WO2005092383A1 - 呼吸器疾患の予防・治療剤 - Google Patents
呼吸器疾患の予防・治療剤 Download PDFInfo
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- WO2005092383A1 WO2005092383A1 PCT/JP2005/006444 JP2005006444W WO2005092383A1 WO 2005092383 A1 WO2005092383 A1 WO 2005092383A1 JP 2005006444 W JP2005006444 W JP 2005006444W WO 2005092383 A1 WO2005092383 A1 WO 2005092383A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the present invention relates to a preventive / therapeutic agent and a diagnostic agent for a respiratory disease, a screening of a preventive / therapeutic agent for a respiratory disease, and the like.
- Chronic obstructive pulmonary disease chronic bronchitis, emphysema, diffuse panbronchiolitis, endogenous asthma, etc. It is thought to get sick.
- Smoking has been shown to be a clear etiology of chronic obstructive pulmonary disease.
- Smoking causes obstructive disorders, the extent of which depends on the number of cigarettes smoked. Specifically, the younger the age at which smoking starts, the easier it is to progress. In addition, a dose correlation between smoking and bronchial gland hyperplasia has been confirmed.
- C0PD chronic obstructive pulmonary disease
- Central respiratory tract lesions show changes in secretory tissue morphology, such as goblet cell hyperplasia, hyperplasia of cells in the submucosal glands, and hypertrophy.
- goblet cell hyperplasia For inflammatory cells, an increase in macrophage II activated T lymphocytes has been shown in the airway mucosa.
- Lesions in the bronchiole region include mucous embolism in the airway lumen, goblet cell dysplasia in the airway epithelium, inflammatory cell infiltration in the airway wall, smooth muscle hypertrophy, and fibrosis.
- Cholesterol 25-hydroxylase (CH25H) (GenBank Accession NO. Orchid-003956) is a type of cholesterol hydroxylase, which converts cholesterol to 25-hydroxycholesterol. It has the activity to convert to sterol (25-HC) (J, Biol. Chem. 273, 34316-
- GDF15 prostate differentiation factor
- PLAB prostate differentiation factor
- AF003934 has an effect of promoting interleukin-8-mediated neutrophil infiltration (J. Immunol. 171: 2057-2065, 2003).
- Matrix Metalloproteinase 19 (Martian P19) (GenBank Accession NO. U38321) is an enzyme having proteolytic enzyme activity (J. Biol. Chem. 272, 4281, p. 4286, 1997).
- the present inventors have conducted intensive studies to solve the above problems, and as a result, found a gene whose expression is significantly increased or decreased in lung tissue of a lung cancer patient with chronic obstructive pulmonary disease (C0PD), As a result of further studies based on this finding, the present invention has been completed.
- C0PD chronic obstructive pulmonary disease
- SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 , SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, a compound having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 60 or SEQ ID NO: 62, or a compound that inhibits the activity of a partial peptide or a salt thereof, or A prophylactic and
- (3c) contains a compound having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 30 or a compound or a salt thereof, which inhibits gene expression of a partial peptide or a salt thereof.
- SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO : 18 SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 3 6 SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO .: 52, SEQ ID NO: 54 SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 or a protein identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 62 or A nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the polynucleotide encoding the
- a diagnostic agent comprising the antibody of (8) above,
- a diagnostic agent for respiratory disease comprising a polynucleotide encoding a protein or
- SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, Sequence SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, Sequence SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 or a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 62 or a portion thereof
- a method for screening a remedy for the prevention and treatment of respiratory diseases which comprises using a
- a respiratory disease 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: 30 or a partial peptide thereof or a salt thereof; Screening methods for prophylactic and therapeutic agents,
- a respiratory disease characterized by containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof or a salt thereof; Prevention. ⁇ Screening kit for therapeutic agents,
- a respiratory disease characterized by using a polynucleotide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a polynucleotide encoding a partial peptide thereof; Screening methods for prophylactic and therapeutic agents,
- SEQ ID NO: 2 1) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, Sequence SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 or SEQ ID NO: 62, or Screenini, a prophylactic and therapeutic agent for respiratory diseases characterized by containing
- a prophylactic respiratory disease comprising a polynucleotide encoding an amino acid sequence-containing protein or a partial peptide thereof.
- SEQ ID NO: 2 For mammals, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 14 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 or an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 62 Compounds or salts thereof that inhibit the activity of contained proteins or partial peptides or salts thereof, or
- the compound is a compound that inhibits the activity of a protein containing the amino acid sequence represented by SEQ ID NO: 2, a partial peptide thereof or a salt thereof, or a compound that inhibits the expression of a gene of the protein.
- SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18.
- SEQ ID NO: 56, SEQ ID NO: 58 Inhibiting the activity of a protein or a partial peptide or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 60 or SEQ ID NO: 62, or Prevent respiratory diseases characterized by inhibiting the expression of protein genes ⁇ Therapeutic method, (26) The above-mentioned (25), which inhibits the activity of the protein containing the amino acid sequence represented by SEQ ID NO: 2, its partial peptide or its salt, or inhibits the expression of the gene of the protein. the method of,
- SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: for manufacturing a preventive and therapeutic agent for respiratory disease : 14, SEQ ID NO: 16, SEQ ID NO: .18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 , SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, a SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 60 or SEQ ID NO: 62 Or a compound that inhibits the activity of a
- the compound is a compound that inhibits the activity of a protein containing the amino acid sequence represented by SEQ ID NO: 2, its partial peptide or a salt thereof, or a compound that inhibits the expression of a gene of the protein.
- a prophylactic or therapeutic agent for respiratory diseases comprising a compound or a salt thereof that promotes the activity of a peptide or a salt thereof;
- (34) 'comprising a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 64 or SEQ ID NO: 66, or a polynucleotide encoding a partial peptide thereof Diagnostic agents for respiratory diseases,
- a method for preventing or treating respiratory diseases which comprises administering an effective amount of a compound or a salt thereof which promotes the activity of the salt, or a compound or a salt thereof which promotes the expression of a gene of the protein.
- (40) promotes the activity of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 64 or SEQ ID NO: 66, a partial peptide thereof or a salt thereof, Or a method for preventing or treating a respiratory disease characterized by promoting expression of a gene for the protein;
- a protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 64 or SEQ ID NO: 66 for producing a prophylactic or therapeutic agent for respiratory disease Or a compound or a salt thereof which promotes the activity of a partial peptide or a salt thereof, or a compound or a salt thereof which promotes the expression of a gene of the protein.
- FIG. 1 is a diagram showing the expression level of the CH25H gene for each group.
- FIG. 2 is a diagram showing the correlation between the CH25H gene expression level and the% -second level (% FEV1).
- ⁇ represents the group
- mouth represents the NE group
- ⁇ represents the NS group
- ⁇ represents the CE1 group
- the drawing represents the CE2A group.
- the vertical axis represents the expression level of the CH25H gene
- the horizontal axis represents the% -second amount (° / .FEVl).
- r (correlation coefficient) 0.36
- P (statistically significant difference) 1.9
- FIG. 3 is a graph showing the correlation between the expression level of the CH25H gene and the% C0 lung diffusion ability (% DLC0).
- ⁇ represents group ⁇
- mouth represents NE group
- ⁇ represents NS group
- ⁇ represents CE1 group
- orchid represents CE2A group
- vertical axis represents CH25H gene expression level
- horizontal axis represents% ⁇ 0 lung Indicates the diffusion ability (% DLC0).
- r (number of correlations) -0.81
- p (statistically significant difference) 0.0002
- FIG. 4 (A) shows the expression levels of the CH25H gene and the CYP27A1 gene in the lungs of mice exposed to cigarette smoke.
- the vertical axis shows the expression level of each gene, and the horizontal axis shows the tobacco smoke exposure period.
- FIG. 4 (B) shows the expression levels of the CH25H gene and the CYP27A1 gene in the bronchoalveolar lavage fluid cells of mice exposed to cigarette smoke.
- the vertical axis shows the expression level of each gene
- the horizontal axis shows the tobacco smoke exposure period.
- FIG. 5 (A) is a graph showing changes in the amount of 25-hydroxycholesterol (25-HC) in lung tissue of mice exposed to tobacco smoke.
- the vertical axis shows the amount of 25-HC
- the horizontal axis shows the number of days of exposure to tobacco smoke.
- Qin represents 25-HC and ⁇ represents control.
- FIG. 5 (B) is a graph showing changes in cholesterol levels in lung tissues of mice exposed to tobacco smoke.
- the vertical axis shows the amount of cholesterol
- the horizontal axis shows the number of days exposed to tapako smoke.
- ⁇ represents 25-HC and ⁇ represents control.
- FIG. 6 (A) is a view showing the expression level of CXCL2 gene when bronchoalveolar lavage fluid cells of a mouse exposed to tobacco smoke were stimulated with LPs and 25-here.
- the vertical axis indicates the expression level of each gene
- the horizontal axis indicates the amounts of LPS and 25-HC added.
- FIG. 6 (B) is a graph showing the expression level of the IL-li3 gene when bronchoalveolar lavage fluid cells of a mouse exposed to tobacco smoke were stimulated with LPS and 25-HC.
- the vertical axis indicates the expression level of each gene, and the horizontal axis indicates the amounts of LPS and 25-HC added.
- FIG. 7 is a graph showing the amount of cytokines in bronchoalveolar lavage fluid after intratracheal administration of 25-HC.
- the vertical axis indicates the amount of cytodynamic force
- the horizontal axis indicates the time after intratracheal administration.
- ⁇ Indicates 25-HC and ⁇ indicates control.
- FIG. 8 is a graph showing the number of neutrophils in bronchoalveolar lavage fluid after intratracheal administration of cholesterol hydroxides.
- the vertical axis indicates neutrophil count
- the horizontal axis indicates cholesterol hydroxide administered intratracheally.
- SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: (, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, Sequence No .: 22 SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40 SEQ ID NO: ⁇ 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: No .: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64 or a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 66 (Hereinafter, sometimes referred to
- any tissue where these cells are present such as the brain, parts of the brain (eg, olfactory bulb, amygdala, basal nucleus, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), Spinal cord, pituitary, stomach, spleen, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular It may be a protein derived from gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, skeletal muscle, etc., or it may be a synthetic protein.
- olfactory bulb amygdala, basal nucleus, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum
- Spinal cord
- SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 , SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, .SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, Sequence
- the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64 or SEQ ID NO: 66 is SEQ ID NO: 2, SEQ ID NO: 4, sequence
- NCBI BLAST National Center for Biotechnology Information Basic Local Alignment search Tool
- SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, Sequence SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, .SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO No .: 38, SEQ ID NO: 40, SEQ ID NO: 4.2, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: No .: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64 or containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 66
- the proteins to be used include,
- the substantially equivalent activity includes cholesterol hydroxylation activity.
- the substantially equivalent activity includes neutrophil infiltration activity via macroleuk through interleukin-8.
- substantially the same activity includes proteolytic enzyme activity.
- substantially homogenous indicates that the properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, the above-mentioned cholesterol hydroxylating activity, neutrophil infiltration activity, and protease activity are the same (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.1 to 10 times). (5 to 2 times), but the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
- Cholesterol hydroxylation activity can be measured by a method known per se, for example, J. Biol.
- the protein of the present invention preferably a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 and a labeled cholesterol substrate are allowed to react with each other.
- the cholesterol hydroxylation activity is measured by measuring the amount of the product (eg, radioactivity).
- Radioactive as a labeled cholesterol substrate Cholesterol labeled with an isotope eg, [ 125 I], [ 131 ⁇ ], [3 ⁇ 4], [ M c], etc.
- the radioactivity is measured according to a known method using a scintillation counter or the like.
- the neutrophil infiltration activity can be measured according to a method known per se, for example, the method described in J. Immunol. 171 vol., Pp. 2057-2065, 2003 or a method analogous thereto.
- the protein of the present invention (preferably, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4) and interleukin 8 are placed in a lower chamber of Transpelle (manufactured by Kojung). Neutrophils are added to the upper chamber and neutrophils are added to the upper chamber, and the number of neutrophils that penetrate the endothelial cell layer and infiltrate from the upper chamber to the lower chamber is measured. The sphere infiltration activity is measured.
- the protease activity can be measured by a method known per se, for example, the method described in J. Biol. Chem. 272, pp. 4281-4286, 1997 or a method analogous thereto.
- Proteolytic activity is measured by measuring the amount of degradation (eg, fluorescence intensity).
- a labeled substrate peptide for example, a substrate peptide (eg, Nma-Pro-Lys_Pro-Leu-Ala-Nva_Trp_) that is labeled with a fluorescent substance (eg, fluorescein, fluoresceinisothiosinate), etc. Lys (Dnp) -N, Nma: N-methyl anthranilic acid and the like are used.
- the measurement of the fluorescence intensity is performed according to a known method, for example, a method using a fluorescence measuring device or the like.
- Examples of the protein used in the present invention include: (i) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 , SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: No .: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64 or SEQ ID NO: 66: 1 or 2 or more (eg, about 1 to 100,
- SEQ ID NO: 40 SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, Torii Column No .: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:
- SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64 or SEQ ID NO: 66 contains one or more amino acid sequences (for example, 1 to: about L00, preferably 1 to About 30 amino acids, preferably about 1 to 10, more preferably about 1 to 5 amino acids, (iii) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 , SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:
- amino acid sequence in which about 30 amino acids are substituted, preferably about 1 to 10, more preferably about 1 to 5 amino acids, and (V) an amino acid sequence obtained by combining them. So-called mucins such as proteins are also included.
