137% and 172%.
この結果から、 TACT-1細胞株のアポトーシス耐性現象のメカニズムの一つが ERK 1/2の活性化増強作用であることが明らかとなった。 参考例 2 5  From these results, it was revealed that one of the mechanisms of the apoptosis resistance phenomenon of the TACT-1 cell line is the activity of enhancing the activation of ERK1 / 2. Reference example 2 5
TACT- 1における P38MAPKリン酸化促進  Enhanced phosphorylation of P38MAPK in TACT-1
参考例 2 3で記載した TACT- 1細胞株のアポ卜一シス耐性現象のメカニズムを調 ベる一環として、 P38MAPKのリン酸化を A31細胞株と比較した。  As a part of investigating the mechanism of the apoptosis resistance phenomenon of the TACT-1 cell line described in Reference Example 23, the phosphorylation of P38MAPK was compared with that of the A31 cell line.
8 X 105個の A31細胞または TACT- 1細胞を、 10%牛胎仔血清 （JRH社） および 50 g/mlゲンタマイシン （Invi trogen社） を含むダルベッコ改変型イーグル最少 培地 （Invi trogen社） 5 mlに懸濁し、 直径 6 cmのペトリディッシュ（BD フアルコ ン社）に播種した。 5%炭酸ガス気流中、 37t:で一晩培養した後、 終濃度 1 z g/ml となるようァニソマイシン （Wako Pure Chemical社） を添加し直ちに培養を継続 した。 ァニソマイシン添加前あるいは添加後 15分、 30分および 60分後の細胞を lmMオルトバナジン (V)酸ナトリウム （Wako Pure Chemical社） を含む PBS 5 mlで 1回洗浄した後、 参考例 1 2に記載の RIPA緩衝液に Phosphatase Inhibi tor Cocktai卜 1を加えた細胞溶解用緩衝液を 0. 2 ml添加し 4°Cで 15分間放置した。 ス クレイパーで細胞破砕液を回収し 4°C、 15000回転で 5分間遠心分離して沈殿物を 除去した。 この細胞破砕液 80 1に 5% 2-メルカプトエタノールを含む 5倍濃縮 SDS- PAGE用サンプルバッファー (Bio Rad社) を 20 1加え、 95°Cで 5分間加熱し た。 なお、 上記細胞溶解用緩衝液を蒸留水で 20倍に希釈した溶液で細胞破砕液 15 1を 20倍希釈し、 Micro BCA protein assay reagent (Pierce社）の処方に準 じてタンパク質濃度を測定し、 タンパク質濃度がほぼ揃っていることを確認した 総タンパク質約 14 gを 5 %— 20 %ポリアクリルアミドグラジェントゲルでの SDS- PAGEに供した後、 PVDF膜に転写した。 転写した膜は 5 %スキムミルクを含む TTBS (0. 1 % Tween 20を含む TBS) で室温、 1時間ブロッキングした後、 5分間の TTBS 洗浄を 3回行った。 その後、 抗 P38MAPK抗体 （Cel l s ignal ing社） または抗リン酸 化 P38MAPK抗体 (Cel l s ignal ing社） を 5% BSA (Sigma社) を含む TTBSで 1000倍 に希釈した一次抗体液を用いて 4°Cで一晩反応させた。 続いて、 5分間の TTBS洗浄 を 3回行った後に、 5 %スキムミルクを含む TTBSで 5000倍に希釈した HRP標識抗ゥ サギ IgG抗体 (Amersham Bioscience社）を添加し室温で 1時間保温した。 再び、 5分 間の TTBS洗浄を 3回行い、 過剰量の抗体を取り去った後に、 ECL plus試薬 8 x 10 5 A31 cells or TACT-1 cells in 5 ml Dulbecco's modified Eagle's minimal medium (Invitrogen) containing 10% fetal calf serum (JRH) and 50 g / ml gentamicin (Invitrogen) And seeded on a Petri dish (BD Huarcon) with a diameter of 6 cm. After culturing overnight at 37 t: in a 5% carbon dioxide gas stream, anisomycin (Wako Pure Chemical) was added to a final concentration of 1 zg / ml, and the culturing was continued immediately. The cells were washed once with 5 ml of PBS containing lmM sodium orthovanadate (V) (Wako Pure Chemical) before or at 15 minutes, 30 minutes and 60 minutes after the addition of anisomycin, and described in Reference Example 12. 0.2 ml of a cell lysis buffer obtained by adding Phosphatase Inhibitor Cocktail 1 to the RIPA buffer was added and left at 4 ° C for 15 minutes. The cell lysate was collected with a scraper and centrifuged at 15,000 rpm at 4 ° C for 5 minutes to remove the precipitate. To the cell lysate 801, 201 1 of a 5-fold concentrated SDS-PAGE sample buffer (Bio Rad) containing 5% 2-mercaptoethanol was added, and heated at 95 ° C for 5 minutes. A cell lysate was prepared by diluting the above cell lysis buffer 20 times with distilled water. 15 Dilute 20 1-fold and measure the protein concentration according to the formulation of Micro BCA protein assay reagent (Pierce), and confirm that the protein concentration is almost the same. After subjecting to SDS-PAGE using a% polyacrylamide gradient gel, it was transferred to a PVDF membrane. The transferred membrane was blocked with TTBS containing 5% skim milk (TBS containing 0.1% Tween 20) for 1 hour at room temperature, and then washed with TTBS three times for 5 minutes. Then, using a primary antibody solution of anti-P38 MAPK antibody (Cell lsignaling) or anti-phosphorylated P38 MAPK antibody (Cells ignaling) diluted 1000-fold with TTBS containing 5% BSA (Sigma). Reaction was carried out overnight at ° C. Subsequently, after washing TTBS three times for 5 minutes, an HRP-labeled anti-heron IgG antibody (Amersham Bioscience) diluted 5000 times with TTBS containing 5% skim milk was added, and the mixture was incubated at room temperature for 1 hour. Perform TTBS washing 3 times again for 5 minutes to remove excess antibody.
