JP2020079708A - Chronic obstructive pulmonary disease biomarker - Google Patents

Abstract

To provide the biomarker of a chronic obstructive pulmonary disease and a method for using the same.SOLUTION: Provided is an inspection method for a chronic obstructive pulmonary disease including a step for detecting at least one kind of protein selected from a protein group (A) in the extracellular vesicles of the body fluid sampled from an examinee. The inspection method for a chronic obstructive pulmonary disease includes a step for determining the examinee is affected by a chronic obstructive pulmonary disease when the amount or concentration of the detected protein is greater than or equal to a cutoff value. In the inspection method for a chronic obstructive pulmonary disease, the cutoff value is 1 to 1.5 times the value of amount or concentration of corresponding protein in the extracellular vesicles of the body fluid sampled from an examinee not affected by a chronic obstructive pulmonary disease.SELECTED DRAWING: None

Description

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

  Chronic obstructive pulmonary disease (COPD) is an inflammatory disease of the lungs mainly due to airflow obstruction caused by smoking and air pollution. COPD is estimated to be the third leading cause of death in the world by 2020, and the number of patients is expected to increase as the population ages. Although COPD is considered to be a systemic disease with various phenotypes, its diagnosis depends only on the evaluation of respiratory function, and therefore the development of biomarkers that can be used for diagnosis, differentiation, treatment, etc. There is an urgent need.

  Moreover, although it is estimated that there are 200 million COPD patients in the world and 5.3 million in Japan, only 5% of the patients receive treatment (Fukuchi et al. Nippon NICE study). In response to this situation, the government has set up the "Next National Health Promotion Campaign Plan" (Continued Health Japan 21) and has listed COPD as a major disease along with cancer, cardiovascular disease, and diabetes. However, since there are no effective screening biomarkers, not only are there many undiagnosed and untreated patients, but there are currently no fundamental and effective therapeutic agents.

  Studies in the ECLIPSE study indicate that fibrinogen, CC16, SP-D, CCL-18, and sRAGE are useful as COPD biomarkers. In this study, COPD patients not only have increased inflammatory biomarkers such as CRP, fibrinogen, IL-6, and IL-8 in the peripheral blood, but the COPD patient group (phenotype) showing chronic inflammation is acute. It has been shown that there are many exacerbations and the prognosis is poor. In addition, high fibrinogen levels have been shown not only to reflect the pathophysiology of COPD but also to correlate with symptoms and exacerbations, and it has been shown that fibrinogen increases even during acute exacerbations. These findings suggest that catching acute inflammation in COPD may be useful in predicting acute exacerbations that affect prognosis, and catching chronic inflammation may be useful in predicting poor prognosis. However, although chronic inflammation is involved in the onset basis of many lifestyle-related diseases including COPD, a method for capturing chronic inflammation has not been established.

  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 because serum containing large amounts of contaminants was used, the only biomarker found was serum contaminants containing prothrombin, which was far from the 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 further COPD chronic obstructive pulmonary disease is not known.

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

  An object of the present invention is to provide a biomarker for chronic 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 chronic obstructive pulmonary disease. As a result of further research based on this finding, the present invention has been completed. That is, the present invention includes the following aspects.

Item 1.
A method of testing for chronic obstructive pulmonary disease, the method comprising:
(1) Protein group (A) in extracellular vesicles of body fluid collected from a subject:
(A) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase/arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosomal protein L8, Ras-related protein Rab-5C, Peroxiredoxin-2, Vesicle-associated membrane protein 3, CD81 antigen, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, And a test method comprising the step of detecting at least one protein selected from the protein group consisting of Calmodulin.

Item 2.
Further, (2) comprising a step of determining that the subject has chronic 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.

Item 3.
Item 2. The cutoff value is 1 to 1.5 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 chronic obstructive pulmonary disease. Inspection method described in.

Item 4.
Item 4. The examination method according to any one of Items 1 to 3, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.