- the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.
- the protein has a N-terminus at the left end (amino terminus) and a C-terminus at the right end (carboxyl terminus) in accordance with the convention of peptide notation.
- Protein that is used in the present invention C-terminal, carboxyl groups (- C00H), carboxylate (- C00-), ami de (- C0NH 2) or may be any of the ester (-C00R).
- R in the ester e.g., methyl, Echiru, n- propyl, isopropyl
- C WINCH 6 alkyl group such as n- butyl, for example, consequent opening pentyl, C 3, such as cyclohexyl cyclo - 8 cycloalkyl group , for example, phenyl, alpha-naphthyl of which C 6 _ 12 Ariru group, for example, C 7 _ 14 benzyl, such as alpha-naphthyl one Cw alkyl group, such as phenyl one Cw ⁇ alkyl group or alpha-naphthylmethyl, such as phenethyl An aralkyl group, a bivaloyloxymethyl group and the like are used.
- the protein used in the present invention has a carboxyl group (or carboxylate) at a position other than the C-terminus
- a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention.
- the ester in this case for example, the above-mentioned C-terminal ester and the like are used.
- an amino group at the N-terminal amino acid residue eg, methionine residue
- a protecting group eg, formyl group, acetyl group, etc., C ', _ 6 alkanol, etc.
- N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation
- Substituent on the side chain of amino acid in the molecule eg, -0H, -SH, Amino group, imidazole group, Or a glycan attached to a compound, which is protected by an appropriate protecting group (for example, a C 6 alkenyl group such as a formyl group or an acetyl group).
- an appropriate protecting group for example, a C 6 alkenyl group such as a formyl group or an acetyl group.
- Complex proteins such as so-called glycoproteins are also included.
- protein used in the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 2, a protein containing the amino acid sequence represented by SEQ ID NO: 4, and a protein containing the amino acid sequence represented by SEQ ID NO: 6.
- a protein containing an amino acid sequence represented by SEQ ID NO: 8, a protein containing an amino acid sequence represented by SEQ ID NO: 8, a protein containing an amino acid sequence represented by SEQ ID NO: 10, SEQ ID NO: 1 A protein containing the amino acid sequence represented by SEQ ID NO: 14; a protein containing the amino acid sequence represented by SEQ ID NO: 16; a protein containing the amino acid sequence represented by SEQ ID NO: 16; A protein containing the amino acid sequence represented by 18; a protein containing the amino acid sequence represented by SEQ ID NO: 20; an amino acid sequence represented by SEQ ID NO: 22 A protein having an amino acid sequence represented by SEQ ID NO: 24; a protein having an amino acid sequence represented by SEQ ID NO: 26; and an amino acid sequence represented by SEQ ID NO: 28 A protein containing the amino acid sequence represented by SEQ ID NO: 30; a protein containing the amino acid sequence represented by SEQ ID NO: 32; and an amino acid sequence represented by SEQ ID NO: 34 A protein containing the amino acid sequence represented
- the partial peptide of the protein used in the present invention is the partial peptide of the protein used in the present invention described above, and preferably has the same properties as the protein used in the present invention described above. Any one may be used. Specifically, a peptide having the first to second amino acid sequences in the amino acid sequence represented by SEQ ID NO: 2 and the first to third amino acids in the amino acid sequence represented by SEQ ID NO: 4 A peptide having the amino acid sequence at position 8 is exemplified. For example, at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, most preferably 200 or more of the constituent amino acid sequences of the protein used in the present invention. Peptides having more than one amino acid sequence are used.
- one or more (preferably about 1 to 10 and more preferably (1 to 5)) amino acids in the amino acid sequence are deleted. Or 1 or 2 or more (preferably: about! To about 20, more preferably about 1 to about 10, and more preferably about 1 to about 5) amino acids are added to the amino acid sequence. Or one or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably a number of (1 to 5)) amino acids in 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 to be inserted or It may be substituted.
- the partial peptide used in the present invention C-terminal carboxyl group (- C00H), carboxylate (- C00-), ami de (- C0NH 2) or it may also be any of the ester (-C00R) Les, .
- the partial peptides used in the present invention include those having a carboxyl group (or carboxylate) in addition to the C-terminus and N-terminal amino acid residues (eg, , Methionine residue) whose amino group is protected by a protecting group, and a glutamine residue formed by cleavage of the N-terminal side in vivo.
- Examples include those in which the group is pyroglutamine-oxidized, those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, and those in which a sugar chain is bound, 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.
- a salt with a physiologically acceptable acid eg, an inorganic acid, an organic acid
- a base eg, an alkali metal salt
- Such salts include, for example, 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, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).
- 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 tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulf
- the protein or its partial peptide or a salt thereof used in the present invention can be produced from the above-mentioned human or warm-blooded animal cells or tissues by a protein purification method known per se, or encodes a protein. It can also be produced by culturing a transformant containing DNA. It can also be produced according to the peptide synthesis method described below.
- the human mammalian tissues or cells are homogenized, and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, Purification and isolation can be achieved by combining chromatography such as ion exchange chromatography.
- a commercially available resin for protein synthesis can usually be 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, 4 -Hydroxymethylmethylphenylacetamide methyl resin, Polyacrylamide resin, 4- (2 ', 4'-Dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2,, 4'Dimethoxyphenyl F moc aminoethyl) phenoxy resin And so on.
- an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods.
- the protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.
- an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain the desired protein or partial peptide or their derivatives. To obtain the amide body.
- the protected amino acid may be added directly to the resin along with a racemization inhibitor (eg, HOB t, HOOB t) or may be pre-protected as a symmetric acid anhydride or HOB t ester or HOOB t ester. Can be added to the resin after activation.
- a racemization inhibitor eg, HOB t, HOOB t
- the solvent used for the condensation of the activated amino acid and the condensation of the protected amino acid can be appropriately selected from solvents known to be usable for the protein condensation reaction.
- acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylvirolidone, halogenated hydrocarbons such as ⁇ methylene chloride, chloroform, and trifluene
- Alcohols such as ethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; and esterols such as methyl acetate and ethyl acetate.
- the reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about 120 ° 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 Yunhydrin reaction, if the condensation is insufficient, it is sufficient to repeat the condensation reaction without removing the protecting group. If sufficient condensation is not 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 amino group of the raw material include, for example, Z, Boc, t-pentyloxycanoleboninole, isobonoreninoleoxycanoleboninole, 4-methoxybenzinole, x11-canylbonyl, C11Z, Br — Z, adamantyloxycarbonyl, trifluoroacetinole, phthaloynole, honoleminole, 2-nitrotropenolinoresnolefuenole, difuenole phosfuinochi oil, Fmoc and the like are used.
- the carboxyl group may be, for example, alkyl esterified (for example, methyl, ethyl, propynole, ptinole, t-butyl, cyclopentynole, cyclohexynole, cycloheptyl, cyclooctyl, 2-adamantyl, etc.
- alkyl esterified for example, methyl, ethyl, propynole, ptinole, t-butyl, cyclopentynole, cyclohexynole, cycloheptyl, cyclooctyl, 2-adamantyl, etc.
- Cyclic alkyl esterification aralkyl esterification (for example, benzyl ester, 4-nitrobenzinoleestenole, 4-methoxybenzinoleestenole, 4-cyclopentinenoestenole, benzhidorinoreesteno)
- the compound can be protected by phenacyl esterification, benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide, or the like.
- the hydroxyl group of serine can be protected, for example, by esterification or etherification.
- Suitable groups for this esterification include, for example, groups derived from carbonic acid such as lower (C 6 ) alkanol groups such as acetyl group, aroyl groups such as benzoyl group, benzyloxycarbonyl group, and ethoxycarbonyl group. Used.
- Examples of a group suitable for etherification include a benzyl group, a tetrahydropyraninole group, and a t-butyl group.
- protecting group for the phenol 3 ⁇ 4k hydroxyl group of tyrosine for example, Bz1, C1-Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used.
- Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-1,2,3,6-trimethinolebenzenesulfonyl, DNP, benzyloxymethinole, Bum, Boc, Trt, and Fmoc. Is used.
- activated carboxyl groups in the raw materials include, for example, corresponding acid anhydrides, azides, and activated esters [alcohols (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitrophenol) , Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N- And esters with hydroxyphthalimide and H ⁇ B t).
- activated amino group of the starting material for example, a corresponding phosphoric acid amide is used.
- 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 or methanesulfonic acid.
- a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid.
- the elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about 120 ° C. to 40 ° C.
- 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.
- a peptide (protein) chain is added to the amino group side of the desired chain.
- the protein or partial peptide from which only the ⁇ -amino group protecting group at the C-terminal of the peptide chain has been removed and the protein or partial peptide from which only the C-terminal carboxyl group protecting 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 group can be removed to obtain the desired crude protein or peptide.
- the crude protein or peptide can be purified using various known purification means, and the main fraction can be freeze-dried to obtain an amide of the desired protein or peptide.
- the desired protein can be obtained in the same manner as the amide of protein or peptide. It is possible to obtain an ester of quality or a peptide.
- the partial peptide or a salt thereof used in the present invention can be produced according to a per se known peptide synthesis method, or by cleaving a protein used in the present invention with an appropriate peptide.
- 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, a partial peptide or amino acid capable of constituting the partial peptide used in the present invention is condensed with a residual portion, and when the product has a protecting group, the protecting group is eliminated to produce the desired peptide. can do.
- Examples of the known condensation method and elimination of the protecting group include the methods described in the following (i) to (V).
- the polynucleotide encoding the protein used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the protein used in the present invention.
- it is DNA.
- the DNA include a genomic DNA, a genomic DNA library, the aforementioned cDNA derived from cells and tissues, the aforementioned cDNA library derived from cells and tissues, and a vector used for a c library that may be any of synthetic DNAs. , Pacteriophage, plasmid, cosmid, phagemid and the like.
- RT-PCR method Transcriptase Polymerase Chain Reaction
- Examples of the DNA encoding the protein used in the present invention include: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19. SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 3.3, SEQ ID NO: 35, SEQ ID NO: 37.
- SEQ ID NO: 39 SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO : 51, SEQ ID NO: 53, SEQ ID NO: 55.
- SEQ ID NO: 28 SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO : 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 6
- Any DNA can be used as long as it encodes a protein having substantially the same properties as the protein having the amino acid sequence represented by SEQ ID NO: 64 or SEQ ID NO: 66.
- SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, Sequence SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, .SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, Sequence No.
- SEQ ID NO: 5.9, SEQ ID NO: 61, SEQ ID NO: 63 or SEQ ID NO: 65 are examples of DNA that can hybridize with the nucleotide sequence under high stringent conditions, for example, SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO : 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 4'7, SEQ ID NO: 49 , SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO:
- Hybridization is performed by a method known per se or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab.
- reaction 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 include, for example, a sodium concentration of about 19-4 Om'M, preferably about 19-20 mM, and a temperature of about 50-70 ° C, preferably about 60 ° C. The condition of ⁇ 65 ° C is shown. In particular, the sodium concentration is about 19 mM and the temperature is about 65. The same case is most preferable.
- DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 2 a DNA containing the base sequence represented by SEQ ID NO: 1 or the like is represented by SEQ ID NO: 4.
- DNA having the nucleotide sequence represented by SEQ ID NO: 3 encodes the protein having the amino acid sequence represented by SEQ ID NO: 6
- the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 8 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 5 or the like.
- the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 10 is, for example, DNA having the nucleotide sequence represented by SEQ ID NO: 9 or the like. , Array number No .: DNA containing the amino acid sequence represented by 12 3 ⁇ 4 As a DNA encoding the protein, DNA containing the base sequence represented by SEQ ID NO: 11 or the like is represented by SEQ ID NO: 14 Examples of the DNA encoding the protein containing the amino acid sequence include a DNA containing the base sequence represented by SEQ ID NO: 13 and a protein containing the amino acid sequence represented by SEQ ID NO: 16.
- Examples of the DNA to be encoded include DNA having the base sequence represented by SEQ ID NO: 15 and DNA having the amino acid sequence represented by SEQ ID NO: 18 ⁇
- the DNA encoding the protein includes DNA having the nucleotide sequence represented by SEQ ID NO: 17, and the like.
- the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 20 is SEQ ID NO: DNA containing the nucleotide sequence represented by SEQ ID NO: 19, etc. contains the nucleotide sequence represented by SEQ ID NO: 21 as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 22
- DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 24 includes DNA having the base sequence represented by SEQ ID NO: 23, and the like.
- DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 25 is DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 28, etc.
- You Examples of the DNA include a DNA containing the base sequence represented by SEQ ID NO: 27, and a DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 30 is represented by SEQ ID NO: 29
- Examples of the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 32 include DNA having the nucleotide sequence represented by SEQ ID NO: 32, and the like.
- DNA encoding the protein having the amino acid sequence represented by 34 DNA containing the base sequence represented by SEQ ID NO: 33, etc., containing the amino acid sequence represented by SEQ ID NO: 36
- DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 38 is the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 38;
- the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 40 includes the DNA having the nucleotide sequence represented by SEQ ID NO: 40, etc.
