KR20180073304A - Biomarker for predicting asthma and method of prediction for asthma using the same - Google Patents

Abstract

The present invention relates to a biomarker for predicting asthma and to a method for predicting asthma using the same. A kit for predicting the prognosis of asthma containing an agent for measuring the expression level of annexin A1 of the present invention is used to measure the prognosis of asthmatic patients and the severity of diseases. Furthermore, the kit for predicting the prognosis of asthma can be used to screen an asthma induction or exacerbation factor.

Description

TECHNICAL FIELD The present invention relates to a biomarker for predicting asthma and a method for predicting asthma using the same.

The present invention relates to a biomarker for predicting asthma and a method for predicting asthma using the same.

Asthma is an allergic disease caused by allergic inflammation of the bronchi in the lungs. When the bronchial inflammation occurs, the bronchial mucosa is swollen and the bronchial muscle is cramped, causing the bronchus to become narrowed or clogged, resulting in shortness of breath. These asthma is caused by the combined genetic and environmental factors, and allergic reactions inherited from parents are caused by the interaction of the allergic constituents and the surrounding asthma inducers. Because of the combined effects of genetic factors and growing environmental factors, asthma patients need continuous self-management.

The treatment of asthma is to avoid the recurrence of the symptoms if there is a symptom first, and to avoid the recurrence of the drug, long-term use of medication, asthma, . There is also an immunotherapy method in which allergic constitution is improved by injecting a small amount of a causative substance. If allergic inflammation of asthma is cured correctly, most of the bronchi will recover to normal, but if not treated and managed continuously, the narrowed bronchus becomes hardened as it does not expand to its original size, so continuous treatment and steady self-management are necessary.

On the other hand, Annexin is a group of cellular proteins that bind to calcium and phospholipids. It is the name of a protein that has been called to unify proteins called lipocortin, calfuxin, endonexin, etc. more than 20 years ago . In humans, annexin I, II, III, IV, V, VI, VII, VIII and XIII have been reported.

Annexin A1 is a protein belonging to the annexin I group with a molecular weight of 37 kDa. Annexin A1, a mediator of glucocorticoid action, acts as a downstream regulator of inflammation in cells of the innate immune system and is involved in many cellular processes such as inhibition of inflammation and membrane fusion of cells. Specifically, it specifically binds to G-protein coupled formyl peptide receptor located in the in vivo calcium and cell membrane, inhibits the synthesis of eicosanoids, inhibits leukocyte migration, It has been reported that stimulation of induction leads to anti-inflammatory effect. It has also been reported to inhibit activity through specific interactions with substances acting as mediators in inflammatory reactions such as phospholipase (Ferlazzo V et al, Int Immunopharmacol , 3: 1363-1369, 2003).

 Ferlazzo V, et al, Int Immunopharmacol, 3: 1363-1369, 2003.

The present invention investigated the overexpression of Annexin A1 in asthma-induced mouse models to predict prognosis of asthma. In addition, the amount of annexin A1 expressed between asthma patients and normal subjects was compared, and the amount of annexin A1 expression in asthmatic patients was periodically monitored. As a result, it was confirmed that annexin A1 is overexpressed in patients with asthma and that the amount of annexin A1 is decreased when the asthma patient's condition worsens. Thus, by developing a biomarker for predicting asthma and a kit for predicting asthma prognosis, Thereby completing the invention.

According to the above object, the present invention provides a kit for predicting asthma prognosis, which comprises an agent for measuring the expression level of annexin A1.

(A) measuring the amount of annexin A1 expression in a sample separated from an individual; (b) comparing the expression amount of annexin A1 with the amount of annexin A1 of a normal control sample; And (c) confirming that the expression level of the annexin A1 is higher than the expression level of annexin A1 of the control group.

Further, the present invention relates to a method for the treatment of asthma, comprising: (a) measuring the amount of annexin A1 expression periodically in asthmatic patients; And (b) confirming that the expression level of annexin A1 is reduced compared to the previous measurement time.

The present invention also provides a DNA microarray chip for predicting asthmatic prognosis in which a gene of annexin A1 or a complementary nucleic acid thereof is integrated.

Further, the present invention relates to a method for screening a compound of the present invention, comprising the steps of: (a) (b) measuring the amount of annexin A1 expression in the sample isolated from the normal mouse; And (c) determining the sample as an asthma inducing factor when the expression level of the annexin A1 is higher than the expression level of annexin A1 of the normal control group.