(Amersham Bioscience社） を用いて p38MAPKタンパク質およびリン酸化 p38MAPK タンパク質に相当するバンドを検出した。 A31細胞では P38MAPKタンパク質のリン 酸化はァニソマイシン添加後 30分でようやく微かに認められたのに対し、 TACT - 1 細胞ではァニソマイシ 添加後 15分で明らかに強く認められ、 P38MAPK夕ンパク 質のリン酸化、 即ち活性化が速やかに誘導されることが判明した。  (Amersham Bioscience) was used to detect bands corresponding to p38MAPK protein and phosphorylated p38MAPK protein. In A31 cells, phosphorylation of P38 MAPK protein was only slightly observed at 30 minutes after the addition of anisomicin, whereas in TACT-1 cells, it was clearly stronger at 15 minutes after the addition of anisomicis, and phosphorylation of P38 MAPK protein was observed. That is, it was found that activation was quickly induced.
この結果から、 TACT-1細胞株のアポトーシス耐性現象のメカニズムの一端を P38MAPKの活性化増強作用が担っていることが示唆された。 参考例 2 6  These results suggest that the activation of P38MAPK plays a role in one of the mechanisms of the apoptosis resistance phenomenon of the TACT-1 cell line. Reference Example 2 6
NAV2タンパク質 （配列番号： 4 9 ) を発現する動物細胞用発現ベクターの構築 を行った。 ヒト脳 Marathon-Ready cDNA (CL0NTECH社） を鎳型とし、 2種のプライ マー 〔プライマー 1 1 (配列番号： 5 2 ) およびプライマー 1 2 (配列番号： 5 3 ) 〕 を用いて PCR反応を行い、 NAV2 (配列番号： 4 9 ) の 1番目から 1 8 8 9 番目までのアミノ酸配列をコードする cDNAのクロ一ニングを行った。 該反応にお ける反応液の組成は、 上記 CDNAI Iを铸型として使用し、 PfuTurbo Hotstart DNA Polymerase (STRATAGENE社） 2. 5ϋ、 プライマー 1 1 (配列番号： 5 8 ) およ びプライマー 1 2 (配列番号： 5 9 ) を各 1. 0 M、 dNTPsを 200 Μ、 および 2XGC Buf fer I (TaKaRa Bio社) を 25 L加え、 50μ の液量とした。 PCR反応は、 94 · 1分の後、 94°C · 20秒、 58°C · 15秒、 72°C · 10分のサイクルを 40回繰り返し、 続 けて 72°C · 10分の反応を行った。 該 PCR反応産物をァガロースゲルにて電気 ¾動 後、 Gel Extraction Kit (QIAGEN社） を用いて精製した。 これをプラスミドべク ター pCR4Blunt- T0P0 (Invitrogen社） へサブクローニングし、 大腸菌 T0P10 (Invitrogen社） に導入後、 cDNAを持つクローンをカナマイシンを含む L B寒天 培地中で選択した。 個々のクローンの配列を解析した結果、 NAV2タンパク質 （配 列番号： 49) の 1番目から 1889番目までのアミノ酸配列をコードする cDNA の塩基配列を得た。 ここで得られたプラスミドベクタ一を pCR4Blunt- T0P0- NAV2 - 5' と命名した。 An expression vector for animal cells expressing the NAV2 protein (SEQ ID NO: 49) was constructed. Using human brain Marathon-Ready cDNA (CL0NTECH) as type III, PCR was performed using two primers [Primer 11 (SEQ ID NO: 52) and Primer 12 (SEQ ID NO: 53)]. The cDNA encoding the amino acid sequence from the first to the 189th amino acid sequence of NAV2 (SEQ ID NO: 49) was cloned. The composition of the reaction solution used in the reaction was as follows: using the above cDNA II as type I, PfuTurbo Hotstart DNA Polymerase (STRATAGENE) 2.5ϋ, primer 11 (SEQ ID NO: 58) and primer 12 ( SEQ ID NO: 59) was added to each of 1.0 M, dNTPs at 200 µl, and 2 L of 2XGC Buf fer I (TaKaRa Bio) to give a 50 µl volume. The PCR reaction is 94 After one minute, a cycle of 94 ° C for 20 seconds, 58 ° C for 15 seconds, and 72 ° C for 10 minutes was repeated 40 times, followed by a reaction at 72 ° C for 10 minutes. The PCR reaction product was electrophoresed on an agarose gel and purified using a Gel Extraction Kit (QIAGEN). This was subcloned into a plasmid vector pCR4Blunt-TOP0 (Invitrogen), introduced into E. coli T0P10 (Invitrogen), and clones having cDNA were selected in LB agar medium containing kanamycin. As a result of analyzing the sequence of each clone, the nucleotide sequence of the cDNA encoding the amino acid sequence from the 1st to the 1889th of the NAV2 protein (SEQ ID NO: 49) was obtained. The plasmid vector obtained here was named pCR4Blunt-TOP0-NAV2-5 '.
