CN116102629A

CN116102629A - Mycobacterium tuberculosis T cell epitope polypeptide and application thereof

Info

Publication number: CN116102629A
Application number: CN202211074402.7A
Authority: CN
Inventors: 张珺; 汪峰; 胡鹏高; 邹亚平
Original assignee: Wuhan Zhongji Biotechnology Co ltd
Current assignee: Wuhan Zhongji Biotechnology Co ltd
Priority date: 2022-09-03
Filing date: 2022-09-03
Publication date: 2023-05-12
Anticipated expiration: 2042-09-03
Also published as: CN116102629B

Abstract

The application discloses a mycobacterium tuberculosis T cell epitope polypeptide and application thereof, wherein the T cell epitope polypeptide is derived from proteins coded by Rv3878 and Rv3879c genes of a mycobacterium tuberculosis H37Rv genome, can effectively detect mycobacterium tuberculosis infection, can detect mycobacterium tuberculosis infection with specificity and sensitivity up to 100%, and has higher specificity and sensitivity compared with the existing T-SPOT.TB kit. The method for detecting the mycobacterium tuberculosis infection can distinguish active tuberculosis infection from inactive tuberculosis infection, provides a guiding basis for clinically treating active tuberculosis and inactive tuberculosis, and has important application significance.

Description

Mycobacterium tuberculosis T cell epitope polypeptide and application thereof

Technical Field

The application belongs to the technical field of biomedical inspection, and particularly relates to a mycobacterium tuberculosis T cell epitope polypeptide and application thereof.

Background

Mycobacterium tuberculosis (M.tuberculosis), commonly known as Mycobacterium tuberculosis (tubercle bacillus), is the causative agent of tuberculosis. Mycobacterium tuberculosis can invade a susceptible body through injury of the respiratory tract, digestive tract or skin, causing tuberculosis of various tissues and organs, among which tuberculosis is most caused through the respiratory tract. Tuberculosis is more common because mycobacterium tuberculosis can be inhaled through droplets of droplets or dust containing bacteria.

Mycobacterium tuberculosis is a facultative intracellular parasitic bacterium, the immunity is mainly cell immunity mainly based on T cells, and CD4T cells are main immune cells. The body can produce antibody to mycobacterium tuberculosis, but the antibody has no protection effect. The activated CD4T cells release cytokines to stimulate macrophages, and the macrophages kill and clear mycobacterium tuberculosis, so that the focus inflammation disappears and the organism is recovered. The clearance of the focus is related to the size of the focus, when the focus is too large and forms a fibrous capsule, macrophages cannot enter to kill bacteria, and the bacteria can enter a resting state in the focus to generate latent infection.

The tuberculin test (pptest) is a test for determining whether or not a body can cause hypersensitivity to mycobacterium tuberculosis by performing a skin test using tuberculin. The conventional test takes 2 PPD with 5 units respectively to inject into the skin of two forearms, and the redness and the swelling with the hardness exceeding 5mm are positive and the strong positive is greater than or equal to 15mm after 48-72 hours, so that the kit has significance for clinical diagnosis. If the PPD-C side is greater than the BCG-PPD side, the infection is caused. In contrast, BCG-PPD is greater than PPD-C, possibly due to BCG vaccination.

Negative reactions indicate no infection with Mycobacterium tuberculosis, but the following should be considered: (1) at the initial stage of infection, hypersensitivity reaction can occur after more than 4 weeks after the infection of the mycobacterium tuberculosis; (2) elderly people; (3) patients with severe tuberculosis or other infectious diseases, such as cellular hypoimmunity caused by measles; (4) acquired cell hypoimmunity such as acquired immune deficiency syndrome or tumor. To exclude false negative, sterile Phytohemagglutinin (PHA) injection, 0.1ml containing 10 μg was added to the domestic unit for skin test. If 24 hours of redness and swelling are greater than PHA skin, the cell immunity is normal, and if no reaction or no reaction exceeds PHA skin, the cell immunity is low.

The cell immunity mediated tubercle bacillus gamma interferon release test (T-cell interferon gamma release assays, TIGRA, also called IFNGRA or GRA) is the IFN-gamma detection release reaction of whole blood or peripheral blood mononuclear cells of a tested person to specific antigens of mycobacterium tuberculosis by adopting enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot (ELISPOT) method to quantitatively detect in recent years, and is used for diagnosing latent infection of tuberculosis. IFN-gamma is a cytokine secreted by Thl cells, can reflect the Thl cell immunity condition of tuberculosis of organisms, and is closely related to the antigen content of tuberculosis bacteria in vivo. T cells sensitized with Mycobacterium tuberculosis antigens can produce high levels of IFN-gamma when encountering the same antigen, so that the T cells are used for diagnosing latent tuberculosis infection, and the specificity is stronger than that of PPD.

