High-specificity diagnostic reagent and kit for mycobacterium tuberculosis infection

Abstract

The invention relates to a high-specificity diagnostic reagent and kit for mycobacterium tuberculosis infection, and relates to the technical field of tuberculosis diagnostic products. The fusion protein contains a first fusion protein and a second fusion protein; the first fusion protein is a protein ESAT-6-CFP-10 encoded by a mycobacterium tuberculosis genome RD1 region, and the second fusion protein is a protein CFP-21-MPT-64 encoded by a mycobacterium tuberculosis genome RD2 region. According to the invention, tuberculosis specific RD2 region antigens CFP21 and MPT64 and RD1 region antigens ESAT-6 and CFP-10 are combined to be applied to diagnosis of mycobacterium tuberculosis infection for the first time, by virtue of the difference degree of RD1 and RD2 in mycobacterium antigen cross reactivity, immunogenicity advantages of the two antigens are concentrated, epitopes of the antigens are increased, human MHC limitation and polymorphism are overcome, and advantage complementation is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of Mycobacterium tuberculosis diagnostic products, and more specifically, to a highly specific diagnostic reagent and kit for Mycobacterium tuberculosis infection, and in particular to the use of Mycobacterium tuberculosis specific gene coding regions RD1 and RD2 and their antigens in the preparation of tuberculosis diagnostic reagents. Background Art

[0002] Tuberculosis (TB) is one of the major infectious diseases that seriously endangers human health in my country and the world. The 2022 Global Tuberculosis Annual Report released by WHO shows that about 1 / 4 of the world's population is infected with Mycobacterium tuberculosis (M.tb), which is a latent tuberculosis infection (LTBI) population; about 10 million new active tuberculosis cases are reported each year, and nearly 2 million deaths. The number of deaths caused by TB ranked first in the ranking of single-cause infectious disease causes of death before the COVID-19 pandemic. TB is listed as one of my country's major infectious diseases.

[0003] TB is a chronic infectious disease mainly caused by M.tb transmitted through the respiratory tract. Once infected, most individuals will establish an asymptomatic infection state, i.e. LTBI, which will accompany them for life. 5-10% of infected people may have their immune systems suppressed or damaged during their lifetime, such as co-infection with HIV, diabetes or use of immunosuppressive drugs, and the latent bacteria in their bodies may be reactivated and develop into active tuberculosis. Active tuberculosis includes forms such as pulmonary tuberculosis and extrapulmonary tuberculosis. Since active pulmonary tuberculosis is significantly contagious, early detection, early diagnosis and early isolation of active pulmonary tuberculosis patients are of great significance for the effective prevention and control of the spread and prevalence of TB. Sensitive and specific diagnostic methods are required for the diagnosis of pulmonary tuberculosis, extrapulmonary tuberculosis and LTBI. Unfortunately, the current clinical methods for diagnosing TB have obvious defects, resulting in the fact that this ancient disease has not been effectively controlled to date.

[0004] Traditional laboratory diagnostic techniques for TB mainly rely on bacteriological diagnosis and imaging. Direct microscopic examination of sputum smears with acid-fast staining is low-cost, simple, rapid, and easy to promote. However, its disadvantages are low sensitivity, only about 40%, and lack of specificity. It cannot distinguish M.tb in the specimen from nearly 200 other non-tuberculous mycobacteria of the genus Mycobacterium, and cannot diagnose LTBI. Although the isolation, culture, and identification of M.tb is the gold standard for diagnosing TB, with a sensitivity of 70%, the diagnosis takes up to 6-8 weeks and requires high conditions, such as a professional Class II biosafety laboratory. PCR-based molecular diagnostic techniques, such as GeneXpert (Sunnyvale, Cepheid, CA, USA) Detection of M.tb nucleic acid in specimens has high specificity and sensitivity and takes only 2-3 hours, but it cannot diagnose LTBI and requires special equipment. The main disadvantage of bacteriological diagnosis technology is that dead or alive M.tb must be present in clinical specimens. Imaging technology such as X-ray and CT assist in the diagnosis of tuberculosis is rapid and sensitive, but it requires special equipment and has poor diagnostic specificity due to the lack of etiological evidence. It can only be diagnosed after typical clinical symptoms, and it is difficult to diagnose extrapulmonary tuberculosis and LTBI.

[0005] Once M.tb is not present in clinical specimens, the above-mentioned bacteriological diagnostic techniques will misdiagnose or miss the diagnosis. In this case, immunological techniques such as serological tests, tuberculin skin test (TST) and interferon gamma release assays (IGRAs) can be applied. Serological tests such as ELISA and gold label detect anti-tuberculosis antibodies. Due to the high false negative and false positive rates, WHO has clearly proposed to stop using such methods as diagnostic tools for active tuberculosis. TST uses purified protein derivatives (PPD) prepared from M.tb or BCG vaccine strains for skin tests to detect the production of delayed-type hypersensitivity reactions in the skin and assist in the diagnosis of LTBI and tuberculosis. It has high sensitivity, low cost, simple operation, and it takes 3 days to observe the results. However, some antigens in the TST active ingredient PPD are cross-reactive antigens of the genus Mycobacterium, which exist in M.tb, as well as in the BCG vaccine strain or some non-tuberculous mycobacteria. Therefore, BCG vaccination, infection or carriage of non-tuberculous mycobacteria can significantly affect the diagnostic results of TST, resulting in a high false positive rate and poor specificity of TST diagnosis, and the inability to distinguish whether the patient has been immunized with BCG or infected with M.tb or non-tuberculous mycobacteria.

