Application of Mycobacterium tuberculosis T cell epitope protein Rv1566c-444

Abstract

The present invention relates to the field of molecular biology and immunology technology, and specifically discloses an application of a Mycobacterium tuberculosis T cell epitope protein Rv1566c-444. The present invention provides an application of a Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 in the preparation of tuberculosis detection reagents and vaccines; wherein the amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is shown in SEQ ID NO.4. The Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 of the present invention can reduce false negatives caused by the stimulation of T cells by intrinsic protein antigens of the test kit, thereby improving the detection sensitivity. The cellular immune response is good, and it can be widely used in related fields such as auxiliary diagnosis of tuberculosis, epidemiological monitoring and infection screening, and provides a new idea for the development of new anti-tuberculosis vaccines.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology and immunology, and in particular to an application of Mycobacterium tuberculosis T cell epitope protein Rv1566c-444. Background Art

[0002] Tuberculosis is a zoonotic disease caused by Mycobacterium tuberculosis. It has high morbidity, prevalence and mortality, and is prone to drug resistance and multidrug resistance, which is increasing. Rapid point-of-care diagnostic tests, new vaccines or effective preventive treatments are the priorities to end the tuberculosis epidemic.

[0003] Early diagnosis of tuberculosis is crucial to the control of tuberculosis. The lack of rapid, sensitive and specific diagnostic tools poses a huge challenge to the prevention and treatment of tuberculosis. The protective immune mechanism against tuberculosis is still not fully understood. Cellular immunity, especially the immune response of T lymphocytes in the circulation and infection site, is believed to play an important role. Therefore, whether the body's T cell immune response can be effectively stimulated, especially CD4 + T cell immune response is the key to the body's success in fighting tuberculosis infection. T lymphocytes sensitized by pathogens will release interferon-γ after being stimulated by the same antigen again. High levels of interferon-γ response can be used to evaluate cell-mediated host immune response, thus indicating infection with the pathogen. In recent years, immunological methods have played an increasingly important role in the diagnosis of tuberculosis or latent tuberculosis infection (LTBI). With the development of genomics, T cell-based in vitro interferon-γ release assays (IGRA) have appeared on the market (LoBue PA, 2012, JAMA 308:241–2), which can be used for auxiliary diagnosis of tuberculosis and screening of tuberculosis infection. Commercial IGRA reagents that use the tuberculosis-specific antigen 6kDa early secretory antigen target [ESAT-6] and culture filtrate protein 10 [CFP-10] encoded in the RD1 region of the Mycobacterium tuberculosis genome as stimuli, such as the QuantiFERON-TB Gold test detected by enzyme-linked immunosorbent assay (ELISA) technology and the T-SPOT.TB test detected by solid-phase enzyme-linked immunospot (ELISPOT) technology, have been used to detect tuberculosis-specific T lymphocytes that release gamma interferon in peripheral blood, and can show high sensitivity and specificity.

[0004] Bacillus Calmette-Guérin (BCG) is the only anti-tuberculosis vaccine approved for human use. It has been shown to induce protective immunity in humans, but it only has limited immune protection against childhood tuberculosis. The development strategy of new anti-tuberculosis vaccines is mainly to prevent tuberculosis infection as a substitute for BCG and a heterologous booster vaccine after BCG primary immunization, or to prevent latent infection, or as a therapeutic vaccine. Recombinant protein subunit vaccines with mixed adjuvants have attracted much attention due to their good safety and clear composition. In mouse models, tuberculosis antigen Ag85B can induce a strong Th1 immune response against Mycobacterium tuberculosis attack (Ahmad F, 2017, Front Immunol 8:1608), so the immunodominant antigen Ag85B is one of the important antigens that can be used in the development of new anti-tuberculosis vaccines.

[0005] The immunogenicity of tuberculosis antigens plays a decisive role in the development of tuberculosis diagnostic reagents and new anti-tuberculosis vaccines based on the immune response caused by immunodominant antigens. Because of the unique intracellular survival mode of Mycobacterium tuberculosis, the protective immune response against Mycobacterium tuberculosis infection in tuberculosis patients is mainly mediated by T cells. Therefore, tuberculosis antigens that can induce strong T cell immune responses can be used as candidate components of tuberculosis diagnostic reagents and new anti-tuberculosis vaccines. Antigenic epitopes are part of antigens and are the core basis of antigenicity; antigens bind to corresponding lymphocyte surface receptors through antigenic epitopes to sensitize lymphocytes and induce immune responses, that is, the specificity of immune responses is directed against antigenic epitopes, not against the entire antigen. Antigenic epitopes play a decisive role in the process of antigen-induced immune responses. The T cell epitope of an antigen is a special chemical group that determines whether the antigen can induce a T cell response in the human body. Protein antigens rich in T cell epitopes have great potential in the development of tuberculosis diagnostic reagents and new anti-tuberculosis vaccines.

[0006] Although the current interferon-γ release test analysis technology has advantages in improving the specificity of diagnosing Mycobacterium tuberculosis infection in late-stage or BCG-vaccinated populations or those exposed to non-tuberculous mycobacteria, it cannot accurately distinguish between latent tuberculosis infection and active tuberculosis. Moreover, this technology mainly uses complete antigens as the corresponding stimulus source, the composition of the antigen components is relatively complex, the sensitivity and specificity need to be improved, and the corresponding reagent cost is relatively high, which is not suitable for large-scale application under the current circumstances.

[0007] Although BCG plays a very important role in preventing active tuberculosis in children under 5 years old, the protection of BCG generally only lasts for 5 to 10 years. The protection of BCG gradually weakens over time, so that it has almost no protective effect against adult tuberculosis. All new anti-tuberculosis vaccines are still in the clinical trial stage, and none of them can really be put on the market. Therefore, the demand for new tuberculosis vaccines is very urgent. Because the immunogenicity of the protein antigens contained in the tuberculosis diagnostic reagents used on the market and the existing anti-tuberculosis vaccines in the research stage still cannot induce the required sufficient immune protection response, finding antigens with good immunogenicity is crucial for the development of tuberculosis diagnostic reagents and new vaccines. Summary of the invention

[0008] The purpose of the present invention is to provide an application of a Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 of the Mycobacterium tuberculosis antigen protein Rv1566c T cell epitope.

