JP2020079707A - Obstructive pulmonary disease biomarker - Google Patents

Abstract

To provide the biomarker of an obstructive pulmonary disease such as bronchial asthma and a method for using the same.SOLUTION: Provided is an inspection method that includes a step for detecting at least one kind of protein selected from a protein group in the extracellular vesicles of the body fluid sampled from an examinee, and further includes a step for determining the examinee is affected by an obstructive pulmonary disease when the amount or concentration of the protein detected in the preceding step is greater than or equal to a cutoff value.SELECTED DRAWING: None

Description

  The present invention relates to a biomarker for obstructive pulmonary disease, its use and the like.

  It is estimated that the number of patients with bronchial asthma (BA) reaches 300 million, and the increase in the prevalence thereof poses a social and economic burden. Bronchial asthma is a chronic airway inflammation, but the key molecules that control inflammation and airway remodeling are not fully understood. On the other hand, although chronic obstructive pulmonary disease (COPD) is estimated to be the third leading cause of death in the world by 2020, many patients have not yet been diagnosed because it depends only on assessment of respiratory function. The diagnosis is left as it is, and the pathological condition is insufficiently elucidated.

  Obstructive pulmonary diseases such as bronchial asthma and COPD are caused not by individual gene and protein mutations themselves, but by dysregulation of the cell-molecular network formed by their interaction. It is thought that. In fact, there are various phenotypes of asthma and COPD characterized by airflow obstruction and inflammation, which may make it more difficult to elucidate the pathology of these diseases. Furthermore, although there is a high rate of overlapping syndromes in which both diseases coexist, attention is being paid to the method of differentiation and detailed pathological conditions are unknown.

  COPD is estimated to have 200 million patients worldwide, 5.3 million in Japan, of which only 5% are treated. On the other hand, asthma, which is estimated to have 200 million patients worldwide, has been declining in the number of deaths due to the spread of inhaled steroids, but the number of cases continues to increase as the population ages. Recently, ACOS, which is associated with COPD and asthma, has been attracting attention because of its poor prognosis and 20% of COPD patients. Furthermore, asthma has various phenotypes (phenotypes) and endotypes, and thus personalized medicine is required. Under such circumstances, treatments targeting molecules such as IL-5, IL-13, and IgE that induce Th2 type inflammation have been developed one after another (In Japan, IgE zorea and IL-5R AB Noucara). Has clinical application). Although treatments targeting Th2-type inflammation are being developed one after another overseas, almost no indicators (companion biomarkers) have been developed to judge treatment selection or efficacy except for periostin. .

  Of course, there is also an urgent need to develop biomarkers that show disease activity in mice. This is because high-throughput screening is impossible because the asthma model evaluation method has to rely on the primitive method of measuring the respiratory function of each mouse in a major pharmaceutical company that develops drug discovery.

  Although serum is considered to be an ideal test sample because it can be repeatedly collected non-invasively, 99% or more of the protein contained in it is a large amount of protein (albumin, etc.) that is regarded as a contaminant from the viewpoint of biomarkers. Therefore, from the viewpoint of proteomics, its usefulness as a test material is low. For example, in a recent paper (non-patent document 1) on a biomarker capable of distinguishing COPD and asthma, analysis of dozens of patients was attempted, but the analysis was performed by an old mass spectrometry (MS) technique using two-dimensional electrophoresis. Not only that, but since serum containing large amounts of contaminants was used, the only biomarker found was serum contaminants containing prothrombin, and it is difficult to call it a COPD-specific biomarker.

  Extracellular vesicles are membrane vesicles secreted from cells, which carry intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of the cell, and are responsible for information transmission between cells locally and systemically. ing. Although extracellular vesicles have been attracting attention as a test sample for cancer in recent years, their usefulness as a test sample for respiratory diseases and obstructive pulmonary diseases such as bronchial asthma is not known.

Am J Respir Crit Care Med. 2011 Jun 15;183(12):1633-43.

  It is an object of the present invention to provide a biomarker for obstructive pulmonary disease such as bronchial asthma and a method for using the biomarker. Preferably, the present invention aims to provide a biomarker capable of differentiating between bronchial asthma and chronic obstructive pulmonary disease (COPD) and their complications, and a method of using the same. More preferably, it is an object to provide a biomarker that reflects the pathological condition of obstructive pulmonary disease and a method for using the biomarker.

  As a result of intensive studies in view of the above problems, the present inventor has found that a specific protein group in extracellular vesicles of a body fluid collected from a subject is useful as a biomarker for obstructive pulmonary disease. As a result of further research, it was found that these biomarkers can be used to distinguish between bronchial asthma and chronic obstructive pulmonary disease (COPD) and their complications. It was also found that some biomarkers reflect the pathophysiology of obstructive pulmonary disease. As a result of further research based on these findings, the present invention has been completed. That is, the present invention includes the following aspects.

Item 1.
A method of testing for obstructive pulmonary disease, the method comprising:
(1) Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from a subject:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and a step of detecting at least one protein selected from the group consisting of proteins consisting of Adenylyl cyclase-associated protein 1, Inspection method.

Item 2.
Furthermore, (2) comprising the step of determining that the subject suffers from obstructive pulmonary disease, when the amount or concentration of the protein detected in the step (1) is a cutoff value or more, The inspection method described in 1.

Item 3.
The step (2) is
(2a) The subject suffers from bronchial asthma when the amount or concentration of at least one protein selected from the group consisting of the protein group (A) and the protein group (C) is a cutoff value or more. Process to determine that
(2b) When the amount or concentration of at least one protein selected from the group consisting of the protein group (B) and the protein group (C) is a cutoff value or more, the subject has chronic obstructive lung Determining that the patient is suffering from a disease,
(2c) When the amount or concentration of at least one protein selected from the protein group (A) is equal to or higher than the cutoff value, the subject has bronchial asthma without chronic obstructive pulmonary disease. Process to determine that
(2d) When the amount or concentration of at least one protein selected from the protein group (B) is equal to or higher than the cutoff value, the subject is suffering from a chronic obstructive pulmonary disease not associated with bronchial asthma. And (2e) the amount or concentration of at least one protein selected from the protein group (C) is equal to or higher than the cutoff value, the subject has bronchial asthma and chronic obstructive property. Item 3. The test method according to Item 2, which is at least one step selected from the group consisting of the steps of determining to be affected by both lung diseases.

Item 4.
The cut-off value is 2.5 to 6 times the value of the amount or concentration of the corresponding protein in the extracellular vesicles of the body fluid collected from a subject not suffering from obstructive pulmonary disease, Item 2 or The inspection method described in 3.

Item 5.
Item 5. The test method according to any one of Items 1 to 4, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.

Item 6.
The protein (A) group is (A1) 14-3-3 protein theta, Thrombospondin-1, Platelet glycoprotein 4, Pyridoxal phosphate phosphatase PHOSPHO2, Integrin beta-2, Collagen alpha-2(VI) chain, Proteoglycan 4, Glycophorin-. C, Versican core protein, Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, and Serum amyloid A-2 protein, and the protein (B) group is ( B1) Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, Complement C1q subcomponent subunit B, Laminin subunit alpha-5, Interferon-induced transmembrane protein 2, Laminin subunit gamma-1, Serglycin, Thrombospondin -1, Alpha-2-macroglobulin, Collagen alpha-1(I) chain, Platelet factor 4, Vesicle-associated membrane protein 3, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Collagen alpha -2(I) chain, Multimerin-1, Ras-related protein Rab-7a, Serine incorporator 3, Basigin, and a group consisting of C4b-binding protein,
Item 6. The inspection method according to any one of Items 1 to 5.

Item 7.
The protein (A1) group is (A2) Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, and Serum amyloid A-2 protein, and the protein (B1 ) Group is a group consisting of (B2) Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, and Complement C1q subcomponent subunit B,
Item 6. The inspection method according to Item 6.

Item 8.
Item 8. The examination method according to any one of Items 1 to 7, wherein the subject is a mouse.

