KR102660742B1 - Recombinant antigen protein for diagnosing latent tuberculosis infection and use thereof - Google Patents

Abstract

The present invention relates to an antigen for diagnosing latent tuberculosis and its use. Specifically, when peripheral blood mononuclear cells isolated from a patient with latent tuberculosis are stimulated with K3 antigen selected from the Korean tuberculosis strain ( M. tuberculosis K strain), It was confirmed that the secretion of interferon gamma was significantly increased in the tuberculosis infection group (latent tuberculosis group and active tuberculosis group) compared to the normal control group, and that the secretion of interferon gamma was significantly increased in the latent tuberculosis group compared to the active tuberculosis group. It can be useful not only for diagnosis but also for diagnosing latent tuberculosis by distinguishing it from active tuberculosis.

Description

Recombinant antigen protein for diagnosing latent tuberculosis infection and use thereof}

The present invention relates to a recombinant antigen protein and its use for diagnosing latent tuberculosis.

Tuberculosis is a respiratory disease that causes chronic infection by infecting the lower respiratory tract with Mycobacterium tuberculosis . In Korea, the incidence of tuberculosis has continued to decrease through national-level tuberculosis control, and the number of new patients has decreased to 28,000, but it still records the highest incidence rate among OECD member countries ( Global Tuberculosis Report 2017, WHO; Kim et al., 2015) .

When tuberculosis bacteria first infect the human body through the respiratory tract, they are killed by the immune system, develop after proliferation (primary tuberculosis), or remain in a latent state within the granuloma and then become reactivated by risk factors. Menstruation appears. In general, it is known that about 5% of people infected with tuberculosis bacteria develop the disease within two years, and 5 to 10% remain latent for a long period of time and develop the disease as the tuberculosis bacteria proliferate due to risk factors.

Latent tuberculosis infection (LTBI) is not transmitted because the bacteria are not germinated, but there is a possibility that the bacteria can be reactivated for some reason, putting people at high risk of developing tuberculosis. Latent tuberculosis has no clinical symptoms, is negative on chest X-ray, and cannot be confirmed by tuberculosis laboratory diagnostic methods such as smear tests or PCR tests.

Mycobacterium tuberculosis is an intracellular bacterium and is highly related to T-cell mediated immune response and interferon-γ (IFN-γ). Therefore, an immunodiagnosis method using the detection of immune substances such as interferon gamma secreted from T cells that detect Mycobacterium tuberculosis antigen is being used to diagnose latent tuberculosis. The methods currently used to diagnose latent tuberculosis are the Tuberculin skin test (TST, delayed skin test using tuberculin antigen) or the Mycobacterium tuberculosis-derived antigen-stimulated interferon secretion test (IFN-γ releasing assay, IGRA), which is an in vitro diagnostic method. The IGRA method stimulates whole blood or peripheral blood mononuclear cells with tuberculosis-specific antigens ESAT-6 and CFP-10 (or up to TB7.7) after blood collection, and stimulates the response effector memory T-cells to secrete This is a method to determine tuberculosis infectiousness by quantitatively measuring interferon gamma.

TST can detect latent tuberculosis infection, but it cannot distinguish between BCG vaccination or NTM (nontuberculous mycobacteria) disease, and additional tests such as radiography, sputum, and culture are required to confirm latent tuberculosis infection. . Therefore, the IGRA test is currently being conducted on citizens aged 5 years or older, but the IGRA test also has the problem of not being able to distinguish between latent tuberculosis and active tuberculosis. As it is difficult to differentiate between latent and active tuberculosis, in Korea, patients positive for latent tuberculosis are recommended to take anti-tuberculosis drugs for at least 3 months at all ages to prevent the development of active tuberculosis, so the burden of drug treatment is very high. .

The mechanism of latent infection with Mycobacterium tuberculosis is complex and unclear and cannot be defined by a single factor. In order to improve the sensitivity and specificity of the IGRA method, in addition to the Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 used, latent tuberculosis and active tuberculosis are distinguished. Therefore, in addition to new diagnostic antigens and interferon gamma, it is necessary to explore various immunosecretory substances that can be used as biomarkers for diagnosing latent tuberculosis.

Prior art for diagnosing latent tuberculosis includes the Republic of Korea Patent No. 10-1665849 on an antigen peptide composition and use thereof for the diagnosis of latent tuberculosis infection and active tuberculosis infection, and active tuberculosis and latent tuberculosis used in conjunction with interferon gamma release analysis. Although Korean Patent Publication No. 10-2016-0139894 discloses a distinguishing biomarker and its use, a method for distinguishing and diagnosing latent tuberculosis and active tuberculosis using the antigen protein of the present invention has not been disclosed.

Accordingly, the present inventors used AIB48604, a membrane protein of the Korean tuberculosis bacterium Mycobacterium tuberculosis K strain, to transform peripheral blood monocytes from normal control, latent tuberculosis patient groups, and active tuberculosis patient groups into a novel recombinant antigen protein. When stimulated, IFN-γ and G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, MIP-1α, TGF- The present invention was completed by confirming that the expression of α and TNF-β was significantly different from the normal control group and the active tuberculosis patient group, and that latent tuberculosis could be diagnosed by distinguishing it from active tuberculosis.

The purpose of the present invention is to provide a novel antigen and its use for diagnosing latent tuberculosis by distinguishing it from active tuberculosis and normal controls.

To achieve the above purpose,

The present invention provides a composition for diagnosing tuberculosis comprising a recombinant antigen protein consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention includes the steps of 1) contacting a recombinant antigen protein consisting of the amino acid sequence of SEQ ID NO: 1 with an isolated sample of a subject or an isolated sample of a normal control;

2) measuring the level of cytokine secretion in a separated sample of the subject;

3) determining tuberculosis if the level of cytokine secretion in the subject's sample is higher than that of the normal control group as a result of the measurement in step 2) above; providing a method of providing information for diagnosing tuberculosis, including the step.

Additionally, the present invention provides a kit for diagnosing tuberculosis, including a recombinant antigen protein consisting of the amino acid sequence of SEQ ID NO: 1 and an antibody against a cytokine.

The present invention relates to an antigen for diagnosing latent tuberculosis and its use. Specifically, when peripheral blood mononuclear cells isolated from a patient with latent tuberculosis are stimulated with K3 antigen selected from the Korean tuberculosis strain ( M. tuberculosis K strain), It was confirmed that the secretion of interferon gamma was significantly increased in the tuberculosis infection group (latent tuberculosis group and active tuberculosis group) compared to the normal control group, and that the secretion of interferon gamma was significantly increased in the latent tuberculosis group compared to the active tuberculosis group. It can be useful not only for diagnosis but also for diagnosing latent tuberculosis by distinguishing it from active tuberculosis.