The key to the detection of mycobacterium tuberculosis by the ELISPOT method is the selection of antigens for stimulating T cells. Appropriate T cell antigen stimulation may enhance the sensitivity and specificity of the kit. The antigen currently used in the T-SPOT.TB kit is a polypeptide fragment from the CFP10 and ESAT6 proteins expressed by the Rv3874 and Rv3875 genes, respectively, of the RD-1 region of the Mycobacterium tuberculosis genome. The RD-1 region gene is unique to Mycobacterium tuberculosis and is critical for its pathogenicity. The sensitivity and specificity of the current T-SPOT.TB kit for detecting the infection of the mycobacterium tuberculosis reach 95.3 percent and 94.1 percent respectively.

Although the existing T-SPOT.TB kit achieves satisfactory effects on the sensitivity and specificity of diagnosis of tuberculosis, the kit cannot distinguish active tuberculosis from inactive tuberculosis (both active tuberculosis and inactive tuberculosis are diagnosed as positive by the kit). The clinic treatment mode of active tuberculosis and inactive tuberculosis is quite different, so that the application of the kit is limited to a certain extent.

Disclosure of Invention

The first aspect of the present application provides a mycobacterium tuberculosis T cell epitope polypeptide, the amino acid sequence of which is shown in SEQ ID No.1-SEQ ID No.30, and the T cell epitope polypeptide is derived from the proteins encoded by Rv3878 and Rv3879c genes of the mycobacterium tuberculosis H37Rv genome. The T cell epitope polypeptide can effectively detect the mycobacterium tuberculosis infection, including active tuberculosis infection and inactive tuberculosis infection (i.e. tuberculosis latency infection), the specificity and sensitivity of the mycobacterium tuberculosis infection detection can reach 100%, and compared with the existing T-SPOT.TB kit, the T cell epitope polypeptide has higher specificity and sensitivity.

In a second aspect, the present application provides a test kit comprising a T cell epitope polypeptide as described above and means for recognition of said T cell epitope polypeptide, the means for recognition of the T cell epitope polypeptide comprising an IFN- γ antibody. The detection kit has higher specificity and sensitivity for detecting the mycobacterium tuberculosis infection.

The detection kit further comprises a positive control and a negative control, wherein the positive control is added with a positive quality control, and the positive quality control comprises one of staphylococcal enterotoxin (staphylo-coccal enterotoxinB, SEB), phytohemagglutinin (PHA), CD4 monoclonal antibody, CEF peptide library or a mixture of PMA and ionomycin (or A23187). Wherein CEF is a mixture of epitope peptides of MHC class I molecules of T cells derived from 32 Cytomegalovirus (CMV), epstein-Barr virus (EBV) and influenza virus (InflLlenzavirus), the stimulatory response is a specific response similar to the stimulatory effect of in vitro administration of specific peptide fragments or antigens, and the response to CEF is different in different humans. PHA was chosen as the positive quality control in the examples of the present application. The negative control, i.e., the blank, is usually without human antigen component, and medium or other buffer is added, as is the case in the examples herein.

In a third aspect the present application provides a method of detecting a mycobacterium tuberculosis infection, the detection method of the present application being based on a cell-mediated immune response, using the T cell epitope polypeptide described above in contact with T cells of a mycobacterium tuberculosis host; determining whether the T cell recognizes said T cell epitope polypeptide by detecting the level of cytokine IFN- γ antibodies secreted from the T cell.

Detection methods for detecting cytokines secreted from T cells are methods commonly used in the art, such as ELISA, ELISPOT, intracellular cytokine staining (including flow cytometry, etc.), immunoblotting, T cell proliferation assays, and the like. The specific embodiments of the present application are described using ELISPOT detection methods as examples. Other detection methods may be inspired by the ELISPOT detection method to achieve the same or similar effect.

Furthermore, the detection method can also distinguish active tuberculosis infection from inactive tuberculosis infection, and is realized by the following steps: and drawing an ROC curve for the T cells capable of recognizing the T cell epitope polypeptides according to the number and average spot area of generated spots, determining a cut-off value, and judging whether the T cells are infected active tuberculosis or infected inactive tuberculosis according to the cut-off value.

T cells in the assay methods of the present application, after infection with mycobacterium tuberculosis, can be detected in the peripheral blood, alveolar lavage fluid, hydrothorax, cerebrospinal fluid, lymph nodes, and other tissues containing T cells of the host.

Recognition in the detection method of the application refers to activation of T cells induced by specific binding of MHC-peptide complex and T cell receptor, and after T cell activation, related cytokines such as IFN-gamma begin to be secreted, and the state of T cell activation can be reflected by detecting the change of the content of the cytokines.

In the embodiment of the application, the ELISPOT detection method is adopted to detect IFN-gamma secreted by T cells, the IFN-gamma is combined with an IFN-gamma monoclonal antibody fixed on a solid carrier to form a primary antibody complex, then combined with a second IFN-gamma antibody coupled with biotin, and then specifically combined with biotin by using enzyme-linked streptavidin. Finally, spots are formed on the solid phase carrier through chromogenic substrates of the enzyme, each spot represents one T cell secreting IFN-gamma, the number of spots can be represented by the number of activated T cells, and the average spot area can represent the average level of IFN-gamma secretion by the T cells. In a fourth aspect, the present application provides an application of a mycobacterium tuberculosis T cell epitope polypeptide in preparing a kit for detecting mycobacterium tuberculosis infection.