[0006] A new immunological method developed in the past 20 years is the interferon-γ release assay (IGRAs). Once the body is infected with M.tb, the M.tb antigen will sensitize the body's T cells after 3-4 weeks, and the sensitized T cells will exist in the body of the infected person and even in the peripheral blood for a long time, while people who have not been infected with M.tb do not have T cells sensitized by M.tb antigens. These sensitized T cells present in peripheral blood mononuclear cells (PBMCs) are stimulated with M.tb antigens or peptides in vitro. The antigen presenting cells (APCs) in the PBMCs process these antigens or peptides, degrade them into peptide fragments, and bind to MHC class II molecules on the surface of the APCs to form MHC-peptide complexes. The complexes are recognized by the T cell receptors (TCR) on the surface of the sensitized T cells in the PBMCs, causing the sensitized T cells to be reactivated and rapidly secrete interferon-γ (IFN-γ). Therefore, based on the difference in the amount of IFN-γ, people infected with M.tb can be distinguished from those who are not infected. This is the technical principle of IGRAs. Based on this principle, the QuantiFERON-TB Gold test (QFT-GIT, Cellestis Limited, Carnegie, Victoria, Australia) and T-SPOT.TB test (Oxford Immunotec Limited, Abingdon, United Kingdom) have been successfully developed abroad and can be used to diagnose active tuberculosis and latent tuberculosis infection. The specific method of QFT-GIT is to first collect the peripheral whole blood of the subject, stimulate the peripheral whole blood with the RD1 region antigens ESAT-6 and CFP-10 of M.tb, and then test the IFN-γ content in the serum by IFN-γ ELISA quantitative method; T-SPOT.TB first collects the peripheral whole blood of the subject and separates the PBMCs therein, and after ESAT-6 or CFP-10 peptide stimulation, uses ELISPOT technology to detect the number of T cells that secrete IFN-γ. The US CDC, UK NIHCE, EU CDC, Japan and China all recommend IGRAs for the screening of LTBI and the diagnosis of active tuberculosis in all age groups and high-risk groups. It is also an immunological diagnostic technology for screening LTBI recommended for promotion and application by WHO.

[0007] Antigens or peptides as stimuli are the core and key to the specificity and sensitivity of IGRAs. Undoubtedly, the specificity of IGRAs diagnostic results is subject to the degree of antigenic cross-reactivity of the stimulating antigens or peptides used. At present, all IGRAs kits at home and abroad cannot do without the stimuli of the low molecular weight proteins ESAT-6 and CFP-10 or their peptides, which were discovered in the earliest stage and encoded by the RD1 region of the M.tb genome. Studies at an earlier stage have revealed that the genomes of most non-tuberculous mycobacteria species widely present in BCG and the environment lack the RD1 region, so it is preliminarily assumed that the cross-reactivity of the RD1 region antigen is low. However, through subsequent research and Blastn homology sequence alignment, ESAT-6 is not only found in TB pathogens, namely the Mycobacterium tuberculosis complex (such as Mycobacterium tuberculosis, Mycobacterium marinum and Mycobacterium africanum, etc.), but also in 12 other Mycobacterium species with more than 90% amino acid sequence homology, including Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium attenuatum, Mycobacterium lacus, Mycobacterium simiae, Mycobacterium shinjukuense, Mycobacterium kubicae, Mycobacterium riyadhense, Mycobacterium simulans, Mycobacterium vicinigordonae, Mycobacterium shottsii, Mycobacterium decipiens, etc., especially Mycobacterium ulcerans and Mycobacterium marinum, which are very common pathogens that cause non-tuberculous mycobacterial diseases in clinical practice.CFP-10 is not only found in TB pathogens. There are 14 other Mycobacterium species with amino acid sequence homology exceeding 90% including Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium szulgai, Mycobacterium gordonae, Mycobacterium lacus, Mycobacterium liflandii, Mycobacterium shinjukuense, Mycobacterium asiaticum, Mycobacterium basiliense, Mycobacterium kubicae, Mycobacterium paragordonae, Mycobacterium riyadhense, and Mycobacterium angelicum, especially Mycobacterium ulcerans, Mycobacteriummarinum, Mycobacterium kansasii, Mycobacterium szulgai, Mycobacterium gordonae, Mycobacterium lacus, Mycobacterium liflandii, Mycobacterium shinjukuense, Mycobacterium asiaticum, Mycobacterium basiliense, Mycobacterium kubicae, Mycobacterium paragordonae, Mycobacterium riyadhense, and Mycobacterium angelicum. gordonae is a very common pathogen that causes non-tuberculous mycobacterial disease in clinical practice. Therefore, since the ESAT-6 and CFP-10 antigens used in the current IGRAs kits have a wide range of antigenic cross-reactivity in the genus Mycobacterium, a positive IGRAs test result means that the diagnosis may be caused by the above-mentioned non-tuberculous mycobacterial infection in addition to Mycobacterium tuberculosis infection. In fact, my country has conducted several epidemiological surveys on the infection rate or carrier rate of non-tuberculous mycobacteria in the national population, revealing that the infection positive rate of the Chinese population has reached 20%. At present, all IGRAs technologies based on ESAT-6 and CFP-10 derived peptides or antigens on the market have no doubt that their diagnostic specificity will be significantly affected by these antigenic cross-reactive bacteria and have defects, but there is still no newer technology to solve this problem.

[0008] A large number of studies have revealed the efficiency of IGRAs in clinical applications. A meta-analysis by Sester et al. showed that the overall sensitivity and specificity of T-SPOT.TB in diagnosing active tuberculosis were 81% (95% CI: 78%-81%) and 59% (95% CI: 56%-62%), respectively, and the overall sensitivity and specificity of QFT-GIT were 80% (95% CI: 75%-84%) and 79% (95% CI: 75%-82%), respectively. In another meta-analysis of diagnostic tests from low- and middle-income countries, Metcalfe et al. reported that in adult patients suspected of tuberculosis, the overall sensitivity and specificity of T-SPOT.TB for diagnosing active tuberculosis were 83% (95% CI: 63% to 94%) and 61% (95% CI: 40% to 79%), respectively, and the overall sensitivity and specificity of QFT-GIT were 69% (95% CI: 52% to 83%) and 52% (95% CI: 41% to 63%), respectively. These studies have clearly confirmed that the sensitivity and specificity of QFT-GIT and T-SPOT.TB diagnosis are still not ideal. More clinical trials have also shown that the sensitivity and specificity of the IGRAs test kits independently developed in China are not as high as or only comparable to similar foreign test kits. The current clinical application of IGRAs has not been able to objectively evaluate and reflect the specificity of such kits established based on the principle of using antigens or peptides derived from the RD1 region as stimuli, and there is a significant problem of false positive diagnosis caused by non-tuberculous mycobacterium infection.