Item 9.
Protein group (A), protein group (B), and protein group (C):
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and adenylyl cyclase-associated protein 1 containing at least one protein selected from the group consisting of protein groups, occlusion Drug for natural lung disease.

Item 10.
Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from animals treated for obstructive pulmonary disease induction:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1 The amount or concentration of at least one protein selected from the group consisting of protein groups is used as an index. A method for screening an animal model for obstructive pulmonary disease.

Item 11.
Item 11. The screening method according to Item 10, comprising a step of selecting the animal as an obstructive pulmonary disease model animal when the value of the index is equal to or higher than a cutoff value.

Item 12.
Protein group (A), protein group (B), and protein group (C):
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and contains at least one protein inhibitor selected from the group consisting of proteins consisting of Adenylyl cyclase-associated protein 1, A preventive or therapeutic agent for obstructive pulmonary disease.

Item 13.
Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from an animal treated with a test substance:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1 The amount or concentration of at least one protein selected from the group consisting of protein groups is used as an index. A method for screening an active ingredient of a preventive or therapeutic agent for obstructive pulmonary disease.

Item 14.
If the value of the index is lower than the amount or concentration of the corresponding protein in extracellular vesicles of body fluid collected from an animal not treated with the test substance, the test substance is used to prevent obstructive pulmonary disease or Item 14. The screening method according to Item 13, comprising a step of selecting as an active ingredient of a therapeutic agent.

Item 15.
Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from an animal treated with a test substance:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1 The amount or concentration of at least one protein selected from the group consisting of protein groups is used as an index. A method for evaluating the induction or exacerbation of obstructive pulmonary disease.

  According to the present invention, a biomarker for obstructive pulmonary disease can be provided. By using the biomarker, examination of obstructive pulmonary disease, determination of the degree of eosinophil infiltration, screening of animal models of obstructive pulmonary disease, etc. can be performed more simply, more efficiently, and at lower cost. You can In addition, by using the biomarker, it is possible to distinguish between bronchial asthma and chronic obstructive pulmonary disease (COPD) and their complications. Furthermore, by using the biomarker, prevention or treatment of obstructive pulmonary disease, screening of active ingredients of a prophylactic or therapeutic agent for obstructive pulmonary disease, etc. may be possible.

  In this specification, the expressions "containing" and "including" include the concepts of "containing", "including", "consisting essentially of" and "consisting solely of".

1. Method for examining obstructive pulmonary disease The present invention, in one aspect thereof, is a method for examining obstructive pulmonary disease,
(1) Detecting at least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from a subject The present invention relates to an inspection method including a step (step 1) (in the present specification, it may be referred to as the “inspection method for pulmonary obstructive pulmonary disease of the present invention”). This will be described below.

1-1. Process (1)
The “obstructive pulmonary disease” to be tested is not particularly limited, and specific examples thereof include bronchial asthma, COPD, diffuse panbronchiolitis, bronchiolitis obliterans, and the like.

  The subject is a target organism of the test method of the present invention, and its biological species is not particularly limited. Examples of the biological species of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, with preference given to mice.

  The state of the subject is not particularly limited. As a subject, for example, a specimen unknown whether it is suffering from obstructive pulmonary disease, has already been determined by another method to be suffering from bronchial asthma, specimen unknown whether it is suffering from COPD, A sample that has already been determined to have COPD by another method, but it is unknown whether it has bronchial asthma, or a sample that has already been determined to have bronchial asthma and COPD by another method. , A sample that has already been determined to have no obstructive pulmonary disease by another method, a sample under treatment for obstructive pulmonary disease, and the like.

  The body fluid is not particularly limited. Examples of the body fluid include whole blood, serum, plasma, spinal fluid, saliva, synovial fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, etc., preferably whole blood, serum , Plasma, and cerebrospinal fluid, and more preferably whole blood, serum, and plasma. As the body fluid, one type may be used alone, or two or more types may be used in combination.

  Body fluid can be collected from a subject by methods known to those skilled in the art. For example, whole blood can be collected by collecting blood using a syringe or the like. Serum is a portion obtained by removing blood cells and a specific blood coagulation factor from whole blood, and can be obtained, for example, as a supernatant after coagulating whole blood. Plasma is a portion obtained by removing blood cells from whole blood, and can be obtained, for example, as a supernatant when subjected to centrifugation under conditions in which whole blood is not coagulated.

  Extracellular vesicles are not particularly limited as long as they are membrane vesicles secreted and released from cells. Extracellular vesicles are usually defined as membrane vesicles that carry out intracellular or systemic cell-to-cell information transfer by transporting intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of the cell. It Examples of extracellular vesicles include exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, secretory microvesicles, and the like.

  Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to known methods or according to known methods. Examples of the method for purifying, separating, concentrating extracellular vesicles include ultracentrifugation (eg pellet down method, sucrose cushion method, density gradient centrifugation etc.), method using immunoaffinity carrier, gel filtration method, field Flow fractionation method, FACS method and the like can be mentioned. Further, purification, separation, concentration, etc. of extracellular vesicles can be performed using a commercially available kit. These methods may be used alone or in combination of two or more.

  The detection target in step (1) is at least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C) (herein, these are collectively referred to as “ Sometimes referred to as "target protein").

  Protein group (A) is (A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8 , Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2, Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1 , Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3-A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2 880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras- related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1 , Early endosome antigen 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, G lycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase , Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and Protein FAM177A1.

  The protein group (A) is a bronchial asthma-specific biomarker, and by using this as an index, it is possible to distinguish bronchial asthma not associated with chronic obstructive pulmonary disease.

  Among the protein group (A), from the viewpoint of more reflecting the disease state (higher correlation with the degree of eosinophil infiltration), preferably 14-3-3 protein theta, Thrombospondin-1, Platelet glycoprotein 4, Pyridoxal phosphate phosphatase PHOSPHO2, Integrin beta-2, Collagen alpha-2(VI) chain, Proteoglycan 4, Glycophorin-C, Versican core protein, Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein and the like can be mentioned.

  Among the protein group (A), from the viewpoint that the amount ratio to the normal sample is higher (including the case where the control expression level cannot be confirmed), Ig kappa chain VV region MOPC 41, Ig kappa chain V-III are preferable. region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent , Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphat e 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, Protein FAM177A1 and the like, more preferably Ras -related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin -related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine -rich secretory protein 3, Protein FAM177A1 and the like, and more preferably Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy c hain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, Protein FAM177A1 and the like can be mentioned.

  Among the protein group (A), from the viewpoint that the amount ratio to the normal sample is higher (not included when the expression level of the control cannot be confirmed), Ig kappa chain VV region MOPC 41, Ig kappa chain V-III are preferable. region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent , Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein and the like, and more preferably Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, etc. And more preferably Serum amyloid A-1 protein, Serum amyloid A-2 protein, and the like.

  Among the protein group (A), Amyloid beta is preferable from the viewpoint of more reflecting the disease state (higher correlation with the degree of infiltration of eosinophils) and the higher ratio of the amount to the normal sample. A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein and the like can be mentioned.

  Protein group (B) includes Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog. 3, Complement C1r-A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5 , Lymphocyte antigen 6E, Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2, F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain , Vesicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related pro tein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3 , Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1- antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40 , Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain , Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1 are protein groups.

  The protein group (B) is a biomarker specific to chronic obstructive pulmonary disease, and by using this as an index, it is possible to distinguish chronic obstructive pulmonary disease not associated with bronchial asthma.

  Among the protein group (B), from the viewpoint of more reflecting the disease state (higher correlation with the mean alveolar diameter), preferably, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r- A subcomponent, Biglycan, Complement C1q subcomponent subunit B, Laminin subunit alpha-5, Interferon-induced transmembrane protein 2, Laminin subunit gamma-1, Serglycin, Thrombospondin-1, Alpha-2-macroglobulin, Collagen alpha-1(I) chain , Platelet factor 4, Vesicle-associated membrane protein 3, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Collagen alpha-2(I) chain, Multimerin-1, Ras-related protein Rab- 7a, Serine incorporator 3, Basigin, C4b-binding protein and the like.

