WO2005030805A1 - Complexe de proteines et utilisation - Google Patents

Complexe de proteines et utilisation 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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English (en)
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/fr

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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.

Abstract

L'invention concerne une protéine conjuguée et un agent préventif/thérapeutique pour le traitement du cancer ou de trouble neurodégénératif, entre autres. On décrit un composé, ou son sel, capable d'inhiber la conjugaison entre (a) une protéine qui contient une séquence d'acides aminés identique ou sensiblement identique à la séquence d'acides aminés SEQ ID NO. 45, ou son peptide partiel, ou un sel correspondant et (b) une protéine qui contient une séquence d'acides aminés identique ou sensiblement identique à la séquence d'acides aminés SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50 ou SEQ ID NO. 51, ou son peptide partiel, ou un sel correspondant. Le produit est utile, par exemple, comme agent préventif/thérapeutique pour le traitement du cancer, entre autres. Par ailleurs, on décrit un composé, ou son sel, capable de promouvoir la conjugaison considérée, utile, par exemple, comme agent préventif/thérapeutique pour le traitement de trouble neurodégénératif, entre autres.
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Citations (6)

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WO2001029221A2 (fr) * 1999-10-20 2001-04-26 Zymogenetics, Inc. Nouvelles proteines et polynucleotides codant ces proteines
JP2002191363A (ja) * 1999-07-29 2002-07-09 Herikkusu Kenkyusho:Kk 全長ｃＤＮＡ合成用プライマー、およびその用途
WO2003000113A2 (fr) * 2001-06-20 2003-01-03 Genentech, Inc. Compositions et procedes destines au diagnostic et au traitement de tumeurs
WO2003087768A2 (fr) * 2002-04-12 2003-10-23 Mitokor Cibles pour une intervention therapeutique identifiee dans le proteome mitochondrial
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JP2004201674A (ja) * 2002-08-21 2004-07-22 Takeda Chem Ind Ltd 新規タンパク質および癌の予防・治療剤

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JP2002191363A (ja) * 1999-07-29 2002-07-09 Herikkusu Kenkyusho:Kk 全長ｃＤＮＡ合成用プライマー、およびその用途
WO2001029221A2 (fr) * 1999-10-20 2001-04-26 Zymogenetics, Inc. Nouvelles proteines et polynucleotides codant ces proteines
WO2003000113A2 (fr) * 2001-06-20 2003-01-03 Genentech, Inc. Compositions et procedes destines au diagnostic et au traitement de tumeurs
WO2003087768A2 (fr) * 2002-04-12 2003-10-23 Mitokor Cibles pour une intervention therapeutique identifiee dans le proteome mitochondrial
JP2004201674A (ja) * 2002-08-21 2004-07-22 Takeda Chem Ind Ltd 新規タンパク質および癌の予防・治療剤
WO2004048938A2 (fr) * 2002-11-26 2004-06-10 Protein Design Labs, Inc. Procedes de detection du sarcome des tissus mous, compositions et procedes de criblage des modulateurs du sarcome des tissus mous

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