次に、 ヒト脳 Marathon- Ready cDNA (CL0NTECH社） を錶型とし、 2種のプライマ 一 〔プライマ一 1 3 (配列番号： 60) およびプライマー 14 (配列番号： 6 1) 〕 を用いて PCR反応を行い、 NAV2 (配列番号： 49) の 1 796番目から 2 429番目までのアミノ酸配列をコードする cDNAのクロ一ニングを行った。 該反 応における反応液の組成は、 上記 cDNAl zlを鍀型として使用し、 PfuTurbo Hotstart DNA Polymerase (STRATAGENE社） 2.5U、 プライマ一 13 (配列番号： 60) およびプライマー 14 (配列番号： 6 1) を各 1.0 M、 dNTPsを 200 Μ、 および 2xGC Buffer I (TaKaRa Bio社) を 25μί加え、 50μίの液量とした。 PCR反 応は、 94°C · 1分の後、 94T · 20秒、 58°C · 15秒、 72°C · 10分のサイクルを 40回 繰り返し、 続いて 72°C · 10分の反応を行った。 該 PCR反応産物をァガロースゲル にて電気泳動後、 Gel Extraction Kit (QIAGEN社） を用いて精製した。 これをプ ラスミドベクター pCR4Blimt- T0P0 (Invitrogen社） へサブクローニングし、 大腸 菌 TOP10 (Invitrogen社） に導入後、 cDNAを持つクローンをカナマイシンを含む LB寒天培地中で選択した。 個々のクローンの配列を解析した結果、 NAV2タンパ ク質 （配列番号： 49) の 1796番目から 2429番目までのアミノ酸配列を コ一ドする cDNAの塩基配列を得た。 ここで得られたプラスミドベクタ一を pCR4Blunt-T0P0-NAV2-3' と命名した。  Next, the human brain Marathon-Ready cDNA (CL0NTECH) was used as type II, and PCR was performed using two primers (primer 13 (SEQ ID NO: 60) and primer 14 (SEQ ID NO: 61)). Was carried out to clone a cDNA encoding the amino acid sequence from 1796 to 2429 of NAV2 (SEQ ID NO: 49). The composition of the reaction solution used in the reaction was as follows: cDNA lzl was used as type I, 2.5 U of PfuTurbo Hotstart DNA Polymerase (STRATAGENE), Primer 13 (SEQ ID NO: 60) and Primer 14 (SEQ ID NO: 61) Was added to each of 1.0 M, 200 μl of dNTPs, and 25 μl of 2 × GC Buffer I (TaKaRa Bio) to give a liquid volume of 50 μl. In the PCR reaction, a cycle of 94 ° C for 1 minute, 94T for 20 seconds, 58 ° C for 15 seconds, 72 ° C for 10 minutes was repeated 40 times, followed by a reaction at 72 ° C for 10 minutes. went. The PCR reaction product was electrophoresed on an agarose gel and purified using a Gel Extraction Kit (QIAGEN). This was subcloned into the plasmid vector pCR4Blimt-TOP0 (Invitrogen) and introduced into Escherichia coli TOP10 (Invitrogen), and the clone having the cDNA was selected in LB agar medium containing kanamycin. As a result of analyzing the sequence of each clone, the nucleotide sequence of the cDNA encoding the amino acid sequence from position 1796 to position 2429 of the NAV2 protein (SEQ ID NO: 49) was obtained. The obtained plasmid vector was designated as pCR4Blunt-T0P0-NAV2-3 ′.
次に、 pCR4Blunt- T0P0- NAV2- 3' 、 および p3XFLAG- CMV- 14 (Sigma社） を制限酵 素 EcoRV、 および Xbalにて処理した。 それぞれの DNA断片をァガロースゲルにて電 気泳動後、 Gel Extraction Kit (QIAGEN社） を用いて精製した。 次に、 ここで得 られた DNA断片を DNA Ligat ion Ki t ver. 2 (TaKaRa Bio社) を用いてライゲーシ ヨン反応を行った後、 大腸菌 T0P10 (Invi trogen社）に導入し、 アンピシリンを含 む LB寒天培地中で選択した。 個々のクローンの配列を解析した結果、 NAV2タンパ ク質 （配列番号： 4 9 ) の 1 7 9 6番目から 2 4 2 9番目までのアミノ酸配列を コードする cDNAの塩基配列を有するベクター P3XFLAG- CMV- 14- NAV2- 3 ' を得た。 次に、 pCR4Blimt- T0P0- NAV2- 5 ' 、 および p3XFLAG- CMV- 14- NAV2- 3' を制限酵素 Not I、 および EcoRVにて処理した。 それぞれの DNA断片をァガロースゲルにて電気 泳動後、 Gel Extract ion Ki t (QIAGEN社） を用いて精製した。 ここで得られた DNA断片を DNA Ligat ion Ki t ver. 2 (TaKaRa Bio社） を用いてライゲーシヨン反 応を行った後、 大腸菌 T0P10 (Invi trogen社）に導入し、 アンピシリンを含む LB寒 天培地中で選択した。 個々のクローンの配列を解析した結果、 NAV2タンパク質Next, pCR4Blunt-TOP0-NAV2-3 'and p3XFLAG-CMV-14 (Sigma) were treated with restriction enzymes EcoRV and Xbal. Each DNA fragment was subjected to electrophoresis on agarose gel, and then purified using Gel Extraction Kit (QIAGEN). Then, get here After performing a ligation reaction using DNA Ligat ion Kit ver. 2 (TaKaRa Bio), the obtained DNA fragment was introduced into E. coli T0P10 (Invitrogen) and selected in LB agar medium containing ampicillin. did. As a result of analyzing the sequences of the individual clones, a vector P3XFLAG-CMV having the nucleotide sequence of cDNA encoding the amino acid sequence from the 176th position to the 249th position of the NAV2 protein (SEQ ID NO: 49) was obtained. -14- NAV2-3 'was obtained. Next, pCR4Blimt-TOP0-NAV2-5 'and p3XFLAG-CMV-14-NAV2-3' were treated with restriction enzymes NotI and EcoRV. Each DNA fragment was electrophoresed on an agarose gel and purified using Gel Extraction Kit (QIAGEN). The DNA fragment obtained here was subjected to a ligation reaction using DNA Ligation Kit ver. 2 (TaKaRa Bio), and then introduced into E. coli T0P10 (Invitrogen), and LB agar medium containing ampicillin was added. Selected in. As a result of analyzing the sequence of each clone, the NAV2 protein
(配列番号： 4 9 ) をコードする cDNAの塩基配列を有する動物細胞用発現べクタ 一 p 3XFLAG- CMV- 14-NAV2を得た。 実施例 1 An animal cell expression vector p3XFLAG-CMV-14-NAV2 having the nucleotide sequence of cDNA encoding (SEQ ID NO: 49) was obtained. Example 1