- Examples of the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 42 include DNA having the base sequence represented by SEQ ID NO: 41, represented by SEQ ID NO: 44
- the DNA encoding the protein containing the amino acid sequence includes DNA having the nucleotide sequence represented by SEQ ID NO: 43, and the like encodes the protein containing the amino acid sequence represented by SEQ ID NO: 46 ′
- Examples of the DNA include a DNA containing the base sequence represented by SEQ ID NO: 45, and a protein containing the amino acid sequence represented by SEQ ID NO: 48 DNA containing the nucleotide sequence represented by SEQ ID NO: 47 is represented by SEQ ID NO: 49.
- DNA encoding the protein comprising the amino acid sequence represented by SEQ ID NO: 50 is represented by SEQ ID NO: 49.
- Examples of DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 52 include DNA having the nucleotide sequence represented by SEQ ID NO: 51 include DNA having the nucleotide sequence represented by SEQ ID NO: 51
- the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 54 includes DNA containing the base sequence represented by SEQ ID NO: 53, and the like.
- the DNA encoding the protein containing DNA includes the nucleotide sequence represented by SEQ ID NO: 55, such as DNA.
- DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 58 is SEQ ID NO: No .:
- DNA containing the nucleotide sequence represented by 57 is, for example, DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 60 is a DNA encoding the nucleotide sequence represented by SEQ ID NO: 59.
- Examples of the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 62 include DNA having the base sequence represented by SEQ ID NO: 61;
- the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 63 includes the DNA having the base sequence represented by SEQ ID NO: 63, and the DN encoding the protein having the amino acid sequence represented by SEQ ID NO: 66.
- A a DNA having the base sequence represented by SEQ ID NO: 65 or the like 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.
- any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used.
- Examples of the DNA encoding the partial peptide used in the present invention include:
- SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 , SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, Sequence SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, DNA containing a part of the DNA containing the base sequence represented by SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63 or SEQ ID NO: 65, or (ii) SEQ ID NO: : 1,
- SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: No .: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO : 57, SEQ ID NO: 59, SEQ ID NO: 61, DNA capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 63 or SEQ ID NO: 65 have the same significance as described above.
- DNAs that completely encode the proteins and partial peptides used in the present invention may be simply abbreviated to the proteins of the present invention.
- a DNA fragment which is amplified by PCR using a synthetic DNA primer containing a part of the nucleotide sequence encoding the protein of the present invention, or a DNA incorporated into an appropriate vector is used as a part of the protein of the present invention.
- selection can be performed by hybridization with a DNA fragment encoding the entire region or labeled with synthetic DNA. Hybridization can be performed according to, 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.
- the nucleotide sequence of DNA is converted by PCR, a known kit such as Mutan TM -super Express Km (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.), etc., using the 0DA-LA PCR method. , Gapped duplex method, Kunkel method and the like, 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 a suitable synthetic DNA adapter.
- Examples of the expression vector for the protein of the present invention include (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) connecting the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by
- the vector examples include plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), and plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC1). 94), yeast-derived plasmids (eg, pSH19, pSH15), Batateliophages such as ⁇ phage, animal viruses such as retro-inoles, vaccinia-inoles, and baculovirus, as well as pAl_ll and pXTL , PRc / CMV, pRc / RSV, pcDNAI / Neo, and the like.
- E. coli eg, pBR322, pBR325, pUC12, pUC13
- Bacillus subtilis eg, pUB110, pTP5, pC1.
- yeast-derived plasmids eg, pSH19,
- 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 mentioned.
- CMV (site megalovirus) promoter, SRa promoter It is preferable to use such as.
- trp promoter When the host is Eshierihia genus bacterium, trp promoter, lac promoter, rec A promoter, XP L promoter one, lpp promoter, T7 promoter, if the host is Bacillus, SPO l promoter, SPO 2 promoter
- yeast such as the PENP promoter, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter and the like are preferable.
- yeast such as the PENP promoter, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter and the like are preferable.
- 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 polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like.
- a selection marker for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin phosphorus gene (hereinafter sometimes abbreviated as Amp r), neomycin resistant gene (hereinafter sometimes abbreviated as Ne 0 1, G418 resistance).
- dhfr dihydrofolate reductase
- MTX metalhotrexate
- Amp r ampicillin phosphorus gene
- Ne 0 1, G418 resistance neomycin resistant gene
- a signal sequence suitable for the host is added to the N-terminal of the protein of the present invention. If the host is Escherichia, the PhoA signal sequence, OmpA.signal sequence, etc., if the host is Bacillus, the ⁇ -amylase signal sequence, If the host is yeast, the MFa * signal sequence, SUC2 signal sequence, etc., if the host is an animal cell, the inulin signal sequence, a-interferon signal sequence, antibody molecule, signal sequence, etc. Available. Using the vector containing the DNA encoding the protein of the present invention thus constructed, 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' saptilus (Bacillus).
- subtilis M I 14 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)] and the like are used.
- yeast examples include Saccharomyces cerevisiae AH 22, AH22R—, ⁇ 87-11 A, DKD—5D, 20 B—12, and Schizosaccharomyces pombe NC YC 1913, NCYC. 2036, Pichia pastoris K M71 and the like are used.
- insect cells for example, when the virus is Ac NPV, larvae derived from the night larva moth (Spodoptera frugiperda cell; Sf Itoda), MG1 cells derived from the midgut of Trichoplusia ni, and Trichoplusia ni Egg-derived High Five TM cells are used.
- Sf cells for example, S f 9 cells (ATCC CRL 1711), S f 21 cells (Vaughn, J. L., et al., In Vivo, 13, 213-217, (1977)) and the like are used.
- insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
- animal cells for example, monkey cells COS-7, Vero, Chinese Muster cell CHO (hereinafter, CH_ ⁇ cells for short), DHF r gene defect Chiyaini over hamster cell CHO (hereinafter, CHO (dhfr -) cell), mouse L cells, mouse A t T-2 0, mouse myeloma cells, Mouse ATDC 5 cells, rat GH 3, human FL cells, etc. are used.
- Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
- a liquid medium is suitable as a medium for culturing, and a carbon source necessary for growth of the transformant is contained therein.
- Nitrogen sources inorganic substances and others.
- carbon sources include glucose, dextrin, soluble starch, and sucrose.
- nitrogen sources include ammonium salts, nitrates, and corn chips.
- Liquor, peptone, potato zein, meat extract, soybean meal, and potato extract examples of the inorganic or organic substance such as a liquid and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like.
- yeast extract, vitamins, growth promoting factors and the like may be added.
- the pH of the medium is preferably about 5-8.
- a culture medium for cultivating Escherichia bacteria for example, glucose, M9 medium containing noric acid [Miller, Journal of Experiments in Molecular
- an agent such as 36-indolylacrylic acid can be added to make the promoter work efficiently.
- the cultivation is usually carried out at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
- the cultivation is usually carried out at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring may be applied.
- the medium When culturing a transformant in which the host is yeast, the medium may be, for example, Burkholder's minimum medium (Bostian, KL et al., Pro Natl. Acad. Sci. USA, 77, 4505 (1980)). And an SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)].
- the pH of the medium is adjusted to about 5-8.
- the cultivation is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added as necessary.
- Grace's Insect Medium (Grace, ⁇ ⁇ C.C., Nature), 195, 788 (1962)) may be used to which immobilized 10% 1 serum or the like is appropriately added.
- the pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
- examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [ Virology, 8 volumes, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199 volumes, 519 (1967)], 19.9 medium
- the pH is about 6-8.
- Culture is usually performed at about 30 to 40 ° C for about 15 to 60 hours, and aeration and / or agitation are added as necessary.
- the protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
- the following method Can be performed.
- cells or cells are collected by a known method, suspended in an appropriate buffer, and then subjected to ultrasonication, lysozyme and Z or freezing. After the cells or cells are ruptured by thawing or the like, a method of obtaining a crude protein extract by centrifugation or filtration is appropriately used.
- the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X100 TM.
- the protein contained in the culture supernatant or extract obtained in this manner can be purified 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.
- the protein obtained by force When the protein obtained by force is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto. It can be converted to the free form or other salts by the method or a method analogous thereto.
- the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein-modifying enzyme before or after purification.
- an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, anoreginyl 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 antibody against the protein or partial peptide or a salt thereof used in the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention. Good.
- An antibody against the protein or partial peptide used in the present invention or a salt thereof (hereinafter sometimes simply referred to as the protein of the present invention in the description of the antibody) uses the protein of the present invention as an antigen, It can be produced according to a known antibody or antiserum production method.
- the protein of the present invention is administered to a warm-blooded animal at a site capable of producing an antibody upon administration by itself or together with a carrier or diluent.
- Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration.
- the administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
- Examples of the warm-blooded animal to be used include monkeys, egrets, 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 obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared.
- the antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then 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 Koehler and Milstein [Nature, 256, 495 (1975)].
- the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
- PEG polyethylene glycol
- the myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used.
- the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is 10 to 80. /.
- Cell fusion can be carried out efficiently by adding the mixture at a concentration of about 10 to 40 ° C, preferably 30 to 37 ° C, for 1 to 10 minutes.
- Various methods can be used to screen monoclonal antibody-producing hybridomas.
- the hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which protein antigens are adsorbed directly or together with a carrier.
- an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mouse) or protein A labeled with a radioactive substance or enzyme, and a monoclonal antibody bound to the solid phase
- a monoclonal antibody bound to a solid phase is prepared by adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, adding a protein labeled with a radioactive substance, an enzyme, or the like. And a method for detecting antibody.
- 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
- a serum-free medium for hybridoma culture SFM-101, 'Nissui Pharmaceutical Co., Ltd.
- the cultivation temperature is usually 20 to 40 ° (: preferably about 37 ° C.
- the cultivation time is usually 5 days to 3 weeks, preferably 1 to 2 weeks.
- the cultivation is usually 5% carbon dioxide.
- the antibody titer of the hybridoma culture supernatant can be measured in the same manner as in 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) '' Adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen binding Solid phase or active adsorbent such as protein A or protein G Specific purification method for obtaining an antibody by dissociating the bond.
- immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE) '' Adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen binding Solid phase or active adsorbent such as protein A or protein G
- immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipit
- 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 an antibody-containing substance against the antibody and separating and purifying the antibody.
- the type of carrier protein and the mixing ratio between carrier and hapten depend on the hapten immunized by cross-linking with carrier. Any antibody may be cross-linked at any ratio as long as the antibody can be efficiently produced.For example, serum albumin ⁇ ⁇ thyroglobulin, hemocyanin, etc. may be used in a weight ratio of hapten 1 to hapten 1. A method of coupling at a ratio of about 0.1 to 20, preferably about 1 to 5 is used.
- Various condensing agents can be used for the hapten and the carrier capri / g, but an active ester reagent containing a daltaraldehyde carbodiimide, a maleimide active ester, a thiol group, or a dithioviridyl group is used.
- Can be The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
- 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 the 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.
- the 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.
- Polynucleotide encoding protein or partial peptide used in the present invention (Eg, in the description of antisense polynucleotide, these DNAs may be abbreviated as the DNA of the present invention) or complementary to or substantially complementary to the base sequence
- An antisense polynucleotide having a unique base sequence or a part thereof has a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof, and the expression of the DNA Any antisense polynucleotide may be used as long as it has an action capable of suppressing the activity, but antisense DNA is preferable.
- the nucleotide sequence substantially complementary to the DNA of the present invention is, for example, the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). And about 70% or more, preferably about 80% or more, more preferably about 9%
- Nucleotide sequences having 0% or more, and most preferably about 95% or more homology are exemplified.
- the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention for example, An antisense polynucleotide having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with the complementary strand of the nucleotide sequence near the start codon).
- an antisense polynucleotide that directs RNA degradation by RN a .se H it is at least about 70%, preferably at least about 80%, complementary to the entire nucleotide sequence of the DNA of the present invention including introns. More preferably, antisense polynucleotides having a homology of about 90% or more, most preferably about 95% or more are suitable.
- SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: .19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 , SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63 or SEQ ID NO: 65
- SEQ ID NO: 23 SEQ ID NO: 25, Sequence SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39.
- SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63 or SEQ ID NO: 65 A nucleotide sequence complementary to the nucleotide sequence of DNA containing the nucleotide sequence represented by Or a part thereof, more preferably an antisense polynucleotide, more preferably SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: : 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, Sequence No .: 33, SEQ ID No .: 35, SEQ ID No .: 37, SEQ ID No .: 39, Rooster system IJ number: 41, Rooster system number: 43, Rooster system number: 45, Rooster system IJ number: 47, Roo
- An antisense polynucleotide is usually composed of about 10 to 40, preferably about 15 to 30 bases.
- the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are, for example, chemically modified phosphate residues such as phosphorothioate, methylphosphonate, and phosphorodithionate. It may be substituted by a group.
- the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-0-methylation, and the base (pyrimidine, purine) may also be chemically modified. As long as it hybridizes to DNA having the nucleotide sequence represented by SEQ ID NO: 2. Anything, These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
- an antisense polynucleotide capable of inhibiting the replication or expression of the protein gene of the present invention is designed and synthesized based on the nucleotide sequence information of the cloned or determined DNA encoding the protein.