  Among the protein group (B), Cytohesin-2, Erythrocyte band 7 integral membrane protein, and Alpha-2 are preferable from the viewpoint of a higher amount ratio with respect to a normal sample (including cases where the expression level of the control cannot be confirmed). -antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha- 2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E, Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2, F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin , Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vesicle-associated membrane protei n8, CD81 antigen, Alpha-1-antitrypsin 1-5, Alpha-synuclein, Calcium-transporting ATPase type 2C member 1 and the like, more preferably Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2 -antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1 And the like, and more preferably, Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Alpha-synuclein, Calcium-transporting ATPase type 2C member 1 and the like.

  Among the protein group (B), from the viewpoint of a higher amount ratio with respect to a normal sample (not included when the expression level of the control cannot be confirmed), preferably Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E, Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2, F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 an tigen, Alpha-1-antitrypsin 1-5 and the like, more preferably Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c Examples include oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent and the like, and more preferable examples include Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin and the like. Is mentioned.

  Among the protein group (B), from the viewpoint of more reflecting the disease state (higher correlation with the average alveolar diameter) and the higher ratio of the amount to the normal sample, preferably Alpha-2- Antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, Complement C1q subcomponent subunit B, etc.

  Protein group (C) is a protein group consisting of Collagen alpha-1(XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1. is there.

  The protein group (C) is a biomarker specific to both bronchial asthma and chronic obstructive pulmonary disease. By using this as an index, bronchial asthma (or bronchial asthma combined type) with chronic obstructive pulmonary disease is used. Chronic obstructive pulmonary disease) can be distinguished.

  Among the protein group (C), from the viewpoint of higher correlation with the degree of eosinophil infiltration, preferably, Interferon-induced transmembrane protein 3, Collagen alpha-1(XVIII) chain, Fibulin-2, Adenylyl cyclase -associated protein 1 and the like, and Collagen alpha-1 (XVIII) chain, Complement C1q subcomponent subunit C and the like are preferable from the viewpoint of higher correlation with mean alveolar diameter.

  Among the protein group (C), from the viewpoint that the amount ratio to the normal sample is higher (including the case where the expression level of the control cannot be confirmed), Collagen alpha-1(XVIII) chain, Carboxypeptidase N subunit 2, Examples include Complement C1q subcomponent subunit C, Fibulin-2, Adenylyl cyclase-associated protein 1 and the like.

  Among the protein group (C), from the viewpoint that the amount ratio to the normal sample is higher (not included even when the expression level of the control cannot be confirmed), Collagen alpha-1(XVIII) chain, Carboxypeptidase N subunit 2, preferably Complement C1q subcomponent subunit C etc. are mentioned.

  In the case of mouse, the proteins of the protein groups (A) to (C) are proteins specified by UniProtKB accession numbers shown in Tables 1 to 4 in Examples described later. In the case of other species, it is the ortholog of the protein identified by the UniProtKB accession number shown in Tables 1-5.

  The number of target proteins in step (1) may be only one type, or may be a combination of two or more types. By combining more (for example, 2, 5, 10, 20, 40, 80, 120 or more) detection targets, tests for obstructive pulmonary disease, bronchial asthma and chronic obstructive pulmonary disease It becomes possible to perform the discrimination of the above, the discrimination of these complications, and the like with a more personality.

  Detection is usually performed by measuring the amount or concentration of the protein of interest. The “concentration” is not limited to the absolute concentration, but may be a relative concentration, a weight per unit volume, or raw data measured to know the absolute concentration.

  The method for detecting the target protein is not particularly limited as long as it is a method capable of specifically detecting part or all of the target protein. Specific examples of the detection method include a mass spectrometry method for detecting a peptide constituting a target protein, an immunological measurement method using an antibody that specifically recognizes the target protein, and the like. The amino acid sequence information of the target protein can be obtained by searching the database of EBI (http://www.ebi.ac.uk/IPI/IPIhelp.html) based on the UniProtKB accession number.

  Suitable examples of the immunological measurement method include immunohistochemical staining method, ELISA method, EIA method, RIA method, and Western blotting method.

Mass spectrometry is a method in which a peptide sample is made into gaseous ions by using an ion source (ionization), and the peptide sample ionized by moving in a vacuum in the analysis section by using electromagnetic force or by a time difference of flight is subjected to mass charge. It refers to a measurement method that uses a mass spectrometer that can be separated according to the ratio and can be detected.Ionization methods using an ion source include EI method, CI method, FD method, FAB method, MALDI method, and ESI method. Etc. can be appropriately selected, and methods for separating the ionized peptide sample in the analysis unit include magnetic field deflection type, quadrupole type, ion trap type, time of flight (TOF) type, Fourier transform A separation method such as an ion cyclotron resonance type can be appropriately selected. In addition, tandem mass spectrometry (MS/MS) or triple quadrupole mass spectrometry, which is a combination of two or more mass spectrometry methods, can be used. When the sample contains a phosphorylated peptide, the sample can be concentrated using iron ion-immobilized affinity chromatography (Fe-IMAC) before introducing the sample into the mass spectrometer. Further, by using liquid chromatography (LC) or HPLC, the peptides constituting the target protein can be separated and purified and used as a sample. Moreover, the detection unit and the data processing method can be appropriately selected. In addition, when detecting and quantifying a peptide that constitutes a target protein by mass spectrometry by mass spectrometry, a peptide that is labeled with a stable isotope having a known concentration and that has the same amino acid sequence as the peptide is used as an internal standard. can do. Such a stable isotope-labeled peptide is a stable isotope-labeled peptide containing at least one of ¹⁵ N, ¹³ C, ¹⁸ O, and ² H in which one or more of the amino acids in the peptide constituting the target protein to be detected. If so, the type, position, number, etc. of amino acids can be appropriately selected, and such stable isotope-labeled peptides can be prepared by using the F-moc method (Amblard., et al. Methods) using amino acids labeled with stable isotopes. Mol Biol. 298:3-24 (2005)) can be chemically synthesized by an appropriate means, such as iTRAQ (registered trademark) reagent, ICAT (registered trademark) reagent, ICPL (registered trademark) reagent, NBS (registered trademark). It can also be prepared using a labeling reagent such as a trademark reagent.

  According to the test method of the present invention including the step (1), it is possible to provide the amount and/or concentration of the target protein, which is a detection index for obstructive pulmonary disease, and thereby assist detection of obstructive pulmonary disease and the like. can do. Further, according to the inspection method of the present invention including the step (1), it is possible to provide the amount and/or concentration of the target protein as a detection index, and thereby assist in the evaluation of the degree of eosinophil infiltration. You can also Since eosinophil infiltration is involved in bronchial asthma activity (hypersensitivity, reversibility, etc.) and acute exacerbation of COPD, the evaluation of eosinophil infiltration is used to judge the possibility of acute exacerbation and the poor prognosis group. It can be used to identify Also, although eosinophil infiltration is an important factor involved in obstructive disease, how can it be evaluated? There was no known method other than the complicated method of measuring exhaled NO. By using the biomarker of the present invention, eosinophil infiltration can be evaluated more easily and efficiently than before without such complicated work.

  The test results obtained by the test method of the present invention including the step (1) include elucidation of the pathophysiology of obstructive pulmonary disease, prognosis prediction of obstructive disease (respiratory function group, acute exacerbation), stratification of subjects, and treatment It can be used for selection (personalized medicine, treatment response), refractory in obstructive pulmonary disease (particularly bronchial asthma), evaluation of remodeling, differentiation of histological type and phenotype of obstructive pulmonary disease, and the like.

1-2. Process (2)
The inspection method of the present invention further includes, as an aspect,
(2) including a step (step 2) of determining that the subject has an obstructive pulmonary disease when the amount or concentration of the protein detected in the step (1) is a cutoff value or more Preferably. According to the inspection method of the present invention including the step 2, it is possible to determine obstructive pulmonary disease.