Figure 1 is a diagram confirming that the expression of K3, K6, and K12 recombinant proteins was induced through an induction process at 37°C with 0.1mM IPTG for 4 hours.
Figure 2 shows peripheral blood mononuclear cells (PBMC) collected from normal control (HC), latent tuberculosis (LTBI), and active tuberculosis (aTB) groups as positive control (PHA) and comparison antigen (PPD). When stimulated, the level of interferon gamma secretion was measured by ELISA. PPD, an antigen for conventional tuberculosis diagnosis, showed no significant difference in the level of interferon gamma secretion between the normal control group and the latent tuberculosis group, distinguishing latent tuberculosis from active tuberculosis. This is a province that confirmed that it cannot be diagnosed.
Figure 3 shows the latency when peripheral blood mononuclear cells (PBMC) collected from latent tuberculosis group (LTBI) were stimulated with a comparative antigen (PPD) and six candidate antigens (K3, K6, K12, K4, K10, K11). This is a diagram measuring the level of interferon gamma secretion in the tuberculosis group (LTBI) using ELISA.
Figure 4 shows stimulation of peripheral blood mononuclear cells (PBMC) collected from active tuberculosis (aTB) with a comparison antigen (PPD) and six candidate antigens (K3, K6, K12, K4, K10, K11). This is a diagram measuring the level of interferon gamma secretion in active tuberculosis (aTB) using ELISA.
Figure 5 shows the ratio of the number of cells secreting interferon gamma (spot forming cell, SFC) measured in the case where PBMCs were not stimulated (Blank) and when PBMCs were stimulated with PHA, a positive control (PHA), ELISPOT This method confirms activity by measuring the basal secretion level of interferon gamma in unstimulated cells and interferon gamma secretion after PHA stimulation.
Figure 6 is a diagram confirming that interferon gamma secretion significantly increased in latent tuberculosis group (LTBI) upon PBMC stimulation with recombinant protein antigens and peptides of K3 and K13.
Figure 7 is a measurement of the number of cells secreting interferon gamma (spot forming cells, SFC) when PBMCs were stimulated with ESAT-6 and CFP-10, existing tuberculosis diagnostic antigens, and showed tuberculosis infection compared to the normal control group. The level of interferon gamma secretion was significantly increased in the group, making it possible to diagnose tuberculosis. However, no significant difference was observed between the levels of interferon gamma secretion between the latent tuberculosis group and the active tuberculosis group, so latent tuberculosis and active tuberculosis cannot be diagnosed separately. This province has confirmed that there is no such thing.
Figure 8 is a measurement of the number of cells secreting interferon gamma (spot forming cells, SFC) when PBMCs were stimulated with K3 antigen. The level of interferon gamma secretion was significantly increased in the tuberculosis infection group compared to the normal control group. Not only can tuberculosis be diagnosed, but a significant difference was observed in the level of interferon gamma secretion between the latent tuberculosis group and the active tuberculosis group, confirming that latent tuberculosis can be diagnosed by distinguishing it from active tuberculosis.
Figure 9 is a measurement of the number of cells secreting interferon gamma (spot forming cells, SFC) when PBMCs were stimulated with K13 antigen and K13 peptide. It increased in the tuberculosis infection group compared to the normal control group, but was not statistically significant. This is the province that confirmed that tuberculosis cannot be diagnosed.
Figure 10 shows the stimulation index (SI), which is a value calculated by calculating the number of cells stimulated by the antigen and secreting interferon gamma relative to the number of PBMC cells not stimulated by the antigen in the K3 recombinant antigen and the comparative antigens ESAT-6 and CFP-10. As a measurement of the value, K3 confirms that the SI value is higher in the latent tuberculosis group than in the active tuberculosis group, so latent tuberculosis can be diagnosed by distinguishing it from active tuberculosis.
Figure 11 shows PBMCs extracted from healthy control (HC), latent TB infection (LTBI), and active TB (aTB) groups stimulated with test antigen or comparison antigen, G-CSF, GM -The secretion level of CSF, IFN-γ, IL-1α, IL-1β, and IL-1RA cytokines was measured. There was a significant difference in secretion levels between the latent tuberculosis group and the active tuberculosis group, so latent tuberculosis and active tuberculosis were diagnosed separately. This is a province that has confirmed that it is a capable biomarker.
Figure 12 shows PBMCs extracted from healthy control (HC), latent TB infection (LTBI), and active TB (aTB) groups stimulated with test antigen or comparison antigen, IL-6, IL -8, This is a measure of the secretion level of MIP-1α, TGF-α, and TNF-β cytokines, and shows a significant difference in secretion levels between the latent tuberculosis group and the active tuberculosis group, making it possible to distinguish latent tuberculosis from active tuberculosis and diagnose it. This is the province that confirmed that it was a marker.
Figure 13 shows PBMCs extracted from the normal control (HC), latent TB infection (LTBI), and active TB (Active TB, aTB) groups were stimulated with test antigen or comparison antigen, and secreted cytokines and Table analyzing chemokines, 11 types of cytokines (G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, MIP-1α, TGF- It has been confirmed that α, TNF-β) is a biomarker that can be used to distinguish between latent tuberculosis and active tuberculosis.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for diagnosing tuberculosis comprising a recombinant antigen protein consisting of the amino acid sequence of SEQ ID NO: 1.

The tuberculosis is latent tuberculosis infection (LTBI) or active tuberculosis (aTB).

Latent tuberculosis infection (LTBI) refers to a person who is infected with tuberculosis bacteria but has no clinical symptoms of tuberculosis, tests negative for tuberculosis such as bacteriological and radiological tests, and cannot spread the virus to others.

Active tuberculosis is a condition in which the tuberculosis bacteria that can cause tuberculosis actively proliferate rather than incubate. A sputum test (smear test, culture test, nucleic acid amplification test (PCR)) may be positive, or a radiology test (chest X-ray test) may be positive. or CT, etc.) refers to tuberculosis in which active lesions are observed and includes recurrent tuberculosis.

Recurrent tuberculosis refers to cases where a person who previously suffered from tuberculosis develops tuberculosis again after treatment, and includes both cases of relapse due to drug resistance and cases of relapse due to the patient stopping medication at will. In other words, it refers to a tuberculosis patient who is currently diagnosed with tuberculosis but has had tuberculosis at least once before.

It is known that 77% of tuberculosis bacteria isolated in Korea belong to the Beijing family (Park YK, et al. J. Microbiol. Methods 63:165-172, 2005), and the number of tuberculosis bacteria occurring in groups in middle and high schools is known to be high. As a result of examining the restriction fragment length polymorphism (RFLP) profile, a unique group of strains was discovered in about 18.4% of the cases, which was named K-strain and similar strains were named K-family (Kim SJ, et al. Int J. Tuberc. 5:824-830, 2001). Approximately 46% of tuberculosis strains isolated from relapsed multidrug resistance patients hospitalized at Masan National Tuberculosis Hospital belong to the K-family.

The recombinant antigen protein consisting of the amino acid sequence of SEQ ID NO: 1 is derived from the Korean tuberculosis strain (K-strain).

The recombinant antigen protein is a membrane protein of Mycobacterium tuberculosis and has antigenicity.

The recombinant antigen protein can stimulate interferon-gamma (IFN-γ) secretion, and can significantly stimulate interferon-gamma (IFN-γ) secretion, especially in patients with latent tuberculosis.

Interferon gamma (IFN-γ) is one of the interferon molecular species and has no homology to interferon α or β, so it is classified as type II interferon and is induced by treatment of T lymphocytes with mitogens or specific antigens.

The recombinant antigen protein induces a cytokine response, that is, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1α, Secretion of one or more cytokines selected from the group consisting of IL-1β, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP-1α), TGF-α, and TNF-β. By deriving the diagnosis, latent tuberculosis or active tuberculosis can be distinguished.

Granulocyte colony-stimulating factor (G-CSF), also called colony-stimulating factor 3, is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream, and functions as a cytokine and hormone.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) functions as a growth factor for white blood cells, stimulates stem cells to produce granulocytes (neutrophils, basophils, eosinophils) and monocytes, and stimulates macrophages. It is a type of cytokine that appears in immune/infection responses because it rapidly increases in number to fight infection.

Interleukin-1α (IL-1α), also known as hematopoietin 1, is a cytokine of the interleukin 1 family encoded by the IL1α gene in humans. Generally, IL-1α is responsible for promoting inflammation and fever and sepsis.

Interleukin-1β (IL-1β) is a cytokine of the interleukin 1 family encoded by the IL1β gene in humans.

Interleukin-1receptor antagonist (IL-1RA) binds to the cell surface interleukin-1 receptor (IL-1R), the same receptor that binds interleukin 1 (IL-1), preventing IL-1 from signaling to the corresponding cells. It is an agent.

Interleukin-6 (IL-6) is both an anti-inflammatory cytokine and an anti-inflammatory myokine.

Interleukin-8 (IL-8) is a chemokine produced by macrophages and cell types such as epithelial cells, airway smooth muscle cells, and endothelial cells.