- Such nucleotides can hybridize with the RNA of the protein gene of the present invention, and can inhibit the synthesis or function of the RNA, or through the interaction with the protein-related RNA of the present invention.
- the expression of the protein gene of the present invention can be regulated and controlled.
- Polynucleotides complementary to the selected sequence of the protein-related RNA of the present invention can be used in vivo and in vitro. It is useful for regulating and controlling gene expression, and is also useful for treating or diagnosing diseases.
- the term "corresponding" means having homology or being complementary to a particular sequence of nucleotides, base sequences or nucleic acids, including genes. “Corresponding” between a nucleotide, base sequence or nucleic acid and a peptide (protein) refers to the amino acid of a peptide (protein) as directed by the nucleotide (nucleic acid) sequence or its complement. Usually pointing.
- 5 'end hairpin loop of protein gene 5' end 6—baseba 'repeat, 5' end untranslated region, polypeptide translation start codon, protein code region, ⁇ RF translation stop codon, 3 'end untranslated
- the region, the 3 'end paring mouth region, and the 3' end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected as a target.
- the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region can be said to be "antisense” if the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target is.
- Antisense polynucleotides are polynucleotides containing 2-dexoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids or synthetic sequence specific nuclei) Acid polymer) or other polymer containing a special bond, provided that the polymer contains nucleotides with a configuration that allows base pairing or base attachment as found in DNA or RNA. And the like.
- RNA hybrids can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified polynucleotides). Nucleotides), and those with known modifications, such as those with labels, caps, methylated, and one or more natural nucleotides as analogs known in the art.
- Substituted, modified with an intramolecular nucleotide such as one having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, , Phosphorothioate, phosphorodithioate, etc., such as proteins (nucleases, nuclease inhibitors, proteins) Has side-chain groups such as syn, antibody, signal peptide, poly-L-lysine, etc.
- an intramolecular nucleotide such as one having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, , Phosphorothioate, phosphorodithioate, etc., such as proteins (nucleases, nuclease inhibitors, proteins)
- nucleoside may include not only those containing purine and pyrimidine bases, but also those having other modified heterocyclic bases.
- Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or ethers, amines, etc. And the like.
- the antisense polynucleotide of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
- modified nucleic acids include sulfuric acid derivatives of nucleic acids ⁇ thiophosphate derivatives, and polynucleoside amides ⁇ ⁇ ⁇ those that are resistant to degradation of oligonucleoside amides, but are not limited thereto. is not.
- the antisense nucleic acid of the present invention can be preferably designed in the following manner: to make the antisense nucleic acid more stable in the cell, to enhance the cell penetration of the antisense nucleic acid, Greater affinity for the target sense strand and, if toxic, lower toxicity of the antisense nucleic acid.
- the antisense nucleic acids of the present invention may contain altered or modified bran, bases, bonds, and may be provided in special forms such as ribosomes and microspheres, applied by gene therapy, It could be given in additional form.
- it can be used in an additional form, such as polylysine, such as polylysine, which acts to neutralize the charge of the phosphate skeleton, enhances interaction with cell membranes, and increases nucleic acid uptake
- Hydrophobic substances such as lipids (eg, phospholipids, cholesterol, etc.) are included.
- Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
- These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond.
- Other groups include capping groups specifically located at the 3 'end or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNAse. Examples of such capping groups include hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene dalicol, but are not limited thereto.
- the inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention.
- the nucleic acid can be applied to cells by various methods known per se.
- the protein or partial peptide of the present invention or a salt thereof (hereinafter, the present invention)
- the protein of the present invention or a polynucleotide encoding the protein or partial peptide of the present invention eg, DNA (hereinafter sometimes abbreviated as the DNA of the present invention)) of the present invention; Antibodies against the protein or partial peptide or a salt thereof (hereinafter, sometimes abbreviated as the antibody of the present invention), and antisense polynucleotides of the DNA of the present invention (hereinafter, abbreviated as the antisense polynucleotide of the present invention) Use).
- SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18 , SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO : 58, SEQ ID NO: 60 or SEQ ID NO: 62, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence, a partial peptide thereof, or a salt thereof is abbreviated as Protein A of the present
- Protein B of the present invention a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 64 or SEQ ID NO: 66, a partial peptide thereof or a salt thereof is referred to as Protein B of the present invention. May be abbreviated.
- compounds that inhibit the activity of protein A of the present invention or salts thereof include, for example, respiratory diseases (eg, Prevention of chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.) it can.
- respiratory diseases eg, Prevention of chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- it is an agent for preventing or treating chronic obstructive pulmonary disease.
- the protein A of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein A of the present invention.
- a host transformed with a vector containing the DNA encoding the protein of the present invention described above is used.
- the host for example, animal cells such as COS 7 cells, CHI cells, and HEK293 cells are preferably used.
- a transformant in which the protein A of the present invention is expressed on a cell membrane or in a cell by culturing by the method described above is preferably used.
- the method of culturing cells capable of expressing the protein A of the present invention is the same as the above-described method of culturing the transformant of the present invention.
- 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 activity of the protein A of the present invention is reduced by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (i).
- the test compound to be tested is a compound that inhibits the activity of the protein A of the present invention, and the activity of the protein A of the present invention in the case (ii) is about 20% or more as compared with the case (i).
- a test compound that preferably increases by 30% or more, more preferably about 50% or more, can be selected as a compound that promotes the activity of the protein A of the present invention.
- Examples of the protein A of the present invention include, for example, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a partial peptide thereof, or a salt thereof (the protein A 1 of the present invention) The case of (abbreviated) is described below.
- the protein A1 of the present invention produces 25-hydroxycholesterol from cholesterol in alveolar macrophages, and the 25-hydroxycholesterol forms an inflammatory site (eg, CXCL2, IL- 1; 3), which promotes the production of Neutrophil infiltration into the lungs is increased, and the pathology of chronic obstructive pulmonary disease progresses. Therefore, compounds or salts thereof that inhibit the activity of protein A1 of the present invention include, for example, respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchiolitis Asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.).
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchiolitis Asthma, cystic fibrosis, irritable pneumonia, pulmonary fibro
- screening method using the protein A1 of the present invention include: (a) a cholesterol hydroxylating activity of the protein A1 of the present invention (a cell capable of producing the same); and (iia) the presence of a test compound.
- the cholesterol hydroxylating activity can be measured according to a method known per se, for example, the method described in J. Biol. Chera. 273, pp. 34316-34327, 1998 or a method analogous thereto.
- a reaction between the protein A1 of the present invention and labeled cholesterol and (iib) a reaction between the protein A1 of the present invention and labeled cholesterol in the presence of a test compound.
- the cholesterol hydroxylating activity in each case is measured, and a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein A1 of the present invention is screened.
- This reaction is performed in an appropriate buffer.
- Cholesterol hydroxylation activity is performed by separating the product from the substrate by thin-layer chromatography and measuring the amount of the product (eg, radioactivity). The measurement of radioactivity is performed according to a known method using a scintillation counter or the like.
- Examples of the above-mentioned protein A1 of the present invention include, for example, protein A1 produced by culturing cells having the ability to produce protein A1 of the present invention, and having the ability to produce protein A1 of the present invention.
- Cells and the like are used.
- the nucleotide sequence represented by SEQ ID NO: 1 is inserted into a (commercially available) expression vector for animal cells, introduced into animal cells (eg, COS cells), and expressed.
- Cells in which the base sequence represented is introduced into an expression vector for animal cells and introduced into animal cells (eg, COS cells) are used.
- a radioactive isotope e.g., [125 1], [131 1], [3 ⁇ 4], [14 C], [32 P], [33 P], [], etc.
- fluorescent substances e.g., Cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience), etc.) 2- 0X3— 1, 3-diazol), BODIPY (boron-dipyrromethene), etc.), enzymes (eg, ⁇ -galactosidase, ⁇ -darcosidase, al-rho-phosphatase, phosphoxidase, malate dehydrogenase, etc.) ), Luminescent substances (eg, noreminol, luminol derivative, noreciferin, lucigenin, etc.), piotin, lanthanide element, etc. are used.
- protein of the present invention examples include, for example, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 30 or a partial peptide thereof, or a salt thereof (protein A2 of the present invention).
- protein A2 of the present invention a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 30 or a partial peptide thereof, or a salt thereof (protein A2 of the present invention).
- the following is a description of a screening method using.
- the protease activity may be measured by a method known per se, for example, the method described in J. Biol. Chem. 272, pp. 4281-4286, 1997 or a method analogous thereto.
- the reaction between (id) the protein A2 of the present invention and a labeled substrate peptide is reacted with (iid) the protein A2 of the present invention and a substrate peptide in the presence of a test compound.
- the proteolytic activity of each case is measured, and a compound or a salt thereof that regulates (preferably inhibits) the activity of protein A2 of the present invention is screened.
- This reaction is performed in an appropriate buffer.
- Proteolytic activity is measured by measuring the amount of substrate peptide degradation (eg,-fluorescence intensity).
- a labeled substrate peptide for example, a substrate peptide (eg, Nma-Pro-Lys-Pro-Leu-Ala-Nva-Trp) labeled with a fluorescent substance (eg, fluorescamine, fluorescein isothiothionate, etc.) -Lys (Dnp -NH ⁇ Nma: N-methyl anthranilic acid, etc.) which is used.
- the measurement of the fluorescence intensity is performed according to a known method, for example, a method using a fluorescence measurement device or the like.
- nucleotide sequence represented by SEQ ID NO: 29 is inserted into a commercially available expression vector for prokaryotic cells, introduced into prokaryotic cells (eg, Escherichia coli), expressed, and then refolded to have an activity. Obtained as purified protein.
- proteins or salts thereof that promote the activity of protein B of the present invention include, for example, respiratory diseases (e.g., chronic obstruction). It can be used as a prophylactic / therapeutic agent for inflammatory lung diseases (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc. Preferably, it is an agent for preventing or treating chronic obstructive pulmonary disease.
- respiratory diseases e.g., chronic obstruction
- inflammatory lung diseases chronic bronchitis, emphysema
- diffuse panbronchiolitis bronchial asthma
- cystic fibrosis irritable pneumonia
- pulmonary fibrosis etc.
- it is an agent for preventing or treating chronic obstructive pulmonary disease.
- the protein B of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates (preferably promotes) the activity of the protein B of the present invention.
- a host transformed with a vector containing the above-mentioned D ⁇ ⁇ encoding the protein of the present invention is used.
- the host for example, animal cells such as COS 7 cells, CHO cells, and HEK293 cells are preferably used.
- a transformant in which the protein B of the present invention is expressed on a cell membrane or in a cell by culturing by the method described above is preferably used.
- the method for culturing cells capable of expressing the protein B of the present invention is the same as the above-described method for culturing the transformant of the present invention.
- Test compounds include, for example, peptides, proteins, antibodies, non-peptide compounds Products, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.
- the activity of the protein B of the present invention is about 20% or more, preferably 30% or more, more preferably about 50% as compared with the case of the above (i').
- a test compound that reduces the above can be selected as a compound that inhibits the activity of protein B of the present invention.
- a compound that regulates (preferably inhibits) the expression of the gene encoding the protein A of the present invention can be used, for example, for respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc. ] It can be used as a preventive and therapeutic agent. Preferably, it is a prophylactic or therapeutic agent for chronic obstructive pulmonary disease.
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- It can be used as a preventive and therapeutic agent.
- it is a prophylactic or therapeutic agent for chronic obstructive pulmonary disease.
- the protein A1 of the present invention produces 25-hydroxycholesterol from cholesterol in alveolar macrophages, and the 25-hydroxycholesterol is inflammatory zygote force-in (eg, CXCL2, IL-1). / 3), which promotes neutrophil infiltration into the respiratory tract and progresses the pathology of chronic obstructive pulmonary disease. Therefore, compounds or salts thereof that inhibit the expression of the gene encoding protein A1 of the present invention include, for example, respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiol Inflammation, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.).
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiol Inflammation, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary
- a compound that regulates (preferably promotes) the expression of the gene encoding the protein B of the present invention can be used, for example, for respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc. ] It can be used as a preventive and therapeutic agent. Preferably, it is an agent for preventing or treating chronic obstructive pulmonary disease. Therefore, the polynucleotide of the present invention (eg, DNA)
- the screening method includes (iii) culturing cells capable of producing the protein of the present invention, and (iv) culturing cells capable of producing the protein used in the present invention in the presence of the test compound.
- a method of measuring and comparing the expression levels of the above genes eg, the amount of the protein of the present invention or the amount of mRNA encoding the protein.
- Test compounds and cells having the ability to produce the protein of the present invention include the same cells as described above.
- the amount of the protein is measured by a known method, for example, using an antibody that recognizes the protein of the present invention, and analyzing the protein present in a cell extract or the like according to a method such as Western analysis, ELISA, or a method analogous thereto. Can be measured.
- the amount of mRNA is measured by a known method, for example, as a probe, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: :, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 1, 3, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 1, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, Sequence SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63 or a nucleic acid containing SEQ ID NO: 65
- a test compound that increases the gene expression level in the case (iv) above by about 20% or more, preferably 30% or more, and more preferably about 50% or more as compared with the case (iii) above As a compound that promotes the expression of the gene encoding the protein of the present invention, a test compound that inhibits about 20% or more, preferably 30% or more, and more preferably about 50% or more of the expression of the gene encoding the protein of the present invention, It can be selected as a compound to be suppressed.