More specifically, the step 2 is divided into, for example, the following (2a) to (2e).
(2a) The subject suffers from bronchial asthma when the amount or concentration of at least one protein selected from the group consisting of the protein group (A) and the protein group (C) is a cutoff value or more. Process to determine that
(2b) When the amount or concentration of at least one protein selected from the group consisting of the protein group (B) and the protein group (C) is a cutoff value or more, the subject has chronic obstructive lung Determining that the patient is suffering from a disease,
(2c) When the amount or concentration of at least one protein selected from the protein group (A) is equal to or higher than the cutoff value, the subject has bronchial asthma without chronic obstructive pulmonary disease. Process to determine that
(2d) When the amount or concentration of at least one protein selected from the protein group (B) is equal to or higher than the cutoff value, the subject is suffering from a chronic obstructive pulmonary disease not associated with bronchial asthma. And (2e) the amount or concentration of at least one protein selected from the protein group (C) is equal to or higher than the cutoff value, the subject has bronchial asthma and chronic obstructive property. The step of determining that the patient has both lung diseases.

  The step (2) is more preferably at least one step selected from the group consisting of the above steps 2a to 2e.

  The cut-off value can be appropriately set by those skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, etc., and was, for example, collected from a subject not suffering from obstructive pulmonary disease. Based on the amount and/or concentration of the target protein in the extracellular vesicles of the body fluid, it can be set to a value determined in each case or a value determined in advance. The cut-off value is, for example, the amount and/or concentration of the protein of interest in extracellular vesicles of body fluid collected from a subject not suffering from obstructive pulmonary disease (in the case of multiple subjects, the average value, the central value, Value), for example, 1 to 10 times, preferably 2 to 8 times, and more preferably 2.5 to 6 times.

  In a more preferred embodiment of step (2), the amount or concentration of the protein detected in step (1) is not less than the cutoff value by using a biomarker that reflects the disease state of obstructive pulmonary disease. In this case, it can be determined that the subject is suffering from obstructive pulmonary disease, which is more severe. In this case, by setting the cutoff value to a value based on the amount and/or concentration of the target protein in extracellular vesicles of body fluid collected from a subject suffering from obstructive pulmonary disease, You can evaluate whether you have Alternatively, when the subject is a subject undergoing treatment for obstructive pulmonary disease, the treatment can be performed by setting the cutoff value to a value based on the amount and/or concentration of the target protein in a past sample of the same subject, for example. The effect can be determined.

  Furthermore, in a more preferred embodiment of step (2), by using a biomarker correlated with the degree of eosinophil infiltration, the amount or concentration of the protein detected in step (1) above is used as an indicator. The extent of eosinophil infiltration can be determined. More specifically, for example, a calibration curve based on a correlation between the degree of eosinophil infiltration (the number of eosinophil infiltration within a certain area) and the biomarker amount or concentration is prepared in advance, and the measured biomarker is measured. The degree of eosinophil infiltration can be determined from the amount or concentration of γ and the calibration curve.

2. When it is determined that the subject is suffering from obstructive pulmonary disease by the inspection method of the present invention including the step (2) of diagnosing obstructive pulmonary disease with higher accuracy, the test method of the present invention further comprises: By combining the steps of applying a diagnosis by a doctor of obstructive pulmonary disease, it is possible to diagnose obstructive pulmonary disease with higher accuracy. In addition, since the test method of the present invention can detect obstructive pulmonary disease more accurately, by combining the above-described steps with the test method of the present invention, the patient suffers from “obstructive pulmonary disease more efficiently and more accurately. Can be diagnosed.

3. When it is determined that the subject has an obstructive pulmonary disease by the inspection method of the present invention including the treatment step (2) for obstructive pulmonary disease, further to the inspection method of the present invention, or the above "2. As described in "Diagnosis of Obstructive Pulmonary Disease with Higher Accuracy", when the patient is diagnosed as suffering from obstructive pulmonary disease, a combination of the test method of the present invention and the step of applying a diagnosis by a doctor is used. On the other hand, further, (3) a subject diagnosed or diagnosed as suffering from obstructive pulmonary disease can be treated with the disease by performing a step of treating the disease. It will be possible. Further, since the test method of the present invention can detect obstructive pulmonary disease more accurately, a step for the test method of the present invention or a combination of the test method of the present invention and the step of applying a diagnosis by a doctor By combining the three, a subject suffering from obstructive pulmonary disease can be treated more efficiently and more reliably.

  The method for treating obstructive pulmonary disease is not particularly limited, but typically includes medication treatment. The drug used for drug treatment is not particularly limited, but examples thereof include anticholinergic drugs such as spiriva, seabri, enclasse, echlira, atrovent, and telsigan; β2-agonists such as cerevent, ombreth, auxis, sartanol, and meptin; ultibro, anoro. , An anticholinergic/β2 stimulant combination such as Spiolt; a steroidal drug such as Cuvar, flutide, palmicote, Orvesco, and Azmanex; a steroidal drug such as adair, symbicort, relvea, a β2 stimulant combination. The medicine can be used alone, in combination of two kinds, or in combination of three or more kinds.

Four. In one aspect thereof, the present invention provides a detection agent for at least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C). The present invention also relates to a test drug for obstructive pulmonary disease, which may be referred to as “the test drug of the present invention” herein. This will be described below.

  The protein group (A), protein group (B), protein group (C), obstructive pulmonary disease and the like are the same as defined in the above “1. Method for examining obstructive pulmonary disease”.

  The detection agent is not particularly limited as long as it can specifically detect the target protein. Examples of the detection agent include an antibody against the target protein. The detection agent may be labeled. Examples of the label include fluorescent substances, luminescent substances, dyes, enzymes, gold colloids, radioisotopes and the like. Further, the detection agent (particularly antibody) may be in a state of being adsorbed on a base material (for example, a plastic base material such as a microwell plate).

  The antibody is not particularly limited as long as it selectively (specifically) recognizes the target protein. Here, “selectively (specifically) recognizes” means that the target protein can be specifically detected by, for example, Western blotting or ELISA, but is not limited thereto, Any substance can be used as long as it can be determined that the detected substance is derived from the target protein.

  Antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, or some of the above antibodies having antigen binding properties such as Fab fragments and fragments produced by Fab expression libraries. The antibody of the present invention also includes an antibody having an antigen-binding property with respect to a polypeptide having at least continuous 8 amino acids, preferably 15 amino acids, and more preferably 20 amino acids in the amino acid sequence of the target protein.

  The method for producing these antibodies is already well known, and the antibody of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology, Chapters 11.12 to 11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, a target protein expressed and purified in Escherichia coli or the like according to a conventional method is used, or an oligopeptide having a partial amino acid sequence of the target protein is synthesized according to a conventional method. Then, it is possible to immunize a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method. On the other hand, in the case of a monoclonal antibody, a non-human animal such as a mouse is immunized with a target protein expressed in Escherichia coli or the like and purified according to a conventional method, or an oligopeptide having a partial amino acid sequence of the target protein, and the resulting spleen cells are It can be obtained from hybridoma cells prepared by cell fusion with myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Sections 11.4 to 11.11).

  The target protein used as an immunizing antigen for the production of antibodies is based on known gene sequence information, DNA cloning, construction of each plasmid, transfection into a host, culture of transformants, and recovery of the protein from the culture. It can be obtained by the operation of. These operations are performed according to methods known to those skilled in the art or methods described in literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)) and the like. Can be done by

  Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding a target protein in a desired host cell is prepared, introduced into a host cell for transformation, and the transformant is cultured. By recovering the target protein from the obtained culture, the protein as an immunogen for producing the antibody of the present invention can be obtained. The partial peptide of the target protein can also be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.

  Moreover, the antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of the target protein. The oligo(poly)peptide used for producing such an antibody is not required to have a functional biological activity, but it is desirable that it has immunogenic properties similar to those of the target protein. An oligo(poly)peptide preferably having this immunogenic property and consisting of at least 8 consecutive amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the target protein can be exemplified.

  Antibodies against such oligo(poly)peptides can also be produced by increasing the immunological reaction with various adjuvants depending on the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surface treatments such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Active substances, human adjuvants such as BCG (bacillus Calmette-Guerin) and Corynebacterium parvum are included.