(A) treating the sample with an asthma-inducing mouse; (b) measuring the amount of annexin A1 expression in a sample isolated from an asthmatic mouse; And (c) determining the sample as an asthma exacerbation factor when the expression level of the annexin A1 is decreased as compared with the previous measurement time.

The prognosis and disease severity of asthmatic patients can be measured using an asthma prognosis prediction kit containing the agent for measuring the expression amount of annexin A1 of the present invention. Furthermore, the kit for predicting the prognosis of asthma can screen for asthma induction or exacerbation factors.

FIG. 1A is a photograph showing the protein expression of annexin A1 through hematoxylin-eosin stain and immunohistochemical staining using lung tissue cells of normal and asthma-induced mice.
FIG. 1B is a graph comparing the amount of protein expression of annexin A1 by immunohistochemical staining in lung tissue cells of normal mice and asthma-induced mice, measuring the protein absorbance of stained annexin A1 (* p <0.05 ).
2A is a photograph showing the amount of protein expression of annexin A1 and FPR2 through Western blot in lung tissue cells of normal mouse and asthma-induced mouse.
FIG. 2b is a graph comparing the amount of protein expression of annexin A1 by measuring the absorbance of HRP labeled with annexin A1 after Western blotting of lung tissue cells of normal mice and asthma-induced mice (* p < 0.05).
FIG. 2C is a graph comparing the amount of FPR2 protein expressed by measuring the absorbance of HRP labeled with FPR2 protein after Western blotting of lung tissue cells of normal mouse and asthma-induced mouse (* p <0.05).
FIG. 3A is a photograph showing the amount of protein expression of annexin A1 in western blot in bronchoalveolar lavage fluid (BALF) of normal mice and asthma-induced mice.
FIG. 3B is a graph comparing the amount of protein expression of annexin A1 measured by measuring the absorbance of HRP labeled with annexin A1 after Western blotting of bronchoalveolar lavage fluid (BALF) of normal mice and asthma-induced mice * p < 0.05).
FIG. 4A is a photograph showing the amount of protein expression of annexin A1 Phospho Try21 in lung tissue of normal mouse and asthma-induced mouse through Western blotting.
FIG. 4B is a graph comparing the protein expression levels of Annexin A1 Phospho Try21 by measuring the absorbance of HRP labeled with the protein of Annexin A1 Phospho Try21 after Western blotting lung tissue cells of normal mice and asthma-induced mice (* p < 0.05).
FIG. 5 is a graph comparing the expression levels of annexin A1 in plasma collected from healthy subjects, asthmatic patients and asthma patients.

The present invention provides a kit for predicting asthma prognosis, which comprises an agent for measuring the expression level of annexin A1.

The term " annexin A1 " as used herein refers to a unique sequence of about 70 amino acid sequences comprising the 'GXGTDE' motif, called the endonexin fold, which is repeated four times Is a protein belonging to the annexin I group having a conserved domain.

The gene and protein information of the annexin A1 can be easily confirmed through a known database such as the NCBI gene bank, for example, but not limited to annexin A1 which is Gene ID: 301 of the NCBI gene bank. The annexin A1 may have the amino acid sequence of SEQ ID NO: 1 and may have the nucleotide sequence of SEQ ID NO: 2, but is not particularly limited thereto.

As described above in the specific examples of the present invention, Annexin A1 is overexpressed in asthmatic patients and decreases in the expression level upon worsening of the disease, so that it can be used as a composition for predicting asthmatic prognosis.

The agent for measuring the expression level of annexin A1 may be one which detects mRNA or protein of annexin A1.

Specifically, the agent for measuring the mRNA expression level of annexin A1 may be a probe or a primer that specifically binds Annexin A1 gene.

The term " agent for measuring the amount of mRNA expression of annexin A1 " used in the present invention means a agent used in a method for measuring the amount of mRNA expression of annexin A1 contained in a sample. For example, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), northern blotting, , A gene chip analysis method, or the like, or a primer or a probe that specifically binds to the annexin A1 gene.

The term " primer " as used herein refers to a nucleic acid sequence having a short free 3 'hydroxyl group and capable of forming a base pair with a complementary template, Refers to a short nucleic acid sequence that functions as a starting point. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

The term " probe " used in the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides, which can specifically bind to a gene or mRNA. An oligonucleotide, A probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection, but the present invention is not limited thereto.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modifications between nucleotides such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of the present invention can also be modified using labels that can directly or indirectly provide a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

Specifically, the agent for measuring the amount of protein expression of annexin A1 may be an antibody specific to annexin A1 protein.