[0009] Specifically, the technical solution of the present invention is as follows:

[0010] In the first aspect, the present invention provides an application of a Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 in the preparation of a tuberculosis detection reagent; wherein the amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is as shown in SEQ ID NO.4, or is an amino acid sequence having the same immunogenicity and the same antigenicity formed by replacing, deleting or adding one or several amino acids in the sequence.

[0011] In a second aspect, the present invention provides an application of a Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 in the preparation of a tuberculosis vaccine; wherein the amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is as shown in SEQID NO.4, or is an amino acid sequence with the same immunogenicity and the same antigenicity formed by replacing, deleting or adding one or several amino acids in the sequence.

[0012] In a third aspect, the present invention provides an application of a Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 in the preparation of a drug for treating tuberculosis; wherein the amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is as shown in SEQ ID NO.4, or is an amino acid sequence having the same immunogenicity and the same antigenicity formed by replacing, deleting or adding one or several amino acids in the sequence.

[0013] The present invention has found that the T cell epitope protein expressed by the gene Rv1566c (RipD) from Mycobacterium tuberculosis has a fairly good performance in the serological diagnosis of tuberculosis. A total of 29 human T cell epitopes were obtained by predicting it with computer software. As a potential immunodominant T antigen protein, Rv1566c has good immunogenicity in theory. The 29 human T cell epitopes predicted in Rv1566c can be divided into six epitope concentration regions. Among the corresponding six epitope concentration region peptides synthesized, only peptide segment 6 (D6) produces a positive reaction in the ELISPOT test of the population, and the diagnostic sensitivity of peptide segment 6 in the population is only 5.8%. In order to make Rv1566c more immunodominant, the present invention intercepts all human T cell epitopes in Rv1566c and deletes redundant sequences to form a T cell epitope protein Rv1566c-444, which is expected to replace Rv1566c and exert better immunogenicity. The present invention finally obtained the scheme of the present invention on the basis of full analysis, verification and comparative study of tuberculosis protein antigens and their epitopes. Through experimental research, it was found that the T cell epitope protein Rv1566c-444 of the present invention has the same diagnostic performance as Rv1566c in diagnosing active tuberculosis using ELISPOT, the sensitivity of Rv1566c-444 in the population is 44.44%, and the diagnostic sensitivity of Rv1566c is 30.56%. The T cell epitope protein Rv1566c-444 greatly improves the sensitivity of diagnosis, exceeding peptide segment 6 and Rv1566c. Surprisingly, Rv1566c-444 also detected a tuberculosis infection case that was misdiagnosed as negative by the T-SPOT.TB kit. When Rv1566c-444 was combined with the peptides (ESAT-6, CFP-10, Rv3615c) provided by the T-SPOT.TB kit for diagnosis, the diagnostic sensitivity of the T-SPOT.TB kit was increased from 94.44% to 97.36%, and this case was not detected by Rv1566c. In addition, Rv1566c-444 can induce an immune response in mice that is significantly stronger than that of Rv1566c. The detection reagent based on the T cell epitope T cell epitope protein of the present invention can be widely used in related fields such as auxiliary diagnosis of tuberculosis, development of new anti-tuberculosis vaccines, epidemiological monitoring and infection screening.

[0014] In the present invention, the nucleotide sequence encoding the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is shown in SEQ ID NO.3.

[0015] Gene Rv1566c-444n comes from Rv1566c gene and contains all 29 human T cell epitopes in Rv1566c gene. The sequence of gene Rv1566c contains 693 bases and the sequence of gene Rv1566c-444n contains 444 bases.

[0016] In a fourth aspect, the present invention provides a tuberculosis diagnostic reagent, which includes a Mycobacterium tuberculosis T cell epitope protein Rv1566c-444, or a DNA molecule encoding the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444, or a recombinant protein produced by a recombinant bacterium containing the DNA molecule; the amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is as shown in SEQ ID NO.4, or it is an amino acid sequence with the same immunogenicity and the same antigenicity formed by replacing, deleting or adding one or several amino acids in the sequence, and the nucleotide sequence of the DNA molecule is as shown in SEQ ID NO.3.

[0017] In a fifth aspect, the present invention provides a tuberculosis-specific interferon gamma (IFN-γ) release test kit containing the above-mentioned tuberculosis diagnostic reagent.

[0018] In the present invention, the kit further comprises:

[0019] ①Primary antibody: mouse IgG monoclonal antibody against human or animal IFN-γ;

[0020] ②Enzyme-labeled reagents: horseradish peroxidase-labeled monoclonal antibodies of different subtypes of IgG against human or animal IFN-γ;

[0021] ③ Standard product: positive raw material containing IFN-γ;

[0022] ④ Culture plate: A 96-well microwell reaction plate containing PVDF membrane or nitrocellulose membrane or enzyme-linked immunosorbent assay. The positive control wells contain nonspecific stimulating antigens for tuberculosis, and the negative control wells contain PBS or basal fluid.

[0023] In a sixth aspect, the present invention provides a tuberculosis vaccine, comprising an effective ingredient and an adjuvant, wherein the effective ingredient comprises the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444, or a DNA molecule encoding the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444, or a recombinant protein produced by a recombinant bacterium containing the DNA molecule; the amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is as shown in SEQ ID NO.4, or an amino acid sequence with the same immunogenicity and the same antigenicity formed by replacing, deleting or adding one or several amino acids in the sequence, and the nucleotide sequence of the DNA molecule is as shown in SEQ ID NO.3.

[0024] In the seventh aspect, the present invention provides an anti-tuberculosis drug, the active ingredients of which include a polyclonal antibody prepared by immunizing experimental animals with the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 as an immunogen and an adjuvant, or a monoclonal antibody that recognizes the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 antigen prepared by immunizing experimental animals with the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 as an immunogen and an adjuvant using hybridoma technology or DNA recombination technology, or a therapeutic biological product prepared by using the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 as an immunogen and an adjuvant; the amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is as shown in SEQ ID NO.4, or it is an amino acid sequence with the same immunogenicity and antigenicity formed by replacing, deleting or adding one or several amino acids in the sequence.