Item 5.
The protein (A) group is (A1) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor A group consisting of H-related protein 5 and Cytochrome b-245 heavy chain,
Item 5. The inspection method according to any one of Items 1 to 4.

Item 6.
The protein (A) group is (A2) Fibulin-1, Integrin beta-2, CD99 antigen, Peroxiredoxin-2, Clusterin, Prostaglandin G/H synthase 1, Vesicle-associated membrane protein 3, Ras-related protein Rab-5C, And a group consisting of EGF-containing fibulin-like extracellular matrix protein 1,
Item 5. The inspection method according to any one of Items 1 to 4.

Item 7.
Item 7. The test method according to any one of Items 1 to 6, wherein the subject is a human.

Item 8.
Protein group (A):
(A) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase/arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosomal protein L8, Ras-related protein Rab-5C, Peroxiredoxin-2, Vesicle-associated membrane protein 3, CD81 antigen, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, And a test agent for chronic obstructive pulmonary disease, which comprises a detection agent for at least one protein selected from the protein group consisting of: and Calmodulin.

Item 9.
Protein group (A):
(A) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase/arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosomal protein L8, Ras-related protein Rab-5C, Peroxiredoxin-2, Vesicle-associated membrane protein 3, CD81 antigen, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, And a prophylactic or therapeutic agent for chronic obstructive pulmonary disease, which comprises an inhibitor of at least one protein selected from the protein group consisting of Calmodulin.

Item 10.
Protein group (A) in extracellular vesicles of body fluid collected from an animal treated with a test substance:
(A) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase/arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosomal protein L8, Ras-related protein Rab-5C, Peroxiredoxin-2, Vesicle-associated membrane protein 3, CD81 antigen, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, And a method for screening an active ingredient of a prophylactic or therapeutic agent for chronic obstructive pulmonary disease, which uses as an index the amount or concentration of at least one protein selected from the protein group consisting of Calmodulin.

Item 11.
When 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 chronic obstructive pulmonary disease. Alternatively, the screening method according to Item 10, including a step of selecting the active ingredient as a therapeutic agent.

Item 12.
Protein group (A) in extracellular vesicles of body fluid collected from an animal treated with a test substance:
(A) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase/arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosomal protein L8, Ras-related protein Rab-5C, Peroxiredoxin-2, Vesicle-associated membrane protein 3, CD81 antigen, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, And a method for evaluating the inducing or aggravating malignancy of chronic obstructive pulmonary disease, which uses as an index the amount or concentration of at least one protein selected from the protein group consisting of Calmodulin.

  According to the present invention, a biomarker for chronic obstructive pulmonary disease can be provided. By using the biomarker, tests for chronic obstructive pulmonary disease and the like can be performed more easily, more efficiently, and at lower cost. Furthermore, by using the biomarker, it may be possible to prevent or treat chronic obstructive pulmonary disease, screen an active ingredient of a preventive or therapeutic agent for chronic obstructive pulmonary disease, and the like.

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

1. Examination method of chronic obstructive pulmonary disease The present invention, in one aspect thereof, a method of examining chronic obstructive pulmonary disease,
(1) In an extracellular vesicle of a body fluid collected from a subject, including a step (step 1) of detecting at least one protein selected from the protein group (A), a test method (herein, Sometimes referred to as "the method for examining chronic obstructive pulmonary disease of the present invention". This will be described below.

1-1. Process (1)
The “chronic obstructive pulmonary disease” to be examined is not particularly limited and may be either emphysema type or non-emphysema type.

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

  The state of the subject is not particularly limited. As the subject, for example, it is unknown whether or not the patient suffers from chronic obstructive pulmonary disease, it has already been determined by another method that the patient suffers from acute pulmonary inflammatory disease, but chronic obstructive pulmonary disease and acute Pulmonary inflammatory disease specimen already determined by another method, chronic obstructive pulmonary disease specimen already determined by another method, treatment of chronic obstructive pulmonary disease The sample and the like in the inside.

  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 the step (1) is at least one protein selected from the protein group (A) (in the present specification, these may be collectively referred to as “target protein”).