以下は、 公知の酵母ツーハイブリッド法（The Yeas t Two-Hybrid System, Oxford Univers i ty Press, Bartel and Fields, 1997年等）に準じて行った。 配列番号： 4 5で示されるアミノ酸配列からなるペイトタンパク質をコードす る cDNAは、 上記参考例で得られたプラスミド p3xFLAG-TACT427- Aから PCR法を用い て増幅した。 得られた cDNAを、 組み換えによって、 酵母の発現べクタ一 pGBT. Qに 導入した。 pGBT. Qは、 pGBT. Cに近縁の誘導体 (Nat. Genet. 12巻、 72- 77頁、 1996年）であり、 ポリリンカ一部位を修飾してシークェンス用 M13プライマ一を揷 入したものである。 この新しいコンストラクトを、 トリブトファン合成能力の有 無により、 酵母の PNY200 株中で直接選択した （この株の遺伝型： MATひ tr 1- 901 leu2-3, 112 ura3-52 his3-200 ade2 gal4A gal80 A ) 。 この酵母細胞中 で、 ペイ卜タンパク質は、 転写因子 Gal4 タンパク質 （GenBank NPJ15076) の DNA結合ドメイン （アミノ酸 1番目〜 147番目） の C末端側に結合した融合タンパク 質として産生された。 プレイライブラリ一を、 酵母の BK100株 （この株の遺伝 型： MATa trp卜 901 leu2-3, 112 ura3-52 is3-200 gal4A gal80 A LYS2 : :GAL- HIS3 GAL2-ADE2 met2 : :GAL7TlacZ ) に形質転換し、 ロイシンを合成させる能力 の有無によって選択した。 この酵母細胞中では、 各 cDNAは転写因子 Gal4タンパ ク質 （GenBank NP— 015076) の転写促進ドメイン （アミノ酸 768番目〜 881番目） の C末端側に結合した融合タンパク質として産生された。 次いで、 ベイトタンパ ク質を発現する PNY200細胞 （接合型 ΜΑΤ θ! ) を、'プレイライブラリ一中のプレイ タンパク質を発現する BK100細胞 （接合型 MATa) と接合させた。 その結果得られ る、 ペイトタンパク質と相互作用するプレイタンパク質を発現する二倍体の酵母 細胞を、 トリプトファン、 ロイシン、 ヒスチジンおよびアデニンの合成能力によ つて選択した。 それぞれのクローンから DNAを調製し、 エレクト口ポレーシヨン 法によって大腸菌の KC8株に形質転換し、 次いで細胞を、 トリブトファンを含ま ない （ベイトプラスミド選択用） 、 またはロイシンを含まない （プレイライブラ リーのプラスミド選択用） アンピシリン含有培地上で選択した。 両プラスミドの DNAを調製し、 ジデォキシヌクレオチド ·チェーン夕一ミネーシヨン法で配列を 決定した。 ベイト cDNAインサートの同一性を確認し、 プレイライブラリーのプラ スミドから得られる cDNAインサートを、 公知のヌクレオチドおよびタンパク質デ 一夕ベースと照合調査する BLASTプログラムを使用して同定した。 The following was carried out according to a known yeast two-hybrid method (The Yeast Two-Hybrid System, Oxford University Press, Bartel and Fields, 1997, etc.). A cDNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 45 was amplified from the plasmid p3xFLAG-TACT427-A obtained in the above Reference Example by PCR. The resulting cDNA was recombinantly introduced into yeast expression vector pGBT.Q. pGBT.Q is a derivative closely related to pGBT.C (Nat. Genet. 12, 72-77, 1996), which is obtained by modifying a polylinker at one position and introducing an M13 primer for sequence. is there. This new construct was selected directly in the yeast strain PNY200, depending on its ability to synthesize tryptophan (genotype of this strain: MAT tr1-901 leu2-3, 112 ura3-52 his3-200 ade2 gal4A gal80 A ). In this yeast cell, the protein was produced as a fusion protein bound to the C-terminal side of the DNA-binding domain (amino acids 1 to 147) of the transcription factor Gal4 protein (GenBank NPJ15076). The play library was used for the yeast BK100 strain (genotype of this strain: MATa trp 901 leu2-3, 112 ura3-52 is3-200 gal4A gal80 A LYS2:: GAL- HIS3 GAL2-ADE2 met2:: GAL7 T lacZ) and selected for its ability to synthesize leucine. In this yeast cell, each cDNA was produced as a fusion protein linked to the C-terminal side of the transcription promoting domain (amino acids 768 to 881) of the transcription factor Gal4 protein (GenBank NP-015076). Next, PNY200 cells expressing the bait protein (conjugated ΜΑΤθ!) Were conjugated to BK100 cells expressing the prey protein in the prey library (conjugated MATa). The resulting diploid yeast cells expressing the prey protein interacting with the pate protein were selected for their ability to synthesize tryptophan, leucine, histidine and adenine. DNA is prepared from each clone and transformed into the E. coli KC8 strain by the electoporation method. Cells are then transformed without tributofan (for bait plasmid selection) or without leucine (for plasmid selection in the pre-library library). For selection on ampicillin-containing medium. The DNAs of both plasmids were prepared, and the sequences were determined by the dideoxynucleotide chain-initiation method. The identity of the bait cDNA insert was confirmed, and the cDNA insert obtained from the prey library plasmid was identified using the BLAST program, which checks against known nucleotide and protein databases.
結果を以下に記載する。  The results are described below.
ヒト乳癌細胞株とヒ卜前立腺癌細胞株の混合 cDNAライブラリーより、 FLNA (配 列番号： 4 6 ) の 2206番目〜 2355番目または 2141番目〜 2351番目までのアミノ酸 配列をコードする cDNA断片を、 ヒト海馬 c D NAライブラリーからは FLNA (配列 番号： 4 6 ) の 2162番目〜 2412番目のアミノ酸配列をコードする cDNA断片が得ら れた。  From a mixed cDNA library of a human breast cancer cell line and a human prostate cancer cell line, a cDNA fragment encoding the amino acid sequence of nucleotides 2206 to 2355 or 2141 to 2351 of FLNA (SEQ ID NO: 46) was obtained. A cDNA fragment encoding the amino acid sequence at positions 2162 to 2412 of FLNA (SEQ ID NO: 46) was obtained from the human hippocampus cDNA library.
ヒト乳癌細胞株とヒト前立腺癌細胞株の混合 cDNAライブラリーより、 FLNB (配 列番号： 4 7 ) の 1960番目〜 2299番目または 2156番目〜 2314番目までのアミノ酸 配列をコードする cDNA断片が、 ヒト海馬 cDNAライブラリーより、 FLNB (配列番 号： 4 7 ) の 2174番目〜 2565番目のアミノ酸配列をコードする cDNA断片が得られ た。  From a mixed cDNA library of a human breast cancer cell line and a human prostate cancer cell line, a cDNA fragment encoding the amino acid sequence of positions 1960 to 2299 or 2156 to 2314 of FLNB (SEQ ID NO: 47) was obtained from human. From the hippocampus cDNA library, a cDNA fragment encoding the amino acid sequence at positions 2174 to 2565 of FLNB (SEQ ID NO: 47) was obtained.