The term " agent for measuring the protein expression level of annexin A1 " used in the present invention means a preparation used for determining the presence or the degree of expression of the protein expressed from the annexin A1 gene in a sample. Specifically, it may be an antibody that specifically binds to the protein of the gene, but is not particularly limited thereto.

The antibody may be an antibody that specifically binds to the protein of annexin A1, and may include a polyclonal antibody, a monoclonal antibody, or a recombinant antibody. Antibodies specifically binding to annexin A1 can be easily prepared using techniques known in the art.

Monoclonal antibodies can be prepared using the hybridoma method (Kohler and Milstein, European Jounal of Immunology, 6: 511-519, 1976), or a phage antibody library (Clackson et al, Nature, 352: 624-628, 1991; Marks et al . , J. Mol . Biol , 222: 581-597, 1991). The antibody prepared by the above method can be separated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

Methods for determining the amount of the target protein bound to the antibody using the antibody include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Protein Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Fluorescence Activated Cell Sorter Chip, and the like, but are not limited thereto.

The kit may be used for predicting asthmatic prognosis by measuring the amount of annexin A1 mRNA or protein expression from a sample of a subject for prediction of asthmatic prognosis, but is not limited thereto. Also, primers or probes and antibodies for measuring the level of mRNA or protein of annexin A1, as well as one or more other component compositions, solutions or devices suitable for the assay method may be included.

Specifically, the kit for predicting asthma prognosis of the present invention may include essential elements necessary for performing RT-PCR. The RT-PCR kit includes a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration varying), deoxynucleotides (dNTPs), Taq polymerase (s), as well as respective primer pairs specific for the Annexin A1 gene And enzymes such as reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.

In addition, the kit of the present invention can include essential elements necessary for performing ELISA. Specifically, the kit is an ELISA kit and may include antibodies specific for the Annexin Al protein. The antibody may be an antibody having high specificity and affinity for the annexin A1 protein and little cross-reactivity to other proteins, for example, a monoclonal antibody, a polyclonal antibody or a recombinant antibody. The ELISA kit may also include antibodies specific for the control protein. The ELISA kit can be used to detect a bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e.g., conjugated to an antibody) and other substrates capable of binding to the substrate or antibody Materials, and the like.

The present invention also provides a method for providing asthma prediction information using the kit for predicting asthma prognosis. Specifically, the method comprises: (a) measuring the amount of annexin A1 expression in a sample isolated from an individual; (b) comparing the expression amount of annexin A1 with the amount of annexin A1 of a normal control sample; And (c) confirming that the expression level of annexin A1 is higher than the expression level of annexin A1 of the control group.

The step of measuring the expression level of annexin A1 may be to measure the mRNA or protein of annexin A1.

The term " sample " used in the present invention refers to a sample, which is a biomarker for asthma prediction, in which the amount of mRNA or protein expressed by annexin A1 is different, Blood, serum, plasma, saliva, or sputum, and the like.

The step of measuring the mRNA of the annexin A1 can be measured by various methods. Specifically, a primer or a probe that specifically binds to the annexin A1 gene can be used.

Specifically, the step of measuring the mRNA may include a reverse transcription polymerase chain reaction (RT-PCR), a competitive RT-PCR, a real-time quantitative RT-PCR, But are not limited to, those determined by RNase protection method, northern blotting or gene chips.

The step of measuring the protein of the annexin A1 can be measured by various methods, and specifically, the antibody that specifically binds to the annexin A1 protein is used .

In addition, the step of measuring the protein of Annexin A1 can be performed by Western blotting, ELISA, radioimmunoassay, radioimmunoprecipitation, Oucheroni immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, FACS, protein chip, but is not limited thereto.

Through these analysis methods, it is possible to compare the formation amount of the antigen-antibody complex in the normal control group with the formation amount of the antigen-antibody complex in asthmatic patients, and judge whether the expression level of the biomarker gene for asthma prediction is increased or decreased , The prognosis of asthmatic patients can be predicted.

The term " antigen-antibody complex " in the present invention refers to a combination of a biomarker protein for asthma prediction and an antibody specific thereto, and the amount of the antigen-antibody complex formed is quantitatively determined through the size of a signal of a detection label .

Such detection labels may be selected from the group consisting of enzymes, chromophores, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are not necessarily limited thereto. When an enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Glucoamylase, terazo, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokutase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decar &Lt; / RTI &gt; beta-lactamase, and the like. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, and fluorescein. Ligands include, but are not limited to, biotin derivatives.