- the screening kit of the present invention contains the protein used in the present invention, a cell capable of producing the protein used in the present invention, a polynucleotide encoding the protein, and the like.
- Compounds or salts thereof obtained using the screening method or the screening kit of the present invention include the test compounds described above, for example, peptides, proteins, antibodies, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, and plants. It is a compound selected from an extract, an animal tissue extract, plasma, or the like, or a salt thereof, and is a compound or a salt thereof that regulates the activity (eg, scavenger receptor activity, etc.) of the protein of the present invention.
- a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein ⁇ of the present invention a compound or a salt thereof that regulates (preferably inhibits) the expression of a gene encoding the protein A of the present invention
- the compound or its salt that regulates (preferably promotes) the activity and the compound or its salt that regulates (preferably promotes) the expression of the gene encoding the protein B of the present invention have low toxicity, for example, respiratory tract.
- a prophylactic or therapeutic agent for diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- it is a prophylactic / therapeutic agent for chronic obstructive pulmonary disease and the like.
- 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, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). ), Syrups, emulsions, suspensions and the like.
- a powerful composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
- Injections are used as parenteral compositions for parenteral administration.
- Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, and joints. Includes dosage forms such as internal injections.
- Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections.
- aqueous liquid for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used.
- Propylene glycol, polyethylene glycol), non-ionic surfactants eg polysorbate 80, HCO-50 (polyoxyethylene (50mol) adduct of hydrogenated castor oil)].
- oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
- the prepared injection solution is usually filled in a suitable ampoule.
- a suppository for rectal administration is prepared by mixing the above antibody or a salt thereof with a conventional suppository base.
- 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, etc., and usually 5 to 500 mg per dosage unit dosage form, especially for injections.
- 5 to 500 mg per dosage unit dosage form especially for injections.
- 5-100 mg, in other dosage forms 10-250 mg of the above compound.
- compositions may contain other active ingredients as long as an undesirable interaction is not caused by blending 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 (eg mice, rats, puppies, sheep, pigs, puppies, pests, birds, cats, dogs, monkeys). , Chimpanzees, etc.) can be administered orally or parenterally. .
- the dose of the above compound or its salt varies depending on its action, target disease, subject to be administered, route of administration, and the like.
- a compound that inhibits the activity of protein A of the present invention for the purpose of treating emphysema Or when its salt is administered orally generally in an adult (with a body weight of 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1 to 100 mg of the compound or its salt per day is used.
- the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like.
- the activity of the protein A of the present invention is inhibited for the treatment of emphysema.
- the compound or a salt thereof When a compound or a salt thereof is administered to an adult (with a body weight of 60 kg) usually in the form of an injection, the compound or a salt thereof is used in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day. It is convenient to administer about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg.
- Antibodies to the protein of the present invention (hereinafter sometimes abbreviated as the antibodies of the present invention) ) Can specifically recognize the protein of the present invention, and thus can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by sandwich immunoassay.
- the protein of the present invention can be quantified using a monoclonal antibody against the protein 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 protein 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-antigen corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any method can be used as long as the amount of the complex is detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. Good.
- nephrometry a competitive method, an immunometric method, and a sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
- 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]. As the above enzymes, Those which are stable and have a large specific activity are preferred.
- the fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
- the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
- a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
- physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used.
- 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, may be performed simultaneously, or may be performed at staggered times.
- the labeling agent and the method of insolubilization can be in accordance with those described above.
- the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
- the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the protein of the present invention binds. That is, for example, when the antibody used in the primary reaction and the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the ⁇ -terminal is used.
- the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry.
- a competition method after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation)
- the labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified.
- a soluble antibody is used as an antibody
- B / F separation is performed using a polyethylene glycol
- a liquid phase method using a second antibody against the antibody or a solid phase antibody is used as the first antibody.
- a solid phase method using a soluble first antibody and a solid phase 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 the labeled antibody, and then the force for separating the solid phase and the liquid phase, or
- the antigen is allowed to react with an excess amount of the labeled antibody, and then 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.
- the amount of the label in either phase is measured to determine the amount of the antigen in the test solution.
- nephelometry the amount of insoluble sediment resulting from an 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.
- the protein measuring system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to the ordinary conditions and procedures in each method. For details of these general technical means, reference can be made to reviews, documents, etc.
- the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
- respiratory diseases eg, chronic obstruction Lung disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- respiratory diseases eg, chronic obstruction Lung disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue.
- a subject such as a body fluid or a tissue.
- detecting the protein of the present invention in each fraction during purification, and analyzing the behavior of the protein of the present invention in test cells, etc. Can be used.
- the DNA of the present invention can be used, for example, in humans or warm-blooded animals (for example, rats, mice, guinea pigs, egrets, birds, sheep, pigs, horses, cats, cats, dogs) by using them as probes. , Monkeys, chimpanzees, etc.) can detect abnormalities (genetic abnormalities) in the DNA or mRNA encoding the protein of the present invention or partial peptides thereof, for example, damage to the DNA or mRNA. It is useful as a diagnostic agent for a gene, such as a mutation or a decrease in expression, or an increase or excessive expression of the DNA or mRNA.
- the above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization ⁇ PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proceedings of the National Academy of Sciences of the United states of America, Vol. 86, pp. 2766-2770 (1989)).
- Northern hybridization detects overexpression or reduction. If DNA mutation is detected by PCR_SSCP method, for example, respiratory disease [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma , Cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.].
- respiratory disease eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma , Cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- the antisense polynucleotide of the present invention which can complementarily bind to a polynucleotide (eg, DNA) encoding the protein A of the present invention and suppresses the expression of the DNA, has low toxicity, and has a low toxicity in vivo.
- a polynucleotide eg, DNA
- the function of the protein A of the invention or the DNA encoding the protein can be suppressed, for example, respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis , Bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.].
- it is a prophylactic / therapeutic agent for chronic obstructive pulmonary disease and the like.
- the antisense polynucleotide When used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered according to a method known per se.
- the above antisense polynucleotide is introduced alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc. Or it can be administered orally or parenterally to mammals (eg, rats, egrets, sheep, pigs, dogs, cats, dogs, monkeys, etc.).
- the antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as a scavenger to promote ingestion, and administered with a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be aerosolized and administered topically into the trachea as an inhalant.
- the antisense polynucleotide is formulated alone or together with a carrier such as ribosome (injection), and is used for intravenous, subcutaneous, and respiratory tract administration. It may be administered to the affected area of the lung.
- the dose of the antisense polynucleotide varies depending on the target disease, the subject of administration, the route of administration, and the like.
- the antisense polynucleotide of the present invention may be used for the treatment of emphysema.
- administering a polynucleotide generally, for an adult (body weight 60 kg), about 0.1 to 100 mg of the antisense polynucleotide is administered daily.
- RNA containing a part of the RNA encoding the protein A of the present invention e.g., siRNA (small) for a polynucleotide encoding the protein A of the present invention
- shRNA small (short) hairpin RNA
- ribozymes containing a part of RNA which encodes protein A of the present invention.
- Gene expression can be suppressed, and the function of the protein A of the present invention or the DNA encoding the same in vivo can be suppressed; for example, respiratory diseases (eg, chronic obstructive pulmonary disease) (Chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.].
- respiratory diseases eg, chronic obstructive pulmonary disease
- Chronic bronchitis, emphysema Chronic bronchitis, emphysema
- diffuse panbronchiolitis bronchial asthma
- cystic fibrosis irritable pneumonia
- pulmonary fibrosis etc.
- it is an agent for preventing or treating chronic obstructive pulmonary disease.
- the double-stranded RNA can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
- the ribozyme can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS, in Molecular Medicine, 7, 221, 2001). For example, it can be produced by linking a known ribozyme to a part of the RNA encoding the protein A of the present invention. As a part of the RNA encoding the protein A of the present invention, a portion (RNA fragment) close to the cleavage site on the RNA of the present invention, which can be cleaved by a known ribozyme, can be mentioned.
- RNA or ribozyme When the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide. (5) a drug containing the antibody of the present invention
- the antibody of the present invention may be used, for example, for respiratory diseases (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.) It can be used as a preventive and therapeutic agent. Preferably, it is an agent for preventing or treating chronic obstructive pulmonary disease.
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- it is an agent for preventing or treating chronic obstructive pulmonary disease.
- the antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition.
- the pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
- Such compositions are provided in dosage forms suitable for oral or parenteral administration.
- compositions for oral administration include solid or liquid dosage forms, specifically, tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, and forcepsenoles (software). Capsenoles), syrups, emulsions, suspensions and the like.
- Powerful compositions are prepared by known methods and contain carriers, diluents or excipients usually used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
- Injections are used as parenteral compositions for parenteral administration.
- Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, and the like. Dosage forms.
- Such injections are prepared according to known methods, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections.
- aqueous liquid for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyano Reconore (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, HCO-50
- Oily liquids include, for example, sesame oil and soybean oil, and benzyl benzoate, benzyl alcohol, etc. are used in combination as dissolution aids.
- the prepared injection solution is usually filled in an appropriate ampoule, and a suppository for rectal administration is prepared by mixing the above antibody or a salt thereof with a usual suppository base. It is.
- compositions are conveniently prepared in dosage unit forms to be compatible with the dosage of the active ingredient.
- Examples of powerful dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), and suppositories. It is preferable that 5 to 500 mg of the above-mentioned antibody is usually contained per dosage unit dosage form, especially 5 to 100 mg for injection, and 10 to 250 mg for other dosage forms.
- compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
- the prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity, and is used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in humans or mammals (eg, rats, egrets, It can be administered orally or parenterally (eg, intravenously) to sheep, pigs, pigs, cats, cats, dogs, monkeys, etc.).
- the dosage varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, when used for the treatment or prevention of pulmonary emphysema in adults, the antibody of the present invention is usually administered in a single dose.
- the antibody of the present invention may be used, for example, for respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia , 'Pulmonary fibrosis, etc.].
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia , 'Pulmonary fibrosis, etc.
- protein B of the present invention decreases in the lung with the progress of chronic obstructive pulmonary disease. If the protein B of the present invention or the polynucleotide encoding the same is abnormal or deficient, for example, respiratory diseases (eg, chronic obstructive pulmonary disease)
- respiratory diseases eg, chronic obstructive pulmonary disease
- the protein B of the present invention or a polynucleotide encoding the same may be used, for example, for respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, Cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.).
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, Cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.).
- Protein B of the present invention or a polynucleotide encoding the same for example, When there is a patient in which the protein B of the present invention or a polynucleotide encoding the same is reduced or deficient in a living body, (a) administering the polynucleotide to the patient, By expressing the protein B of the present invention in a (mouth) cell and then transplanting the cell into a patient; or (c) transforming the protein B of the present invention into the cell By administering to a patient or the like, the role of the protein B of the present invention in the patient can be sufficiently or normally exerted.
- the DNA may be used alone or in a suitable vector such as a reticulovirus vector, an adenovirus vector, or an adenoviral vector. After insertion into the vector, it can be administered to humans or warm-blooded animals according to conventional means.
- the DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and then administered using a gene gun or a catheter such as a hydrogel catheter.
- the protein B of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it is purified to at least 90%, preferably at least 95%, more preferably at least 98%, further preferably at least 99%. It's better to use something! / ,.
- the protein B of the present invention can be used, for example, as a sugar-coated tablet, capsule, elixir, microcapsule, etc., orally, or with water or another pharmaceutically acceptable liquid, if necessary. It can be used parenterally (preferably subcutaneous administration) in the form of an injection such as a sterile solution or suspension.
- the protein B of the present invention may be combined with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc., in a unit dosage required for the generally accepted formulation practice. It can be manufactured by mixing in the form. The amounts of the active ingredients in these preparations U are adjusted so that an appropriate dose in the specified range can be obtained.
- a vector into which the above polypeptide (eg, DNA) has been inserted is also formulated in the same manner as described above, and is usually used parenterally.
- the preparations obtained in this way are safe and have low toxicity, for example, in humans or warm-blooded animals (for example, rats, mice, guinea pigs, egrets, birds, sheep, pigs, etc.). To animals, cats, dogs, cats, dogs, monkeys, etc.).
- the dosage of the protein B of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like.For example, when the protein B of the present invention is administered parenterally for the purpose of treating emphysema, it is generally In an adult (assuming a body weight of 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the protein B is administered per day. In the case of other animals, it is also possible to administer the amount in terms of weight per 60 kg. ⁇
- prophylactic / therapeutic agent for respiratory diseases comprising a compound having a function of regulating cholesterol hydroxylating activity or a salt thereof” of the present invention
- a “compound having an activity of regulating cholesterol hydroxylation activity” is a compound having an activity of regulating cholesterol hydroxylation activity (eg, peptide, protein, antibody, non-peptide compound, synthetic compound, fermentation product) , Cell extract, plant extract, animal tissue extract, plasma, etc.). Bronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.]. Preferably, it is an agent for preventing or treating chronic obstructive pulmonary disease.