ヒト海馬 cDNAライブラリ一より、 FLNC (配列番号： 4 8 ) の 2295番目〜 2705番 目までのアミノ酸配列をコードする cDNA断片が得られた。 ヒト海馬 cDNAライブラリーより、 NAV2 (配列番号： 49) の 68番目〜 286番目 までのアミノ酸配列をコードする cDNA断片が得られた。 From one human hippocampus cDNA library, a cDNA fragment encoding the amino acid sequence from nucleotides 2295 to 2705 of FLNC (SEQ ID NO: 48) was obtained. From the human hippocampus cDNA library, a cDNA fragment encoding the amino acid sequence from position 68 to position 286 of NAV2 (SEQ ID NO: 49) was obtained.
ヒト乳癌細胞株とヒト前立腺癌細胞株の混合 cDNAライブラリーより、 BTBD2 (配列番号： 50) の 201番目〜 525番目までのアミノ酸配列をコードする cDNA断 片が、 ヒト海馬 cDNAライブラリーより、 BTBD2 (配列番号： 50) の 215番目〜 506番目までのアミノ酸配列をコードする cDNA断片が得られた。  From a mixed cDNA library of a human breast cancer cell line and a human prostate cancer cell line, a cDNA fragment encoding the amino acid sequence from position 201 to position 525 of BTBD2 (SEQ ID NO: 50) was obtained from the human hippocampus cDNA library. A cDNA fragment encoding the amino acid sequence from position 215 to position 506 of (SEQ ID NO: 50) was obtained.
ヒト乳癌細胞株とヒト前立腺癌細胞株の混合 cDNAライブラリーより、 RAB3IL1 (配列番号： 51) の 282番目〜 356番目までのアミノ酸配列をコードする cDNA断 片が得られた。 実施例 2  From a mixed cDNA library of a human breast cancer cell line and a human prostate cancer cell line, a cDNA fragment encoding the 282nd to 356th amino acid sequence of RAB3IL1 (SEQ ID NO: 51) was obtained. Example 2
TACT427- Aタンパク質、 および NAV2タンパク質の局在性検討 （細胞染色） ヒト胎児腎臓由来 HEK293細胞 1.5X106個を 10%牛胎仔血清 （JRH社） を含むダ ルべッコ改変型イーグル最少培地 （Invitrogen社） 9 mlに懸濁し、 直径 10cmのぺ トリディッシュに播種した。 5%炭酸ガス気流下、 37°Cで一晚培養した後、 予め FuGene6トランスフエクシヨン試薬 18 1 (Roche Diagnostics社） および OPTI- MEM I (Invitrogen社） と混合し室温で 15分間放置しておいた pcDNA3.1 (+) - TACT427-A 3 g、 および p3XFLAG- CMV- 14 - NAV2 3 /igを添加し同条件で培養を継 続した。 1日後に細胞 5X103個を 8ゥエル ポリ- D-リジンコ一トカルチヤ一スライ ド （BDファルコン社） に播種した。 5%炭酸ガス気流下、 37 で一晩培養した後、 細胞を PBSで洗浄し、 10%中性ホルマリン緩衝液を加え室温で 30分間固定した。 その後 PBSで 0.1%に希釈した TritonX- 100を加え室温で 5分放置した後、 再び PBS で洗浄し、 更に 1% BSAを含む PBSを加え 4°Cで 24時間放置した。 次に 1% BSAを含 む PBSで lO^g/mlとなるよう希釈した AS- 2482 (参考例 1 1にて作製） 、 およびマ ウス抗 FLAG M2抗体 （Sigma社） を加え、 室温で 45分間反応させた。 その後、 PBS で洗浄し、 続いて 1% BSAを含む PBSで 10 g/mlとなるよう希釈した Alexa488標識 抗ゥサギ IgG抗体 (Molecular Probes社) 、 および Alexa546標識抗マウス IgG抗体 (Molecular Probes社） を加え、 室温で 45分間反応させてから PBSで洗浄し、 蛍 光顕微鏡により観察した。 その結果、 TACT427- Aタンパク質、 および NAV2タンパク質の細胞内局在が一致 していることが明らかとなった。 実施例 3 Localization of TACT427-A protein and NAV2 protein (cell staining) 1.5 × 10 6 human fetal kidney-derived HEK293 cells were modified with Darbecco's modified Eagle's minimal medium containing 10% fetal bovine serum (JRH) ( (Invitrogen) and seeded on a 10-cm diameter dish. After culturing at 37 ° C in a 5% carbon dioxide gas stream, mix with FuGene6 transfection reagent 181 (Roche Diagnostics) and OPTI-MEM I (Invitrogen) in advance and leave at room temperature for 15 minutes. Then, 3 g of pcDNA3.1 (+)-TACT427-A and p3XFLAG-CMV-14-NAV23 / ig were added, and the culture was continued under the same conditions. One day later, three 5 × 10 3 cells were seeded on an 8-well poly-D-lysine culture slide (BD Falcon). After culturing overnight at 37 in a 5% carbon dioxide gas stream, the cells were washed with PBS, 10% neutral formalin buffer was added, and the cells were fixed at room temperature for 30 minutes. Thereafter, TritonX-100 diluted to 0.1% with PBS was added, and the mixture was allowed to stand at room temperature for 5 minutes. After washing with PBS again, PBS containing 1% BSA was added, and the mixture was allowed to stand at 4 ° C for 24 hours. Next, AS-2482 (prepared in Reference Example 11) diluted to 10 g / ml with PBS containing 1% BSA and mouse anti-FLAG M2 antibody (Sigma) were added, and the mixture was added at room temperature. Allowed to react for minutes. After washing with PBS, Alexa488-labeled anti-Peagle IgG antibody (Molecular Probes) and Alexa546-labeled anti-mouse IgG antibody (Molecular Probes) diluted with PBS containing 1% BSA to 10 g / ml were used. In addition, the mixture was reacted at room temperature for 45 minutes, washed with PBS, and observed with a fluorescence microscope. As a result, it was clarified that the TACT427-A protein and the NAV2 protein had the same intracellular localization. Example 3