  The test agent of the present invention may be in the form of a composition. The composition may contain other components as necessary. Other components include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, and fragrances. , Chelating agents and the like.

  The test agent of the present invention may be in the form of a kit. The kit may contain, in addition to the above-mentioned detection agent or the above-mentioned composition containing the same, one that can be used for detection of a target protein in extracellular vesicles of a body fluid of a subject. Specific examples of such substances include various reagents (eg, secondary antibody, buffer, etc.), instruments (eg, instruments for purification, separation, concentration of extracellular vesicles (eg, columns)) and the like.

Five. Method for Screening Animal Model for Obstructive Pulmonary Disease In one embodiment of the present invention, the protein group (A) and the protein group (B) in extracellular vesicles of body fluid collected from an animal subjected to obstructive pulmonary disease induction treatment , And a method for screening an animal model for obstructive pulmonary disease, which uses as an index the amount or concentration of at least one protein selected from the group consisting of the protein group (C) (in the present specification, "the model animal screening of the present invention "Method" may be shown.) This will be described below.

  For body fluids, extracellular vesicles, protein group (A), protein group (B), protein group (C), obstructive lung disease, measurement of the amount or concentration of the target protein, etc., see “1. Obstructive lung disease” above. The inspection method” is the same as the definition in “Inspection method”.

  The animal is an animal to be screened, and its biological species is not particularly limited as long as it is an animal other than human. Examples of the biological species of the animal include various mammalian animals such as monkey, mouse, rat, dog, cat, rabbit and the like, preferably mouse.

  The obstructive pulmonary disease-inducing treatment is not particularly limited as long as it is a treatment capable of inducing obstructive pulmonary disease. For example, in the case of inducing bronchial asthma, for example, OVA (ovalbumin);, inducer such as allergens of mites, pollen, insects, etc. are administered (eg, nasal administration, oral administration, inhalation administration, transbronchial administration, abdominal administration) The induction treatment is performed by intraperitoneal administration, intravenous administration, transdermal administration, and the like, preferably by intraperitoneal administration and intranasal administration) or by gene modification treatment. For example, in the case of inducing chronic obstructive pulmonary disease, for example, elastase, smoking exposure, tobacco extract, LPS + tobacco extract and the like inducer administration (for example, nasal administration, oral administration, inhalation administration, transbronchial administration, The induction treatment is carried out by intraperitoneal administration, intravenous administration, transdermal administration, etc., preferably nasal administration) or by gene modification treatment.

  More specifically, the model animal screening method of the present invention selects the animal as an obstructive pulmonary disease model animal (or an obstructive pulmonary disease model candidate animal) when the value of the above-mentioned index is not less than the cutoff value. Including the step of

  The cutoff value can be appropriately set by those skilled in the art from the viewpoint of the accuracy of screening, and for example, the amount of the target protein in the extracellular vesicles of the body fluid collected from the animal not subjected to obstructive pulmonary disease induction treatment and Based on the density, it may be a value determined in each case or a value determined in advance. The cut-off value is, for example, the amount and/or concentration of the protein of interest in extracellular vesicles of body fluid collected from an animal not treated with obstructive pulmonary disease (in the case of multiple subjects, the average value, the median value). Etc.), for example, 1 to 10 times, preferably 2 to 8 times, and more preferably 2.5 to 6 times.

6. Preventive or therapeutic agent for obstructive pulmonary disease The present invention, in one aspect thereof, suppresses at least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C) The present invention relates to a prophylactic or therapeutic agent for obstructive pulmonary disease containing an agent (in the present specification, sometimes referred to as the "agent of the present invention"). This will be described below.

  The protein group (A), protein group (B), protein group (C) and the like are the same as the definitions in "1. Method for examining obstructive pulmonary disease".

  As the inhibitor, an expression inhibitor of the target protein is preferably mentioned.

  The expression inhibitor of the target protein is not particularly limited as long as it can suppress the expression level of the target protein, its mRNA and the like. For example, gene-specific small interfering RNA (siRNA) of the target protein, gene-specific of the target protein MicroRNA (miRNA), gene-specific antisense nucleic acid of target protein, expression vectors thereof; gene-specific ribozyme of target protein; gene-gene editing agent of target protein by CRISPR/Cas system.

  Incidentally, the expression suppression, the expression level of the target protein, its mRNA and the like, for example, 1/2, 1/3, 1/5, 1/10, 1/20, 1/30, 1/50, 1/100. , 1/200, 1/300, 1/500, 1/1000, 1/10000 or less, which means that the expression level of these is set to 0.

  The gene siRNA of the target protein is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of the gene encoding the target protein. In one embodiment, the siRNA preferably has a length of, for example, 18 bases or more, 19 bases or more, 20 bases or more, or 21 bases or more. The siRNA preferably has a length of, for example, 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less. It is assumed that the upper limit value and the lower limit value of the siRNA length described here are arbitrarily combined. For example, the lower limit is 18 bases, the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases in length; the lower limit is 19 bases, the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases A certain length; the lower limit is 20 bases, the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases; the lower limit is 21 bases, the upper limit is 25 bases, 24 bases, 23 bases, or 22 Combinations of lengths that are bases are envisioned.

  The siRNA may be shRNA (small hairpin RNA). shRNA can be designed such that a part thereof forms a stem-loop structure. For example, in shRNA, if the sequence of a certain region is sequence a and the complementary strand to sequence a is sequence b, these sequences are present in one RNA strand such that sequence a, spacer, sequence b are arranged in this order. And can be designed to be 45-60 bases in length. Sequence a is a sequence of a partial region of the base sequence encoding the target protein to be targeted, and the target region is not particularly limited, and any region can be used as a candidate. The length of the sequence a is 19 to 25 bases, preferably 19 to 21 bases.

  The gene-specific siRNA of the protein of interest may have an additional base at the 5'or 3'end. The length of the additional base is usually about 2 to 4 bases. The additional base may be DNA or RNA, but the use of DNA may improve the stability of nucleic acid in some cases. Examples of such additional base sequences include ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3'. Examples include, but are not limited to, sequences such as', uuuuu-3'.

  The siRNA may have a protruding sequence (overhang) at the 3'end, and specific examples thereof include those to which dTdT (dT represents deoxythymidine) is added. In addition, it may be a blunt end with no terminal addition. The siRNA may have different numbers of bases in the sense strand and the antisense strand, and for example, the “asymmetrical interfering RNA (where the antisense strand has a protruding sequence (overhang) at the 3′ end and the 5′ end) ( aiRNA)". A typical aiRNA has an antisense strand of 21 bases, a sense strand of 15 bases, and an overhang structure of 3 bases at each end of the antisense strand.

  The position of the target sequence of the gene-specific siRNA of the protein of interest is not particularly limited, but in one embodiment, it is targeted from a region other than the 5'-UTR and the start codon to about 50 bases and a region other than the 3'-UTR. It is desirable to choose a sequence. Regarding the candidate group of the selected target sequence, whether or not there is homology in the continuous sequence of 16 to 17 bases in the mRNA other than the target was determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ It is preferable to check the specificity of the selected target sequence by using homology search software such as ). Regarding the target sequence whose specificity was confirmed, a sense strand having a 3′ end overhang of TT or UU at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or Double-stranded RNA consisting of an antisense strand having a 3'-terminal overhang of UU may be designed as siRNA. Further, for shRNA which is a precursor of siRNA, an arbitrary linker sequence capable of forming a loop structure (for example, about 5-25 bases) is appropriately selected, and the sense strand and the antisense strand are linked via the linker sequence. It can be designed by connecting.

The sequences of siRNA and/or shRNA can be searched using search software provided for free on various websites. Examples of such sites include the following.
SiRNA Target Finder provided by Ambion (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) Insert design tool for pSilencer (registered trademark) Expression Vector (http://www.ambion.com/ (jp/techlib/misc/psilencer_converter.html) GeneSeer (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) provided by RNAi Codex.

  siRNA, the sense strand and the antisense strand of the target sequence on mRNA are respectively synthesized by a DNA/RNA automatic synthesizer, and after denaturing in a suitable annealing buffer at about 90 to about 95° C. for about 1 minute, It can be prepared by annealing at about 30 to about 70° C. for about 1 to about 8 hours. It can also be prepared by synthesizing shRNA that is a precursor of siRNA and cleaving it with an RNA cleaving protein dicer.