  Protein group (A) is (A) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase/arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosomal protein L8, Ras-related protein Rab-5C, Peroxiredoxin-2, Vehicle-associated membrane protein 3, CD81 antigen, Guanine nucleotide-binding protein G(I)/G(S)/G( T) A group of proteins consisting of subunit beta-1 and calmodulin.

  The protein group (A) is a biomarker specific to chronic obstructive pulmonary disease, and by using this as an index, chronic obstructive pulmonary disease can be distinguished.

  Among the protein group (A), from the viewpoint of a higher amount ratio to a normal sample, preferably Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI , Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain and the like, more preferably Apolipoprotein (a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M , Prostaglandin G/H synthase 1 and the like.

  Of the protein group (A), from the viewpoint of being associated with inflammation and being a membrane protein, preferably Fibulin-1, Integrin beta-2, CD99 antigen, Peroxiredoxin-2, Clusterin, Prostaglandin G/H synthase 1, vesicle-associated membrane protein 3, Ras-related protein Rab-5C, EGF-containing fibulin-like extracellular matrix protein 1 and the like.

  In the case of human, the protein of the protein group (A) is a protein specified by UniProtKB accession number shown in Table 1 in the below-mentioned Example. In the case of other species, it is the ortholog of the protein identified by the UniProtKB accession number shown in Table 1.

  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, 30 or more) detection targets, it becomes possible to more accurately carry out tests such as chronic obstructive pulmonary disease.

  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, sandwich 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 the concentration of the target protein, which is a detection index for chronic obstructive pulmonary disease, thereby detecting chronic obstructive pulmonary disease, etc. Can be assisted.

  The test results by the test method of the present invention including the step (1) include treatment effect determination, elucidation of pathological condition of chronic obstructive pulmonary disease, prognosis prediction of chronic obstructive pulmonary disease (respiratory dysfunction group, acute exacerbation), patient stratification , Selection of treatment method (personalized medicine, treatment response), refractory in chronic obstructive pulmonary disease, evaluation of remodeling, differentiation of histological type and phenotype of chronic obstructive pulmonary disease, etc. ..

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

  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 is, for example, collected from a subject not suffering from chronic obstructive pulmonary disease. Based on the amount and/or the concentration of the target protein in the extracellular vesicles of the body fluid, the value can be set to a predetermined value or a predetermined value. The cutoff value is, for example, the amount and/or concentration of the target protein in the extracellular vesicles of the body fluid collected from a subject not suffering from chronic obstructive pulmonary disease (in the case of a plurality of subjects, an average value, The value may be, for example, 1 to 1.5 times the median value).

  In a preferred embodiment of step (2), when the subject is a subject under treatment for chronic obstructive pulmonary disease, the cut-off value is, for example, the amount and/or concentration of the protein of interest in past samples of the same subject. The therapeutic effect can be determined by setting the value based on

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

3. When the subject is determined to have chronic obstructive pulmonary disease by the inspection method of the present invention including the treatment step (2) for chronic obstructive pulmonary disease, further to the inspection method of the present invention, or When the patient is diagnosed as suffering from chronic obstructive pulmonary disease as described in "2. Diagnosis of chronic obstructive pulmonary disease with higher accuracy", a step of applying the examination method of the present invention and the diagnosis by a doctor In addition to the combination of (3) the subject determined or diagnosed as suffering from chronic obstructive pulmonary disease, by performing a step of treating the disease, the disease of the subject Can be treated. In addition, the test method of the present invention can detect chronic obstructive pulmonary disease more accurately, and therefore, for the test method of the present invention, or for the combination of the test method of the present invention and the step of applying a diagnosis by a doctor. By combining Step 3, the subject suffering from chronic obstructive pulmonary disease can be treated more efficiently and more reliably.

  The method for treating chronic 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, Azmanex; The medicine can be used alone, in combination of two kinds, or in combination of three or more kinds.