[0009] On the other hand, the sensitivity of T-SPOT.TB in diagnosing active tuberculosis is higher than that of QFT-GIT, reaching more than 80%, but this result is still objectively limited by the technology's own peptide stimuli. T-SPOT.TB uses ESAT-6 or CFP-10 peptides as stimuli. The T cell epitopes covered by the peptides are narrow and affected by the epitope conformation. The epitopes may be destroyed when processed by APC. At the same time, due to the restriction and polymorphism of human MHC, these peptides have significant differences in their stimulation reactivity in patients from different regions, races or genders, which weakens their diagnostic sensitivity. Although some test kits have improved the diagnostic sensitivity by combining more TB antigens such as Rv3615c and TB7.7, the effect is not significant.

[0010] Therefore, the sensitivity and specificity issues of IGRAs, which are currently commercially used at home and abroad, have been significantly ignored in the absence of kits with higher sensitivity and specificity. As a result, IGRAs are more widely used only in non-TB endemic countries; due to the poor clinical diagnostic performance of IGRAs, countries with a high TB ​​burden often do not use IGRAs as one of the objective indicators for diagnosis. How to improve the diagnostic performance of IGRAs technology so that it can be included as an objective indicator for diagnosis in countries with a high TB ​​burden is not only a very important scientific issue, but also has significant practical value.

[0011] The key to solving the above problems is to find antigens that are more specific than the antigens encoded in the RD1 region. We found that the RD2 region genes in the M.tb genome only exist in the M.tb complex group, and are not present in the BCG Pasteur strain and Danish strain commonly vaccinated in the Chinese population, as well as the Copenhagen strain, Tice strain, Glaxo strain, Prague strain, Philippine strain, etc., nor in non-tuberculous mycobacterium species with amino acid sequence homology exceeding 90%. Therefore, the antigen encoded in the RD2 region has a higher degree of specificity than the antigen encoded in the RD1 region, and the degree of antigen cross-reactivity between different species of Mycobacterium is lower. The RD2 region genes include 12 open reading frames, from Rv1978 to Rv1988. We expressed these genes and purified the corresponding antigens through genetic engineering; PMBCs were collected from active TB, LTBI and uninfected healthy people, and PMBCs were stimulated with these antigens respectively. Based on the interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) technology, two of the antigens, MPT64 (Rv1980c) and CFP21 (Rv1984c), were screened as strong T cell reactive antigens.

[0012] However, adding more antigens will make the antigen composition of the IGRAS kit diagnostic reagents complex, increase the cost of the clinical diagnostic kit, and make it unsuitable for widespread promotion in my country and other countries with a high TB ​​burden. Summary of the invention

[0013] The present invention is based on the diagnostic technology of sensitized T cells releasing interferon-γ, comprehensively applies the difference in the antigenic cross-reactivity between different species of bacteria of the genus Mycobacterium in the RD1 region and RD2 region antigens of the Mycobacterium tuberculosis genome, and combines the RD1 region antigens ESAT-6 (Rv3875) and CFP-10 (Rv3874), and the RD2 region antigens CFP21 (Rv1984c) and MPT64 (Rv1980c), so as to achieve the purpose of improving the specificity of the IGRA diagnostic kit and increasing the detection sensitivity at the same time.

[0014] According to a first aspect of the present invention, a fusion protein composition is provided for use in preparing a reagent for detecting infection with Mycobacterium tuberculosis, wherein the fusion protein composition comprises a first fusion protein and a second fusion protein; the first fusion protein is a fusion protein rEC composed of the protein ESAT-6 and the protein CFP-10 encoded in the RD1 region of the Mycobacterium tuberculosis genome, and the second fusion protein is a fusion protein rCM composed of the protein CFP-21 and the protein MPT-64 encoded in the RD2 region of the Mycobacterium tuberculosis genome;

[0015] The sequence of the fusion protein rEC is shown in SEQ ID NO:5, and the sequence of the fusion protein rCM is shown in SEQ ID NO:6.

[0016] Preferably, the first fusion protein and the second fusion protein serve as antigen stimulators.

[0017] Preferably, when both the first fusion protein antigen detection and the second fusion protein antigen detection are positive, the sample is positive for Mycobacterium tuberculosis infection;

[0018] When the first fusion protein antigen test is negative and the second fusion protein antigen test is positive, the sample is positive for Mycobacterium tuberculosis infection.

[0019] Preferably, when the first fusion protein antigen test is positive and the second fusion protein antigen test is negative, the sample is positive for non-tuberculous mycobacterium infection.

[0020] Preferably, when both the first fusion protein antigen detection and the second fusion protein antigen detection are negative, the sample is excluded from Mycobacterium tuberculosis infection.

[0021] According to another aspect of the present invention, a reagent for detecting Mycobacterium tuberculosis infection is provided, wherein the kit is a fusion protein composition, and the fusion protein composition comprises a first fusion protein and a second fusion protein; the first fusion protein is a fusion protein rEC composed of the protein ESAT-6 and the protein CFP-10 encoded in the RD1 region of the Mycobacterium tuberculosis genome, and the second fusion protein is a fusion protein rCM composed of the protein CFP-21 and the protein MPT-64 encoded in the RD2 region of the Mycobacterium tuberculosis genome;

[0022] The sequence of the fusion protein rEC is shown in SEQ ID NO:5, and the sequence of the fusion protein rCM is shown in SEQ ID NO:6.

[0023] According to another aspect of the present invention, a kit for detecting Mycobacterium tuberculosis infection is provided, the kit comprising a fusion protein composition, the fusion protein composition comprising a first fusion protein and a second fusion protein; the first fusion protein is a fusion protein rEC composed of the protein ESAT-6 encoded by the RD1 region of the Mycobacterium tuberculosis genome and the protein CFP-10, and the second fusion protein is a fusion protein rCM composed of the protein CFP-21 encoded by the RD2 region of the Mycobacterium tuberculosis genome and the protein MPT-64;

[0024] The sequence of the fusion protein rEC is shown in SEQ ID NO:5, and the sequence of the fusion protein rCM is shown in SEQ ID NO:6.

[0025] Preferably, the kit is an ELISA kit.