[0025] The beneficial effects of the present invention are at least:

[0026] The present invention uses peripheral blood mononuclear cells from the same test population to diagnose infection using IGRA technology, and finds that protein Rv1566c and / or the antigen T cell epitope T cell epitope protein Rv1566c-444 contained therein can successfully diagnose tuberculosis infection. Compared with the T-SPOT.TB test kit, Rv1566c-444 also detects a tuberculosis infection case that was misdiagnosed as negative by the T-SPOT.TB test kit. The present invention provides a T cell epitope protein antigen that is different from the antigen used in previous test kits. Compared with previous detection methods, the present invention can reduce false negatives caused by stimulation of T cells by inherent antigens of the test kit, thereby improving detection sensitivity.

[0027] When the same adjuvant DDA and Poly I:C mixture was used to immunize BALB / c mice for immunogenicity testing, the protein Rv1566c immunization group could induce a cellular immune response equivalent to that of the Ag85B immunization group, and the protein Rv1566c-444 immunization group induced a cellular immune response superior to that of the protein Rv1566c immunization group.

[0028] The present invention uses a prokaryotic expression system to express purified protein, is suitable for large-scale commercial production, and has low cost. In addition, the present invention predicts the human T cell epitopes of the Rv1566c gene through computer software, thereby further selecting the epitope concentration sequence Rv1566c-444n, and the recombinant protein Rv1566c-444 expressed by the sequence has a diagnostic effect superior to that of the parent protein and a cellular immune response caused in a mouse model because the redundant sequence of the original protein itself is deleted, which reduces the cost of full-length protein synthesis and overcomes the shortcomings of insufficient immunogenicity caused by insufficient concentration of single antigen protein epitopes and the presence of redundant sequences.

[0029] The present invention verifies that Rv1566c and / or the T cell epitope protein Rv1566c-444 contained therein can be used for specific detection of tuberculosis infection, provides an antigen-derived T cell epitope protein, and applies the T cell epitope protein to the detection of tuberculosis cases and healthy volunteers. The detection reagent based on the present invention can be widely used in related fields such as auxiliary diagnosis of tuberculosis, epidemiological monitoring and infection screening, and provides a new idea for the development of new anti-tuberculosis vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Distribution of six T cell epitope-enriched domains in Rv1566c.

[0031] Figure 2 The present invention is an SDS-PAGE analysis electrophoresis pattern of the purification and expression of the recombinant proteins Rv1566c-444 and Rv1566c of the present invention, M: pre-stained color protein standard molecular weight, sample 1 is the pET32a-Rv1566c-444 transformed into Escherichia coli without induction; samples 2 and 3 are the supernatant and precipitate of the induced pET32a-Rv1566c-444 after ultrasonic centrifugation; sample 4 is the purified recombinant protein Rv1566c-444; sample 5 is the pET32a-Rv1566c transformed into Escherichia coli without induction; samples 6 and 7 are the supernatant and precipitate of the induced pET32a-Rv1566c after ultrasonic centrifugation; sample 8 is the purified recombinant protein Rv1566c.

[0032] Figure 3 This is the ELISPOT response analysis of recombinant proteins Rv1566c-444 and Rv1566c in tuberculosis patients and healthy volunteers. A is the scatter plot of effector T cells produced by subjects stimulated by recombinant proteins, and B is the ROC curve of recombinant proteins. T-SPOT.TB represents the T-SPOT.TB kit, which was purchased from Beijing Tongsheng Times Biotechnology Co., Ltd.

[0033] Figure 4The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows:

[0034] Figure 5 It is the level of antigen-specific cytokines released by spleen cells of immunized mice. Among them, A is the comparison result of IFN-γ cytokine level, B is the comparison result of IL-6 cytokine level, and C is the comparison result of IL-4 cytokine level. Stimulated by represents the stimulus source, Immunized by represents the immune source, and antigens / DP is the mixed emulsion of antigen and DP adjuvant.

[0035] Figure 6 CD3 in spleen of mice immunized with different proteins + CD4 + T cells and CD3 + CD8 + The proportion of T cells and the CD4 secretion of different cytokines + or CD8 + Among them, A is the proportion of CD3 T cells in the spleen cells of mice in different immunization groups. + CD4 + T cells and CD3 + CD8 + T cell ratio statistics, B is CD4 + Statistical results of the ratio of IFN-γ / TNF-α / IL-4 produced by T cells, C is CD8 + Statistical results of the ratio of IFN-γ / TNF-α / IL-4 produced by T cells.

[0036] In each figure (if there is a statistically significant difference between the two groups compared), * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0038] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0039] The present invention uses computer technology to analyze the human T cell epitopes of the Rv1566c gene of Mycobacterium tuberculosis, and further selects Rv1566c-444n containing all 29 human T cell epitopes in the Rv1566c gene. Specifically, the technical solution of the present invention is to use computer technology to analyze the human T cell epitopes of the Rv1566c gene, divide the 29 T cell epitopes into six epitope concentration regions, identify the diagnostic efficacy of epitope polypeptide 6, and then select all sequences containing the T cell epitope region, namely Rv1566c-444n. Using H37Rv genomic DNA as a template, the full sequence of the Rv1566c gene and the T cell epitope region sequence Rv1566c-444n were amplified by polymerase chain reaction (PCR) technology, and then connected to the pET-32a plasmid after double restriction digestion, transformed into Escherichia coli BL21 (DE3), and protein expression was induced by isopropyl β-d-thiogalactoside (IPTG). After purification, the proteins Rv1566c and Rv1566c-444 were firstly detected in tuberculosis cases and healthy volunteers using the existing T cell gamma interferon release test technology to evaluate the sensitivity and specificity of the antigen and epitope peptide for tuberculosis detection. Subsequently, the proteins Rv1566c and Rv1566c-444 were mixed with adjuvants dimethyl trihexadecylammonium (DDA) and polyinosinic acid (Poly I:C) to immunize BALB / c mice for protein immunogenicity detection.