TACT427-Aタンパク質、 および FLNタンパク質の局在性検討 （細胞染色） ヒト胎児腎臓由来 HEK293細胞 1. 5 X 106個を 10%牛胎仔血清 （JRH社） を含むダ ルべッコ改変型イーグル最少培地 （Invi trogen社） 9 mlに懸濁し、 直径 10cmのべ トリディッシュに播種した。 5%炭酸ガス気流下、 37°Cで一晩培養した後、 予め FuGene6トランスフエクシヨン試薬 18 1 (Roche Diagnost ics社） および OPTI- MEM I (Invi trogen社） と混合し室温で 15分間放置しておいた pcDNA3. 1 (+) -TACT427-A protein, and localization study of FLN protein (cell staining) Da Le Beck co Modified Eagle containing human fetal kidney derived HEK293 cell 1. 5 X 10 6 cells with 10% fetal bovine serum (JRH Co.) The cells were suspended in 9 ml of a minimal medium (Invitrogen) and seeded on a 10-cm diameter dish. After culturing overnight at 37 ° C in a 5% carbon dioxide gas stream, mix with FuGene6 transfection reagent 181 (Roche Diagnostics) and OPTI-MEM I (Invitrogen) in advance, and leave at room temperature for 15 minutes. PcDNA3.1 1 (+)-
TACT427-A を添加し同条件で培養を継続した。 1日後に細胞 5 X 103個を 8ゥェ ル ポリ- D-リジンコ一トカルチヤ一スライド （BDファルコン社） に播種した。 5%炭酸ガス気流下、 37°Cで一晩培養した後、 細胞を PBSで洗浄し、 10%中性ホル マリン緩衝液を加え室温で 30分間固定した。 その後 PBSで 0. 1 %に希釈した Tri tonX- 100を加え室温で 5分放置した後、 再び PBSで洗浄し、 更に 1 % BSAを含む PBSを加え 4°Cで 24時間放置した。 次に 1 % BSAを含む PBSで 10 z g/mlとなるよう希 釈した AS- 2482 (参考例 1 1にて作製） 、 およびマウス抗 FLN抗体 （CHEMIC0N社） を加え、 室温で 45分間反応させた。 その後、 PBSで洗浄し、 続いて 1 % BSAを含む PBSで 10 g/mlとなるよう希釈した Alexa488標識抗ゥサギ IgG抗体 （Molecular Probes社） 、 および Alexa546標識抗マウス IgG抗体 （Molecular Probes社） を加 え、 室温で 45分間反応させてから PBSで洗浄し、 蛍光顕微鏡により観察した。 その結果、 TACT427- Aタンパク質、 および FLNタンパク質の細胞内局在が一致し ていることが明らかとなった。 TACT427-A was added, and the culture was continued under the same conditions. One day later, 5 × 10 3 cells were seeded on an 8-well poly-D-lysine culture slide (BD Falcon). After culturing overnight at 37 ° C in a 5% carbon dioxide gas stream, the cells were washed with PBS, and 10% neutral formalin buffer was added, and fixed at room temperature for 30 minutes. Thereafter, TritonX-100 diluted to 0.1% with PBS was added, and the mixture was allowed to stand at room temperature for 5 minutes. After washing with PBS again, PBS containing 1% BSA was added, and the mixture was allowed to stand at 4 ° C for 24 hours. Next, AS-2482 (prepared in Reference Example 11) diluted to 10 zg / ml with PBS containing 1% BSA and mouse anti-FLN antibody (CHEMIC0N) were added, and reacted at room temperature for 45 minutes. Was. After that, the cells were washed with PBS, and then diluted with PBS containing 1% BSA to a concentration of 10 g / ml using Alexa488-labeled anti-Peacock IgG antibody (Molecular Probes) and Alexa546-labeled anti-mouse IgG antibody (Molecular Probes). In addition, the mixture was reacted at room temperature for 45 minutes, washed with PBS, and observed with a fluorescence microscope. As a result, it was clarified that the TACT427-A protein and the FLN protein had the same intracellular localization.
産業上の利用可能性 Industrial applicability
(a) 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に同一の アミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはその塩およ び （b) 配列番号： 4 6、 配列番号： 4 7、 配列番号： 4 8、 配列番号： 4 9、 配列番号： 5 0または配列番号： 5 1で表されるアミノ酸配列と同一もしくは実 質的に同一のァミノ酸配列を含有するタンパク質もしくはその部分べプチドまた はその塩を含有する複合体の形成を阻害する化合物またはその塩は、 例えば、 癌(a) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, a partial peptide thereof, or a salt thereof; and (b) SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or identical or real amino acid sequence to SEQ ID NO: 51 A compound or a salt thereof that inhibits the formation of a complex containing a qualitatively identical amino acid sequence or a partial peptide thereof or a complex containing a salt thereof includes, for example, cancer
(例、 大腸癌、 乳癌、 肺癌、 前立腺癌、 食道癌、 胃癌、 肝臓癌、 胆道癌、 脾臓癌、 腎癌、 膀胱癌、 子宮癌、 精巣癌、 卵巣癌、 甲状腺癌、 腌臓癌、 脳腫瘍、 血液腫瘍 など） などの予防 ·治療剤として、 上記複合体の形成を促進する化合物またはそ の塩は、 例えば神経変性疾患 （例、 アルツハイマー病 （家族性アルツハイマー病、 若年性アルツハイマー病、 孤発性アルツハイマー病など） など） などの予防 ·治 療剤として有用である。 (E.g., colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, ovarian cancer, thyroid cancer, kidney cancer, brain tumor Compounds or salts thereof that promote the formation of the above complex as prophylactic or therapeutic agents for, for example, hematologic malignancies, etc., include, for example, neurodegenerative diseases (eg, Alzheimer's disease (familial Alzheimer disease, juvenile Alzheimer's disease, It is useful as an agent for prevention and treatment of Alzheimer's disease).