[0026] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0027] (1) In order to improve the defects of low specificity and insufficient sensitivity of the existing IGRAs detection technology, the present invention adopts a method of adding epitopes, and the RD1 region antigens ESAT-6 (Rv3875) and CFP-10 (Rv3874), and the RD2 region antigens MPT64 (Rv1980c) and CFP21 (Rv1984c) are fused and expressed, and purified to prepare the RD1 region antigen and the RD2 region antigen; during the detection process, the antigens processed by APC in PBMCs will produce more and richer T cell epitopes than a single antigen or epitope and overcome the MHC polymorphism and restriction of the population, thereby improving the sensitivity of diagnosis. More importantly, the RD1 region antigen and the RD2 region antigen are combined as stimuli respectively, and the difference in the degree of antigen cross-reactivity between the two antigens in the mycobacterium bacterial species is utilized to improve the specificity of diagnosis. Finally, the ELISPOT technology established in this project by combining RD1 region antigen and RD2 region antigen to diagnose Mycobacterium tuberculosis infection can not only improve the sensitivity of diagnosis, but also improve the specificity of diagnosis. At the same time, it has three major capabilities: diagnosing Mycobacterium tuberculosis infection, identifying non-tuberculous mycobacterium infection, and even accurately excluding Mycobacterium tuberculosis infection.

[0028] (2) The present invention combines tuberculosis-specific RD2 region antigens CFP-21 and MPT-64 with RD1 region antigens ESAT-6 and CFP-10 for the diagnosis of Mycobacterium tuberculosis infection for the first time. By taking advantage of the difference in the cross-reactivity between RD1 and RD2 in mycobacterium antigens, the immunogenicity advantages of the two types of antigens are concentrated, the antigen epitopes are increased, and the deficiencies of the destruction of peptide epitopes when APC processes peptides, human MHC restriction and polymorphism, etc. in the traditional technology are overcome, so as to achieve complementary advantages, thereby avoiding the deficiencies of similar kits at home and abroad that only use RD1 antigen or peptide as a stimulus; due to the combination of 4 antigens, false negatives caused by low-frequency single antigen or peptide-specific T cells can be reduced; the number of spots in the well will be significantly increased, and the detection results will be clearer compared with the single antigen or peptide well. These improvements ultimately improve the sensitivity and specificity of the detection; at the same time, it has the three major capabilities of diagnosing Mycobacterium tuberculosis infection, distinguishing non-tuberculous mycobacterium infection, and even accurately excluding Mycobacterium tuberculosis infection. In addition, the combined antigen format reduces stimuli, thereby simplifying the detection design and operation process, significantly reducing the cost of the test kit, and facilitating its promotion and application.

[0029] (3) More importantly, because the antigen cross-reactivity of RD1 and RD2 antigens has significant differences between different species of Mycobacterium, in the present invention, when both the antigen RD1 well and the RD2 well react positively, or the RD2 well reacts positively, it is diagnosed as positive infection with Mycobacterium tuberculosis; when the antigen RD1 well is positive and the RD2 well reacts negatively, it is diagnosed as positive infection with non-tuberculous mycobacteria; when both the antigen RD1 well and the RD2 well react negatively, the diagnosis of Mycobacterium tuberculosis infection can be excluded. The above diagnostic features are functions that the IGRAs currently used in clinical practice do not have.

[0030] (4) The present invention uses the difference in the degree of antigenic cross-reactivity between different species of Mycobacterium in the RD1 region and RD2 region antigens of the Mycobacterium tuberculosis genome, combines the RD1 region antigens ESAT-6 (Rv3875) and CFP-10 (Rv3874), and the RD2 region antigens CFP21 (Rv1984c) and MPT64 (Rv1980c) as stimuli, and combines the ELISPOT technology to detect the number of T cells that secrete gamma interferon or the number of T cells that secrete interferon-inducible protein 10 that are specific to tuberculosis antigens, so as to achieve the purpose of improving the specificity of the IGRA diagnostic kit and increasing the detection sensitivity at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The schematic diagram is a structural diagram of the recombinant protein particle constructed by the present invention and a purification and identification diagram of its expression product.

[0032] Figure 2It shows the head-to-head test results of 24 persons suspected of being infected with Mycobacterium tuberculosis by the kit of the present invention and T-SPOT.TB.

[0033] Figure 3 and Figure 4 The results of head-to-head testing of different stimulation antigens by the kit of the present invention and T-SPOT.TB in 24 clinically suspected patients with Mycobacterium tuberculosis infection were further disclosed in detail.

[0034] Figure 5 The results of head-to-head testing of 24 patients with difficult-to-diagnose tuberculosis using the kit of the present invention and domestic reagents on the market are shown.

[0035] Figure 6 and Figure 7 It shows the head-to-head test results of 24 patients with difficult-to-diagnose tuberculosis using the fusion protein reagent of the present invention and domestic diagnostic reagents on the market, as well as other laboratory test results of the patients. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] In order to improve the specificity of detection, in addition to using RD1 region antigens ESAT-6 (SEQ ID NO: 1) and CFP-10 (SEQ ID NO: 2), the present invention particularly found that RD2 region antigens only exist in TB pathogens such as Mycobacterium tuberculosis and Mycobacterium bovis, and are missing in BCG strains immunized by the Chinese population and other bacteria of the genus Mycobacterium. Combined with Mycobacterium tuberculosis RD2 region antigens, it is more specific for diagnosis than RD1 region antigen alone. Preferred RD2 region antigens are CFP21 (SEQ ID NO: 3) (Rv1984c) and MPT64 (SEQ ID NO: 4) (Rv1980c), which can stimulate the peripheral blood of tuberculosis patients and LTBI patients to produce high levels of IFN-γ. Combined with RD1 region antigens and RD2 region antigens for IGRAs, it can diagnose Mycobacterium tuberculosis infection, identify non-tuberculous mycobacterium infection, and exclude Mycobacterium tuberculosis infection.

[0038] Secondly, in order to improve the sensitivity of detection, the present invention proposes to use recombinant protein form, connect the genes of ESAT-6 and CFP-10, as well as CFP21 and MPT64 together, clone them into Escherichia coli expression vectors for engineering expression, and then further purify the fusion proteins ESAT-6-CFP-10 (SEQ ID NO: 5) and CFP21-MPT64 (SEQ ID NO: 6) on a large scale. The antigens prepared by this strategy concentrate the advantages of the immunogenicity of the two antigens, increase the epitopes of the antigens, and overcome the restriction and polymorphism of human MHC, achieving complementary advantages, thereby avoiding the shortcomings of similar kits at home and abroad that use ESAT-6 and CFP-10 antigens or peptides as stimuli, and significantly improving the sensitivity of detection.