Macrophage inflammatory protein (MIP-1α) is a chemokine belonging to the chemotactic cytokine family.

TGF-α is a member of the epidermal growth factor family and is a mitogenic polypeptide. It is activated when it binds to a receptor with protein kinase activity for cell signaling.

TNF-α (tumor necrosis factor-alpha) refers to a cytokine that is included in the inflammatory response and is a member of the acute-phase protein.

In addition, the present invention includes the steps of 1) contacting a recombinant antigen protein consisting of the amino acid sequence of SEQ ID NO: 1 with an isolated sample of a subject or an isolated sample of a normal control;

2) measuring the level of cytokine secretion in a separated sample of the subject;

3) determining tuberculosis if the level of cytokine secretion in the subject's sample is higher than that of the normal control group as a result of the measurement in step 2); Provides a method of providing information for tuberculosis diagnosis, including.

The method can detect latent tuberculosis and active tuberculosis separately.

The samples include whole blood, serum, and plasma.

In the above method, a normal control group is a person or a sample derived from a person who has no clinical symptoms of tuberculosis, no active lesions were found on chest X-ray, and a negative IGRA (QuantiFERON-TB Gold In-Tube; QFT-IT) test. .

In the above method, markers according to the immune response to the antigen: IFN-γ, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, MIP- 1α, TGF-α, and TNF-β can be detected using various known methods.

Methods based on the formation of antigen-antibody complexes or immune responses using the above-mentioned cytokines as antigens are used, and these methods include, for example, radial immunodiffusion, immunoelectrophoresis, or immunoprecipitation analysis including reverse current electrophoresis. , RIA (Radioimmunoassay), FACS, or ELISA (Enzyme Linked Immunosorbent Assay).

The ELISA method is used in the information provision method, and in this case, a capture antibody and/or detection antibody that specifically binds to the cytokine may also be used.

The capture antibody used to detect the marker according to the present application may be a commercially available antibody that specifically recognizes the cytokine, and may be labeled with a substance that can be detected visually or using various image detection equipment. and, for example, those described herein may be used.

The detection antibody that can be used in the above method binds specifically to human IgG, and the detection antibody may be labeled with a substance that can be detected visually or using various image detection equipment.

As labeling substances, peroxidase such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, and urease ), catalase, asparginase, ribonuclease, malate dehydrogenase, staphylococcal nuclease, triose phosphate isomerase The presence of specific substrates such as triose phospate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase. It may be labeled as an enzyme that catalyzes a chemical reaction under the sun and can emit detectable color reaction or light, but is not limited to this.

The labeling material according to the present application is a chromophore used in viroluminescence, chemiluminescence, electroluminescence, electrochemiluminescence, and photoluminescence that emits light of a different wavelength from the irradiated light by irradiation of light. . Examples of proteins include green fluorescent protein; Organic compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorecamine. Including but not limited to this.

Detection antibodies and/or capture antibodies according to the present application include, for example, a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; radioactive substances; or materials containing nanoparticles such as colloidal gold particles or colored latex particles, but are not limited thereto.

Additionally, the present invention provides a kit for diagnosing tuberculosis, including a recombinant antigen protein consisting of the amino acid sequence of SEQ ID NO: 1 and an antibody against a cytokine.

The kit can detect latent tuberculosis and active tuberculosis.

The kit is for ELISA or FACS analysis.

The cytokines include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1α, IL-1β, IL- At least one selected from the group consisting of 1RA, IL-6, IL-8, macrophage inflammatory protein (MIP-1α), TGF-α, and TNF-β, by inducing secretion of the above-mentioned cytokines It can be diagnosed by distinguishing between latent tuberculosis and active tuberculosis.

In the kit, the capture antibody is a microwell plate such as a 96 microwell plate, beads or particles containing colloidal gold particles or colored latex particles, or cellulose, nitrocellulose, polyethersulfone, polyvinylidine, fluoride, nylon, It can be attached to membranes such as charged nylon and polytetrafluoroethylene. The method of attaching or coating can be a known method, for example, refer to those described in the Examples herein.

The kit can be used in a sandwich-type immunoassay method such as ELISA (Enzyme Linked Immuno Sorbent Assay), RIA (Radio Immuno Assay), etc. This method involves attaching a sample to a capture antibody bound to a solid substrate, such as a bead, membrane, slide, or microwell plate made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, or nitrocellulose. After adding the sample in contact with the antigen according to the present application, labeling substances capable of direct or indirect detection, such as radioactive substances such as ³ H or ¹²⁵ I as described above, fluorescent substances, chemiluminescent substances, haptens, biotin, and dig Qualitative or quantitative through binding to antibodies labeled with oxygenin, etc., or conjugated with enzymes such as horseradish peroxidase, alkaline phosphatase, and malate dehydrogenase, which can develop color or luminescence through interaction with a substrate. It can be detected. Additionally, the immunoassay method is Enzyme Immunoassay, ET Maggio, ed, CRC Press, Boca Raton, Florida, 1980; It is described in Gaastra, W, Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol 1, Walker, JM ed, Humana Press, NJ, 1984. ELISA kits are reagents capable of detecting bound antibodies, such as secondary detection antibodies labeled with the above-mentioned substances such as chromophores, enzymes (e.g. conjugated with antibodies), and substrates used for detection. etc. may be additionally included.

The method or kit may be used in the form of an array or chip containing a microarray. Capture antibodies can be attached to the surface of a support such as glass or nitrocellulose, and array fabrication techniques are described, for example, in Schena et al, 1996, Proc Natl Acad Sci USA 93(20):10614-9; Schena et al, 1995, Science 270(5235):467-70; and US Pat Nos 5,599,695, 5,556,752 or 5,631,734. The fluorescence intensity can be measured using a scanning confocal microscope, available from, for example, Affymetrix, Inc. or Agilent Technologies, Inc.

Additionally, the method or kit is used in the form of a dip stick rapid kit depending on the analysis mode.

In the case of dipstick, it is a technology widely used in the POCT (Point of Care Treatment) field, and the capture antibody that binds to the cytokine according to the present application is bound to a substrate such as nitrocellulose, such as the antigen according to the present application. When immersed in a sample in contact with a sample, the sample moves through the substrate by capillary action and develops color when it binds to the antibody in the substrate.

Additionally, the kit can be used for lateral flow analysis depending on the analysis mode. Lateral flow assay is a method of quantitatively or qualitatively measuring specific substances contained in a sample, such as specific nucleic acids or proteins. For example, a nitrocellulose membrane with a capture antibody bound to a specific location. This is a method of detecting a specific protein in the sample through an antigen-antibody reaction by moving a sample in contact with an antigen according to the present application using a chromatography method (development medium), for example.

The kit may also include instructions on how to use the marker according to the present disclosure. Additionally, it may further include antibodies specific to the control group or reagents capable of detecting bound antibodies.

In a specific example of the present invention, a total of 14 candidate antigen groups were selected by analyzing the protein sequence of M. tuberculosis K strain, a representative domestic tuberculosis strain, and selecting interferon gamma secretion promoting substances (see Table 1).

Among the proteins predicted to be antigens, proteins K3, K4, K6, and K12 verified through genetic analysis, protein antigens K10, K11 expressed during the latent period of Mycobacterium tuberculosis, and K13, which has antigenicity in tuberculosis patients through immunoproteomics analysis, were selected as evaluation antigens. In addition, the level of interferon gamma secretion was confirmed by stimulating the above-mentioned evaluation antigen group in peripheral blood mononuclear cell samples isolated from normal control, latent tuberculosis, and active tuberculosis group patients. As a result, K3 antigen was higher in the tuberculosis infection group (latent tuberculosis group) compared to the normal control group. group and active tuberculosis group), and secretion increased in the latent tuberculosis group compared to the active tuberculosis group, confirming that latent tuberculosis can be distinguished and diagnosed (see Figures 3, 4, 6, and 8).