- the prophylactic / therapeutic agent is produced in the same manner as described above.
- the present invention relates to a DNA encoding an exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention). And a non-human mammal having the same.
- Non-human mammal having the exogenous DNA of the present invention or its mutant DNA (hereinafter referred to as The DNA transgenic animal of the present invention is abbreviated to unfertilized eggs, fertilized eggs, germ cells containing spermatozoa and their progenitor cells, etc., preferably for embryo development in the development of non-human mammals. (Preferably single cell or fertilized egg cell stage and generally before the 8-cell stage), calcium phosphate method, electric pulse method, ribofusion method, aggregation method, microinjection method, particle gun method, DEAE-dextran method
- the target DNA can be produced by transferring the target DNA, for example.
- exogenous DNA of the present invention intended for somatic cells, organs of living organisms, tissue cells, and the like can be transferred by the DNA transfer method and used for cell culture, tissue culture, and the like.
- the DNA-transferred animal of the present invention can also be produced by fusing the cells with the above-mentioned germ cells by a cell fusion method known per se.
- Non-human mammals include, for example, porcupines, pigs, higgins, goats, porcupines, dogs, cats, monoremots, hamsters, mice, rats, and the like.
- a rat eg, Wistar, SD, etc.
- Examples of the “mammal” in the recombinant vector that can be expressed in mammals include human and the like in addition to the above-mentioned non-human mammals.
- the exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from the mammal, not the DNA of the present invention originally possessed by a non-human mammal.
- mutant DNA of the present invention those in which a mutation (for example, mutation) has occurred in the base sequence of the original DNA of the present invention, specifically, addition or deletion of a base, substitution with another base, etc. DNA that has been used is used, and abnormal DNA is also included.
- a mutation for example, mutation
- the abnormal DNA means a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.
- the exogenous DNA of the present invention may be derived from a mammal of the same species or a different species as the animal of interest. Transferring the DNA of the present invention to a target animal Thus, it is generally advantageous to use the DNA as a DNA construct downstream of a promoter capable of being expressed in animal cells.
- a promoter capable of being expressed in animal cells For example, when the human DNA of the present invention is transferred, it is derived from various mammals having the DNA of the present invention having a high homology to the human DNA (eg, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.).
- DNA constructs eg, vectors to which the human DNA of the present invention is bound downstream of various promoters capable of expressing the same DNA can be microinjected into fertilized eggs of target mammals, for example, mouse fertilized eggs.
- a DNA transgenic mammal that highly expresses the DNA of the present invention can be created.
- Examples of the expression vector of the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a Batateriophage such as ⁇ phage, a retrovirus such as Moroni leukemia virus, a vaccinia virus or Animal viruses such as baculovirus are used.
- a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
- promoter that regulates DNA expression examples include, for example, i) a promoter of DNA derived from a virus (eg, Simian virus, cytomegaloinoles, Moroni leukemia ⁇ inores, JC ⁇ inores, breast cancer virus, poliovirus, etc.); Ii) Promoters from various mammals (humans, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.), for example, anolebumin, insulin II, peroplaskin II, elastase, erythropoietin, endothelin muscle creatine kinase , Glial fibrillary acidic protein, glutathione S-transferase, platelet-derived growth factor, keratin K1, 10 ⁇ : ⁇ : 14, collagen type I and type II, cyclic AMP-dependent protein kinase j3 I subunit, dist Fin, tartrate-resistant alkaline phosphata
- cytomegalovirus promoter that can be highly expressed throughout the whole body, a human peptide chain elongation factor 1a (EF-1 ⁇ ) promoter, a human and a chicken] 3 actin promoter, and the like are preferable.
- EF-1 ⁇ human peptide chain elongation factor 1a
- the vector preferably has a sequence that terminates transcription of the target messenger RN ⁇ in a DNA-transferred mammal (generally referred to as terminator 1).
- terminator 1 a sequence that terminates transcription of the target messenger RN ⁇ in a DNA-transferred mammal.
- the SV40 terminator of the simian virus is preferably used.
- the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc. are used 5 'upstream of the promoter region, between the promoter region and the translation region or for the purpose of further expressing the target exogenous DNA. Connection to the 3 'downstream of the region is also possible depending on the purpose.
- the normal translation region of the protein of the present invention is derived from the liver, kidney, thyroid cells, and fibroblasts derived from humans or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). It is possible to obtain all or part of genomic DNA from DNA and various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cells, and fibroblast-derived RNA as raw materials. I can do it.
- the exogenous abnormal DNA can produce a translation region obtained by mutating the translation region of a normal protein obtained from the above cells or tissues by point mutagenesis.
- the translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering technique in which it is ligated downstream of the above promoter and, if desired, upstream of the transcription termination site.
- the transfer of the exogenous DNA of the present invention at the fertilized egg cell stage can be carried out in the embryo of the target mammal.
- the cell is ensured to be present in all of the somatic cells.
- the presence of the exogenous DNA of the present invention in the germinal cells of the transgenic animal after the DNA transfer indicates that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germinal and somatic cells. Means to do.
- the offspring of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germ cells and somatic cells.
- the non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain exogenous DNA by mating, and should be subcultured as an animal having the DNA in a normal breeding environment. Can be done.
- Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal.
- Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer indicates that all of the offspring of the produced animal contain the exogenous DNA of the present invention in all of its germ cells and somatic cells. Means to have.
- the progeny of this type of animal that has inherited the exogenous DNA of the present invention has an excess of the exogenous DNA of the present invention in all of its germinal and somatic cells.
- the normal DNA of the present invention is highly expressed, and the function of the protein of the present invention is finally enhanced by promoting the function of endogenous normal DNA.
- the disease may develop and can be used as a model animal for the disease.
- using the normal DNA-transferred animal of the present invention it is possible to elucidate the pathological mechanism of the hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible.
- mammals to which the exogenous normal DNA of the present invention has been transferred have increased or decreased symptoms of the free protein of the present invention, and therefore, for example, respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchial bronchi). Inflammation, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.].
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchial bronchi). Inflammation, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- a non-human mammal having the foreign abnormal DNA of the present invention After confirming that the sex DNA is stably retained, the animal can be subcultured in a normal breeding environment as the DNA-bearing animal. Furthermore, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
- a DNA construct with a promoter can be prepared by a usual DNA engineering technique. Transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
- the presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells.
- the progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells.
- a homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing the male and female animals, it is possible to breed so that all progeny have the DNA.
- the abnormal DNA of the present invention is highly expressed, and the function of the protein of the present invention is finally impaired by inhibiting the function of endogenous normal DNA. It may be active refractory and can be used as a model animal for the disease. For example, using the abnormal DNA transgenic animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.
- the abnormal DNA highly expressing animal of the present invention can be used to inhibit the function of the normal protein by the abnormal protein of the present invention (dominant negative action) in the function-inactive refractory disease of the protein of the present invention.
- mammals to which the abnormal DNA of the present invention has been transferred have increased or decreased symptoms of the free protein of the present invention, and therefore, for example, respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchial bronchi). Inflammation, pulmonary emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.].
- cells of DNA-containing tissue are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture. 4) cells by using the cells described in 3) above Drug screening to enhance the function of and
- the protein of the present invention can be identified, its relationship with apoptosis, differentiation, or proliferation, or its signaling mechanism can be examined, and its abnormalities can be examined. It is an effective research material for elucidation.
- a therapeutic agent for a disease associated with the protein of the present invention including a functionally inactive refractory type of the protein of the present invention
- using the DNA-transferred animal of the present invention Using a quantitative method or the like, it is possible to provide an effective and rapid screening method for the therapeutic agent for the disease.
- using the transgenic animal of the present invention or the exogenous DNA expression vector of the present invention it is possible to examine and develop a method for treating DNA associated with the protein of the present invention.
- the present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expressing the DNA of the present invention. That is, the present invention
- the DNA is inactivated by introducing a reporter gene (eg, a B-galactosidase transgene derived from Escherichia coli), so that the reporter gene can be expressed under the control of a promoter for the DNA of the present invention.
- a reporter gene eg, a B-galactosidase transgene derived from Escherichia coli
- Screening for a compound or a salt thereof that promotes or inhibits the promoter activity of D ⁇ ⁇ according to the present invention which comprises administering a test compound to the animal described in paragraph 7) and detecting the expression of a reporter gene. Provide a method.
- a non-human mammal embryonic stem cell in which the DNA of the present invention has been inactivated is an artificially mutated DNA of the present invention possessed by the non-human mammal, which suppresses the expression of the DNA, Alternatively, the DNA substantially does not have the ability to express the protein of the present invention by substantially losing the activity of the protein of the present invention encoded by the DNA (hereinafter referred to as the knockout DNA of the present invention).
- Non-human mammalian embryonic stem cells hereinafter abbreviated as ES cells).
- non-human mammal those similar to the above can be used.
- the method of artificially mutating the DNA of the present invention can be carried out, for example, by deleting a part or all of the DNA sequence by inserting or substituting another DNA with a genetic engineering technique. These mutations can be used, for example, to produce the knockout DNA of the present invention by shifting the codon reading frame or disrupting the function of the promoter or exon.
- Non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated include, for example, isolating the DNA of the present invention from a non-human mammal of interest, and exposing the exon portion to neomycin resistance.
- Gene a drug resistance gene represented by the hygromycin resistance gene, or a reporter gene represented by lac Z (6-galactosidase gene) or cat (chloramphenicol acetyltransferase gene).
- lac Z 6-galactosidase gene
- cat chloramphenicol acetyltransferase gene
- targeting vector Into the chromosome of the animal by, for example, a homologous recombination method, and obtaining the obtained ES cells on a Southern hybridization analysis or a targeting vector using the DNA sequence of or near the DNA of the present invention as a probe. It can be obtained by analyzing the DNA sequence and the DNA sequence of the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector by PCR using primers, and selecting the knockout ES cells of the present invention. .
- ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like for example, those already established as described above may be used, or according to the known Evans and Kaufman method. May be newly established.
- the 129-line ES cells are generally used.
- the immunological background is not clear, a pure line that substitutes them is used for immunological inheritance.
- blastocysts 3.5 days after fertilization are generally used.
- the blastocysts can be efficiently collected by collecting 8-cell embryos and culturing them up to blastocysts. Many early embryos can be obtained.
- Either male or female ES cells may be used, but male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing. .
- An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR.
- this method conventionally, for example G-banding method, it requires about 1 0 6 cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 5 0)
- the primary selection of ES cells at the initial stage of culture can be performed by gender discrimination, and the early selection of male cells can greatly reduce the labor required at the beginning of culture.
- the secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method.
- the number of chromosomes in the obtained ES cells is desirably 100% of the normal number.
- the embryonic stem cell line obtained in this way usually has very good growth potential, but it must be carefully subcultured because it tends to lose its ability to generate individuals.
- LIF (1 to
- trypsin ZE DTA solution (usually 0.001 to 0.5% trypsin / 0.1 to 5 mM EDTA, preferably about 0.1% trypsin / lm
- ES cells can be transformed into various types of cells, such as parietal, visceral, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. (MJ Evans and MH
- the DNA-deficient cell of the present invention obtained by differentiating the ES cell of the present invention is a cell biology of the protein of the present invention in vitro. It is useful in strategic research.
- the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
- non-human mammal those similar to the above can be used.
- the non-human mammal deficient in expression of the DNA of the present invention may be prepared, for example, by introducing the targeting vector prepared as described above into mouse embryonic stem cells or mouse egg cells, and inactivating the targeting vector to inactivate the DNA of the present invention.
- the DNA of the present invention can be knocked down by homologous recombination of the live DNA sequence with the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. .
- the cells in which the DNA of the present invention has been knocked out are derived from the DNA sequence on the southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and from the mouse used for the targeting vector. And the DNA sequence of a nearby region other than the DNA of the present invention can be determined by PCR analysis using primers.
- a cell line in which the DNA of the present invention has been inactivated is cloned by homologous recombination, and the cells are cloned at an appropriate time, for example, at the 8-cell stage.
- the chimeric embryo is injected into a human mammal embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal.
- the produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially altered DNA locus of the present invention.
- all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like.
- the individuals obtained in this manner are usually individuals deficient in the hetero-expression of the protein of the present invention, mated with individuals deficient in the hetero-expression of the protein of the present invention, and obtained from their offspring to obtain the protein of the present invention.
- a homozygous expression deficient individual can be obtained.
- a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into the nucleus of an egg cell by a microinjection method. Compared to non-human mammals, it can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination of the gene.
- the individual in which the DNA of the present invention has been knocked out can be bred in a normal breeding environment after confirming that the DNA has been knocked out in the animal individual obtained by mating. .
- the germline can be obtained and maintained in accordance with a standard method. That is, by crossing male and female animals having the inactivated DNA, homozygous animals having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in a state where one normal individual and plural homozygous animals are obtained. By crossing male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
- the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing the non-human mammal deficient in expression of the DNA of the present invention.
- the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, a disease caused by inactivation of the biological activity of the protein of the present invention. It is useful for investigating the causes of these diseases and studying treatment methods.
- the non-human mammal deficient in expression of the DNA of the present invention can be used for screening for a compound having a therapeutic / preventive effect against a disease caused by deficiency or damage of the DNA of the present invention.