[0039] Finally, the preparation of stimuli using recombinant proteins is simpler than the synthesis of peptides, which significantly reduces the cost of the kit and is conducive to its promotion and application.

[0040] The present invention overcomes the defect of low specificity of existing IGRAs detection technology, provides a method for preparing recombinant proteins in RD1 region and RD2 region, and uses recombinant protein antigens as stimulants, combines ELISPOT technology to detect the number of T cells secreting gamma interferon specific to tuberculosis antigens, or combines other technologies for detection. The recombinant protein used in the detection kit can not only improve the specificity of the IGRAs diagnostic kit, but also increase the detection sensitivity.

[0041] The present invention provides a tuberculosis-specific recombinant protein antigen, wherein the genes of ESAT-6 and CFP-10, as well as CFP21 and MPT64 are connected together, cloned into an Escherichia coli expression vector for engineered expression, and then the fusion proteins ESAT-6-CFP-10 and CFP21-MPT64 are further purified on a large scale to prepare the desired antigen as an antigen stimulator.

[0042] The present invention provides a method for preparing a recombinant protein of a fusion protein for diagnosis of tuberculosis infection, comprising a recombinant protein of an RD1 region antigen ESAT-6-CFP-10 and a recombinant protein of an RD2 region antigen CFP21-MPT64. The amino acid sequence of the fusion protein rEC is shown in SEQ ID NO.5. The amino acid sequence of the fusion protein rCM is shown in SEQ ID NO.6. The antigen in the composition can be a derivative analogue of the above-mentioned antigen with the same antigenicity after substitution, deletion or addition of one or several amino acids in the amino acid sequence.

[0043] In some embodiments, the recombinant protein is engineered to be expressed using an E. coli expression vector.

[0044] The present invention provides an application of a tuberculosis antigen fusion protein in in vitro detection of specific T cell immune response caused by Mycobacterium tuberculosis infection. After human T cells are stimulated by the composition, cytokines secreted by Mycobacterium tuberculosis specific T cells are detected.

[0045] In some embodiments, the detected cytokine is interferon-γ (IFN-γ) or interferon-induced protein 10 (IP-10 or CXCL10).

[0046] In some embodiments, the product for diagnosing tuberculosis infection includes: specific recombinant protein antigens ESAT-6-CFP-10 and CFP21-MPT64, IFN-γ specific antibodies, and IFN-γ specific antibodies with colorimetric markers.

[0047] In some embodiments, the source of human T cells includes: human peripheral blood, cerebrospinal fluid or pleural effusion, etc.

[0048] In some embodiments, the detection method for diagnosing tuberculosis infection is an enzyme-linked immunospot assay (ELISPOT).

[0049] Among them, the kit for enzyme-linked immunosorbent spot assay (ELISPOT) is composed as follows:

[0050] (1) Fusion protein ESAT-6-CFP-10 (SEQ ID NO: 5) and fusion protein CFP21-MPT64 (SEQ ID NO: 6);

[0051] (2) phytohemagglutinin solution or other non-specific positive stimulators of T cells;

[0052] (3) 96-well PVDF membrane plate for ELISPOT;

[0053] (4) human interferon-γ monoclonal antibody (primary antibody), biotin-labeled human interferon-γ monoclonal detection antibody (secondary antibody), horseradish peroxidase-labeled streptavidin and horseradish peroxidase reaction substrate;

[0054] (5) Other reagents and consumables required for ELISPOT assay.

[0055] In some embodiments, the antigen is used for clinical detection of Mycobacterium tuberculosis infection, comprising the following steps:

[0056] 1) The screening criteria for the volunteer cases involved in the present invention are:

[0057] (1) Suspected latent tuberculosis infection:

[0058] ① No clinical evidence of active tuberculosis infection or typical imaging evidence of pulmonary tuberculosis;

[0059] ② No bacteriological evidence of tuberculosis infection in the sample

[0060] ③ No history of autoimmune diseases, infectious diseases, malignant tumors or other diseases or immunosuppressant treatment.

[0061] ④ The clinician's prescription requires T-SPOT.TB diagnosis and the patient has not received any anti-tuberculosis treatment.

[0062] (2) Patients with suspected difficult-to-diagnose tuberculosis:

[0063] ① The patient has clinical symptoms related to active pulmonary tuberculosis, such as cough, weight loss, fever, etc.;

[0064] ②CT imaging evidence;

[0065] ③ Pathogenic examination of tuberculosis in specimens such as sputum or bronchoalveolar lavage fluid, including morphological evidence, serological evidence or molecular biological evidence;

[0066] ④ The clinician's prescription required T-SPOT.TB diagnosis and had not received any anti-tuberculosis treatment. The suspected cases and healthy volunteers were aged between 18 and 60 years old, and the gender was randomly selected in a 1:1 ratio. Finally, this project screened 24 suspected difficult-to-diagnose tuberculosis patients and 24 suspected latent tuberculosis patients.

[0067] 2) Isolation of peripheral blood mononuclear cells (PBMC):

[0068] The PBMC used in the present invention comes from the venous peripheral blood of individuals. The selected individuals are informed by the experimenter of the specific project process and the amount of blood required. After the volunteers agree and sign the informed consent form, the clinical doctor will collect blood from the volunteers. Disposable vacuum blood collection tubes containing heparin anticoagulation are used for blood collection, and about 5mL of blood is collected from each volunteer. Then PBMC is separated:

[0069] (1) Freshly collected peripheral blood was thoroughly mixed with an equal amount of sterile PBS solution in a ratio of peripheral blood: phosphate buffered saline (PBS, pH 7.4): Ficoll separation solution = 1:1:1. The diluted blood was slowly superimposed on the Ficoll separation solution along the wall of a 15 mL centrifuge tube using a dropper. The blood was added gently and slowly, and a clear interface was maintained. The screw cap was tightened.

[0070] (2) Carefully place the 15 mL centrifuge tube in the previous step into the centrifuge, adjust the lifting speed to the lowest, and centrifuge horizontally at 800g for 2 minutes. After centrifugation, the blood can be seen to be divided into 4 layers. The upper layer is serum and RPMI1640 culture medium, and the lower layer is mainly red blood cells and granulocytes. The middle layer is lymphocyte separation fluid. At the interface between the upper and middle layers, there is a white cloud layer mainly composed of mononuclear cells. Then carefully aspirate the cells in the cloud layer into a new centrifuge tube, which are mononuclear cells (PBMCs).