In addition, the K3 antigen has a stimulation index (SI) value, which is a value that calculates the number of cells that secrete interferon gamma stimulated by the antigen relative to the number of PBMC cells that are not stimulated by the antigen, compared to the normal control group in the tuberculosis infected group (latent tuberculosis group). and active tuberculosis group), and secretion increased in the latent tuberculosis group compared to the active tuberculosis group, confirming that latent tuberculosis can be distinguished and diagnosed (see Figure 10).

In addition, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, MIP-1α, TGF-α, and TNF-β 11 types of cytokines showed a significant difference in secretion levels between the latent tuberculosis group and the active tuberculosis group, confirming that it can be used as a biomarker to diagnose latent tuberculosis by distinguishing it from active tuberculosis (see Figures 11, 12, and 13).

Therefore, the recombinant antigen protein of the present invention can be usefully used for diagnosing latent tuberculosis by distinguishing it from active tuberculosis.

Hereinafter, the present invention will be described in detail through the following examples and experimental examples.

However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

<Example 1> Selection process for Mycobacterium tuberculosis-specific antigens that can diagnose latent tuberculosis

Antigen candidates capable of characteristically stimulating interferon gamma secretion by Mycobacterium tuberculosis antigen protein were selected as follows.

<1-1> Selection of Korean-type tuberculosis strain-specific antigen proteins

We attempted to select antigen candidates by performing BLAST or FASTA analysis on the protein sequence of M. tuberculosis K strain, a representative tuberculosis strain in Korea.

Specifically, the protein sequence of M. tuberculosis K obtained from the MtbVeb database was compared with that of various Mycobacteria strains, and 128 K strain-specific proteins were selected.

All 128 selected protein sequences are predicted to be 'secreted proteins' by at least two programs among SignalP, TargetP, and PSORTb, or if the results of SecretomeP or TatP are 'secreted proteins', the proteins are qualified as secreted proteins. considered. In addition, in TMHMM, if the TM value was less than 1, the protein was judged to be secreted, and if the TM value was 2 or more, it was judged to be a surface protein. In addition, if two or more predicted values among TargetP, PSORTb, and Phobius were intracellular proteins, the protein was determined to be an intracellular protein. If it was predicted to be 'Membrane' in a situation where it was considered a secreted protein, the results were judged comprehensively and recognized as a 'secreted protein'. Based on the above criteria, 37 secreted proteins and 2 surface proteins were discovered among 130 proteins specific to domestic standard strains of Mycobacterium tuberculosis.

<1-2> Selection of substances that stimulate interferon gamma secretion

Among 39 proteins (37 secreted proteins and 2 surface proteins) of M. tuberculosis K predicted to be secreted proteins or surface proteins, antigen proteins or antigen functional groups that promote interferon gamma secretion were analyzed.

In order to predict substances that induce the secretion of interferon gamma, T-cell epitopes were extracted using IEDB information extraction and two interferon epitope analysis methods (a method using BLAST and FASTA and a Support Vector Machine (SVM) algorithm). The area was predicted.

As a result, as shown in Table 1 below, 12 antigen protein candidates and 68 antigen effector candidates (34 antigen effector groups derived from IEDB, antigen effector groups predicted by IFNepitope) are substances predicted to ultimately stimulate the secretion of IFN-γ. 34) were selected. Among these, AIB46610, AIB48134, and AIB48604, although none were similar to the IEDB antigen protein, were predicted to be most suitable as antigen substances because the antigen functional groups were predicted in both the IEDB antigen functional group analysis and the IFNepitope antigen functional group analysis. These 12 antigens and T-cell epitope regions have cross-reactivity with M. tuberculosis protein sequences registered in NCBI, so they are expected to be common antigens. Since there is no cross-reaction with the 5 BCG strains, immunological diagnosis is possible. In this case, the possibility of being useful in diagnosing tuberculosis without a non-specific reaction to the BCG strain was confirmed.

In addition to the above 12 antigen proteins, two antigen proteins (K13, K14) derived from M. tuberculosis H37Rv were added as a candidate antigen protein group.

ID Protein ID detail of fuction amino acid length
(aa) interferon gamma secreting molecule IEDB_antigen INFepitope
(ea) IEDB_epitope
(ea) K1 AIB46610 Hypothetical protein 47 No match 8 One K12 AIB48069 Hypothetical protein 1,239 No match 0 5 K2 AIB48134 Membrane protein 599 No match 2 2 K3 AIB48604 Membrane protein 125 No match One 4 K4 AIB48608 PE family proteins 42 No match 0 3 K5 AIB48657 Hypothetical proteins 68 No match 8 No match K6 AIB49348 PhiRv2 phage protein 107 Ag_P9WJ13 One No match K7 AIB49767 Hypothetical protein 46 No match 5 No match K8 AIB50254 Hypothetical protein 132 No match 2 No match K9 AIB50255 RNA-directed RNA polymerase 561 No match One No match K10 Rv3037 Membrane potential protein 210 Ag_P9WLS9 0 12 K11 Rv2029c Glycolysis-related proteins 339 Ag_P9WID3 6 7 K13 Rv0475 Heparin binding hemagglutinin 199 - - - K14 Rv3037c S-adenosylmethione-dependent methyltransferase 358 - - -

<1-3> Selection of final evaluation antigen protein

Among the proteins predicted to be antigens, proteins K3, K4, K6, and K12 verified through genetic analysis, protein antigens K10, K11 expressed during the latent period of Mycobacterium tuberculosis, and K13, which has antigenicity in tuberculosis patients through immunoproteomics analysis, were selected as evaluation antigens. did.

<Example 2> Synthesis and purification of recombinant antigen protein

Among the antigens listed in Table 2 below, K3, K6, and K12 proteins were cloned for recombinant protein production using the E. coli BL21 (DR3) strain expression system. The optimal conditions for protein expression were confirmed, 0.1mM IPTG was added at 37°C, and protein expression was induced through an induction process for 4 hours as shown in Figure 1. It was purified through Ni-NTA, the buffer was changed through a dialysis process, and K3 and K12 were purified with 3M urea buffer, the minimum concentration that does not generate inclusion bodies. Additionally, K6 was purified with 50mM Tris and 200mM KCl buffer. The start codon of K3, K6, and K12 proteins was valine (Val), which was replaced with methionine (Met) during the amplification of the inserted gene to induce smooth expression in the E. coli system.

In the case of K4, due to the size limitation (4 kDa), it was not expressed in both soluble and insoluble forms in the expression vector pET or pGEX labeling His-tag or GST-tag and the BL21 E. coli expression system, and it was not expressed in both soluble and insoluble forms in the E. coli translation system. Cell-free expression based on was attempted, but did not occur. K10 and K11 were not expressed in the E. coli system, and high GC repeats and amino acid mutations were problems, so codon-optimized genes were synthesized considering E. coli codon usage, and recombinant proteins were produced using the E. coli cell-free expression system. An attempt was made to synthesize, but it was not expressed.