配列番号： 4 6、 配列番号： 4 7、 配列番号： 4 8、 配列番号： 4 9、 配列番 号： 5 0または配列番号： 5 1で表されるアミノ酸配列と同一もしくは実質的に 同一のアミノ酸配列を含有する夕ンパク質もしくはその部分べプチドまたはその 塩は、 癌、 神経変性疾患などの予防 ·治療剤のスクリーニング、 癌、 神経変性疾 患などの診断に有用である。  SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 51 or 51 Proteins containing amino acid sequences or partial peptides or salts thereof are useful for screening preventive and therapeutic agents for cancer and neurodegenerative diseases and for diagnosing cancer and neurodegenerative diseases.

Claims

請求の範囲 The scope of the claims
1 . (a) 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に同 一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはその塩、 および （b) 配列番号： 4 6で表されるアミノ酸配列と同一もしくは実質的に同 一のアミノ酸配列を含有するタンパク質もしくはその部分べプチドまたはその塩、 配列番号： 4 7で表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸 配列を含有するタンパク質もしくはその部分ペプチドまたはその塩、 配列番号： 4 8で表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含有 するタンパク質もしくはその部分ペプチドまたはその塩、 配列番号： 4 9で表さ れるァミノ酸配列と同一もしくは実質的に同一のァミノ酸配列を含有する夕ンパ ク質もしくはその部分ペプチドまたはその塩、 配列番号： 5 0で表されるァミノ 酸配列と同一もしくは実質的に同一のァミノ酸配列を含有するタンパク質もしく はその部分ペプチドまたはその塩、 ならびに配列番号： 5 1で表されるアミノ酸 配列と同一もしくは実質的に同一のアミノ酸配列を含有するタンパク質もしくは その部分ペプチドまたはその塩から選ばれる一または二種以上を含有する複合体1. (a) a protein or a partial peptide thereof or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, and (b) a protein represented by SEQ ID NO: 46 A protein containing the same or substantially the same amino acid sequence as the amino acid sequence or its partial peptide or a salt thereof, and an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 47. A protein or a partial peptide thereof or a salt thereof; a protein or a partial peptide thereof or a salt thereof having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 48; A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented, or a fragment thereof. Or a salt thereof, a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 50 or a salt thereof, and SEQ ID NO: 51 Complex containing one or more selected from a protein having the same or substantially the same amino acid sequence as its amino acid sequence or a partial peptide thereof or a salt thereof
2 . 配列番号： 4 5で表されるアミノ酸配列と同一もしくは実質的に同一のァ ミノ酸配列を含有するタンパク質が、 配列番号： 1、 配列番号： 4、 配列番号： 1 0、 配列番号： 1 5、 配列番号： 1 7、 配列番号： 2 0、 配列番号： 2 2、 配 列番号： 2 5または配列番号： 2 7で表されるアミノ酸配列からなるタンパク質 である請求項 1記載の複合体。 2. A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 45 is represented by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: The composite according to claim 1, which is a protein comprising an amino acid sequence represented by 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, or SEQ ID NO: 27. body.
3 . (a) 配列番号： 1 5、 配列番号： 1 7、 配列番号： 2 0、 配列番号： 2 2、 配列番号： 2 5または配列番号： 2 7で表されるアミノ酸配列と同一もしく は実質的に同一のアミノ酸配列を含有するタンパク質、 および （b) 配列番号： 4 6、 配列番号： 4 7、 配列番号： 4 8、 配列番号： 4 9、 配列番号： 5 0また は配列番号： 5 1で表されるアミノ酸配列と同一もしくは実質的に同一のァミノ 酸配列を含有するタンパク質もしくはその部分ペプチドまたはその塩を含有する 請求項 1記載の複合体。  3. (a) SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25 or identical to the amino acid sequence represented by SEQ ID NO: 27 Is a protein containing substantially the same amino acid sequence, and (b) SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: The conjugate according to claim 1, comprising a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by 51, a partial peptide thereof, or a salt thereof.
4. 請求項 1記載の複合体の形成を阻害する活性を有する、 配列番号： 4 5で 表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含有する夕 ンパク質もしくはその部分ペプチドまたはその塩に対する抗体。 4. having an activity of inhibiting the formation of the complex according to claim 1, wherein SEQ ID NO: 45 An antibody against a protein, a partial peptide thereof, or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented.
5. 請求項 1記載の複合体の形成を促進する活性を有する、 配列番号： 45で 表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含有する夕 ンパク質もしくはその部分ペプチドまたはその塩に対する抗体。  5. A protein or a partial peptide thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, which has the activity of promoting the formation of the complex according to claim 1 Antibodies to salts.
6. 配列番号： 45で表されるアミノ酸配列と同一もしくは実質的に同一のァ ミノ酸配列を含有するタンパク質が、 配列番号： 1、 配列番号： 4、 配列番号： 10、 配列番号： 15、 配列番号： 17、 配列番号： 20、 配列番号： 22、 配 列番号： 25または配列番号： 27で表されるァミノ酸配列からなる夕ンパク質 である請求項 4または 5記載の抗体。  6. A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 45 is represented by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 15, 6. The antibody according to claim 4 or 5, which is a protein comprising an amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25 or SEQ ID NO: 27.
7. 請求項 4記載の抗体を含有してなる医薬。  7. A pharmaceutical comprising the antibody according to claim 4.
8. 請求項 5記載の抗体を含有してなる医薬。  8. A pharmaceutical comprising the antibody according to claim 5.
9. 癌細胞のアポ卜一シス促進剤または癌の予防 ·治療剤である請求項 7記載 の医薬。  9. The medicament according to claim 7, which is an apoptosis promoting agent for cancer cells or an agent for preventing or treating cancer.
10. 神経細胞のアポトーシス阻害剤または神経変性疾患の予防 ·治療剤であ る請求項 8記載の医薬。  10. The medicament according to claim 8, which is a neuronal apoptosis inhibitor or an agent for preventing or treating a neurodegenerative disease.
11. 請求項 4または 5記載の抗体を含有してなる診断薬。  11. A diagnostic agent comprising the antibody according to claim 4 or 5.