The protein expression level of the annexin A1 can be measured by an ELISA method. Specifically, an antibody is attached to a solid support, a sample is reacted, followed by labeling with a labeled antibody recognizing the antigen of the antigen-antibody complex, or by labeling the antibody labeled with an antibody recognizing the antigen of the antigen- It can be detected by a sandwich ELISA method in which a secondary antibody is attached and developed enzymatically. Asthma prognosis can be predicted by confirming the degree of complex formation of biomarker protein and antibody for prediction of asthma.

Western blotting using one or more antibodies against the biomarker for prediction of asthma may be used. Specifically, the whole protein is separated from the sample, electrophoresed, the protein is separated according to its size, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the protein produced by the expression of the gene can be confirmed by confirming the amount of the antigen-antibody complex produced by using the labeled antibody, thereby confirming whether the asthmatic disease is worsened or not. The detection method comprises a method of examining the amount of expression of a marker gene in a control group and the expression level of a marker gene in a cell isolated from a patient suffering from asthma. The amount of mRNA or protein expressed can be expressed as the absolute (e.g., / / ml) or relative (e.g., the relative intensity of the signal) difference of the marker protein.

In addition, immunohistological staining using one or more antibodies against the biomarker for predicting asthma may be performed. Specifically, a sample isolated from an asthmatic patient is collected and fixed, and a paraffin-embedded block is prepared by a method well known in the art. These are cut into pieces of several μm thick and attached to a glass slide, and then reacted with a selected one of the above antibodies by a known method. Thereafter, the unreacted antibody is washed and labeled with one of the above-mentioned detection labels, and the labeling of the antibody is read on a microscope.

More specifically, a protein chip in which at least one antibody against the biomarker for predicting asthma is arranged at a predetermined position on a substrate and immobilized at high density can be used. A method of analyzing a sample using a protein chip is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the protein, Asthma can be predicted.

The present invention also provides a method for providing asthma exacerbation information using the asthma prognosis prediction kit. Specifically, the method comprises: (a) measuring the amount of annexin A1 expression periodically in an asthmatic patient; And (b) confirming that the expression level of annexin A1 is reduced compared to the previous measurement time.

The step of measuring the expression level of annexin A1 may be to measure the mRNA or protein of annexin A1.

The process of separating a sample from an individual can be carried out using a known process. The term " sample " used in the present invention is as described above in the method of providing asthma prediction information.

The step of measuring the mRNA of the annexin A1 is as described above in the method of providing the asthma prediction information.

In addition, the step of measuring the protein of Annexin A1 is as described above in the method of providing asthma prediction information.

The present invention also provides a DNA microarray chip for predicting asthmatic prognosis in which a gene of annexin A1 or a complementary nucleic acid thereof is integrated.

The DNA microarray chip of the present invention comprises a conventional microarray except that it contains the gene of annexin A1 of the present invention or a nucleic acid complementary thereto. The DNA microarray chip may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include cDNA corresponding to a quantitative control gene or a fragment thereof. The DNA microarray chip of the present invention can be produced by a method known to a person skilled in the art. A method of fabricating the microarray chip is as follows. In order to immobilize the biomarker on a substrate of a DNA microarray chip using the biomarker as a probe DNA molecule, a micropipetting method using a piezoelectric method or a pin-type spotter ) Is used. The DNA microarray chip substrate may be coated with an activator such as amino-silane, poly-L-lysine and aldehyde. Further, the substrate may be a slide glass, a plastic, a metal, a silicon, a nylon film, or a nitrocellulose film. The kit also comprises a buffer solution used for hybridization, a reverse transcriptase for synthesis of cDNA from RNA, a labeled reagent such as cNTPs and rNTP (pre-mixed or separate feed type), a chemical inducer of fluorescent dye, .

The present invention also provides a method for screening asthmatic inducers using the kit for predicting asthma prognosis. The method comprises: (a) treating the sample with a normal mouse; (b) measuring the expression level of annexin A1 in a sample isolated from a normal mouse; And (c) determining the sample as an asthma inducing factor when the expression level of the annexin A1 is higher than the expression level of annexin A1 of the normal control.

The sample may be a substance expected to cause asthma. Specifically, it may include, but is not limited to, smoking, pollution, smoke, detergent, spray, pollen, dust mites, animal dander and house mold.

The process of separating a sample from an individual can be carried out using a known process. The term " sample " used in the present invention is as described above in the method of providing asthma prediction information.

As described above in the present invention, since Annexin A1 is overexpressed in asthmatic patients, the sample may be an asthma inducer when the amount of annexin A1 significantly increases after treating the sample with normal mice.