[0040] Example 1 Prediction of T cell epitopes and selection of sequences rich in T cell epitopes

[0041] The sequence of the Mycobacterium tuberculosis Rv1566c gene is from the National Center for Biotechnology Information (NCBI), see SEQ ID No. 1, and the corresponding protein sequence is seen in SEQ ID No. 2. T cell epitope prediction software TEpredict and IEDB-AR were used to predict T cell epitopes in the Rv1566c gene that can bind to human HLA-A02 supertype alleles (including HLA-A*0201, *0202, *0203, *0206 motifs). A total of 29 human T cell epitopes were predicted in the Rv1566c gene, which can be divided into six epitope concentration regions. The present invention designs a sequence that retains all 29 T cell epitopes contained in the Rv1566c gene. The targeting DNA sequence is named Rv1566c-444n, see SEQ ID No.3, located at nucleotide positions 109 to 552 of the Rv1566c gene, and the corresponding protein is defined as Rv1566c-444, see SEQ ID No.4. The distribution of the 6 T cell epitope enrichment domains in the Rv1566c gene is shown in Figure 1 .

[0042] Example 2 Verification of T cell epitopes

[0043] The gene sequences of the six epitope concentration regions of Rv1566c were sent to Shanghai Shenggong Biotechnology Co., Ltd. to synthesize the corresponding six epitope peptides. About 20 ml of peripheral blood from patients with pulmonary tuberculosis was collected, and peripheral blood lymphocytes were separated. The six synthesized peptides were used to stimulate peripheral blood lymphocytes from 10 patients with pulmonary tuberculosis. The Mycobacterium tuberculosis effector T cell detection kit (enzyme-linked immunospot assay) purchased from Beijing Jinhao Pharmaceutical was used to perform ELISPOT test according to the instructions. If the ELISPOT result of one tuberculosis patient among the 10 results was positive according to the kit standard, then this epitope peptide was determined to be a positive human T cell epitope peptide. The sixth peptide ( Figure 1 The D6 in the sample was determined as a positive human T cell epitope polypeptide. Subsequently, ELISPOT experiments were performed using blood samples from 51 tuberculosis patients and 55 healthy volunteers to determine the antigenicity of the T cell epitope polypeptide 6 of Rv1566c in the two populations.

[0044] The screening criteria for the volunteer cases involved are: patients diagnosed with pulmonary tuberculosis by clinical symptoms, signs and chest imaging examinations according to the national standard "Diagnostic Criteria for Pulmonary Tuberculosis" (WS288-2008) issued by the Ministry of Health, or tuberculosis patients who are positive or negative by bacteriological examination (sputum smear acid-fast staining microscopy and / or Roche solid culture medium bacteria solid culture); and patients with extrapulmonary tuberculosis (such as bone tuberculosis, renal tuberculosis, intestinal tuberculosis, lymph node tuberculosis, etc.). The screening criteria for healthy volunteers: no clinical symptoms of tuberculosis, no history of close contact with tuberculosis patients, and no other diseases or infections. The selected tuberculosis patients and volunteers are between 15 and 80 years old and are randomly selected from a continuous time sample of visits to the tuberculosis ward.

[0045] The diagnostic efficacy results of T cell epitope peptide 6 of Rv1566c presented in the human population are shown in Table 1 .

[0046] Table 1

[0047]

[0048] Example 3 Gene amplification, expression and purification of recombinant proteins pET-32a-Rv1566c and pET-32a-Rv1566c-444

[0049] Using the genomic DNA of Mycobacterium tuberculosis H37Rv as a template, the full sequence of the Rv1566c gene and the epitope region sequence Rv1566c-444n were amplified by PCR technology. After being digested with EcoRI and hind, they were connected to the pET-32a plasmid and transformed into Escherichia coli BL21 (DE3). 1mM isopropyl β-d-thiogalactoside (IPTG) was used to induce protein expression in the culture medium. After shaking and culturing at 37℃ for 3.5h, the cells were collected by centrifugation at 4℃ (4000rpm, 10min, 4℃). The expression level and form of Rv1566c and Rv1566c-444 in Escherichia coli were detected by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the inclusion body protein was denatured with 8M urea and purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography. Then, the dialysis solution was dialyzed with a low concentration of urea gradient, and finally the dialysis solution was replaced with 20 mM Tris-HCL (pH 8.0) to complete the refolding. Finally, the 0.22 μm filter was used for sterile filtration. SDS-PAGE analysis showed that Rv1566c-444 and Rv1566c proteins were mainly expressed in the form of inclusion body proteins, with molecular weights of approximately 35 kDa and 45 kDa, respectively. Figure 2 .

[0050] Example 4 Development of an ELISPOT test kit for detecting tuberculosis using antigens and / or their T cell epitope protein antigens

[0051] The antigen and its T cell epitope peptide of the present invention can be used for the detection of tuberculosis infection to form a kit for clinical or laboratory diagnosis. The kit is designed based on the double antibody sandwich principle and uses the ELISPOT method to detect the antigen. The experimental process is as follows: the primary antibody coated on the PVDF membrane can bind to IFN-γ in the cell supernatant as a capture antibody, and IFN-γ can be captured and colored by the enzyme-labeled secondary antibody. The two antibodies are monoclonal antibodies that recognize different antigenic epitopes of IFN-γ.

[0052] The basic composition of the kit is as follows:

[0053] 1. The protein antigen Rv1566c prepared in Example 3 / or the T cell epitope protein antigen Rv1566c-444 synthesized in Example 3;

[0054] 2. Primary antibody: mouse IgG monoclonal antibody against human or animal IFN-γ;

[0055] 3. Enzyme-labeled reagents: horseradish peroxidase-labeled monoclonal antibodies of different subtypes of IgG against human or animal IFN-γ;

[0056] 4. Calibrator: positive raw material containing IFN-γ;

[0057] 5. Culture plate: A 96-well microwell reaction plate with a PVDF membrane or a nitrocellulose membrane. The positive control wells contain nonspecific stimulating antigens for tuberculosis (such as PHA, etc.), and the negative control wells contain PBS or basal fluid.

[0058] 6. Other reagents and consumables required for ELISPOT testing.

[0059] Example 5 Protein antigen Rv1566c and / or T cell epitope protein antigen Rv1566c-444 for clinical detection of tuberculosis infection

[0060] 1. Isolation of peripheral blood lymphocytes:

[0061] The screening criteria for volunteer cases involved in the embodiments of the present invention are shown in Example 2.