- the present invention is characterized in that a test compound is administered to a non-human mammal deficient in expression of the DNA of the present invention, and changes in the animal are observed and measured.
- Diseases caused by, for example, respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- a method for screening a compound or a salt thereof having a therapeutic / preventive effect e.g, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis,
- the non-human mammal deficient in DNA expression of the present invention used in the screening method includes the same ones as described above.
- Test compounds include, for example, peptides, proteins, antibodies, non-peptide conjugates, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma. May be a novel compound or a known compound.
- a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and changes in each organ, tissue, disease symptoms, etc. of the animal are used as indicators. Therapeutic and prophylactic effects of test compounds can be tested.
- test compound for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like.
- the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.
- a test compound is administered to a non-human mammal deficient in expression of DNA of the present invention, and the difference between the test compound non-administration group and pulmonary emphysema! / Observationally.
- test compound when administered to a test animal, When the disease symptom of the test animal is improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound has a therapeutic / preventive effect on the above-mentioned diseases. It can be selected as a compound.
- the compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect against a disease caused by a deficiency or damage of the protein of the present invention. It can be used as a safe and low toxic prophylactic and therapeutic agent. Further, a compound derived from the compound obtained by the above-mentioned screening can be used similarly.
- the compound obtained by the screening method may form a salt.
- the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkalis). Salts with metals and the like are used, and especially preferred are physiologically acceptable acid addition salts.
- salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) And succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
- inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
- organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
- succinic acid tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.
- a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention.
- the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs). , Monkeys, etc.).
- the dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like. Administer about 0.1 to: 00 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound per day. When administered parenterally, a single dose of the compound may vary depending on the administration subject, target disease, and the like. When given, about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 30 mg of the compound per day. It is convenient to administer about 10 to about 10 by intravenous injection. other In the case of animals, the dose can be administered in terms of weight per 60 kg.
- the present invention provides a non-human mammal deficient in expression of the DNA of the present invention, which comprises administering a test compound to detect the expression of a reporter gene, thereby promoting the activity of a promoter against the DNA of the present invention.
- a method for screening a compound or a salt thereof that inhibits the inhibition is provided.
- the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention, wherein the DNA of the present invention comprises a reporter gene. Those inactivated and capable of expressing the reporter gene under the control of a promoter for the DNA of the present invention are used. -Examples of test compounds include the same as described above.
- reporter gene the same one as described above is used, and a 6_galactosidase gene (1a.cZ), a soluble alkaline phosphatase gene or a luciferase gene is suitable.
- a tissue expressing the protein of the present invention originally 6_galactosidase is expressed instead of the protein of the invention. Therefore, for example, by staining with a reagent serving as a substrate for ⁇ -galactosidase, such as 5-promo-14-cloth-3-indolyl ⁇ -galatatoviranoside (X-gal), the present method can be easily performed. The state of expression of the protein of the present invention in an animal body can be observed.
- the protein-deficient mouse of the present invention or a tissue section thereof is fixed with dartal aldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X_ga1 at room temperature or at 37 ° C. Around ° C, After reacting for about 30 minutes to 1 hour, the B-galactosidase reaction may be stopped by washing the tissue specimen with a 1 mM EDTA / PBS solution, and the coloration may be observed. Further, mRNA encoding 1 ac ⁇ ⁇ may be detected according to a conventional method.
- PBS phosphate buffered saline
- the compound or a salt thereof obtained by the above-described screening method is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the promoter activity for DNA of the present invention.
- the compound obtained by the screening method may form a salt, and the salt of the compound may be a physiologically acceptable acid (eg, an inorganic acid, etc.) or a base (eg, an alkali metal, etc.) And the like, and a physiologically acceptable acid addition salt is particularly preferable.
- salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used.
- the compound of the present invention which promotes or inhibits the activity of the promoter for DNA, or a salt thereof can regulate the expression of the protein of the present invention and regulate the function of the protein.
- obstructive pulmonary disease chronic bronchitis, emphysema
- diffuse panbronchiolitis bronchial asthma
- cystic fibrosis irritable pneumonia
- pulmonary fibrosis etc.
- preventive and therapeutic agents for chronic obstructive pulmonary disease are preferred.
- a drug containing a compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing a protein of the present invention or a salt thereof.
- the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or mammals (e.g., rats, mice, guinea pigs, egrets, sheep, pigs, horses, cats, cats, Dogs, monkeys, etc.).
- mammals e.g., rats, mice, guinea pigs, egrets, sheep, pigs, horses, cats, cats, Dogs, monkeys, etc.
- the dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like.
- the dose for the DNA encoding the protein A of the present invention may be different.
- a compound that inhibits oral motor activity is orally administered, generally, in an adult (assuming a body weight of 60 kg) emphysema patient, the compound is used in an amount of about 0.1 to 100 mg, preferably about 1.0 to 100 mg per day.
- the single dose of the compound varies depending on the administration subject, target disease, and the like.For example, the compound that inhibits the promoter activity for DNA is usually in the form of an injection.
- the dose When administered to an adult (assuming a body weight of 60 kg) emphysema patient, about 0.01 to 30 mg, preferably about 0.1 to 20 mg , more preferably about 0.1 to 10 mg of the compound per day is injected intravenously. It is convenient to administer. In the case of other animals, the dose can be administered in terms of 60 kg.
- the non-human mammal deficient in DNA expression of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention. It can greatly contribute to the investigation or prevention of various diseases caused by DNA expression deficiency.
- genes encoding various proteins are ligated downstream thereof and injected into an egg cell of an animal to produce a so-called transgenic animal (gene transfer). Animal), it becomes possible to specifically synthesize the protein and examine its effects in the living body. Furthermore, by linking an appropriate reporter gene to the above-mentioned promoter portion and establishing a cell line in which this is expressed, a low-molecular-weight molecule capable of specifically promoting or suppressing the ability of the protein itself of the present invention to produce in the body can be obtained. It can be used as a search system for compounds.
- bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the relevant field, and examples thereof are described below.
- optical isomer for an amino acid the L-form is indicated unless otherwise specified.
- DNA Deoxyribonucleic acid
- RNA ribonucleic acid
- mRNA messenger ribonucleic acid
- d ATP deoxyadenosine triphosphate
- dTTP deoxythymidine triphosphate
- dGTP deoxyguanosine triphosphate
- CTP deoxycytidine triphosphate
- a la Alanine
- Pro Proline A sn: Asparagine
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 5 is shown.
- SEQ ID NO: 11 shows the amino acid sequence of a protein translated from the nucleotide sequence represented by 11.
- amino acid sequence of a protein translated from the base sequence represented by SEQ ID NO: 15 is shown.
- 1 shows the nucleotide sequence of IER3.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 17 is shown.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 21 is shown.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 21 is shown.
- [SEQ ID NO: 23] 1 shows the nucleotide sequence of IL 1 RN.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 23 is shown.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 25 is shown.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 27 is shown.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 29 is shown.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 31 is shown.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 35 is shown.
- 1 shows the base sequence of LDLR.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 37 is shown.
- 1 shows the nucleotide sequence of TNFRSF 10B.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 39 is shown.
- FIG. 2 shows the nucleotide sequence of TNFRSF 12A.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 41 is shown.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 43 is shown.
- Amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 45 Indicates.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 49 is shown.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 51. is shown.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 53 is shown.
- 1 shows the nucleotide sequence of TNC.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 55 is shown.
- SEQ ID NO: 58 The amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 57 is shown.
- amino acid sequence of a protein translated from the nucleotide sequence represented by SEQ ID NO: 59 is shown.
- SEQ ID NO: 61 This shows the amino acid sequence of a protein translated from the nucleotide sequence represented by 1.
- amino acid sequence of a protein translated from the base sequence represented by SEQ ID NO: 65 is shown.
- the base sequence of primer 1 used for detection of the expression level of the CH25H gene is shown.
- the base sequence of primer 2 used for detection of the expression level of the CH25H gene is shown.
- [SEQ ID NO: 70] 7 shows the base sequence of primer 4 used in Example 5.
- Example 7 shows the amino acid sequence of the antigen peptide used in Example 5.
- Lung cancer patients who needed pulmonary resection provided a sample of the lung that had been removed during lung resection surgery as research material. Lung samples were provided by the Tohoku University Ethics Committee and obtained informed consent from patients.
- FEV Patients with FVC of 70% and% FEV ⁇ 80% are mild (stage I) C0PD, FEV Patients with FVC of 70% and SiW FEV, and 80% are moderate (stage IIA) C0PD, FEV FVC iW In addition, patients with SO ⁇ FEV, especially 50%, were diagnosed as moderate (stage I IB) C0PD.
- a lung cancer patient with FEV / FVC ⁇ 70 and no symptoms such as chronic cough and sputum was diagnosed as non-C0PD (non-C0PD).
- Lung cancer patients were divided into non-C0PD and no-smoker group (group II, 12 cases), non-C0PD and ex-smoker group (NE group, 6 cases), non-C0PD and smoker group (NS group, 5 cases), Stage I was classified into Oki group (CE1 group, 7 cases), stage I IA C0PD group (CE2A group, 6 cases), and stage I IB C0PD group (CE2B group, 2 cases).
- RNA samples after lung surgery for lung cancer patients with C0PD were frozen in liquid nitrogen and crushed with a frozen tissue crusher.
- the cells were immersed in Isogen (manufactured by Futabajin Co., Ltd.) corresponding to 10 times the wet weight, and total RNA was prepared according to the attached protocol.
- total RNA of group ⁇ 5 cases
- NE group 3 cases
- NS group 2 cases
- CE1 group 3 cases
- CE2A group 2 cases
- each gene is expressed as the expression value when the median expression value of all genes of each oligonucleotide microarray is set to 1, the average value is taken between groups, and the numerical value is used as the gene expression value for each gene. Comparisons were made between groups.
- CSF3 colony stimulating factor 3 (granulocyte) 0.12 0.02 0.54 4.22 1.46 17.9
- TNFAIP3 tumor necrosis factor alpha-induced protein 3 0.91 0.98 2.36 5.08 5.80 4.4
- TNFAIP6 tumor necrosis factor alpha-induced protein 6 0.19 0.29 0.73 1.02 1.76 4.0
- TNFRSF10B tumor necrosis factor receptor superfamily member 10b 0.74 0.79 1.30 2.39 1.71 2.4
- TNFRSF12A tumor necrosis factor receptor superfamily member 1 A 1.75 1.56 2.14 5.39 4.89 2.9 AP3 8 mitogen-activated protein kinase kinase kinase 8 0.21 0.20 0.29 0.56 0.64 2.6
- ADAMTS1 a disintegrin-like and metalloprotease (reprolysin type)
- TFPI2 tissue factor pathway inhibitor 2 0.34 1.81 0.65 0.93 2.22 1.7
- TNC ESTs 0.25 0.31 0.65 0.73 1.02 2.43 endothelial differentiation, sphingolipid G-protein— coupled
- GPR73L1 G protein-coupled receptor 73— like 1 0.20 0.30 0.41 0.51 0.60 2.01
- HIMAP2 DKFZP586D0824 protein 0.95 0.93 0.85 0.40 0.39 0.43
- CSF3 (NMJ00759) (SEQ ID NO: 5), R (Martial One 014470) (SEQ ID NO: 7),
- TNFAIP3 (NMJ06290) (SEQ ID NO: 13), TNFAIP6 (NM_007115) (SEQ ID NO: 15), IER3 (NM_003897) (SEQ ID NO: 17), GADD45A (NMJ01924) (SEQ ID NO: 19), GADD45B (AF087853) ( (SEQ ID NO: 21), IL1RN (NM_1738 1) (SEQ ID NO: 23), S0CS2 (NM_003877) (SEQ ID NO: 25), S0CS3 (NMJ03955) (SEQ ID NO: 27), Rei P190J38321) (SEQ ID NO: 29) ), DUSP2 (NMJ04418) (SEQ ID NO: 31), DUSP5 (U16996) (SEQ ID NO: 33), STC1 (U46768) (SEQ ID NO: 35), LDLR (NMJ00527) (SEQ ID NO: 37), TNFRSF10B ( AF016266) (SEQ ID NO:
- HIMAP2 (NM_015660) (SEQ ID NO: 63) and SSTR1 (Marauder-001049) (SEQ ID NO: 65) were detected as genes whose expression decreased with the progression of C0PD disease (Table 3).
- SSTR1 Marauder-001049
- Example 1 In order to determine whether the expression of the gene whose expression was fluctuated in Example 1 was related to the C0PD disease state, correlation analysis of the expression level and respiratory function (% -second amount,% co lung diffusion ability) was performed for each gene. went.