 Also, the present invention provides a method for screening asthma exacerbation factors using the kit for predicting asthma outcome. The method comprises: (a) treating the sample with an asthma-inducing mouse; (b) measuring the amount of annexin A1 expression in a sample isolated from an asthmatic mouse; And (c) determining the sample as an asthma exacerbation factor when the expression level of the annexin A1 is decreased compared to the previous measurement time.

The sample may be a substance that is expected to exacerbate asthma. Specifically, it may include, but is not limited to, smoking, pollution, smoke, detergent, spray, pollen, dust mites, animal dander and house mold.

The process of separating a sample from an individual can be carried out using a known process. The term " sample " used in the present invention is as described above in the method of providing asthma prediction information.

As described above in the present invention, since Annexin A1 is reduced when asthma patient's condition becomes worse, when the amount of annexin A1 expression is significantly decreased after treating the sample with asthma-induced mice, the sample exhibits asthma exacerbation factor .

The asthma-inducing mice can be produced through specific examples of the present invention, but the present invention is not limited thereto.

Example  1. In the mouse model Annexin  A1 expression level

Example  1-1. Modeling asthma-inducing mice

Females of 6-week-old BALB / c mouse species were emulsified with 50 μg of 98% purity egg albumin (Sigma-Aldrich, St Louis, MO) in 100 μl of D-PBS for 14 days and 10 mg of hydroxyl aluminum The solution was injected intraperitoneally to sensitize albumin. From day 21 to day 23, the mice were injected into the spleen with 50 μl of D-PBS plus 150 μl of 90% purity albumin (Sigma-Aldrich, St Louis, MO). Control mice were treated with saline.

The experimental and control mice were anesthetized with 2.5 mg / kg of tilatamin and lysozyme (Bayer Korea Co, Seoul, Korea) and then treated with 0, 5, 20 and 100 mg / The reaction was evaluated by injection. When airway hyperresponsiveness was assessed, asthma-induced mice were increased at an injection concentration of 100 mg / ml compared to normal mice.

On day 24, bronchoalveolar lavage fluid (BALF) and lung tissue cells were collected. The collected bronchoalveolar lavage fluid (BALF) was centrifuged and the supernatant was stored at -20 ° C. The cells were resuspended and counted. Cells were then differentiated using a cytospin slide at a density of 500 cells / mouse (Diff-Quick-stining). Lung tissue cells were fixed with 4 vol% phosphate buffered paraformaldehyde and v-embedded in paraffin-embedded tissue to a thickness of 4 ㎛, and treated with hematoxylin and eosin staining and immunohistochemical staining.

Example  1-2. In immunohistochemical staining of asthma-induced mouse lung tissue Annexin  A1 Confirm the expression level

Paraffin-treated lung tissue samples were treated with ethanol series for deparaffinization and rehydration. After that, methanol treated with 1.4 vol.% Hydrogen peroxide was treated and reacted for 30 minutes to block intracellular peroxidase reaction. To prevent nonspecific binding, 1.5 vol% horse serum was treated and treated overnight with anti-rabbit annexin A1 (1: 200, Thermo Fisher, Rockford, Ill.). The next day samples were transferred to ABC Kit (Vector Laboratories, Burlingame, Calif.). After staining with a liquid DAB + substrate kit (Golden Bridge International Inc, Mukilteo, WA), hematoxylin was treated and stained for contrast.

As a result, the protein expression amount of annexin A1 of asthma-induced mouse lung tissue cells was larger than that of annexin A1 of normal mouse lung tissue cells (FIGS. 1A and 1B).

Example  1-3. Western Blat  In asthmatic-induced mouse lung tissue cells Annexin  A1 and FPR2  Check the expression level

The extracted lung tissue cells of asthma-induced mice were subjected to protein extraction and dissolution in 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5 mM EDTA and 100 mM PMSF in distilled water Lt; / RTI &gt; After centrifugation at 14000 rpm at 4 ° C for 30 minutes, the supernatant was recovered. The proteins of the extracted mouse lung tissue cells were transferred to a PVDF membrane after SDS-PAGE. The PVDF membrane was blocked with 5 vol% skim milk for 2 hours at room temperature and then incubated with anti-rabbit anexin A1 (1: 1000, Thermo Fisher, Rockford, Ill.) Or anti- Nexin A1 phospho Tyr21 (1: 500, GeneTex, Inc., North America) was treated and allowed to react overnight at 4 ° C. HRP-labeled secondary antibody was treated and analyzed using a chemiluminescence image analyzer (EzWestLumi plus Western Blot Detection System, ATTO, Japan).