For proteins K4, K10, and K11, for which it is difficult to produce recombinant proteins, 20 mer 10 mer-overlapped peptides were designed and synthesized for the protein to be analyzed. The peptides to be used in the study were synthesized and purified by HPLC to obtain 5 mg peptides with a purity of ~80%, and were dissolved in endotoxin-free tertiary distilled water or DMSO to prepare at 1 mg/mL or 5 mg/mL.

antigen
name NCBI product Size (aa) origin sequence number K3 membrane protein 125 Mtb K SEQ ID NO: 1 K4 PE family proteins 42 Mtb K SEQ ID NO: 2 K6 PhiRv2 phage protein 107 Mtb K SEQ ID NO: 3 K10 Rv1733c
Possible transmembrane 210 Mtb H37Rv SEQ ID NO: 4 K11 Rv2029c
Probable phospho-
fructokinase 339 Mtb H37Rv SEQ ID NO: 5 K12 PE family proteins 304 Mtb K SEQ ID NO: 6 K13 Rv0475
Heparin-binding hemagglutinin (HBHA) 199 Mtb H37Rv SEQ ID NO: 7

<Experimental Example 1> Collection of clinical specimens and preparation of samples

<1-1> Collection of clinical specimens

Whole blood from 35 people in each group of healthy control (HC), latent TB infection (LTBI), and active TB (aTB) group with the definitions in Table 3 below was collected at Chonnam National University Hospital. It was collected through joint research with . Some were used for basic tests and latent tuberculosis tests, and the remaining blood was centrifuged using a ficoll-density gradient to extract peripheral blood mononuclear cells (PBMC), 1×10 ⁶ cells/vial. After dispensing, it was stored in a LN ₂ and nitrogen high-pressure gas tank at -180°C.

division Justice Clinical information normal control
(Healthy control, HC) ㆍNo known contact history with tuberculosis patients
ㆍNo symptoms or signs of tuberculosis
ㆍNo past tuberculosis lesions on chest X-ray
ㆍNegative IGRA test at the time of sample collection
ㆍNo underlying disease (autoimmune disease, infectious disease, diabetes, chronic respiratory disease, chronic kidney disease, tumor, chronic liver disease, immunosuppressant treatment) ㆍ≥ 18 y (excluding pregnant women)
ㆍBasic test, IGRA test, tuberculosis test information
ㆍAge and gender
ㆍBCG vaccination history
ㆍWithin 2 weeks of taking tuberculosis medication latent tuberculosis group
(Latent TB infection, LTBI) ㆍHas a history of contact with a patient with active tuberculosis
ㆍNo clinical or radiological evidence of active tuberculosis
ㆍPositive in IGRA test (or TST) Active tuberculosis group
(Active TB, aTB) ㆍConfirmed TB: When confirmed positive by Xpert MTB/RIF test
ㆍHighly probable TB: Clinically confirmed tuberculosis, negative in the culture test and Excluding confirmed cases)

<1-2> Preparation of samples

Among the peripheral blood mononuclear cells (cryopreserved PBMC, cPBMC) stored at -180°C in Experimental Example 1, cells stored within 3 months to 1 year were used considering cell viability and activity. The number of cPBMCs processed did not exceed 2 per time, and the total number of experiments per day did not exceed 10. After thawing the cPBMCs, slowly add 1 ml of CTL-anti-agglutination wash to a cryovial, take the sample, transfer it to a tube containing 8 ml of CTL-anti-agglutination wash culture medium, and wash by centrifugation at 330 × g for 10 minutes. did. After removing the supernatant, the cell pellet was loosened by tapping, 10 ml of CTL-test culture medium was added, and the cells were rested for 18 hours to enhance cell activity before antigen stimulation. After 18 hours, the cells were sedimented by centrifugation at 330 × g for 10 minutes, and the supernatant was removed. The cell pellet was again released by tapping, 0.5 to 1 ml CTL-test culture was added, and live cells were counted using tryptophan blue staining.

<Experimental Example 2> Primary screening of antigen using ELISA

ELISA was performed on a total of 7 latent tuberculosis diagnostic candidate antigens, K3, K4, K6, K10, K11, K12, and K13 in Table 1 above, and found that the latent tuberculosis group or the active tuberculosis group secreted interferon gamma significantly compared to the normal control group. Antigens with increasing levels were first screened.

<2-1> Measurement of interferon gamma secretion according to treatment of PPD (tuberculin purified protein derivative) test antigen and phytohemagglutinin (PHA) positive control group

The cPBMCs of Experimental Example 1-2 were dispensed into a 96-well plate at 1×10 ⁵ to 5×10 ⁵ cells/100 μl/well, cultured for 2 hours, and 10 μg/ml of phytohemagglutinin (PHA) as a positive control , for comparison. Purified protein derivatives (tuberculin, PPD) 5㎍/㎖ (Staten Serum Institute, Denmark) were used as antigens, and CTL-test cultures were treated as negative controls.

Test antigen (PPD) or positive control (PHA) was treated at 10 ㎍/㎖ each, stimulated for 48 hours, and 100 ㎕ of the culture supernatant was taken and interferon gamma (IFN-γ) was quantitatively measured by ELISA. ELISA was conducted using the Human IFN-γ DuoSet ELISA kit (R&D system), and the 540 nm measurement value was subtracted from the 450 nm measurement value, and IFN-γ was determined based on the result obtained after subtracting the blank value and the duplicated standard concentration value. Quantified. After removing the supernatant, the remaining cells were subjected to MTT assay to measure cell viability.

As a result, as shown in Figure 2 and Table 4 below, when not stimulated with antigen, the interferon gamma secretion level of PBMC was 130.7 ± 130.7± in the normal control group (HC), latent tuberculosis group (LTBI), and active tuberculosis group (aTB), respectively. The level of interferon gamma secretion in active tuberculosis was high at 256.8 pg/ml, 168.5±370.6 pg/ml, and 302.3±1104.6 pg/ml. The levels of interferon gamma secretion in response to PHA stimulation were 628.7±396.1 pg/ml, 590.8±362.6 pg/ml, and 2629±3403.4 pg/ml in the normal control group (HC), latent tuberculosis group (LTBI), and active tuberculosis group (aTB), respectively. . The levels of interferon gamma secretion in response to PPD stimulation were 331.4.7±387.0 pg/㎖, 362.2.±368.9 pg/㎖, and 762.2±1010.5 pg in the normal control group (HC), latent tuberculosis group (LTBI), and active tuberculosis group (aTB), respectively. It was confirmed that secretion in response to PPD stimulation was high in some samples classified as normal controls at /ml.

The above results suggest that PPD, an existing antigen for diagnosing tuberculosis, does not show a significant difference in the level of interferon gamma secretion between the normal control group and the latent tuberculosis group, and therefore cannot diagnose latent tuberculosis.

pg/ml Normal control (HC) Latent tuberculosis (LTBI) Active tuberculosis (TB) Antigen non-stimulation 130.7±256.8 168.5±370.6 302.3±1104.6 PHAs 628.7±396.1 590.8±362.6 2629±3403.4 PPD 331.4.7±387.0 362.2.±368.9 762.2±1010.5

<2-2> Measurement of interferon gamma secretion amount according to treatment of candidate antigen

The seven candidate antigens in Table 1 (recombinant proteins (K3, K6, K12, K13) or peptide mixtures (K4, K10, K11)) were treated at 10 μg/ml each, then the recombinant proteins were treated for 48 hours, and the peptide mixtures were incubated for 48 hours. After stimulation for 24 hours, 100 ㎕ of culture supernatant was taken and interferon gamma (IFN-γ) was quantitatively measured using ELISA. ELISA was conducted using the Human IFN-γ DuoSet ELISA kit (R&D system), and the 540 nm measurement value was subtracted from the 450 nm measurement value, and IFN-γ was determined based on the result obtained after subtracting the blank value and the duplicated standard concentration value. Quantified. After removing the supernatant, the remaining cells were subjected to MTT assay to measure cell viability.

As a result, as shown in Figures 3 and 4, the secretion of interferon gamma for PPD increased in the active tuberculosis group (612.2 ± 1256.4 pg/ml) compared to the latent tuberculosis group (357.3 ± 374.1 pg/ml).

Additionally, the secretion of interferon gamma in response to recombinant protein K3 antigen was decreased in the active tuberculosis group (-165.4±1149.7 pg/ml) compared to the latent tuberculosis group (81.1±394.9 pg/ml).

In addition, the secretion of interferon gamma in response to K12 antigen was not observed to increase in the latent tuberculosis group, but decreased in the active tuberculosis group (-235.4±1115.5 pg/ml).