  The gene-specific miRNA of the protein of interest is arbitrary as long as it inhibits the translation of the gene encoding the protein of interest. For example, miRNAs may pair with the 3'untranslated region (UTR) of the target and inhibit its translation rather than cleaving the target mRNA like siRNA. The miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), and mature miRNA. The length of miRNA is not particularly limited, the length of pri-miRNA is usually several hundred to several thousand bases, the length of pre-miRNA is usually 50 to 80 bases, and the length of mature miRNA is usually 18 ~ 30 bases. In one embodiment, the gene-specific miRNA of the protein of interest is preferably pre-miRNA or mature miRNA, more preferably mature miRNA. Such gene-specific miRNA of the target protein may be synthesized by a known method or may be purchased from a company that provides synthetic RNA.

  The gene-specific antisense nucleic acid of the target protein is a nucleic acid containing a base sequence complementary or substantially complementary to the base sequence of mRNA of the gene encoding the target protein, or a part thereof, and is specific to the mRNA. It is a nucleic acid having a function of suppressing target protein synthesis by forming a stable and stable double chain and binding. The antisense nucleic acid may be DNA, RNA, or a DNA/RNA chimera. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA is recognized by the endogenous ribonuclease H (RNase H) to cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the gene of the target protein. The intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the gene of the target protein using a homology search program such as BLAST or FASTA.

  The length of the target region of the gene-specific antisense nucleic acid of the target protein is not limited as long as the antisense nucleic acid hybridizes with the result that the translation into the target protein is inhibited. The gene-specific antisense nucleic acid of the target protein may be the entire sequence or a partial sequence of mRNA encoding the target protein. Considering easiness of synthesis, antigenicity, intracellular transferability and the like, an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferable, but not limited thereto. More specifically, 5'end hairpin loop of the gene of the target protein, 5'end untranslated region, translation initiation codon, protein coding region, ORF translation termination codon, 3'end untranslated region, 3'end palindromic region Alternatively, the 3′-end hairpin loop or the like may be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.

  The gene-specific antisense nucleic acid of the target protein not only hybridizes with the mRNA of the gene of the target protein or the early transcription product to inhibit translation into a protein but also binds to these genes that are double-stranded DNA. It may be one capable of forming a triplex and inhibiting transcription into RNA (antigene).

The nucleotide molecules constituting the gene-specific siRNA of the target protein, the gene-specific miRNA of the target protein, and the gene-specific antisense nucleic acid of the target protein described above have stability (chemical and/or counterenzyme) and specific activity ( Various chemical modifications may be included in order to improve (affinity with RNA). For example, in order to prevent the degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is changed to, for example, phosphorothioate (PS), methylphosphonate, or phosphorodithioate. It may be replaced with a chemically modified phosphate residue such as a phosphorodithioate. In addition, the hydroxyl group at the 2'position of the sugar (ribose) of each nucleotide is replaced with -OR (R=CH ₃ (2'-O-Me), CH ₂ CH ₂ OCH ₃ (2'-O-MOE), CH ₂ CH ₂ NHC(NH)NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN, etc.). Further, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. May be given. In addition, a part of the nucleotide molecule constituting siRNA or miRNA may be replaced with a natural DNA.

  Gene-specific siRNA of the target protein, gene-specific miRNA of the target protein, and gene-specific antisense nucleic acid of the target protein are targets of mRNA or early transcript based on the cDNA sequence or genomic DNA sequence of the gene of the target protein. It can be prepared by determining the sequence and using a commercially available DNA/RNA automatic synthesizer to synthesize a sequence complementary thereto. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a known method.

  For the gene-specific siRNA of the target protein, the gene-specific miRNA of the target protein, or the gene-specific antisense nucleic acid expression vector of the target protein, the gene-specific siRNA of the target protein, the gene-specific miRNA of the target protein, or the target There is no particular limitation as long as the gene-specific antisense nucleic acid of the protein is incorporated in an expressible state. Typically, the expression vector comprises a promoter sequence and a gene-specific siRNA of the protein of interest, a gene-specific miRNA of the protein of interest, or a coding sequence of a gene-specific antisense nucleic acid of the protein of interest (and optionally A transcription termination signal sequence), and optionally other sequences. The promoter is not particularly limited and includes, for example, CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter and other RNA polymerase II (pol II) type promoters; mouse and human U6-snRNA promoters, Examples include RNA polymerase III (polIII) type promoters such as human H1-RNase P RNA promoter and human valine-tRNA promoter. Among them, the polIII type promoter is preferable from the viewpoint that short RNA can be accurately transcribed. preferable. The other sequence is not particularly limited, and various known sequences that can be contained in the expression vector can be adopted. Examples of such sequences include an origin of replication, a drug resistance gene, and the like. Moreover, the types of drug resistance genes and types of vectors can be exemplified as described above.

  Another example of the gene expression inhibitor of the target protein is a gene-specific ribozyme of the target protein. The term “ribozyme” in a narrow sense means RNA having an enzymatic activity of cleaving a nucleic acid, but in the present application, it also includes DNA as long as it has a sequence-specific nucleic acid cleaving activity. The most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known. The hammerhead type exhibits enzymatic activity with about 40 bases, and several bases at both ends adjacent to the part that takes a hammerhead structure (about 10 bases in total) are made into sequences complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has an advantage that it does not attack genomic DNA because it uses only RNA as a substrate. When the mRNA of the gene of the target protein has a double-stranded structure by itself, by using a hybrid ribozyme in which an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase is linked, the target sequence is single-stranded. [Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcript to the cytoplasm, it should be a hybrid ribozyme further linked with a tRNA-modified sequence. Also available [Nucleic Acids Res., 29(13): 2780-2788 (2001)].

  The application target of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. ..

  The form of the agent of the present invention is not particularly limited and may be a form usually used in each application depending on the application of the agent of the present invention.

  Examples of the form include tablets (eg, disintegrating buccal tablets, chewable tablets, effervescent tablets, troches, and jelly-like drops) when the use is a medicine, health promoting agent, nutritional supplement (supplement, etc.), etc. ), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jelly preparations suitable for oral ingestion Formulations suitable for parenteral ingestion (oral formulation), nasal drops, inhalants, rectal suppositories, intercalates, enemas, jellies, injections, patches, lotions, creams, etc. Oral formulation form).

  As the form, when the application is a food composition, liquid, gel-like or solid food, such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, baby milk powder , Cake mixes, powdered or liquid dairy products, breads, cookies and the like.

  As a form, when the application is an oral composition, for example, liquid (solution, emulsion, suspension, etc.), semisolid (gel, cream, paste, etc.), solid (tablet, particulate, capsule, Film agents, kneaded products, molten solids, waxy solids, elastic solids, etc.), more specifically, dentifrices (toothpaste, liquid toothpaste, liquid toothpaste, powdered toothpaste, etc.), mouthwash, Examples thereof include coating agents, patches, fresheners in the mouth, and foods (eg, chewing gum, tablet confectionery, candy, gummies, films, troches, etc.).

  The agent of the present invention may further contain other components, if necessary. The other component is not particularly limited as long as it is a component that can be blended in, for example, a medicine, a food composition, an oral composition, a health promoter, a nutritional supplement (supplement, etc.), and for example, a base , Carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents and the like.

  The content of the inhibitor of the target protein of the agent of the present invention is dependent on the type of the inhibitor, use, usage mode, application target, state of application target, etc., but is not limited, and for example, 0.0001 to 100 It can be wt%, preferably 0.001 to 50 wt%.

  The application amount (eg, administration, ingestion, inoculation, etc.) of the composition of the present invention is not particularly limited as long as it is an effective amount that exerts a medicinal effect, and is usually 0.1 to 1000 per day as the weight of the active ingredient. mg/kg body weight. The above-mentioned dose is preferably administered once a day or divided into two to three times a day, and can be appropriately increased or decreased depending on age, disease state, and symptom.