12. 癌または神経変性疾患の診断薬である請求項 11記載の診断薬。  12. The diagnostic agent according to claim 11, which is a diagnostic agent for cancer or a neurodegenerative disease.
13. (a) 配列番号： 45で表されるアミノ酸配列と同一もしくは実質的に 同一のァミノ酸配列を含有するタンパク質もしくはその部分べプチドまたはその 塩、 および （b) 配列番号： 46、 配列番号： 47、 配列番号： 48、 配列番 号： 49、 配列番号： 50または配列番号： 51で表されるアミノ酸配列と同一 もしくは実質的に同一のアミノ酸配列を含有するタンパク質もしくはその部分べ プチドまたはその塩を用いることを特徴とする、 請求項 1記載の複合体の形成を 阻害または促進する化合物またはその塩のスクリーニング方法。  13. (a) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 45 or a partial peptide thereof or a salt thereof, and (b) SEQ ID NO: 46, SEQ ID NO: No .: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 51, or a partial peptide thereof or The method for screening a compound or a salt thereof, which inhibits or promotes the formation of a complex according to claim 1, wherein a salt is used.
14. 配列番号： 45で表されるアミノ酸配列と同一もしくは実質的に同一の アミノ酸配列を含有するタンパク質もしくはその部分ペプチドまたはその塩を産 生する能力を有する細胞を用いることを特徴とする請求項 13記載のスクリー二 ング方法。 14. A cell having an ability to produce a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 45, a partial peptide thereof, or a salt thereof. 13. The screening method according to 13.
15. 配列番号： 46、 配列番号： 47、 配列番号： 48、 配列番号： 49、 配列番号： 50または配列番号： 51で表されるアミノ酸配列と同一もしくは実 質的に同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまた はその塩を産生する能力を有する細胞を用いることを特徴とする請求項 13記載 のスクリーニング方法。 15. Contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 14. The screening method according to claim 13, wherein cells having the ability to produce a protein or a partial peptide thereof or a salt thereof are used.
16. (a) 配列番号： 45で表されるアミノ酸配列と同一もしくは実質的に 同一のァミノ酸配列を含有するタンパク質もしくはその部分べプチドまたはその 塩、 および （b) 配列番号： 46、 配列番号： 47、 配列番号： 48、 配列番 号： 49、 配列番号： 50または配列番号： 51で表されるアミノ酸配列と同一 もしくは実質的に同一のァミノ酸配列を含有するタンパク質もしくはその部分べ プチドまたはその塩を含有することを特徴とする、 請求項 1記載の複合体の形成 を阻害または促進する化合物またはその塩のスクリーニング用キット。  16. (a) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 45, or a partial peptide thereof or a salt thereof; and (b) SEQ ID NO: 46, SEQ ID NO: : 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 or a partial peptide or a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 51 or A kit for screening a compound or a salt thereof, which inhibits or promotes the formation of the complex according to claim 1, characterized by containing a salt thereof.
17. 請求項 1記載の複合体の形成を阻害する化合物またはその塩を含有して なる癌細胞のアポトーシス促進剤または癌の予防 ·治療剤。  17. An agent for promoting apoptosis of cancer cells or an agent for preventing or treating cancer, comprising a compound or a salt thereof that inhibits the formation of the complex according to claim 1.
18. 請求項 1記載の複合体の形成を促進する化合物またはその塩を含有して なる神経細胞のアポ卜一シス阻害剤または神経変性疾患の予防 ·治療剤。  18. A neuronal apoptosis inhibitor or a preventive / therapeutic agent for neurodegenerative disease, comprising the compound for promoting the formation of the complex according to claim 1 or a salt thereof.
19. 配列番号： 46、 配列番号： 47、 配列番号： 48、 配列番号： 49、 配列番号： 50または配列番号： 51で表されるアミノ酸配列と同一もしくは実 質的に同一のアミノ酸配列を含有するタンパク質もしくはその部分ペプチドまた はその塩を用いることを特徴とする、 癌または神経変性疾患の予防 ·治療作用を 有する化合物またはその塩のスクリ一ニング方法。  19. Contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 A method for screening a compound having a prophylactic or therapeutic effect on cancer or a neurodegenerative disease or a salt thereof, which comprises using a protein or a partial peptide thereof or a salt thereof.
20. 請求項 1記載の複合体の形成を阻害することを特徴とする癌細胞のアポ トーシス促進法または癌の予防 ·治療法。  20. A method for promoting apoptosis of cancer cells or a method for preventing and treating cancer, which comprises inhibiting the formation of the complex according to claim 1.
21. 請求項 1記載の複合体の形成を促進することを特徴とする神経細胞のァ ポトーシス阻害法または神経変性疾患の予防 ·治療法。  21. A method for inhibiting apoptosis of neurons or preventing and treating neurodegenerative diseases, which comprises promoting the formation of the complex according to claim 1.
22. 請求項 4記載の抗体の有効量を、 哺乳動物に対して投与することを特徴 とする癌細胞のアポト一シス促進法または癌の予防 ·治療法。  22. A method for promoting apoptosis of cancer cells or a method for preventing and treating cancer, which comprises administering an effective amount of the antibody according to claim 4 to a mammal.
23. 請求項 5記載の抗体の有効量を、 哺乳動物に対して投与することを特徴 とする神経細胞のアポ卜一シス阻害法または神経変性疾患の予防 ·治療法。 23. A method for inhibiting neuronal apoptosis or a method for preventing and treating neurodegenerative diseases, which comprises administering an effective amount of the antibody according to claim 5 to a mammal.
2 4 . 癌細胞のアポト一シス促進剤または癌の予防 ·治療剤の製造のための請 求項 4記載の抗体の使用。 24. Use of the antibody according to claim 4, for the manufacture of an apoptosis promoting agent for cancer cells or an agent for preventing or treating cancer.
2 5 . 神経細胞のアポ卜一シス阻害剤または神経変性疾患の予防 ·治療剤の製 造のための請求項 5記載の抗体の使用。  25. Use of the antibody according to claim 5 for producing an apoptosis inhibitor for neuronal cells or a preventive / therapeutic agent for a neurodegenerative disease.
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