In addition, secretion of interferon gamma for peptide K4 was measured at low concentration in the active tuberculosis group (54.7 ± 285.9 pg/ml), and secretion of interferon gamma was observed for peptide mixture K4, K10, and K11 by stimulation of peripheral blood mononuclear cells. It didn't work.

In addition, the cut-off value of the interferon gamma secretion concentration of peripheral blood mononuclear cells (PBMC) stimulated with PPD and K3 antigen was set and the reactivity was analyzed in percent. Responsiveness was confirmed by setting the low concentration cut-off to 20 pg/ml (weak interferon gamma reactivity; weak IFN-γ response) and the high concentration cut-off to 100 pg/ml (strong interferon gamma reactivity; strong IFN-γ response). As a result, for PPD, in the latent tuberculosis group, 20% (7 samples/35 samples) showed weak interferon gamma reactivity, 62.9% (22 samples/35 samples) showed strong interferon gamma reactivity, and in the active tuberculosis group, 92.9% showed strong interferon gamma reactivity. (26 samples/28 samples) showed strong interferon gamma reactivity. Regarding K3, in the latent tuberculosis group, 45.7% (16 samples/35 samples) showed weak interferon gamma reactivity, 40% (14 samples/35 samples) showed strong interferon gamma reactivity, and in the active tuberculosis group, 57.1% (16 samples) Dog samples/28 samples) showed weak interferon gamma reactivity, and 35.7% (10 samples/28 samples) showed strong interferon gamma reactivity.

In other words, in response to stimulation with K3 antigen, the latent tuberculosis group showed a higher interferon gamma secretion ability than the active tuberculosis group.

<Experimental Example 3> Secondary screening of antigens using ELISpot (Enzyme-Linked ImmunoSpot)

This was performed by referring to the T-SPOT.TB method (Oxford Immunotec) and the Human IFN-γ ELISpot assay method (R&D Systems), which are recognized as diagnostic tests for latent tuberculosis and active tuberculosis. As comparative antigens for diagnosing tuberculosis, the interferon gamma secretion levels of ESAT-6 and CFP-10 peptide antigens and the K3 and K13 antigens in Table 1 above were compared.

Specifically, 50㎕ of ESAT-6 and CFP-10 peptide antigens were used as comparative antigens, and phytohemagglutinin (PHA) was used as a positive control antigen. A 96-well ELISpot plate was treated with 10 μg/ml of positive control (PHA) and recombinant protein or peptide pools for each antigen. The rested PBMCs were dispensed into an antigen-treated 96-well plate at 2 × 10 ⁵ cells/100 μl/well. After 24 hours of antigen stimulation, the culture supernatant was removed and spots of cells secreting interferon gamma were identified. The number of spots was measured using ImmunoSpot S6 Univeral-V. In the case of K13, it was expressed in M. smegmatis and a protein close to its native form was used. Native K13 is methylated after transcription, and it has been reported to have higher interferon gamma reactivity in tuberculosis patients compared to recombinant proteins using the E. coli system (Am J Respir Crit Care Med. 182:848-854.). Before the antigen evaluation test, activity was confirmed by measuring the basal secretion level of interferon gamma in unstimulated cells and interferon gamma secretion after PHA stimulation using the ELISPOT method (spot forming cell, SFC). When evaluating antigens, all measured values were corrected to blank (SFC of unstimulated cells) (test value - blank value).

As a result, as shown in Figure 5, when PBMCs were stimulated with PHA, which is a positive control, interferon gamma secretion was confirmed to increase in all of the normal control group, latent tuberculosis group, and active tuberculosis group, and PBMCs collected from the test subject group It was confirmed that all were active.

In addition, as shown in Figure 6, it was confirmed that interferon gamma secretion was significantly increased in the latent tuberculosis group (LTBI) upon PBMC stimulation with recombinant protein antigens and peptides of K3 and K13.

Additionally, as shown in Figure 7, SFC for ESAT-6 (×10 ⁵ cells) was significantly increased in the tuberculosis infection group (latent tuberculosis group + active tuberculosis group) compared to the normal control group (normal control group = 0.3 ± 0.5). , tuberculosis infection group = 19.5 ± 19.1 [p = < 0.0001]). Compared to the normal control group, interferon gamma secretion was significantly increased in the latent TB group and the active TB group (normal control = 0.3 ± 0.5, latent TB group = 17.9 ± 16.6 [p = <0.0001]; normal control = 0.3 ± 0.5, active Tuberculosis group=21.9±22.7 [p=0.0016]; latent tuberculosis group=17.9±16.6, active tuberculosis group=21.9±22.7 [p=0.9208]).

Additionally, SFC (×10 ⁵ cells) for CFP-10 was significantly increased in the tuberculosis infection group compared to the normal control group (normal control = 0.1 ± 0.3, tuberculosis infection group = 22.3 ± 29.1 [p = <0.0001]; normal Compared to the control group, there was a significant increase in the latent tuberculosis group and the active tuberculosis group, respectively (normal control group = 0.1 ± 0.3, latent tuberculosis group = 17.1 ± 23.1 [p = <0.0001]; normal control group = 0.3 ± 0.5; active tuberculosis group = 29.8 ±35.7[p=0.0007]; latent tuberculosis group=17.1±23.1, active tuberculosis group=29.8±35.7[p=0.3774]).

In addition, as shown in Figure 8, the SFC (×10 ⁵ cells) for recombinant protein K3 was significantly increased in the tuberculosis infection group compared to the normal control group (normal control = 4.6 ± 6.2, tuberculosis infection group = 21.4 ± 24.5 [ p=0.0312]), compared to the normal control group, the latent tuberculosis group and the active tuberculosis group each significantly increased (normal control group=4.6±6.2, latent tuberculosis group=25.3±24.1[p=0.0138]; normal control group=4.6±6.2). , active tuberculosis group = 15.6 ± 24.7 [p = 0.2055]; latent tuberculosis group = 25.3 ± 24.1, active tuberculosis group = 15.6 ± 24.7 [p = 0.1090]).

In addition, as shown in Figure 9, the SFC (×10 ⁵ cells) for recombinant protein K13 increased in the tuberculosis infection group compared to the normal control group, but was not statistically significant (normal control = 8.4 ± 13.6, tuberculosis infection group = 28.8). ±37.4[p=0.0971]), increased in the latent tuberculosis group and active tuberculosis group compared to the normal control group (normal control group=8.4±13.6, latent tuberculosis group=29.7±32.0[p=0.0779]; normal control group=8.4±13.6 , active tuberculosis group = 27.5 ± 45.4 [p = 0.2555]; latent tuberculosis group = 29.7 ± 32.0, active tuberculosis group = 27.5 ± 45.4 [p = 0.5033]).

In summary, the existing tuberculosis diagnostic antigens ESAT-6 and CFP-10 can diagnose tuberculosis by significantly increasing the level of interferon gamma secretion in the tuberculosis infection group compared to the normal control group, but it is possible to diagnose tuberculosis in the latent tuberculosis group and active tuberculosis group. No significant difference was observed between the levels of interferon gamma secretion between groups, so it was not possible to distinguish between latent tuberculosis and active tuberculosis.

However, it was confirmed that the K3 antigen can be used to distinguish active tuberculosis from latent tuberculosis by increasing the secretion of interferon gamma in the latent tuberculosis group.

<Experimental Example 4> Measurement of stimulation index (SI) value, which calculates the number of stimulated cells relative to the number of unstimulated cells

The stimulation index (SI) value was measured for the K3 recombinant antigen and the comparative antigens ESAT-6 and CFP-10, which is a value calculated by calculating the number of cells stimulated by the antigen and secreting interferon gamma relative to the number of PBMC cells not stimulated by the antigen. did. All measurements were corrected to blank (divided by the SFC of unstimulated cells).

As a result, as shown in Figure 10, it was confirmed that K3 had a higher SI value in the latent tuberculosis group compared to the active tuberculosis group. The above results suggest that K3 antigen can not only diagnose tuberculosis, but also distinguish between latent tuberculosis and active tuberculosis.