[0062] Before the start of the trial, the experimenter informed the volunteers / tuberculosis patients of the purpose, significance, experimental process, and the required specimens and their quantity of the specific project. After they agreed and signed the informed consent form, the clinical laboratory technician drew blood from them. In the end, blood samples were collected from 36 tuberculosis volunteers and 44 healthy volunteers in this project. The peripheral venous blood was collected using endotoxin-free heparin anticoagulation vacuum blood collection tubes, and about 20 ml of blood was collected from each volunteer.

[0063] 1) PBMCs were separated using Ficoll-Hypaque separation buffer within 4 hours.

[0064] 2) First, dilute the whole blood with RPMI-1640 medium in a 1:1 ratio and mix well. Add a certain volume of separation solution to the centrifuge tube, spread the diluted blood sample on the surface of the separation solution, and keep the interface between the two liquid surfaces clear. The volume ratio of separation solution, anticoagulant undiluted whole blood, and RPMI 1640 medium is 1:1:1. Centrifuge at room temperature (18-26°C) and 800g for 20 minutes.

[0065] 3) After centrifugation, the bottom of the tube contains red blood cells, the middle layer contains separation fluid, the top layer contains plasma, and between the plasma layer and the separation fluid layer is a white, cloudy layer of mononuclear cells (including lymphocytes and monocytes). Use a pipette to draw up the white, cloudy cell layer and transfer it to a 15 ml sterile centrifuge tube, add RPMI 1640 medium to 10 ml, and centrifuge at 800 g for 10 minutes at room temperature.

[0066] 4) Discard the supernatant, resuspend and add 7 ml of RPMI 1640 medium, and centrifuge at 700 g for 10 minutes.

[0067] 5) Discard the supernatant and add 0.5 ml AIM-V medium to resuspend the pellet.

[0068] 6) Count the cells using an automatic cell counter and prepare 500 μL of AIM-V medium with a cell concentration of 2.5×10 6 / ml cell suspension.

[0069] 2. Preparation of Antigen Protein

[0070] The antigen protein and T cell epitope protein antigen in Example 3 were diluted to a certain concentration using RPIM1640 culture medium containing 10% fetal bovine serum before use.

[0071] 3.ELISPOT detection of antigen-specific T cells

[0072] Using the kit of Example 4, the following reagents were added to the microplate pre-coated with the primary antibody. Four test wells were set for each patient: a positive control well (with 100 μL of phytohemagglutinin PHA as a positive stimulus), a negative control well (with 100 μL of PBS as a negative control), and a test well (with 100 μL of Rv1566c or Rv1566c-444 with a final concentration of 20 μg / ml). 100 μL of the above-diluted PBMC was added to each well to make the number of PBMCs in each well reach 250,000. The antigen and PBMC cells were placed in an incubator at 37° C. and 5% CO2 and cultured for 20 hours.

[0073] 4. Washing the plate and determining the results

[0074] Wash away PBMC cells and antigen stimulators, add 100 μL primary antibody and incubate at room temperature for 1 hour, wash 5 times with PBS, add secondary antibody and incubate at room temperature for 1 hour, wash 5 times with PBS, add substrate and color for 7 minutes in the dark, terminate the color development with purified water, place the culture plate in a vent to dry and observe the number of spots on the plate.

[0075] Example 6 Analysis of the results of using Rv1566c and / or Rv1566c-444 in a diagnostic test kit

[0076] The ELISPOT test for tuberculosis infection was conducted using proteins Rv1566c and / or Rv1566c-444. A total of 36 tuberculosis patients and 44 healthy volunteers who were strictly screened according to the criteria were involved. The results showed that:

[0077] 1. The SFC induced by Rv1566c and / or Rv1566c-444 in tuberculosis patients was higher than that in healthy volunteers (P<0.05, P<0.01). There was no significant difference in the SFC induced by Rv1566c-444 and Rv1566c in tuberculosis patients. Figure 3 A in

[0078] 2. The diagnostic sensitivity of Rv1566c is 30.56%, and the specificity is 90.91%. When SFC>13, it can be diagnosed as tuberculosis infection. The diagnostic sensitivity of protein Rv1566c-444 is 44.44%, and the specificity is 84.09%. When SFC>5, it can be diagnosed as tuberculosis infection (the number of control spots in the positive quality control well must be ≥20). The results are shown in Figure 3 B in

[0079] 3. Rv1566c-444 detected a tuberculosis infection case that was misdiagnosed as negative by the T-SPOT.TB kit. When Rv1566c-444 was used in combination with the peptides (ESAT-6, CFP-10, Rv3615c) provided by the T-SPOT.TB kit, the diagnostic sensitivity of the T-SPOT.TB kit was increased from 94.44% to 97.36%.

[0080] Note: The detection sensitivity is expressed as: (number of tuberculosis patients with positive test results / total number of tuberculosis patients) × 100%; the detection specificity is expressed as: (1-number of healthy volunteers with positive test results / total number of healthy volunteers) × 100%.

[0081] Example 7 Process of using mouse model to immunize antigen

[0082] Specific pathogen-free female BALB / c mice (6 weeks old) were used for the immunization experiment. The mice were divided into 5 groups, with 6 mice in each group. A DP mixture consisting of DDA and Poly I:C was used as an adjuvant. The DP mixed adjuvant is believed to induce a mixed Th1 / Th2 immune response after inoculation of BALB / c mice. DDA and poly I:C were prepared into solutions with a final concentration of 0.5 mg / mL and 2.5 mg / mL using Tris-HCl (pH 8.0). 50 μL of DDA was mixed with 50 μL of poly I:C in a 1:1 ratio, and 100 μg of antigen (Rv1566c-444, Rv1566c, or Ag85B purified in this laboratory) was emulsified in 100 μL of DP mixture to prepare the vaccine. All mice were subcutaneously inoculated with the emulsion on days 1, 14, and 28. The mice in the positive control group were immunized with Ag85B, and the mice in the negative control group were given 200 μL of Tris-HCl (pH 8.0) or 200 μL of DP mixture. BALB / c mice were sacrificed 35 days after the first immunization, and various immune tests were performed to evaluate the immunogenicity of the antigen.