7. Method for screening active ingredient of preventive or therapeutic agent for obstructive pulmonary disease The present invention is, in one aspect thereof, extracellular vesicles of body fluid collected from an animal treated with a test substance, protein group (A), A method for screening an active ingredient of a prophylactic or therapeutic agent for obstructive pulmonary disease, which comprises the amount or concentration of at least one protein selected from the group consisting of protein group (B) and protein group (C) as an index (the present invention) In the specification, it may be referred to as "the method for screening an active ingredient of the present invention"). This will be described below.

  For body fluids, extracellular vesicles, protein group (A), protein group (B), protein group (C), obstructive lung disease, measurement of the amount or concentration of the target protein, etc., see “1. Obstructive lung disease” above. The inspection method” is the same as the definition in “Inspection method”.

  The animal species is not particularly limited. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, with preference given to mice.

  As the test substance, a naturally occurring compound or an artificially produced compound can be widely used. Further, not only the purified compound but also a composition in which various compounds are mixed and an extract of animals and plants can be used. The compound is not limited to low molecular weight compounds, and includes high molecular weight compounds such as proteins, nucleic acids, and polysaccharides.

  More specifically, the method for screening the active ingredient of the present invention is such that the value of the above-mentioned index is the amount or concentration of the corresponding protein in the extracellular vesicles of the body fluid collected from the animal not treated with the test substance (control value). When it is lower than the above), the test substance is selected as an active ingredient of a prophylactic or therapeutic agent for obstructive pulmonary disease (or a candidate substance for an active ingredient of a prophylactic or therapeutic agent for obstructive pulmonary disease).

  The corresponding protein means the same protein as the target protein used as an index.

  “Low” means that the index value is, for example, 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.

8. In one aspect thereof, the present invention provides, in one embodiment thereof, a protein group (A) and a protein group in extracellular vesicles of a body fluid collected from an animal treated with a test substance. (B), and using the amount or concentration of at least one protein selected from the group consisting of protein group (C) as an index, a method for evaluating the induction or exacerbation of obstructive pulmonary disease (herein, Sometimes referred to as "toxicity evaluation method of the present invention"). This will be described below.

  For body fluids, extracellular vesicles, protein group (A), protein group (B), protein group (C), obstructive pulmonary disease, measurement of the amount or concentration of target protein, animal species, test substances, etc. The definitions are the same as those in "1. Method for examining obstructive pulmonary disease" and "7. Method for screening active ingredient of preventive or therapeutic agent for obstructive pulmonary disease".

  More specifically, the toxicity evaluation method of the present invention is characterized in that the value of the above-mentioned index is the amount or concentration (control value) of the corresponding protein in the extracellular vesicles of the body fluid collected from the animal not treated with the test substance. If it is higher than the above, the test substance is determined to have an inducing or malignant state of obstructive pulmonary disease.

  The corresponding protein means the same protein as the target protein used as an index.

  “High” means that the index value is, for example, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times the control value.

  Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Test example 1. Preparation of Serum of Bronchial Asthma Model Mouse As a mouse, 11-week-old C57BL6/J male mouse was used. 20 μg of OVA (Sigma, A5503, GradeV) and 2 mg of Alum (Thermo Imject Alum (#77161 Lot NB168145)) were suspended in 400 μL of PBS and intraperitoneally administered to a mouse anesthetized with 4% isoflurane. Seven days after the first administration, a suspension of OVA (20 μg) and Alum (2 mg) in PBS (400 μL) was intraperitoneally administered again. 21, 22 and 23 days after the first administration, a suspension of OVA (50 μg) in PBS (50 μL) was intranasally administered. Serum was collected 28 days after the first administration. The pathological evaluation was performed, and the serum of the mouse (n=8) in the bronchial asthma state was selected as the serum of the bronchial asthma model mouse. In the pathological evaluation, the number of eosinophil infiltration per fixed area in the blood vessel peripheral region around the bronchus was measured according to a known method. The number of infiltrated eosinophils is a value serving as an activation index for asthma.

Test example 2. Preparation of serum of chronic obstructive pulmonary disease (COPD) model mouse As a mouse, 11-week-old C57BL6/J male mouse was used. To mice anesthetized by inhalation with 4% isoflurane, 40 μL of sterile PBS containing 7.5 U of porcine elastase (manufactured by Elastase Products Company) was administered in 20 μL each in two divided doses. More specifically, 20 μL was dropped and sucked at the first time, allowed to rest for a while, and then dropped again and sucked. Serum was collected 21 days after administration. The pathological evaluation was performed, and the serum of the mouse (n=8) in the state of chronic obstructive pulmonary disease was selected as the serum of the chronic obstructive pulmonary disease model mouse. In the pathological evaluation, the average alveolar diameter was measured according to a known method. This average alveolar diameter is a value that serves as an index of.

Test example 3. Preparation of extracellular vesicle fraction An extracellular vesicle fraction was prepared from the serum obtained in Test Examples 1 and 2 and the serum of a normal mouse (11-week-old C57BL6/J male mouse, n=8). The extracellular vesicle fraction was prepared using an extracellular vesicle purification column (EV-Second, manufactured by GL Sciences).

Test example 4. Proteomics analysis (non-label, LC-MS/MS)
The protein in the extracellular vesicle fraction was quantified by LC-MS/MS analysis (non-label method). Specifically, it was performed as follows.

(Sample preparation)
The extracellular vesicle fraction was reduced with 5 mM TCEP for 30 minutes at 37° C. and alkylated with 25 mM iodoacetamide for 45 minutes at room temperature. Then, the sample was diluted 7-fold with 50 mM ammonium bicarbonate, placed in a 96-well filter plate, and shaken with 5 μL of immobilized trypsin (manufactured by Thermo Fisher Scientific) at 37° C. for 6 hours for digestion. The tryptic digest was desalted using Oasis HLB 96-well μElution Plate (Waters Corporation, USA) and subjected to LC-MS/MS analysis.

(LC/MS/MS analysis)
The dried peptide sample was resuspended in a 2% acetonitrile solution containing 0.1% trifluoroacetic acid and LTQ-Orbitrap-Velos mass spectrometer (Ultimate 3000 RSLC nano-flow HPLC system (DIONEX, USA) equipped with The analysis was performed using Thermo Fisher Scientific). In a 75 μm × 150 mm C18 tip-column (manufactured by Nikyo Technos), solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile) were used at a flow rate of 250 nL/min. Samples were separated on a multi-step linear gradient of 6.4-30% Solvent B in minutes and then 30-95% Solvent B in 10 minutes.

The eluted peptide was ionized at a spray voltage of 2000 V and MS data was acquired by the data dependent fragment method. The measurement scan (survey scan) was performed at m/z 400 to 1600, resolution of 60000, and AGC target value of 1.0×10 ⁶ ion count. The top 20 precursor ions in each measurement scan were subjected to low resolution MS/MS acquisition using a normal CID scan mode with an AGC target value of 5000 ion count in a linear ion trap.

  An attempt was made to identify the protein by conducting a SEQUEST database search using Proteome Discoverer 1.3 software (manufactured by Thermo Fischer Scientific). MS/MS spectra were searched against the mouse protein database UniProt. The search parameters are: Enzyme Name = Semitrypsin, Precursor Mass Tolerance = 3 ppm, Fragment Mass Tolerance = 0.8 Da, Dynamic Modification = Oxidation (Met), Static Modification = Carbamidomethyl (Cys). The peptide was identified when the FDR (false discovery rate) by Peptide Validator activity in SEQUEST Decoy Database Search was less than 1%.