[0071] (3) Place the cells obtained in step 2 into another 15 mL centrifuge tube, add an equal volume of phosphate buffered saline (PBS, pH 7.4) to wash the cells, mix gently, carefully place in a centrifuge, and centrifuge at 600 g for 10 minutes at room temperature (18°C-25°C).

[0072] Carefully discard the supernatant, add 1 mL of 37°C preheated RPIM1640 complete medium containing 5% serum, gently pipette to resuspend, then add preheated 1640 complete medium to 10 mL, carefully place in a centrifuge, and centrifuge at 600g for 10 minutes at room temperature (18°C-25°C).

[0073] (4) Discard the supernatant and resuspend in 1 mL of RPIM1640 complete medium containing 10% serum. Take an appropriate amount and count the cells on a cell counter, and adjust the final concentration to 2.5 × 10 6 cells / mL;

[0074] 3) Antigen preparation

[0075] As described above, the prepared antigen was diluted with RPIM1640 medium containing 10% serum to a final concentration of 0.1 mg / mL.

[0076] 4) ELISPOT detection of specific T cells after combined antigen stimulation

[0077] The ELISPOT plate was coated with anti-human interferon-γ monoclonal antibody diluted in phosphate buffered saline (PBS, pH 7.4) and placed at 4° C. for more than 12 hours. Before adding stimulating antigens and cells, the plate was blocked with RPIM1640 medium containing 10% serum at room temperature for 1 hour.

[0078] PBMC cells from each sample were taken and added to four wells of the ELISPOT plate, with 100 μL of cell solution added to each well.

[0079] The four wells were set as follows: physiological saline negative control well, RD1 antigen solution well, RD2 antigen solution well, and phytohemagglutinin (PHA) positive control well. 50 μL of each solution was added to the four wells.

[0080] Incubate the ELISPOT plate at 37°C, 5% CO2 for 18-24 hours.

[0081] 5) ELISPOT plate washing and result acquisition:

[0082] (1) After the incubation, the cell solution in the wells was discarded, and 200 μL of room temperature deionized water was quickly added to each well for washing twice, and then washed three times with PBST (PBS + 0.05% Tween-20).

[0083] (2) Remove the wash solution, pat dry on absorbent paper, add 100 μL of diluted detection antibody to each well, and incubate at room temperature for 2 hours.

[0084] (3) Remove the detection antibody solution, wash three times with PBST, then add 100 μL of diluted avidin-HRP conjugate to each well and incubate at room temperature for 1 hour.

[0085] (4) Color development: Remove the avidin-HRP conjugate solution, wash three times with PBST, and then wash twice with PBS. Dry firmly on absorbent paper, then add 100 μL of AEC substrate solution to each well, incubate at room temperature for 15 to 30 minutes, and when clear spots are seen, rinse the front and back of the membrane with distilled water to terminate the reaction.

[0086] (5) Dry at 37°C or room temperature, then place the ELISPOT plate on an automatic plate reader to count the number of spots in the reaction wells, adjust the parameters and perform quality control to give the final reaction results.

[0087] The following are specific embodiments

[0088] Example 1 Amplification of target genes in the RD1 region and RD2 region of the present invention and construction of expression vectors

[0089] (1) First, primers were designed to amplify ESAT-6 and CFP10 in the RD1 region and CFP21 and MPT64 in the RD2 region. The PCR reaction conditions were as follows: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 1 minute, 60°C annealing for 1 minute, 72°C extension for 45 seconds, 30 cycles; 72°C extension for 5 minutes. The PCR products were purified using a purification kit.

[0090] (2) Amplify the fusion genes ESAT-6-CFP10 and CFP21-MPT64 and clone them into the pProEXHTb plasmid respectively: Use the gene splicing method, take the PCR product mixture of ESAT-6 (Rv3875), CFP10 (Rv3874) and CFP21 (Rv1984c), MPT64 (Rv1980c) as template, and use the designed upstream primers and downstream primers for amplification. The PCR reaction conditions are as follows: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 1 minute, renaturation at 60°C for 1 minute, extension at 72°C for 90 seconds, 30 cycles; extension at 72°C for 10 minutes. After purification, the PCR product was double-digested with BamHI and HindIII, cloned and constructed into the plasmid pProEXHTb, and the expression vectors pPro610 and pPro2164 were established. The structural schematic diagram of the expression vectors pPro610 and pPro2164 is shown in Figure 1 A, 1B; Enzyme digestion identification of expression vectors pPro610 and pPro2164 is shown in Figure 1 C.

[0091] (3) Efficient expression of recombinant proteins ESAT-6-CFP10 and CFP21-MPT64 in Escherichia coli

[0092] The constructed expression vectors pPro610 and pPro2164 were transformed into Escherichia coli BL21 (DE3) strains and expressed efficiently. The steps are as follows: activate the cells, transfer them into 1 liter of LB medium and shake for 2-3 hours, then induce them with 1mM IPTG at 37℃ for 4 hours. Collect the cells by centrifugation, add 2×SDS loading buffer, and boil in water for 3 minutes. The expression product was confirmed by 12.5% ​​SDS-PAGE electrophoresis.

[0093] (4) Purification of recombinant proteins ESAT-6-CFP10 and CFP21-MPT64

[0094] ESAT-6-CFP10 fusion protein (rEC) and CFP21-MPT64 fusion protein (rCM) were present in the precipitate in the form of inclusion bodies. Protein purification was performed according to the instructions of the Ni-NTA purification system (Invitrogen).