Four. Chronic Obstructive Pulmonary Disease Test Agent In one aspect thereof, the present invention comprises a test agent for chronic obstructive pulmonary disease, which comprises a detection agent for at least one protein selected from protein group (A) (herein, , "Inspection drug of the present invention"). This will be described below.

  The protein group (A), chronic obstructive pulmonary disease and the like are the same as the definitions in "1. Method for testing chronic obstructive pulmonary disease" above.

  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, Chapter 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 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. Section 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. Preventive or therapeutic agent for chronic obstructive pulmonary disease The present invention, in one aspect thereof, comprises a prophylactic or therapeutic agent for chronic obstructive pulmonary disease, which comprises an inhibitor of at least one protein selected from the protein group (A). (In the present specification, it may be referred to as “the agent of the present invention”.). This will be described below.

  The protein group (A) and the like are the same as the definitions in the above “1. Method for examining chronic 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. The types of drug resistance genes and the 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 (intraorally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drop agents, etc.) 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 use is a food composition, liquid, gel-like or solid food, for example, juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, baby milk powder , Cake mix, powdered or liquid dairy products, bread, 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. Other components are not particularly limited as long as they are components that can be blended in, for example, a medicine, a food composition, an oral composition, a health promoting agent, a nutritional supplement (supplement, etc.), , 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.

6. Method for screening active ingredient of preventive or therapeutic agent for chronic obstructive pulmonary disease The present invention is, in one aspect thereof, a protein group (A) in an extracellular vesicle of a body fluid collected from an animal treated with a test substance. Using the amount or concentration of at least one protein selected as an index, a method for screening an active ingredient of a prophylactic or therapeutic agent for chronic obstructive pulmonary disease (herein, "the active ingredient screening method of the present invention" and Sometimes indicated). This will be described below.

  Body fluid, extracellular vesicles, protein group (A), chronic obstructive pulmonary disease, measurement of the amount or concentration of the target protein, etc. are the same as defined in "1. Method for testing chronic obstructive pulmonary disease" above. ..

  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). ) Is lower than the above), the step of selecting the test substance as an active ingredient of a prophylactic or therapeutic agent for chronic obstructive pulmonary disease (or a candidate substance for an active ingredient of a prophylactic or therapeutic agent for chronic obstructive pulmonary disease). Including.

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

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

7. The method for evaluating the induction or exacerbation of chronic obstructive pulmonary disease The present invention, in one embodiment thereof, is selected from protein group (A) in extracellular vesicles of body fluid collected from an animal treated with a test substance. A method for evaluating the inducibility or malignancy of chronic obstructive pulmonary disease using the amount or concentration of at least one protein as an index (herein, sometimes referred to as "the toxicity evaluation method of the present invention"). ) Concerning. This will be described below.

  For body fluids, extracellular vesicles, protein group (A), chronic obstructive pulmonary disease, measurement of the amount or concentration of the target protein, animal species, test substances, etc., see “1. It is the same as the definition in "Test method" and "6. Method for screening active ingredient of preventive or therapeutic agent for chronic 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 higher than the above, the test substance is determined to have an inducing or malignant state of chronic 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 extracellular vesicle fraction Extracellular small vesicles were obtained from the sera of each human subject (10 persons) diagnosed with chronic obstructive pulmonary disease (COPD) and the sera of each healthy human subject (6 persons). The cell fraction was prepared. The extracellular vesicle fraction was prepared by stepwise ultracentrifugation. Specifically, it carried out as follows. The sera obtained in Test Examples 1 and 2 and the sera of normal mice (11-week-old C57BL6/J male mice) were diluted with PBS and centrifuged (2000 g, 4°C, 30 minutes). The supernatant was passed through a 0.22 μm filter and ultracentrifuged (100,000 g, 4° C., 90 minutes). The pellet was washed with PBS and then ultracentrifuged under the same conditions. This washing-ultracentrifugation step was repeated twice in total. The finally obtained pellet was suspended in PBS to obtain an extracellular vesicle fraction.