<Experimental Example 5> Analysis of cytokines and chemokines secreted after antigen stimulation

PBMCs extracted from healthy control (HC), latent TB infection (LTBI), and active TB (aTB) groups are stimulated with test antigen or comparison antigen, and secreted cytokines and chemokines are analyzed. did.

Specifically, 1×10 ⁶ /ml of PBMCs collected from healthy control (HC), latent TB infection (LTBI), and active TB (aTB) groups were cultured in a 96-well plate for 16 hours. After 24 hours of stimulation by adding the ESAT-6+CFP-10 mixture as a test antigen or comparison antigen, 25㎕ of the collected supernatant was taken and 37 types of cytotoxicity (Merk Millipore) were collected using the Milliplex MAP Human Cytokine/Chemokine 37-plex kit. ) Cytokines and chemokines were simultaneously measured using a luminex analyzer. The 37 types of cytokines and chemokines are as follows: EGF, Eotaxin/CCL11, FGF-2/FGF-basic, Fractalkine/CX3CL1, G-CSF, GM-CSF, IFN-α2, IFN-γ, IL-1 α. /IL-1F1, IL-1 beta/IL-1F2, IL-1 ra/IL-1F3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 /CXCL8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IP-10/CXCL10/CRG-2, MCP-1/CCL2, MCP -3/CCL7, MDC/CCL22, MIP-1α/CCL3, MIP-1β/CCL4, sCD40L, TGF-α, TNF-β/LTA, VEGF, Fit-3 Ligand, GRO. The experiment was repeated twice, and biomarkers with statistically significant differences between test groups were analyzed.

As a result, the cytokines that showed significant differences between the normal control group and the latent tuberculosis group in response to K3 antigen stimulation were Eotaxin, FGF-2, Fit-3L, Fractalkine, G-CSF, GM-CSF, GRO, IFN-α2, and IFN- γ, IL-12p70, IL-13, IL-1α, IL-1β, IL-1RA, IL-3, IL-4, IL-6, IL-7, IL-8, MIP-1α, MIP-1β, There were 25 types of TGF-α, TNF-α, TNF-β, and VEGF (p < 0.05), and cytokines that showed significant differences between the latent and active tuberculosis groups, as well as G-CSF, GM-CSF, IFN-γ, and IL. It was confirmed that there were 11 types of -1α, IL-1β, IL-1RA, IL-6, IL-8, MIP-1α, TGF-α, and TNF-β (p < 0.01) (Figures 11, 12, and 13). .

The above results show that 11 types of cytokines (G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, MIP-1α, TGF-α, TNF This suggests that -β) is a biomarker that can be used to distinguish between latent tuberculosis and active tuberculosis.