As a result, the amount of protein expression of annexin A1 and FPR2 in asthma-induced mouse lung tissue cells was larger than that of annexin A1 and FPR2 in normal mouse lung tissue cells (Figs. 2A to 2C).

Example  1-4. Western Blat  In asthma-induced bronchial alveolar lavage fluid Annexin  A1 Confirm the expression level

Bronchoalveolar lavage fluid of the extracted asthma-induced mouse was dissolved in protein extraction solution in which 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.1% SDS, 1 volume% Triton X-100, 0.5 mM EDTA and 100 mM PMSF were dissolved in distilled water Lt; / RTI &gt; After centrifugation at 14000 rpm at 4 ° C for 30 minutes, the supernatant was recovered. The proteins of bronchoalveolar lavage fluid of extracted asthmatic mice were transferred to PVDF membrane after SDS-PAGE. The PVDF membrane was blocked with 5 vol% of skim milk at room temperature for 2 hours and then incubated with anti-rabbit annexin A1 (1: 1000, Thermo Fisher, Rockford, Ill.) Or anti- rabbit annexin A1 phospho Tyr21 (1: 500, GeneTex, Inc, North America) and allowed to react overnight at 4 ° C. HRP-labeled secondary antibody was treated and analyzed using a chemiluminescence image analyzer (EzWestLumi plus Western Blot Detection System, ATTO, Japan).

As a result, the amount of annexin A1 expression in asthma-induced mouse bronchoalveolar lavage fluid was higher than that of annexin A1 in normal mouse bronchoalveolar lavage (FIGS. 3A and 3B).

Example  2. In asthmatic patients Annexin  A1 expression level

Example  2-1. Experimental group  And control group screening and sample preparation

Samples and data of the asthmatic patients used in the present invention were obtained from the Human Resources Bank of Soonchunhyang University Bucheon Hospital. The diagnosis of asthma was made in compliance with the GINA guidelines. As a control group, normal subjects were selected by considering the age, sex, and body mass index of asthmatic patients, and screened for respiratory symptoms, other allergic diseases, and lung capacity. In asthmatic patients, asthmatic patients with a history of asthma more than 2 years were selected, and symptoms such as coarse breathing, wheezing, chest tightness were gradually increased, the frequency of drug use increased, We defined asthma as worse when visiting hospital and emergency room within 3 days. The study of the present invention was conducted with the approval of Institutional Review Board (IRB) of Soonchunhyang University Bucheon Hospital.

Characteristic Control group (healthy) Experimental group (asthmatic patients) (ballast) (Worsening machine) Number of individuals (persons) 25 50 Gender Male Female) 2/23 20/30 age 58.3 ± 6.2 54.9 ± 14.1 Age at onset 47.06 ± 17.25 Two-month period 6.63 ± 3.6 Absorption
(Non-experienced / experienced / smoker) 25/0/0 32/12/6 Body Mass Index (BMI) 24.8 ± 2.61 25.4 ± 3.3 Initial lung function 1 second forced expiratory volume (FEV ₁ %) 115.36 ± 16.59 82.42 + - 21.94 * Effective Spirometer (FVC%) 96.56 ± 14.51 83.24 ± 17.73 * Ballast function in ballast or exacerbator 1 second forced expiratory volume (FEV ₁ %) 85.43 + - 19.82 62.60 ± 18.14 ** Effective Spirometer (FVC%) 84.83 + - 16.68 66.51 ± 16.76 ** Total IgE (kU / L) 106.63 + - 188.7 421.5 699.19 * atopy 1 (4%) 21 (42%) Blood white blood cells / ㎕ 5587.2 ± 1268.0 7768.8 + - 448.9 * 9873.0 ± 4917.1 Eosinophils in blood (%) 2.73 ± 2.26 5.09 + 4.97 * 3.83 ± 5.42 Neutral white blood cells in blood (%) 54.7 ± 12.9 64.6 ± 19.3 **

(* P < 0.1 / ** P < 0.5)

Example  2-2. Enzyme immunoassay ( ELISA )through Annexin  A1 expression level

The expression of Annexin A1 in blood from normal patients and asthmatic patients was analyzed by ELISA (USCN, Wuhan, China) method. The blood of the patient was collected and centrifuged at 2500 rpm for 10 minutes, and the supernatant was recovered. 100 μl of normal patient and asthmatic patients were placed in a plate well coated with Annexin A1, reacted at 37 ° C for 1 hour, added with 100 μl of detection reagents A and B, and incubated at 37 ° C for 1 hour , And reacted for 30 minutes. Subsequently, the wells were washed 5 times, 90 μl of the substrate solution was added, and reacted at 37 ° C for 20 minutes. Then, 50 μl of the stop solution was added. After the addition, the absorbance was measured at a wavelength of 450 nm.