[0083] Example 8 Immunogenicity Detection of Rv1566c and / or Its T Cell Epitope Protein Rv1566c-444

[0084] 1. Body fluid immunogenicity detection:

[0085] A. Separation of serum

[0086] 500 μL of blood was collected from the eyeball of the mouse immunized in Example 7, and the blood was placed in a 37° C. incubator for 2 hours, and then transferred to a 4° C. refrigerator overnight. The next day, the blood was centrifuged at 3000 rpm for 5 minutes, and the supernatant was aspirated.

[0087] B. Serum antibody titer ELISA test

[0088] 1) The ELISA plate was coated with 2 μg / ml Rv1566c-444 or Rv1566c protein at 4°C overnight. The next day, the plate was washed five times with PBST.

[0089] 2) Block with PBS containing 2% BSA at 37°C for 2 hours and wash five times with PBST.

[0090] 3) The serum of each group was diluted by 20000, 40000, 80000, 160000, 320000, 640000, 1280000, and 2560000 times with PBS, 100 μL of the diluted serum was added to each well, incubated at 37°C for 1 hour, and then washed 5 times with PBST.

[0091] 4) Add 5000-fold diluted HRP-labeled IgG, IgG1, and IgG2a antibodies, 100 μL per well, incubate at 37°C for 1 hour, and wash five times with PBST.

[0092] 5) After adding TMB and color development at 37°C for 15 minutes, add 2M sulfuric acid as a stop solution.

[0093] 6) The ELISA reader detects the photometric value of the pipette at a wavelength of 450 nm.

[0094] 7) Judgment standard: OD ≥ 2.1 × OD (negative control) was judged as positive.

[0095] 2. Cellular immunogenicity detection:

[0096] A. ELISA to detect cytokines IFN-γ, L-6, IL-4

[0097] 1) The mice immunized in Example 7 were killed and soaked in 75% alcohol for 5 minutes. The mice were fixed on a foam board in a clean bench, the peritoneum was cut open, the spleen was separated, and placed in a dish containing 1640 culture medium.

[0098] 2) On a 200-mesh nylon mesh, gently grind the mouse spleen with the inner core of a syringe, filter the ground cells through a filter, centrifuge at 1000 r / min for 5 minutes, discard the supernatant, and obtain the cells.

[0099] 3) Gently shake the cells to loosen them, add 2 mL / cell of red blood cell lysis buffer, mix well, incubate in a 37°C incubator for 10 minutes, add 1640 culture medium with 2 times the volume of red blood cell lysis buffer to terminate the reaction, centrifuge at 1000 r / min for 5 minutes, discard the supernatant, and obtain spleen cells.

[0100] 4) Resuspend spleen cells with 2 ml / cell of 1640 medium, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and obtain spleen cells. Measure the cell concentration using a cell counter.

[0101] 5) Dilute spleen cells with 1640 medium containing 10% FBS to a concentration of 2×10 6500 μL of spleen cells were taken from each group and placed in a 24-well culture plate. The spleen cells in the Tris-HCL group and the DP adjuvant group were stimulated with 5 μg / mL of Ag85B, Rv1566c-444 and Rv1566c antigens, respectively. The Ag85B group was incubated with 500 μL of 5 μg / mL Ag85B antigen, the Rv1566c group was incubated with 500 μL of 5 μg / mL Rv1566c antigen, and the Rv1566c-444 group was incubated with 500 μL of 5 μg / mL Rv1566c-444 antigen. 500 μL of sterile PBS and 500 μL of 5 μg / mL phytohemagglutinin (PHA) were added to each group as negative and positive controls, and 2 replicates were performed in each test well. After incubating the spleen cells with the corresponding stimulators in a 37°C, 5% CO2 incubator for 72 hours, the cell supernatant was collected.

[0102] 6) IFN-γ monoclonal antibody, IL-6 monoclonal antibody, and IL-4 monoclonal antibody were diluted 1:500 with carbonate coating solution (pH=9.6), added to a 96-well microplate at 100 μL / well, and coated at 4°C overnight.

[0103] 7) After washing three times with PBTS, pat dry, add 10% FBS in PBS and block for 1 hour at room temperature.

[0104] 8) After washing with PBST for 5 times, dilute the IFN-γ, IL-6, and IL-4 standards to the corresponding concentrations according to the corresponding ratios and add them to each ELISA plate as the standard curve. Add 100 μL of the cell supernatant to be tested to the rest and incubate at room temperature for 2 hours.

[0105] 9) After diluting 5 times with PBST, add 100 μL of IFN-γ, IL-6, IL-4 detection antibodies + HRP-labeled secondary antibodies respectively and incubate at room temperature for 1 hour.

[0106] 10) After washing 7 times with PBST, 100 μL of TMB colorimetric solution was added, incubated at room temperature for 30 minutes, and then 50 μL of stop solution was added to terminate the reaction.

[0107] 11) The absorbance at a wavelength of 450 nm was measured by an enzyme-labeled instrument, and the absorbance at a wavelength of 570 nm was measured as a control.

[0108] 12) Result analysis: A standard curve was prepared based on the cytokine standards, and then the OD values ​​of the test wells were substituted into the formula to calculate the final concentrations of various cytokines in each group of mice.

[0109] B. Flow cytometry analysis of cell typing

[0110] 1) After the mice immunized in Example 7 were treated according to steps 1)-4) of the above ELISA method for detecting cytokines, a protein transport inhibitor whose main component was monensin (purchased from BD) was added to the cells and protein stimulation was performed as described in step 5) above.

[0111] 2) The cells were cultured at 37°C and 5% CO2 for 8 hours, stained with LIVE / DEAD Fixable DEAD Cell Stain, and then surface stained with anti-CD3, CD4, and CD8. Antibodies against IFN-γ, TNF-α, and IL-4 were then used for intracellular staining.

[0112] 3) The spleen cells were fixed with 4% paraformaldehyde and detected by flow cytometry.

[0113] Example 9 Analysis of the results of immunogenicity testing of Rv1566c and / or Rv1566c-444

[0114] 1. Analysis of humoral immunogenicity test results:

[0115] After mice were immunized with Rv1566c and Rv1566c-444 mixed adjuvants, the titers of Rv1566c and Rv1566c-444-specific serum IgG and IgG1 and IgG2a subtypes were determined by ELISA and compared with the titers of Ag85B-specific serum IgG and IgG1 and IgG2a subtypes.