(Label-free quantitative analysis on Expressionist server)
The LC-MS/MS data set was loaded into the Expressionist RefinerMS module (Genedata AG, Switzerland) for data processing and free quantitative analysis. Figure 2 shows the overall workflow of RefinerMS software. A Spectrum Grid was set every 10 data points on the 2D MS chromatogram plane (x = m/z, y = RT). Then, in the first and second Chemical Noise Subtraction, Structure Removal was sequentially performed at RT = 2 scans and m/z = 6 points. Subsequently, in the third Chemical Noise Subtraction, Structure Removal was performed with RT Window = 500 scans and Quantile = 90%. In the 4th Chemical Noise Subtraction, Structure Removal was performed at RT = 2 scans. The signal with intensity below 2000 was then eliminated using Intensity Thresholding. Finally, in the 5th and 6th Chemical Noise Subtraction, Structure Removal was performed at RT = 2 scans and m/z = 5 points, respectively.

  Then, Chromatogram Grid was set every 10 scans on the noise subtraction data, and Chromatogram RT Alignment was performed using the following parameters: m/z Window = 11 points, RT Window = 11 scans, Gap Penalty = 1, RT Search Interval = 2 minutes, Alignment Scheme = pairwise alignment based tree.

  Subsequently, peaks in the temporal averaged chromatogram were detected by the Summed Peak Detection Activity using the following parameters: Summation Window = 20 scans, Overlap = 10, Minimum Peak Size = 6 scans, Maximum Merge Distance = 1 data points, Gap/Peak Ratio = 5, Method = curvature-based peak detection, Peak Refinement Threshold = 5, Consistency Filter Threshold = 1. Finally, the Summed Isotope Clustering Activity was used to group the isotopic peaks from one molecule into isotopic clusters. The parameters at this time are: Minimum Charge = 1, Maximum Charge = 6, Maximum Missing Peaks = 0, First Allowed Gap Position = 10, Ionization = protonation, RT Tolerance = 0.1 minute, m/z Tolerance = 0.01. Da, Minimum Cluster Size Ratio = 0.5.

(Statistical analysis in Expressionist Analyst)
The group of tissue fibrosis disease model mice was compared with the group of normal mice, and by t-test, candidates showing expression levels significantly (P<0.05) apart between the two groups were extracted. Similarly, a group of chronic obstructive pulmonary disease model mice and a group of normal mice were compared, and by t-test, candidates showing expression levels significantly (P<0.05) apart between the two groups were extracted. Next, the minimum combination of biomarker candidates was estimated by the Support Vector Machine-Recursive Feature Elimination (SVM-RFE) algorithm or SVM-SVM algorithm in Expressionist Analyst module (Genedata AG) according to the instruction manual.

(result)
Quantitative data of various proteins were obtained. The protein whose average value in the extracellular vesicle fraction of the bronchial asthma model mouse (bronchial asthma group) was 5 times or more the average value in the extracellular vesicle fraction of the normal mouse (control group), And the expression was not confirmed in the control group, but the expression was confirmed in the bronchial asthma group, and the average value of the extracellular vesicle fraction of chronic obstructive pulmonary disease model mice (COPD group) was Proteins that were more than 3 times the mean value in the outer vesicle fraction (control group) and proteins that could not be confirmed in the control group but were confirmed in the COPD group were used as biomarkers for obstructive pulmonary disease. It was selected because it was judged to have a certain degree of effectiveness. Furthermore, the correlation between the number of eosinophil infiltration and the protein amount in each bronchial asthma group model mouse and the correlation between the average alveolar diameter and the protein amount in each chronic obstructive pulmonary disease model mouse were analyzed, and the correlation coefficient (r Value) was calculated. Tables 1 to 4 show the selected proteins, the quantitative ratio to the control group, and the correlation coefficient.

  Of the protein groups shown in Tables 1 to 4, Collagen alpha-1(XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1 In both the bronchial asthma group and the COPD group, the protein had a certain amount or more compared to the control group.

Claims (15)

A method of testing for obstructive pulmonary disease, the method comprising:
(1) Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from a subject:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and a step of detecting at least one protein selected from the group consisting of proteins consisting of Adenylyl cyclase-associated protein 1, Inspection method. The method further comprises the step of (2) determining that the subject has obstructive pulmonary disease when the amount or concentration of the protein detected in the step (1) is a cutoff value or more. The inspection method according to Item 1. The step (2) is
(2a) The subject suffers from bronchial asthma when the amount or concentration of at least one protein selected from the group consisting of the protein group (A) and the protein group (C) is a cutoff value or more. Process to determine that
(2b) When the amount or concentration of at least one protein selected from the group consisting of the protein group (B) and the protein group (C) is a cutoff value or more, the subject has chronic obstructive lung Determining that the patient is suffering from a disease,
(2c) When the amount or concentration of at least one protein selected from the protein group (A) is equal to or higher than the cutoff value, the subject has bronchial asthma without chronic obstructive pulmonary disease. Process to determine that
(2d) When the amount or concentration of at least one protein selected from the protein group (B) is equal to or higher than the cutoff value, the subject is suffering from a chronic obstructive pulmonary disease not associated with bronchial asthma. And (2e) the amount or concentration of at least one protein selected from the protein group (C) is equal to or higher than the cutoff value, the subject has bronchial asthma and chronic obstructive property. The test method according to claim 2, which is at least one step selected from the group consisting of the steps of determining to be affected by both lung diseases. The cut-off value is 2.5 to 6 times the value of the amount or concentration of the corresponding protein in extracellular vesicles of body fluid collected from a subject not suffering from obstructive pulmonary disease. Or the inspection method described in 3. The test method according to claim 1, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum. The protein (A) group is (A1) 14-3-3 protein theta, Thrombospondin-1, Platelet glycoprotein 4, Pyridoxal phosphate phosphatase PHOSPHO2, Integrin beta-2, Collagen alpha-2(VI) chain, Proteoglycan 4, Glycophorin-. C, Versican core protein, Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, and Serum amyloid A-2 protein, and the protein (B) group is ( B1) Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, Complement C1q subcomponent subunit B, Laminin subunit alpha-5, Interferon-induced transmembrane protein 2, Laminin subunit gamma-1, Serglycin, Thrombospondin -1, Alpha-2-macroglobulin, Collagen alpha-1(I) chain, Platelet factor 4, Vesicle-associated membrane protein 3, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Collagen alpha -2(I) chain, Multimerin-1, Ras-related protein Rab-7a, Serine incorporator 3, Basigin, and a group consisting of C4b-binding protein,
The inspection method according to claim 1. The protein (A1) group is (A2) Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, and Serum amyloid A-2 protein, and the protein (B1 ) Group is a group consisting of (B2) Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, and Complement C1q subcomponent subunit B,
The inspection method according to claim 6. The test method according to claim 1, wherein the subject is a mouse. Protein group (A), protein group (B), and protein group (C):
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s occlusivity, including ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1 Test agent for natural lung disease. Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from animals treated for obstructive pulmonary disease induction:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1 The amount or concentration of at least one protein selected from the group consisting of protein groups is used as an index. A method for screening an animal model for obstructive pulmonary disease. The screening method according to claim 10, comprising a step of selecting the animal as an obstructive pulmonary disease model animal when the value of the index is equal to or higher than a cutoff value. Protein group (A), protein group (B), and protein group (C):
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and contains at least one protein inhibitor selected from the group consisting of proteins consisting of Adenylyl cyclase-associated protein 1, A preventive or therapeutic agent for obstructive pulmonary disease. Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from an animal treated with a test substance:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1 The amount or concentration of at least one protein selected from the group consisting of protein groups is used as an index. A method for screening an active ingredient of a preventive or therapeutic agent for obstructive pulmonary disease. If the value of the index is lower than the amount or concentration of the corresponding protein in extracellular vesicles of body fluid collected from an animal not treated with the test substance, the test substance is used to prevent obstructive pulmonary disease or The screening method according to claim 13, comprising a step of selecting as an active ingredient of a therapeutic agent. Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from an animal treated with a test substance:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2(VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880/PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc-13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c , Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1 , TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BR CA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine/threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2(IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2(VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1(I) chain, Vehicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vehicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2(I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3 , Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein , And a protein group consisting of Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1(XVIII) chain, Carboxypeptidase N s ubunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1 The amount or concentration of at least one protein selected from the group consisting of protein groups is used as an index. A method for evaluating the induction or exacerbation of obstructive pulmonary disease.