- CH25H (SEQ ID NO: 1), PLAB (SEQ ID NO: 3), CSF3 (SEQ ID NO: 5), H06 (SEQ ID NO: 7), SFN (SEQ ID NO: 9), SSB1 (SEQ ID NO: 1) 1), TNFAIP3 (SEQ ID NO: 13), TNFAIP6 (SEQ ID NO: 15), IER3 (SEQ ID NO: 17), GADD45A (SEQ ID NO: 19), GADD45B (SEQ ID NO: 21), IL1RN
- SEQ ID NO: 23 S0CS2 (SEQ ID NO: 25), S0CS3 (SEQ ID NO: 27), MMP19 (SEQ ID NO: 29), DUSP2 (SEQ ID NO: 31), DUSP5 (SEQ ID NO: 33), STC1 ( SEQ ID NO: 35), LDLR (SEQ ID NO: 37), TNFRSF10B (SEQ ID NO: 39), TNFRSF12A (SEQ ID NO: 41), MAP3K8 (SEQ ID NO: 43), EGR1
- CH25H Changes in the expression of CH25H (SEQ ID NO: 1) were measured using a whole lung resection sample [Marauder group (12 cases), NE group (6 cases), NS group (5 cases), CE1 group (7 cases), .CE2A group (6 Example)] was used for quantitative RT-PCR.
- cDNA was synthesized by a reverse transcription reaction in a 50 ⁇ l reaction solution using a TaqMan Gold RT-PCR Kit (manufactured by Applied Biosystems). After diluting the reaction solution 2.5 times with distilled water, use 2 ⁇ l of the solution to perform real-time analysis using ABI PRISM 7900 seauence detection system (Applied Biosystems) and QuantiTect SYBR Green PCR Kit (QIAGEN). The Ct value of each gene was measured by quantitative PCR.
- CH25H is a type of cholesterol hydroxylase. Therefore, how the expression of CYP3A4, CYP7AK, CYP46 and CYP27A1, which are cholesterol hydroxylases other than CH25H, are changed in C0PD patients, is shown from the GeneChip data shown in Example 1 for each gene. The expression values were examined by extracting and comparing. As a result, expression of CYP3A4, CYP7AK, CYP46. And CYP27A1 did not change in C0PD patients. This indicates that the expression fluctuated only in CH25H with the progression of the C0PD disease state.
- the tissue distribution of CH25H, CYP3A4, CYP7AK, CYP46 and CYP27A1 was analyzed using Human MTC Panel I, Human MTC Panel II, Human Immune System MTC PaneL Human Blood.
- CH25H was specifically expressed in the lung. Apart from CH25H, only CYP27A1 was highly expressed in the lung.
- mouse CH25H gene in lung and bronchial lavage fluid cells exposed to cigarette smoke was examined for expression fluctuation.
- mice exposed to cigarette smoke were prepared by exposing C57BL / 6 mice (7 weeks old, male) to cigarette smoke under the following conditions.
- For exposure to cigarette smoke use Kentucky reference c igaret te 2R4F, which was cut with a filter, as 3% diluted smoke, and 150 puf fs / 15min ⁇ interval / 15min ⁇ 150 puf fs / 15inin ⁇ interval / 15min ⁇ 150 puf fs / 15min per day ⁇ Exposure was performed at interval / 15min ⁇ 150 puf fs / 15min (40 cigarettes). After 2 or 3 days of exposure, the lungs were removed and total RA was prepared according to the method described in Example 1.
- mice CH25H and mouse CYP27A1 genes were measured according to the real-time quantitative PCR method described in Example 1.
- Mice exposed to cigarette smoke for 3 days were tracheally intubated under pentobarbital anesthesia, and 0.5 ml of PBS was injected into the lung three times and collected.
- Changes in the expression of mouse CH25H and mouse CYP27A1 in inflammatory cells in the bronchial lavage fluid were also analyzed.
- Each probe was used by selecting a corresponding probe from Assays on demand gene expression product (manufactured by Applied Biosystems).
- the expression level of each gene was calculated as a relative expression value to Rodent GAPDH by the comparative Ct value method.
- mice exposed to tobacco smoke were the same as C57BL / 6 mice (6 weeks old, male) under the same conditions as in Example 1. Exposure to tobacco smoke. The days of exposure to cigarette smoke were 1, 3, and 9, and 24 hours after each final exposure, the mice were sacrificed by an overdose of pentobarbi and the lungs were removed. Lungs were stored at -80 ° C until measurement of 25-HC and cholesterol levels.
- 25-HC and cholesterol levels are determined by LC / MS / MS (API 4000, Applied
- PyroBest polymerase was used for human genes, using the lung Marathon cDNA library (manufactured by Clontech) as type II, and for mouse genes, using the spleen Marathon cDNA library (manufactured by Clontech) as type II.
- Ex-Tad polymerase manufactured by Takara Shuzo
- each full-length gene was amplified according to the attached manual.
- the PCR product was inserted into pCR Bluntn T0P0 vector (Invitrogen) according to the attached manual (pCRII Bluntll III-MH25H and pCRII Bluntll TOPO-mCH25H).
- mice CH25H gene insertion plasmid (pCRII Bluntll TOPO-mCH25H) constructed in (1) above was digested with BamHI and ⁇ , respectively, and a Linear DNA having a T7 promoter binding site and a Linear DNA having an SP6 promoter binding site downstream of the mouse CH25H gene.
- mice CH25H antisense RNA and sense RNA were prepared according to the attached protocol.
- antisense RNA and sense RA were also prepared for the MMP-12 gene.
- lungs were excised from mice exposed to cigarette smoke for 3 months, fixed with 4% paraformaldehyde, cut into ⁇ ⁇ sections using a cryostat, and attached to APS-coated slide glasses.
- the sample attached was used as a sample.
- Hybridization was carried out using In situ hybridization reagents (manufactured by Futtsubon Gene) according to the attached protocol. Detection of the RA probe was performed using a DIG detection kit (Roche Diagnostics).
- CH25H-expressing cells were alveolar macrophages.
- the expression cells were identified by immunostaining using the CH25H antibody.
- a peptide (SEQ ID NO: 73; manufactured by MBL) synthesized based on the method described in Lund et al. (The Journal of Biological Chemistry vol.273, pp.34316-34327, 1998) was immunized together with KHL into Egret. The serum after the fifth immunization was purified by a peptide column to prepare an anti-CH25H antibody.
- lungs were extracted from mice exposed to cigarette smoke for 3 months, and slices were prepared with a cryostat to obtain samples. After air drying, fixed for 15 minutes in Ma Irudohorumu were 1 hour at 0.3ffl 2 0 2 / MeOH for 30 minutes reacted Block Ace (Snow Brand Milk Co., Ltd.).
- an anti-mouse F4 / 80 antibody (UK-Serotec) that recognizes macrophages, an AlexaFluor594-labeled anti-rat IgG antibody (Molecular Probe), an anti-mouse CH25H antibody, and a MexaFluor488-labeled anti-Egret IgG
- the reaction was performed for 30 minutes for each antibody (Molecular Probe). After washing with PBS / 0.1 TritonX-100, nuclear staining and encapsulation were performed using VECTASHIELD with DAPI (manufactured by VECTOR), followed by observation with a fluorescence microscope and photographing.
- Bronchoalveolar lavage fluid was collected from the mice exposed to cigarettes for 4 days according to the method described in Example 4 on the day after the final exposure according to the method described in Example 4, and contained alveolar macrophages. cells were seeded to become lx i0 6 cel ls / nil in 96Wel l play. Bok. The next day, the cells were stimulated with LPS (10 ng / ml) and 25-HC (0.3-3 g / ml) and cultured for another 24 hours. Then, according to the method described in Example 1, total RNA was collected from the cells according to the attached manual, and the mRNA levels of inflammatory cytokines CXCL2 and IL-ljS were quantified by real-time quantitative PCR.
- Each probe was used by selecting a corresponding probe from Assays on demand gene expression product (manufactured by Applied Biosystems). The expression level of each gene was calculated as a relative expression value to Rodent GAPDH by the comparative Ct value method.
- mice were injected with physiological saline (10% aqueous ethanol solution) containing 25-H and the same concentration of the solvent used for 25-HC dissolution as a control.
- physiological saline 10% aqueous ethanol solution
- 50 g / 50 / il / mouse intratracheally and bronchoalveolar lavage fluid is collected 3, 6, 12, 24 and 48 hours after administration, and the inflammatory cytodynamic force in the lavage fluid KC and MIP-2 levels were measured using a commercially available ELISA kit.
- COS cells were seeded in 6-well plates at a concentration of 2 x i0 5 cel ls / wel l, the next day, 2 g of the human CH25H expression plasmid (pcDNA-hCH25H) using FuGENE6 (manufactured by Roche Daiagunosute Itsukusu Co.) Introduced according to the attached manual. After further culturing for 2 days, the medium was replaced with a serum-free DMEM medium containing 20 mg / ml of 2-hydroxyprory; 8-cydodextrin, and cultured at 37 ° C for 1 hour.
- pcDNA-hCH25H human CH25H expression plasmid
- FuGENE6 manufactured by Roche Daiagunosute Itsukusu Co.
- the plate was applied to a plate (20 cm ⁇ 20 cm, manufactured by Merck). After that, it was developed with AcOEt / Ph-Me (4: 6), and the conversion of cholesterol to 25-HC was detected by BAS2000 to determine the CH25H enzyme activity (conversion rate). In addition, the activity (conversion rate) of the CH25H enzyme for converting cholesterol to 25-HC without the addition of the test compound was also determined.
- Inhibition rate (%) (1- (conversion rate when test compound is added / conversion rate when test compound is not added))
- test compound having an inhibition rate (%) of 50% or more was selected as a compound having a CH25H inhibitory action.
- SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, Sequence No.
- respiratory disease eg, chronic obstructive pulmonary disease (eg, Chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis chronic obstructive pulmonary disease (eg, chronic bronchitis, emphysema), diffuse pandemic It is useful as a diagnostic marker for bronchitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.].
- respiratory disease eg, chronic obstructive pulmonary disease (eg, Chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.].
- SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30 , SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 or amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 62
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis Etc.
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis Etc.
- respiratory diseases eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis Etc.
- Accelerators, antibodies that promote the activity of the above proteins, the above proteins, the above polynucleotides, and the like include, for example, respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, Bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.].
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US10/594,266 US7981598B2 (en) | 2004-03-26 | 2005-03-25 | Preventive/remedy for respiratory diseases |
JP2006511608A JP4829780B2 (ja) | 2004-03-26 | 2005-03-25 | 呼吸器疾患の予防・治療剤 |
EP05727282A EP1731171A4 (en) | 2004-03-26 | 2005-03-25 | PREVENTATIVE MEDICINE / REMEDY FOR RESPIRATORY DISEASES |
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Cited By (2)
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US7947271B2 (en) | 2004-03-23 | 2011-05-24 | Biogen Idec Ma Inc. | Methods of decreasing tumor volume and reducing tumor burden using TNF-receptor-coupling agents |
US9056908B2 (en) | 2007-08-03 | 2015-06-16 | Abbvie Biotherapeutics Inc. | Therapeutic use of anti-tweak receptor antibodies |
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GB0922377D0 (en) * | 2009-12-22 | 2010-02-03 | Arab Gulf University The | Mutant LDL receptor |
EP3689370A1 (en) | 2012-12-21 | 2020-08-05 | Aveo Pharmaceuticals Inc. | Anti-gdf15 antibodies |
Citations (4)
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WO2002099134A1 (en) * | 2001-06-05 | 2002-12-12 | Auckland Uniservices Limited | Methods and compositions for assessment of pulmonary function and disorders |
WO2003073990A2 (en) * | 2002-03-01 | 2003-09-12 | Children's Hospital Medical Center | Treatment for asthma or allergies |
JP2003274982A (ja) * | 2001-11-02 | 2003-09-30 | Takeda Chem Ind Ltd | 新規タンパク質、そのdnaおよびその用途 |
JP2003325187A (ja) * | 2002-01-21 | 2003-11-18 | Takeda Chem Ind Ltd | 新規タンパク質、そのdnaおよびその用途 |
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US6562609B1 (en) | 1998-10-22 | 2003-05-13 | Board Of Regents, The University Of Texas System | Cholesterol 25-hydroxylase |
WO2003037927A1 (en) | 2001-11-02 | 2003-05-08 | Takeda Chemical Industries, Ltd. | Novel protein, its dna and use thereof |
JP2004121218A (ja) * | 2002-08-06 | 2004-04-22 | Jenokkusu Soyaku Kenkyusho:Kk | 気管支喘息または慢性閉塞性肺疾患の検査方法 |
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WO2002099134A1 (en) * | 2001-06-05 | 2002-12-12 | Auckland Uniservices Limited | Methods and compositions for assessment of pulmonary function and disorders |
JP2003274982A (ja) * | 2001-11-02 | 2003-09-30 | Takeda Chem Ind Ltd | 新規タンパク質、そのdnaおよびその用途 |
JP2003325187A (ja) * | 2002-01-21 | 2003-11-18 | Takeda Chem Ind Ltd | 新規タンパク質、そのdnaおよびその用途 |
WO2003073990A2 (en) * | 2002-03-01 | 2003-09-12 | Children's Hospital Medical Center | Treatment for asthma or allergies |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7947271B2 (en) | 2004-03-23 | 2011-05-24 | Biogen Idec Ma Inc. | Methods of decreasing tumor volume and reducing tumor burden using TNF-receptor-coupling agents |
US9056908B2 (en) | 2007-08-03 | 2015-06-16 | Abbvie Biotherapeutics Inc. | Therapeutic use of anti-tweak receptor antibodies |
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EP1731171A1 (en) | 2006-12-13 |
US7981598B2 (en) | 2011-07-19 |
US20070207462A1 (en) | 2007-09-06 |
JP4829780B2 (ja) | 2011-12-07 |
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