As a result, the expression level of annexin A1 in asthmatic patients was higher than that of normal annexin A1, and the amount of annexin A1 expression was decreased in asthma-exacerbated patients (FIG. 5).

Reference example  1. Statistical Analysis

In order to clearly distinguish the differences between the groups, data were analyzed using mean error range and Mann-Whitney test. A p-value less than 0.05 is considered statistically significant.

<110> Soonchunhyang University Industry Academy Cooperation Foundation <120> BIOMARKER FOR PREDICTING ASTHMA AND METHOD OF PREDICTION FOR          ASTHMA USING THE SAME <130> FPD / 201611-0025 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 346 <212> PRT <213> Homo sapiens <400> 1 Met Ala Met Val Ser Glu Phe Leu Lys Gln Ala Trp Phe Ile Glu Asn   1 5 10 15 Glu Glu Gln Glu Tyr Val Gln Thr Val Ser Ser Lys Gly Gly Pro              20 25 30 Gly Ser Ala Val Ser Pro Tyr Pro Thr Phe Asn Pro Ser Ser Asp Val          35 40 45 Ala Ala Leu His Lys Ala Ile Met Val Lys Gly Val Asp Glu Ala Thr      50 55 60 Ile Ile Asp Ile Leu Thr Lys Arg Asn Asn Ala Gln Arg Gln Gln Ile  65 70 75 80 Lys Ala Ala Tyr Leu Gln Glu Thr Gly Lys Pro Leu Asp Glu Thr Leu                  85 90 95 Lys Lys Ala Leu Thr Gly His Leu Glu Glu Val Val Leu Ala Leu Leu             100 105 110 Lys Thr Pro Ala Gln Phe Asp Ala Asp Glu Leu Arg Ala Ala Met Lys         115 120 125 Gly Leu Gly Thr Asp Glu Asp Thr Leu Ile Glu Ile Leu Ala Ser Arg     130 135 140 Thr Asn Lys Glu Ile Arg Asp Ile Asn Arg Val Tyr Arg Glu Glu Leu 145 150 155 160 Lys Arg Asp Leu Ala Lys Asp Ile Thr Ser Asp Thr Ser Gly Asp Phe                 165 170 175 Arg Asn Ala Leu Leu Ser Leu Ala Lys Gly Asp Arg Ser Glu Asp Phe             180 185 190 Gly Val Asn Glu Asp Leu Ala Asp Ser Asp Ala Arg Ala Leu Tyr Glu         195 200 205 Ala Gly Arg Arg Lys Gly Thr Asp Val Asn Val Phe Asn Thr Ile     210 215 220 Leu Thr Thr Arg Ser Tyr Pro Gln Leu Arg Arg Val Phe Gln Lys Tyr 225 230 235 240 Thr Lys Tyr Ser Lys His Asp Met Asn Lys Val Leu Asp Leu Glu Leu                 245 250 255 Lys Gly Asp Ile Glu Lys Cys Leu Thr Ala Ile Val Lys Cys Ala Thr             260 265 270 Ser Lys Pro Ala Phe Phe Ala Glu Lys Leu His Gln Ala Met Lys Gly         275 280 285 Val Gly Thr Arg His Lys Ala Leu Ile Arg Ile Met Val Ser Ser Ser Ser     290 295 300 Glu Ile Asp Met Asn Asp Ile Lys Ala Phe Tyr Gln Lys Met Tyr Gly 305 310 315 320 Ile Ser Leu Cys Gln Ala Ile Leu Asp Glu Thr Lys Gly Asp Tyr Glu                 325 330 335 Lys Ile Leu Val Ala Leu Cys Gly Gly Asn             340 345 <210> 2 <211> 1411 <212> DNA <213> Homo sapiens <400> 2 ctccctaagg ctggggtgaa acctgctacg gtctgcgcaa gttgactgtt aatgaatttg 60 attctcaggt acactttttc aaaaatggca atggtatcag aattcctcaa gcaggcctgg 120 tttattgaaa atgaagagca ggaatatgtt caaactgtga agtcatccaa aggtggtccc 180 ggatcagcgg tgagccccta tcctaccttc aatccatcct cggatgtcgc tgccttgcat 240 aaggccataa tggttaaagg tgtggatgaa gcaaccatca ttgacattct aactaagcga 300 aacaatgcac agcgtcaaca gatcaaagca gcatatctcc aggaaacagg aaagcccctg 360 gatgaaacac tgaagaaagc ccttacaggt caccttgagg aggttgtttt agctctgcta 420 aaaactccag cgcaatttga tgctgatgaa cttcgtgctg ccatgaaggg ccttggaact 480 gatgaagata ctctaattga gattttggca tcaagaacta acaaagaaat cagagacatt 540 aacagggtct acagagagga actgaagaga gatctggcca aagacataac ctcagacaca 600 tctggagatt ttcggaacgc tttgctttct cttgctaagg gtgaccgatc tgaggacttt 660 ggtgtgaatg aagacttggc tgattcagat gccagggcct tgtatgaagc aggagaaagg 720 agaaagggga cagacgtaaa cgtgttcaat accatcctta ccaccagaag ctatccacaa 780 cttcgcagag tgtttcagaa atacaccaag tacagtaagc atgacatgaa caaagttctg 840 gacctggagt tgaaaggtga cattgagaaa tgcctcacag ctatcgtgaa gtgcgccaca 900 agcaaaccag ctttctttgc agagaagctt catcaagcca tgaaaggtgt tggaactcgc 960 cataaggcat tgatcaggat tatggtttcc cgttctgaaa ttgacatgaa tgatatcaaa 1020 gcattctatc agaagatgta tggtatctcc ctttgccaag ccatcctgga tgaaaccaaa 1080 ggagattatg agaaaatcct ggtggctctt tgtggaggaa actaaacatt cccttgatgg 1140 tctcaagcta tgatcagaag actttaatta tatattttca tcctataagc ttaaatagga 1200 aagtttcttc aacaggatta cagtgtagct acctacatgc tgaaaaatat agcctttaaa 1260 tcatttttat attataactc tgtataatag agataagtcc attttttaaa aatgttttcc 1320 ccaaaccata aaaccctata caagttgttc tagtaacaat acatgagaaa gatgtctatg 1380 tagctgaaaa taaaatgacg tcacaagaca a 1411