[0095] The steps are briefly summarized as follows: 50 mL of bacterial solution was collected by centrifugation and resuspended in 8 mL of guanidine hydrochloride lysis buffer (6 M guanidine hydrochloride, 20 mM sodium phosphate pH 7.8, 500 mM sodium chloride). After ultrasonic disruption of bacteria in an ice bath, the cells were centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was added to Ni-NTA resin pretreated with denaturing binding buffer (8 M urea, 200 mM PBS pH 7.8, 500 mM sodium chloride). Incubate at room temperature for 30 minutes, wash the column twice with 4 mL of denaturing binding buffer (8M Urea, 200 mM PBS pH 7.8, 500 mM NaCl), twice with denaturing washing buffer (8M Urea, 20 mM PBS pH 6.0, 500 mM NaCl), four times with non-denaturing washing buffer (20 mM PBS, 500 mM NaCl, 20 mM imidazole, pH 8.0), and finally elute with 8 mL of non-denaturing elution buffer (20 mM PBS, 500 mM NaCl, 250 mM imidazole, pH 8.0). Take 10 μL of the supernatant at each step of the purification process for 12% SDS-PAGE analysis. The supernatant was placed in a dialysis tape and washed once with 6M, 4M, 2M, 1M, and 0.5M urea wash solutions (each urea wash solution contains 5mMTris-Cl), and finally washed twice with 0M urea (containing 0.6057g Tris-Cl). The dialysate was vacuum freeze-dried to obtain the recombinant protein lyophilized powder. It was stored at -20℃ for future use. The purification process and SDS-PAGE identification of the final purified rEC protein and rCM protein are shown in Figure 1 DF.

[0096] Figure 1 A in FIG. 1 is a schematic diagram of the structure of the expression vector pPro610; Figure 1 B is a schematic diagram of the structure of the expression vector pPro2164; Figure 1 The M in the C represents a DNA molecular weight standard; lanes 1-4 represent (1) PCR products of ESAT-6; (2) PCR products of CFP10; (3) PCR products of ESAT-6-CFP10; (4) products of pPro610 plasmid digestion; lanes 5-8 represent (5) PCR products of CFP21; (6) PCR products of MPT64; (7) PCR products of CFP21-MPT64; (8) products of pPro2164 plasmid digestion; Figure 1 The M in D represents the protein molecular weight standard, lane 1 is the lysis product of the engineered bacteria before IPTG induction, lanes 2-3 represent the lysis products of the engineered bacteria 4h and 6h after IPTG induction, lanes 4-7 represent the removal process of impurity proteins, and lane 8 represents the purified rCM protein. Figure 1The M in E represents the protein molecular weight standard, lanes 1-4 represent the removal process of impurity proteins, and lane 5 represents the purified rEC protein; Figure 1 F in the figure is the final product of purified rCM protein and rEC protein.

[0097] Example 2 Application of fusion protein antigen in clinical detection of Mycobacterium tuberculosis infection

[0098] Peripheral blood was collected from subjects, with inclusion criteria as described above, for a total of 48 subjects. The fusion protein described in Example 1 of the present invention was used to compare the fusion protein with imported diagnostic reagents and domestic diagnostic reagents available on the market.

[0099] The ELISpot kit for the diagnosis of Mycobacterium tuberculosis infection contains the following components:

[0100] (1) the rEC protein and rCM fusion protein described in Example 1;

[0101] (2) phytohemagglutinin (PHA), positive stimulant solution;

[0102] (3) normal saline (NS), negative stimuli;

[0103] (4)ELISPOT 96-well PVDF membrane plate;

[0104] (5) human interferon-γ monoclonal antibody (primary antibody), biotin-labeled human interferon-γ monoclonal detection antibody (secondary antibody), horseradish peroxidase-labeled streptavidin and horseradish peroxidase reaction substrate;

[0105] The concentrations of the rEC and rCM fusion proteins were both 0.1 mg / mL.

[0106] The specific steps include:

[0107] (1) ELISPOT plates were coated with anti-human interferon-γ monoclonal antibodies diluted in phosphate buffer (pH 7.4) for more than 12 hours in advance, placed flat at 4°C, and blocked with RPIM1640 medium containing 10% serum at room temperature for 1 hour before adding stimulating antigens and cells.

[0108] (2) Use lymphocyte separation solution to separate PBMCs, take 100 μL of 2.5×10 5 Cells were added to the ELISPOT plate that had been pre-coated with anti-human interferon-γ monoclonal antibody. Cells were added to 4 wells for each sample, namely the negative control well, the stimulation antigen rEC protein well, the rCM protein well, and the positive control well, and 50 μL of the corresponding solution was added to each well.

[0109] (3) Incubate the ELISPOT plate at 37°C and 5% CO2 for 18-24 hours. After incubation, discard the cell solution in the wells, quickly add 200 μL of room temperature deionized water to each well, wash twice, and then wash three times with PBST (PBS + 0.05% Tween-20).

[0110] (4) Remove the wash solution, pat dry on absorbent paper, add 100 μL of diluted detection antibody to each well, and incubate at room temperature for 2 hours.

[0111] (5) Remove the detection antibody solution, wash three times with PBST, then add 100 μL of diluted avidin-HRP conjugate to each well and incubate at room temperature for 1 hour.

[0112] (6) Color development: Remove the avidin-HRP conjugate solution, wash three times with PBST, and then wash twice with PBS. Dry firmly on absorbent paper, then add 100 μL of AEC substrate solution to each well, incubate at room temperature for 15 to 30 minutes, and when clear spots are seen, rinse the front and back of the membrane with distilled water to terminate the reaction.

[0113] (7) Allow to dry at 37°C or room temperature, then place the ELISPOT plate on an automatic plate reader to count the number of spots in the reaction wells, adjust the parameters, perform quality control, and give the final reaction results.

[0114] (8) Result analysis:

[0115] a. When the number of spots in the negative control well is 0-5 and (the number of spots in the antigen RD1 or RD2 well) - (the number of spots in the negative control well) ≥ 6, the well is judged as a positive well;

[0116] b. When the number of spots in the negative control well is 6-10 and (the number of spots in the antigen RD1 or RD2 well) ≥ 2× (the number of spots in the negative control well), the well is judged as a positive well.

[0117] (9) Interpretation of results:

[0118] a. When both the antigen RD1 and RD2 holes are positive, or the RD2 hole is positive, it indicates that the patient is positive for Mycobacterium tuberculosis infection;

[0119] b. When the antigen RD1 well is positive and the RD2 well is negative, it indicates that the patient is positive for nontuberculous mycobacterium infection.

[0120] c. When the antigen RD1 hole is negative and the RD2 hole is negative, it indicates that the patient can be ruled out of positive Mycobacterium tuberculosis infection.