[0116] The results showed that there was no significant difference in IgG levels between the Rv1566c group and the Ag85B group, but the IgG1 and IgG2a levels in the Rv1566c group were higher than those in the Ag85B group. Figure 4 There was no statistical difference in the ratio of IgG1 to IgG2a induced by Rv1566c-444, Rv1566c and Ag85B. Figure 4 B in

[0117] 2. Analysis of cell immunogenicity test results:

[0118] A. The levels of Th1 cytokine IFN-γ, Th2 cytokines IL-4 and IL-6 produced by spleen cells of mice immunized with Rv1566c and Rv1566c-444 mixed adjuvants after stimulation with immune antigens were detected by ELISA and compared with those of the Ag85B immunization group.

[0119] The results showed that the production of IFN-γ and IL-4 in mice immunized with Rv1566c-444 was significantly increased compared with that in the Rv1566c group and the Ag85B group (P values ​​were all < 0.01). Figure 5A and C in the figure. There was no significant difference in IFN-γ concentration between the Rv1566c group and the Ag85B group. Figure 5 The IL-6 secretion level of the Rv1566c-444 group was significantly higher than that of the Rv1566c and Ag85B groups (P value < 0.0001). Figure 5 B. The IFN-γ / IL-4 ratio of Rv1566c-444 was higher than that of Rv1566c (25.61 vs 23.10, P < 0.05), indicating that the Th1 response induced by Rv1566c was comparable to that of Ag85B, however, the Th1 response induced by Rv1566c-444 was stronger than that of Rv1566c;

[0120] B. Analyze protein-stimulated splenocytes using flow cytometry to detect CD3 + CD4 + T cells and CD3 + CD8 + The proportion of T cells in splenocytes and the CD4 secretion of different cytokines + or CD8 + The proportion of T cells.

[0121] The results showed that CD3 + CD4 + The proportion of T cells was similar to that of the Rv1566c group but significantly higher than that of the Ag85B group (P<0.01). Figure 6 A in the Rv1566c-444 group CD3 + CD8 + The proportion of T cells was significantly higher than that of Rv1566c (P<0.001) and Ag85B groups (P<0.05). Figure 6 A. CD4 in Rv1566c-444-immunized mice compared with Ag85B and Rv1566c groups + TNF-α + The proportion of T cells increased significantly (P<0.05, P<0.0001). Figure 6 B. in CD3 + CD8 + In T cells, the present invention found that Rv1566c-444 group CD8 + The proportion of T cells expressing IFN-γ was similar to that in the Rv1566c group but significantly lower than that in the Ag85B group (P<0.05). + The percentage of T cells expressing IL-4 was lower than that in the Rv1566c group (P<0.05) and the Ag85B group (P<0.01). Figure 6C. The cellular response induced by Rv1566c was comparable to that induced by Ag85B, whereas the cellular response induced by Rv1566c-444 was stronger than that induced by Rv1566c.

[0122] The above results prove that protein Rv1566c and / or Rv1566c-444 can successfully diagnose tuberculosis infection, and Rv1566c-444 can also improve the sensitivity of diagnosis when used in combination with the T-SPOT.TB kit; when used as an immune antigen, the protein Rv1566c immune group can induce a cellular immune response equivalent to the Ag85B immune group, and the protein Rv1566c-444 immune group induces a cellular immune response better than the protein Rv1566c immune group.

[0123] In summary, the T cell epitope protein antigen Rv1566c-444 of the Mycobacterium tuberculosis protein antigen Rv1566c can be used as a supplementary diagnostic reagent for the diagnosis and detection of tuberculosis, and Rv1566c and / or its T cell epitope protein Rv1566c-444 have good immunogenicity, can induce a strong cellular immune response and humoral immune response in the body, and are immunodominant antigens. Compared with Rv1566c, the sequence of the T cell epitope protein Rv1566c-444 contained in it is shorter, and at the same time overcomes the shortcomings of insufficient immunogenicity caused by the lack of concentration of protein epitopes and the presence of redundant sequences in monoclonal antibodies, showing stronger immunogenicity, and can be used for the construction and preparation of tuberculosis vaccines.