<110> Korea Disease Control and Prevention Agency <120> Antigen for diagnosing latent tuberculosis infection and use of that <130> 2020P-12-034 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> K3 <400> 1 Met Gln Arg Gln Ser Leu Met Pro Gln Gln Thr Leu Ala Ala Gly Val 1 5 10 15 Phe Val Gly Ala Leu Leu Cys Gly Val Val Thr Ala Ala Val Pro Pro 20 25 30 His Ala Arg Ala Asp Val Val Ala Tyr Leu Val Asn Val Thr Val Arg 35 40 45 Pro Gly Tyr Asn Phe Ala Asn Ala Asp Ala Ala Leu Ser Tyr Gly His 50 55 60 Gly Leu Cys Glu Lys Val Ser Arg Gly Arg Pro Tyr Ala Gln Ile Ile 65 70 75 80 Ala Asp Val Lys Ala Asp Phe Asp Thr Arg Asp Gln Tyr Gln Ala Ser 85 90 95 Tyr Leu Leu Ser Gln Ala Val Asn Glu Leu Cys Pro Ala Leu Ile Trp 100 105 110 Gln Leu Arg Asn Ser Ala Val Asp Asn Arg Arg Ser Gly 115 120 125 <210> 2 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> K4 <400> 2 Met Gly Ala Gly Thr Pro Ala Gly Gly Ser Ser Lys Ala Gly Leu Val 1 5 10 15 Ser Ala Thr Gly Pro Ala Asp Glu Asp Asp Lys Asp Pro Glu Asp Arg 20 25 30 Arg Asp Gln Pro Ser Gly Glu Arg Leu Ala 35 40 <210> 3 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> K6 <400> 3 Met Thr His Lys Arg Thr Lys Arg Gln Pro Ala Ile Ala Ala Gly Leu 1 5 10 15 Asn Ala Pro Arg Arg Asn Arg Val Gly Arg Gln His Gly Trp Pro Ala 20 25 30 Asp Val Pro Ser Ala Glu Gln Arg Arg Ala Gln Arg Gln Arg Asp Leu 35 40 45 Glu Ala Ile Arg Arg Ala Tyr Ala Glu Met Val Ala Thr Ser His Glu 50 55 60 Ile Asp Asp Asp Thr Ala Glu Leu Ala Leu Leu Ser Met His Leu Asp 65 70 75 80 Asp Glu Gln Arg Arg Leu Glu Ala Gly Met Lys Leu Gly Trp His Pro 85 90 95 Tyr His Phe Pro Asp Glu Pro Asp Ser Lys Gln 100 105 <210> 4 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> K10 <400> 4 Met Ile Ala Thr Thr Arg Asp Arg Glu Gly Ala Thr Met Ile Thr Phe 1 5 10 15 Arg Leu Arg Leu Pro Cys Arg Thr Ile Leu Arg Val Phe Ser Arg Asn 20 25 30 Pro Leu Val Arg Gly Thr Asp Arg Leu Glu Ala Val Val Met Leu Leu 35 40 45 Ala Val Thr Val Ser Leu Leu Thr Ile Pro Phe Ala Ala Ala Ala Gly 50 55 60 Thr Ala Val Gln Asp Ser Arg Ser His Val Tyr Ala His Gln Ala Gln 65 70 75 80 Thr Arg His Pro Ala Thr Ala Thr Val Ile Asp His Glu Gly Val Ile 85 90 95 Asp Ser Asn Thr Thr Ala Thr Ser Ala Pro Pro Arg Thr Lys Ile Thr 100 105 110 Val Pro Ala Arg Trp Val Val Asn Gly Ile Glu Arg Ser Gly Glu Val 115 120 125 Asn Ala Lys Pro Gly Thr Lys Ser Gly Asp Arg Val Gly Ile Trp Val 130 135 140 Asp Ser Ala Gly Gln Leu Val Asp Glu Pro Ala Pro Pro Ala Arg Ala 145 150 155 160 Ile Ala Asp Ala Ala Leu Ala Ala Leu Gly Leu Trp Leu Ser Val Ala 165 170 175 Ala Val Ala Gly Ala Leu Leu Ala Leu Thr Arg Ala Ile Leu Ile Arg 180 185 190 Val Arg Asn Ala Ser Trp Gln His Asp Ile Asp Ser Leu Phe Cys Thr 195 200 205 Gln Arg 210 <210> 5 <211> 339 <212> PRT <213> Artificial Sequence <220> <223> K11 <400> 5 Met Thr Glu Pro Ala Ala Trp Asp Glu Gly Lys Pro Arg Ile Ile Thr 1 5 10 15 Leu Thr Met Asn Pro Ala Leu Asp Ile Thr Thr Ser Val Asp Val Val 20 25 30 Arg Pro Thr Glu Lys Met Arg Cys Gly Ala Pro Arg Tyr Asp Pro Gly 35 40 45 Gly Gly Gly Ile Asn Val Ala Arg Ile Val His Val Leu Gly Gly Cys 50 55 60 Ser Thr Ala Leu Phe Pro Ala Gly Gly Ser Thr Gly Ser Leu Leu Met 65 70 75 80 Ala Leu Leu Gly Asp Ala Gly Val Pro Phe Arg Val Ile Pro Ile Ala 85 90 95 Ala Ser Thr Arg Glu Ser Phe Thr Val Asn Glu Ser Arg Thr Ala Lys 100 105 110 Gln Tyr Arg Phe Val Leu Pro Gly Pro Ser Leu Thr Val Ala Glu Gln 115 120 125 Glu Gln Cys Leu Asp Glu Leu Arg Gly Ala Ala Ala Ser Ala Ala Phe 130 135 140 Val Val Ala Ser Gly Ser Leu Pro Pro Gly Val Ala Ala Asp Tyr Tyr 145 150 155 160 Gln Arg Val Ala Asp Ile Cys Arg Arg Ser Ser Thr Pro Leu Ile Leu 165 170 175 Asp Thr Ser Gly Gly Gly Leu Gln His Ile Ser Ser Gly Val Phe Leu 180 185 190 Leu Lys Ala Ser Val Arg Glu Leu Arg Glu Cys Val Gly Ser Glu Leu 195 200 205 Leu Thr Glu Pro Glu Gln Leu Ala Ala Ala His Glu Leu Ile Asp Arg 210 215 220 Gly Arg Ala Glu Val Val Val Val Ser Leu Gly Ser Gln Gly Ala Leu 225 230 235 240 Leu Ala Thr Arg His Ala Ser His Arg Phe Ser Ser Ile Pro Met Thr 245 250 255 Ala Val Ser Gly Val Gly Ala Gly Asp Ala Met Val Ala Ala Ile Thr 260 265 270 Val Gly Leu Ser Arg Gly Trp Ser Leu Ile Lys Ser Val Arg Leu Gly 275 280 285 Asn Ala Ala Gly Ala Ala Met Leu Leu Thr Pro Gly Thr Ala Ala Cys 290 295 300 Asn Arg Asp Asp Val Glu Arg Phe Phe Glu Leu Ala Ala Glu Pro Thr 305 310 315 320 Glu Val Gly Gln Asp Gln Tyr Val Trp His Pro Ile Val Asn Pro Glu 325 330 335 Ala Ser Pro <210> 6 <211> 304 <212> PRT <213> Artificial Sequence <220> <223> K12 <400> 6 Met Lys Val Leu Gln Ile Ala Ile Tyr Ser Gly Arg Ser His Asp Cys 1 5 10 15 Phe Gly Val Cys Gln Thr Glu Leu Arg Gly Ala Thr Asp Arg Phe Val 20 25 30 Arg Cys Val Arg Ser His Gly Gln Ala Asp Arg Val Pro Gln Cys Asp 35 40 45 Leu Arg Asn Arg Ser Thr Leu Ala Ala Gly Cys Ile Thr Phe Glu Gln 50 55 60 Arg Trp Phe Arg Pro Ser His Arg Ala Ile Arg Arg Lys Val Arg Trp 65 70 75 80 Leu Gln Thr Tyr Phe Cys Gly Phe Ala Gly Leu Leu Thr Arg Ser Gly 85 90 95 Ser Arg Ile Val Leu Leu Ser Gly Ala Ala Gly Pro Gly Phe Ala Ala 100 105 110 Val Val Ala Pro Ala Phe Arg Gly Asp Cys Tyr Gly Gly Val Thr Ser 115 120 125 Val Arg Val Arg Ala Gln Thr His Arg Ser Asn Asn Gln Met Asp Ser 130 135 140 Leu Ser His Asp Pro Ala Ala Gly Asp Ile Gly Ser Gln Leu Val Glu 145 150 155 160 Ile Gly Ser Arg Gly Leu Ala Ala Gly Asn Ala Ala Thr Met Pro Thr 165 170 175 Met Thr Gly Leu Val Pro Ala Gly Gly Glu Glu Val Ser Ala Gln Ala 180 185 190 Val Met Ala Phe Ala Thr Glu Ala Ala Ser Met Ile Ala Ser Asn Thr 195 200 205 Ala Ala Gln Glu Glu Leu Met Arg Ala Gly Thr Ala Leu Thr Asp Ile 210 215 220 Ala Arg Met Tyr Gly Asp Thr Asp Asp Asn Ala Ala Gly Ala Leu Thr 225 230 235 240 Ile Gly Ala Gly Leu Thr Ser Arg His Pro Leu Ala Gly Gly Ser Gly 245 250 255 Ala Ser Ala Gly Ala Gly Leu Met Arg Ala Gly Ser Leu Pro Gly Glu 260 265 270 Ala Gly Ser Ala Ala Arg Thr Pro Leu Met Ala Gln Leu Leu Glu Gly 275 280 285 His Pro Arg Arg Trp His Arg Pro Pro Leu Thr Pro Asp Arg Arg Arg 290 295 300 <210> 7 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> K13 <400> 7 Met Ala Glu Asn Ser Asn Ile Asp Asp Ile Lys Ala Pro Leu Leu Ala 1 5 10 15 Ala Leu Gly Ala Ala Asp Leu Ala Leu Ala Thr Val Asn Glu Leu Ile 20 25 30 Thr Asn Leu Arg Glu Arg Ala Glu Glu Thr Arg Thr Asp Thr Arg Ser 35 40 45 Arg Val Glu Glu Ser Arg Ala Arg Leu Thr Lys Leu Gln Glu Asp Leu 50 55 60 Pro Glu Gln Leu Thr Glu Leu Arg Glu Lys Phe Thr Ala Glu Glu Leu 65 70 75 80 Arg Lys Ala Ala Glu Gly Tyr Leu Glu Ala Ala Thr Ser Arg Tyr Asn 85 90 95 Glu Leu Val Glu Arg Gly Glu Ala Ala Leu Glu Arg Leu Arg Ser Gln 100 105 110 Gln Ser Phe Glu Glu Val Ser Ala Arg Ala Glu Gly Tyr Val Asp Gln 115 120 125 Ala Val Glu Leu Thr Gln Glu Ala Leu Gly Thr Val Ala Ser Gln Thr 130 135 140 Arg Ala Val Gly Glu Arg Ala Ala Lys Leu Val Gly Ile Glu Leu Pro 145 150 155 160 Lys Lys Ala Ala Pro Ala Lys Lys Ala Ala Pro Ala Lys Lys Ala Ala 165 170 175 Pro Ala Lys Lys Ala Ala Ala Lys Lys Ala Pro Ala Lys Lys Ala Ala 180 185 190 Ala Lys Lys Val Thr Gln Lys 195

Claims (15)

It contains a recombinant antigen protein composed of the amino acid sequence of SEQ ID NO: 1, which is a membrane protein of M. tuberculosis K strain,
A composition for diagnosing tuberculosis, wherein the recombinant antigen protein induces a higher level of interferon gamma secretion in the latent tuberculosis group compared to the active tuberculosis group, thereby enabling the detection of latent tuberculosis and active tuberculosis.
delete delete delete delete delete delete delete 1) contacting a recombinant antigen protein consisting of the amino acid sequence of SEQ ID NO: 1 with an isolated sample of a subject or an isolated sample of a normal control;
2) measuring the level of cytokine secretion in an isolated sample of the subject or the normal control group, wherein the cytokine is interferon gamma (IFN-γ); and
3) If, as a result of the measurement in step 2), the secretion level of cytokines in the subject's sample is higher than that of the normal control group, determining tuberculosis, it is characterized in that latent tuberculosis and active tuberculosis can be distinguished and detected. How to provide information for tuberculosis diagnosis.
delete delete Including antibodies against recombinant antigen proteins and cytokines consisting of the amino acid sequence of SEQ ID NO: 1,
The cytokine is interferon gamma (IFN-γ), and is a kit for diagnosing tuberculosis, which can distinguish and detect latent tuberculosis and active tuberculosis.
delete The kit for diagnosing tuberculosis according to claim 12, wherein the kit is for ELIspot, ELISA or FACS analysis.
delete