[0121] d. When the number of spots in the negative control well exceeds 10, the result is "uncertain" and needs to be retested.

[0122] In the results of Example 2, Figure 2 It shows the head-to-head test results of 24 suspected Mycobacterium tuberculosis infected persons by the kit of the present invention and T-SPOT.TB respectively; Figure 3 and Figure 4 The results of head-to-head testing of different stimulation antigens by the kit of the present invention and T-SPOT.TB in 24 clinically suspected patients with Mycobacterium tuberculosis infection were further disclosed in detail, among which patient No. 20 was detected as non-tuberculous mycobacterium infection by the kit of the present invention; Table 1 compares the positive rates of the kit of the present invention and T-SPOT.TB in diagnosing 24 suspected patients with Mycobacterium tuberculosis infection; Table 2 compares the diagnostic results of the kit of the present invention and T-SPOT.TB in 24 suspected patients with Mycobacterium tuberculosis infection, among which 1 / 24 (4.2%) of the patients diagnosed as NTM by the present invention were negative for T-SPOT.TB diagnosis; Table 3 compares the diagnostic performance coincidence rates of the kit of the present invention and T-SPOT.TB.

[0123] Table 1

[0124]

[0125] Table 2

[0126]

[0127]

[0128] Table 3

[0129]

[0130] In the results of Example 2, Figure 5 It shows the head-to-head test results of 24 patients with difficult-to-diagnose tuberculosis using the kit of the present invention and domestic reagents on the market; Figure 6 and Figure 7 It shows the head-to-head test results of 24 patients with difficult-to-diagnose tuberculosis using the fusion protein reagent of the present invention and domestic diagnostic reagents on the market, as well as other laboratory test results of the patients; Figure 6It is disclosed that the clinical CT diagnosis of No. 125 and No. 126 was negative, the domestic kit diagnosed it as positive for tuberculosis infection, and the kit of the present invention diagnosed it as non-tuberculous mycobacterium infection. The clinical CT diagnosis of No. 127 was negative, the domestic kit diagnosed it as negative for tuberculosis infection, and the kit of the present invention diagnosed it as non-tuberculous mycobacterium infection. Table 4 compares the diagnostic positivity rate of the fusion protein reagent of the present invention and the imported diagnostic reagents on the market in 24 patients with difficult-to-diagnose tuberculosis; Table 5 compares the diagnostic results of the fusion protein reagent of the present invention and chest CT in 24 patients with difficult-to-diagnose tuberculosis, among which the patients diagnosed as NTM by the present invention accounted for 3 / 24 ( 12.5%), and the CT results of these 3 patients were all diagnosed as negative; Table 6 shows the statistical diagnosis results of 24 patients by the fusion protein of the present invention and the domestic diagnostic reagents on the market, among which 3 / 24 (12.5%) patients diagnosed as NTM by the present invention; Table 7 compares the diagnostic performance of the fusion protein reagent of the present invention and the domestic diagnostic reagents on the market for 24 patients with difficult-to-diagnose tuberculosis, with CT as the diagnostic standard, among which, the fusion protein used in the present invention has a diagnostic sensitivity of 94.4% for difficult-to-diagnose TB positive patients, a specificity of 66.7%, a positive predictive value of 89.5%, and a negative predictive value of 80.0%, all of which are higher than the domestic diagnostic reagents on the market.

[0131] Table 4

[0132]

[0133] Table 5

[0134]

[0135] Table 6

[0136]

[0137] Table 7

[0138]

[0139] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a fusion protein composition for preparing a reagent for detecting Mycobacterium tuberculosis infection, characterized in that: The fusion protein composition comprises a first fusion protein and a second fusion protein; the first fusion protein is a fusion protein rEC composed of the protein ESAT-6 and the protein CFP-10 encoded in the RD1 region of the Mycobacterium tuberculosis genome, and the second fusion protein is a fusion protein rCM composed of the protein CFP-21 and the protein MPT-64 encoded in the RD2 region of the Mycobacterium tuberculosis genome; The sequence of the fusion protein rEC is shown in SEQ ID NO:5, and the sequence of the fusion protein rCM is shown in SEQ ID NO:

6.

2. The use according to claim 1, characterized in that The first fusion protein and the second fusion protein serve as antigen stimulators.

3. The use according to claim 1, characterized in that When both the first fusion protein antigen detection and the second fusion protein antigen detection are positive, the sample is positive for Mycobacterium tuberculosis infection; When the first fusion protein antigen test is negative and the second fusion protein antigen test is positive, the sample is positive for Mycobacterium tuberculosis infection.

4. The use according to claim 1, characterized in that When the first fusion protein antigen test is positive and the second fusion protein antigen test is negative, the sample is positive for nontuberculous mycobacterium infection.

5. The use according to claim 1, characterized in that When both the first fusion protein antigen detection and the second fusion protein antigen detection are negative, the sample is excluded from Mycobacterium tuberculosis infection.

6. A reagent for detecting Mycobacterium tuberculosis infection, characterized in that: The kit is a fusion protein composition, which includes a first fusion protein and a second fusion protein; the first fusion protein is a fusion protein rEC composed of the protein ESAT-6 and the protein CFP-10 encoded in the RD1 region of the Mycobacterium tuberculosis genome, and the second fusion protein is a fusion protein rCM composed of the protein CFP-21 and the protein MPT-64 encoded in the RD2 region of the Mycobacterium tuberculosis genome; The sequence of the fusion protein rEC is shown in SEQ ID NO:5, and the sequence of the fusion protein rCM is shown in SEQ ID NO:

6.

7. A kit for detecting Mycobacterium tuberculosis infection, characterized in that: The kit comprises a fusion protein composition, which comprises a first fusion protein and a second fusion protein; the first fusion protein is a fusion protein rEC composed of the protein ESAT-6 and the protein CFP-10 encoded in the RD1 region of the Mycobacterium tuberculosis genome, and the second fusion protein is a fusion protein rCM composed of the protein CFP-21 and the protein MPT-64 encoded in the RD2 region of the Mycobacterium tuberculosis genome; The sequence of the fusion protein rEC is shown in SEQ ID NO:5, and the sequence of the fusion protein rCM is shown in SEQ ID NO:

6.

8. The kit according to claim 7, characterized in that The kit is an ELISA kit.

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