Test example 2. Proteomics analysis (TMT label, LC-MS/MS)
TMT label-LC-MS/MS proteomics analysis was performed according to the published literature (Journal of Proteome Research., 2017, 16 (2), pp 1077-1086.). The outline is as follows. A peptide sample obtained by digesting the protein in the extracellular vesicle fraction obtained in Test Example 1 with trypsin was labeled with a TMT 10-plex reagent. The labeled sample was fractionated on an SCX column and analyzed by LC-MS/MS using a mass spectrometer (LTQ-Orbitrap XL, Thermo Fisher Scientific). The obtained raw data was analyzed using an analysis program (Proteome Discoverer ver.1.3 (manufactured by Thermo Fisher Scientific) equipped with Mascot v2.3.1 (manufactured by Matrix Science) for UniProt/SwissProt) to quantify various proteins. I got the data.

  The amount of protein in the extracellular vesicle fraction of COPD patients (COPD group) was significantly higher than the amount in the extracellular vesicle fraction of healthy subjects (control group). It was determined that it had a certain degree of effectiveness as a marker and was selected. Table 1 shows the selected proteins and the quantitative ratio to the control group.

Claims (12)

A method of testing for chronic obstructive pulmonary disease, the method comprising:
(1) Protein group (A) in extracellular vesicles of body fluid collected from a subject:
(A) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase/arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosomal protein L8, Ras-related protein Rab-5C, Peroxiredoxin-2, Vesicle-associated membrane protein 3, CD81 antigen, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, And a test method comprising the step of detecting at least one protein selected from the protein group consisting of Calmodulin. Further, (2) comprising a step of determining that the subject has chronic 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 claim 1. The cut-off value is 1 to 1.5 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 chronic obstructive pulmonary disease, The inspection method described in 2. 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) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor A group consisting of H-related protein 5 and Cytochrome b-245 heavy chain,
The inspection method according to claim 1. The protein (A) group is (A2) Fibulin-1, Integrin beta-2, CD99 antigen, Peroxiredoxin-2, Clusterin, Prostaglandin G/H synthase 1, Vesicle-associated membrane protein 3, Ras-related protein Rab-5C, And a group consisting of EGF-containing fibulin-like extracellular matrix protein 1,
The inspection method according to claim 1. The inspection method according to claim 1, wherein the subject is a human. Protein group (A):
(A) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase/arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosomal protein L8, Ras-related protein Rab-5C, Peroxiredoxin-2, Vesicle-associated membrane protein 3, CD81 antigen, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, And a test agent for chronic obstructive pulmonary disease, which comprises a detection agent for at least one protein selected from the protein group consisting of Calmodulin. Protein group (A):
(A) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase/arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosomal protein L8, Ras-related protein Rab-5C, Peroxiredoxin-2, Vesicle-associated membrane protein 3, CD81 antigen, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, And a prophylactic or therapeutic agent for chronic obstructive pulmonary disease, which comprises an inhibitor of at least one protein selected from the protein group consisting of Calmodulin. Protein group (A) in extracellular vesicles of body fluid collected from an animal treated with a test substance:
(A) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase/arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosomal protein L8, Ras-related protein Rab-5C, Peroxiredoxin-2, Vesicle-associated membrane protein 3, CD81 antigen, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, And a method for screening an active ingredient of a prophylactic or therapeutic agent for chronic obstructive pulmonary disease, which uses as an index the amount or concentration of at least one protein selected from the protein group consisting of Calmodulin. When 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 chronic obstructive pulmonary disease. Alternatively, the screening method according to claim 10, comprising a step of selecting as an active ingredient of a therapeutic agent. Protein group (A) in extracellular vesicles of body fluid collected from an animal treated with a test substance:
(A) Apolipoprotein(a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G/H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase/arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosomal protein L8, Ras-related protein Rab-5C, Peroxiredoxin-2, Vesicle-associated membrane protein 3, CD81 antigen, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, And a method for evaluating the inducing or aggravating malignancy of chronic obstructive pulmonary disease, which uses as an index the amount or concentration of at least one protein selected from the protein group consisting of Calmodulin.