Claims (15)

A kit for predicting asthma prognosis, comprising an agent for measuring the expression level of annexin A1.
The method according to claim 1,
Wherein the agent detects the mRNA or protein of annexin A1.
3. The method of claim 2,
Wherein the agent for measuring the mRNA expression level of the annexin A1 is a probe or primer that specifically binds to the annexin A1 gene.
3. The method of claim 2,
The kit for predicting asthma prognosis, wherein the agent for measuring the amount of protein expression of annexin A1 is an antibody specific to annexin A1 protein.
(a) measuring the expression level of annexin A1 in a sample separated from an individual;
(b) comparing the expression amount of annexin A1 with the amount of annexin A1 of a normal control sample; And
(c) confirming that the expression level of annexin A1 is higher than the expression level of annexin A1 of the control group.
6. The method of claim 5,
Wherein the step of measuring the expression level of annexin A1 measures the mRNA or protein of annexin A1.
The method according to claim 6,
Using the probe or primer that specifically binds to the annexin A1 gene in the step of measuring the mRNA of the annexin A1.
The method according to claim 6,
The method of providing asthma prediction information using an antibody that specifically binds to the annexin A1 protein in the step of measuring the protein of annexin A1.
(a) measuring the expression level of annexin A1 periodically in an asthmatic patient; And
(b) confirming that the expression level of annexin A1 is reduced compared to the previous measurement period.
10. The method of claim 9,
Wherein the step of measuring the expression level of annexin A1 measures the mRNA or protein of annexin A1.
11. The method of claim 10,
The method of providing asthma exacerbation information using a probe or a primer that specifically binds to the annexin A1 gene in the step of measuring the mRNA of the annexin A1.
11. The method of claim 10,
The method of providing asthma exacerbation information using an antibody that specifically binds to the annexin A1 protein in the step of measuring the protein of annexin A1.
A DNA microarray chip for predicting asthma prognosis integrated with genes of Annexin A1 or its complementary nucleic acid.
(a) treating the sample with a normal mouse;
(b) measuring the amount of annexin A1 expression in the sample isolated from the normal mouse; And
(c) determining the sample as an asthma inducer if the expression level of the annexin A1 is higher than the expression level of annexin A1 of the normal control.
(a) treating the sample with asthma-inducing mice;
(b) measuring the amount of annexin A1 expression in a sample isolated from an asthmatic mouse; And
(c) determining the sample as an asthma exacerbation factor when the expression level of the annexin A1 is decreased compared to the previous measurement time.

Images