[0124] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention. Sequence Listing <110> Institute of Infectious Disease Prevention and Control, Chinese Center for Disease Control and Prevention <120> Application of Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 <130> KHP221110480.9 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 693 <212> DNA <213> Artificial Sequence <400> 1 atgaaacgca gcatgaaaag cggctccttc gcgatcggtc tggcaatgat gctcgccccg 60 atggtggccg cgcccggtct tgcggccgca gacccggcca cgcggccggt ggattatcaa 120 cagatcaccg acgtcgtgat cgcgcgcggg ctgtcgcagc gcggcgtgcc gttctcctgg 180 gccggcggcg gcatcagcgg ccccacgcgc ggcaccggta ccggcatcaa caccgtcggg 240 ttcgacgcct ccggtttgat ccagtacgcc tatgccggtg ccgggctaaa gctgccgcgt 300 tcttccggcc agatgtacaa ggttgggcaa aaggtcctgc cgcagcaagc gcgcaagggc 360 gacctgatct tctacggccc cgaaggcacg caaagcgtcg cgttatacct cgggaagggc 420 cagatgctgg aggtgggcga cgtcgtccag gtttcgccgg tgcgcaccaa cggcatgacg 480 ccttacctgg tccgggttct cgggacccag ccgacgcccg tccaacaggc gccggtccag 540 ccagcgccgg tccagcaagc gcccgtccag caagcgcccg tccaacaggc gcccgtccaa 600 caggcgccgg tccaacaggc gccggtccag caagcgcccg tccagcaagc gcccgtccag 660 ccgcctccct tcggcaccgc gcgctcacgc taa 693 <210> 2 <211> 230 <212> PRT <213> Artificial Sequence <400> 2 Met Lys Arg Ser Met Lys Ser Gly Ser Phe Ala Ile Gly Leu Ala Met 1 5 10 15 Met Leu Ala Pro Met Val Ala Ala Pro Gly Leu Ala Ala Ala Asp Pro 20 25 30 Ala Thr Arg Pro Val Asp Tyr Gln Gln Ile Thr Asp Val Val Ile Ala 35 40 45 Arg Gly Leu Ser Gln Arg Gly Val Pro Phe Ser Trp Ala Gly Gly Gly 50 55 60 Ile Ser Gly Pro Thr Arg Gly Thr Gly Thr Gly Ile Asn Thr Val Gly 65 70 75 80 Phe Asp Ala Ser Gly Leu Ile Gln Tyr Ala Tyr Ala Gly Ala Gly Leu 85 90 95 Lys Leu Pro Arg Ser Ser Gly Gln Met Tyr Lys Val Gly Gln Lys Val 100 105 110 Leu Pro Gln Gln Ala Arg Lys Gly Asp Leu Ile Phe Tyr Gly Pro Glu 115 120 125 Gly Thr Gln Ser Val Ala Leu Tyr Leu Gly Lys Gly Gln Met Leu Glu 130 135 140 Val Gly Asp Val Val Gln Val Ser Pro Val Arg Thr Asn Gly Met Thr 145 150 155 160 Pro Tyr Leu Val Arg Val Leu Gly Thr Gln Pro Thr Pro Val Gln Gln 165 170 175 Ala Pro Val Gln Pro Ala Pro Val Gln Gln Ala Pro Val Gln Gln Ala 180 185 190 Pro Val Gln Gln Ala Pro Val Gln Gln Ala Pro Val Gln Gln Ala Pro 195 200 205 Val Gln Gln Ala Pro Val Gln Gln Ala Pro Val Gln Pro Pro Pro Phe 210 215 220 Gly Thr Ala Arg Ser Arg 225 230 <210> 3 <211> 444 <212> DNA <213> Artificial Sequence <400> 3 gtggattatc aacagatcac cgacgtcgtg atcgcgcgcg ggctgtcgca gcgcggcgtg 60 ccgttctcct gggccggcgg cggcatcagc ggccccacgc gcggcaccgg taccggcatc 120 aacaccgtcg ggttcgacgc ctccggtttg atccagtacg cctatgccgg tgccgggcta 180 aagctgccgc gttcttccgg ccagatgtac aaggttgggc aaaaggtcct gccgcagcaa 240 gcgcgcaagg gcgacctgat cttctacggc cccgaaggca cgcaaagcgt cgcgttatac 300 ctcgggaagg gccagatgct ggaggtgggc gacgtcgtcc aggtttcgcc ggtgcgcacc 360 aacggcatga cgccttacct ggtccgggtt ctcgggaccc agccgacgcc cgtccaacag 420 gcgccggtcc agccagcgcc ggtc 444 <210> 4 <211> 148 <212> PRT <213> Artificial Sequence <400> 4 Val Asp Tyr Gln Gln Ile Thr Asp Val Val Ile Ala Arg Gly Leu Ser 1 5 10 15 Gln Arg Gly Val Pro Phe Ser Trp Ala Gly Gly Gly Ile Ser Gly Pro 20 25 30 Thr Arg Gly Thr Gly Thr Gly Ile Asn Thr Val Gly Phe Asp Ala Ser 35 40 45 Gly Leu Ile Gln Tyr Ala Tyr Ala Gly Ala Gly Leu Lys Leu Pro Arg 50 55 60 Ser Ser Gly Gln Met Tyr Lys Val Gly Gln Lys Val Leu Pro Gln Gln 65 70 75 80 Ala Arg Lys Gly Asp Leu Ile Phe Tyr Gly Pro Glu Gly Thr Gln Ser 85 90 95 Val Ala Leu Tyr Leu Gly Lys Gly Gln Met Leu Glu Val Gly Asp Val 100 105 110 Val Gln Val Ser Pro Val Arg Thr Asn Gly Met Thr Pro Tyr Leu Val 115 120 125 Arg Val Leu Gly Thr Gln Pro Thr Pro Val Gln Gln Ala Pro Val Gln 130 135 140 Pro Ala Pro Val 145

Claims

1. A use of a Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 in the preparation of a tuberculosis detection reagent; wherein: The amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is shown in SEQ ID NO.

4.

2. Use of a Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 in the preparation of a tuberculosis vaccine; wherein: The amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is shown in SEQ ID NO.

4.

3. Use of a Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 in the preparation of a drug for treating tuberculosis; wherein: The amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is shown in SEQ ID NO.

4.

4. The use according to any one of claims 1 to 3, characterized in that: The nucleotide sequence encoding the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is shown in SEQ ID NO.

3.

5. A tuberculosis diagnostic reagent, characterized in that: It includes the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444, or a DNA molecule encoding the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444, or a recombinant protein produced by a recombinant bacterium containing the DNA molecule; the amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is shown in SEQ ID NO.4, and the nucleotide sequence of the DNA molecule is shown in SEQ ID NO.

3.

6. A tuberculosis-specific interferon-gamma release test kit containing the tuberculosis diagnostic reagent according to claim 5.

7. The kit according to claim 6, characterized in that The kit also contains: ①Primary antibody: mouse IgG monoclonal antibody against human or animal IFN-γ; ②Enzyme-labeled reagents: horseradish peroxidase-labeled monoclonal antibodies of different subtypes of IgG against human or animal IFN-γ; ③ Standard product: positive raw material containing IFN-γ; ④ Culture plate: A 96-well microwell reaction plate containing PVDF membrane or nitrocellulose membrane or enzyme-linked immunosorbent assay. The positive control wells contain nonspecific stimulating antigens for tuberculosis, and the negative control wells contain PBS or basal fluid.

8. A tuberculosis vaccine, characterized in that: The invention comprises an effective ingredient and an adjuvant, wherein the effective ingredient comprises the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444, or a DNA molecule encoding the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444, or a recombinant protein produced by a recombinant bacterium containing the DNA molecule; the amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is shown in SEQ ID NO.4, and the nucleotide sequence of the DNA molecule is shown in SEQ ID NO.

3.

9. An anti-tuberculosis drug, characterized in that: The effective ingredients include a polyclonal antibody prepared by immunizing experimental animals with the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 as an immunogen and an adjuvant, or a monoclonal antibody that recognizes the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 antigen prepared by immunizing experimental animals with the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 as an immunogen and an adjuvant using hybridoma technology or DNA recombination technology, or a therapeutic biological product prepared by using the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 as an immunogen and an adjuvant; the amino acid sequence of the Mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is shown in SEQID NO.4.

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