Compared with the prior art, the application has the following technical effects:

the T cell epitope polypeptide can effectively detect the mycobacterium tuberculosis infection, including active tuberculosis infection and inactive tuberculosis infection (i.e. tuberculosis latency infection), the specificity and sensitivity of the mycobacterium tuberculosis infection detection can reach 100%, and compared with the existing T-SPOT.TB kit, the T cell epitope polypeptide has higher specificity and sensitivity.

The method for detecting the mycobacterium tuberculosis infection can distinguish active tuberculosis infection from inactive tuberculosis infection, provides a guiding basis for clinically treating active tuberculosis and inactive tuberculosis, and has important application significance.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a graph of ROC distinguishing active binding from latent binding provided in an embodiment of the present application.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects to be solved by the present application more clear, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.

In this application, the term "and/or" describes an association relationship of an association object, which means that there may be three relationships, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.

In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.

It should be understood that, in various embodiments of the present application, the sequence number of each process does not mean that the sequence of execution is sequential, and some or all of the steps may be executed in parallel or sequentially, where the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The weights of the relevant components mentioned in the embodiments of the present application may refer not only to specific contents of the components, but also to the proportional relationship between the weights of the components, and thus, any ratio of the contents of the relevant components according to the embodiments of the present application may be enlarged or reduced within the scope disclosed in the embodiments of the present application. Specifically, the mass described in the specification of the examples of the present application may be a mass unit known in the chemical industry such as μ g, mg, g, kg.

The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the present application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.

EXAMPLE 1 screening of Mycobacterium tuberculosis T cell epitope Polypeptides

In the embodiment of the application, different mycobacterium tuberculosis antigen polypeptide fragments are artificially synthesized by a polypeptide synthesis technology, and the polypeptide fragments are derived from proteins coded by Rv3878 and Rv3879c genes of a mycobacterium tuberculosis H37Rv genome. Wherein SEQ No.1-SEQ No.54 is from the protein encoded by the Rv3878 gene and SEQ No. 55-SEQ No.198 is from the protein encoded by the Rv3879c gene.

TABLE 1 Artificial synthesis of different Mycobacterium tuberculosis antigen polypeptide fragments

/>

/>

/>

/>

/>

/>

/>

In the embodiment of the application, the screening of the mycobacterium tuberculosis T cell epitope polypeptides is to detect specific gamma interferon which stimulates T cell secretion by different polypeptide fragments of SEQ NO.1-SEQ NO.198 by an ELISPOT detection method, and the immune effect of the different polypeptide fragments after T cell stimulation is measured.

The specific detection process of the embodiment of the application is as follows:

PBMCs isolation: selecting 10 tuberculosis patients whole blood samples, and using Ficoll-Paque TM ^Plus+ PBMCs were isolated according to the protocol, layers of white, hazy PBMCs were pipetted and transferred to 15 mL-bottomed centrifuge tubes, 10mL of medium was added, and 600g was centrifuged for 7 minutes. The supernatant was discarded and the cells were resuspended in 1mL of medium. The medium was added to 10mL and centrifuged at 350g for 7 min. The supernatant was discarded and the cells were resuspended in 0.7mL of medium.

Cell count: mu.L of the cell suspension was added to 40. Mu.L of 0.4% (w/v) trypan blue and mixed well. The counting plate is adjusted to a proper position to count the number of cells in the grid. Automatic cytometry may also be used for counting.

Cell concentration adjustment: based on the result of cell count, the cell concentration was adjusted to 2.5X10 ⁶ And each mL.

And (3) detection: the culture plate bar is taken out and restored to room temperature in the embedded culture plate frame. The whole blood of 10 tuberculosis patients is detected by using 200 hole sites respectively. Each sample operates as follows: 50. Mu.L of SEQ NO.1-SEQ NO.198 was added to wells 1-198, respectively, 50. Mu.L of medium was added to well 199 as a blank control, and 50. Mu.L of LPHA was added to well 200 as a positive control. Then 100. Mu.L of a solution containing 2.5X10 s was added to 1-200 wells ⁶ Samples of individual/mL PBMCs were placed in a 5% CO solution ₂ Is cultured in an incubator of (a).

Spot count: the plates were removed from the incubator, the medium was discarded, and washed 3 times with PBS buffer. 50. Mu.L of labeled antibody was added to each well and incubated at 2-8deg.C for 1 hour. The labeled antibody was discarded and washed 3 times with PBS buffer. mu.L of substrate chromogenic solution was added to each well and incubated at room temperature for 7 minutes. The reaction was stopped by discarding the substrate developing solution, thoroughly washing the plate with distilled water, and drying the plate. The number of spots generated is counted using a spot counter or a magnifying glass is counted manually.

Through the detection, the polypeptide fragments of SEQ No.2, SEQ No.9, SEQ No.15, SEQ No.18, SEQ No.19, SEQ No.23, SEQ No.25, SEQ No.36, SEQ No.37, SEQ No.38, SEQ No.42, SEQ No.43, SEQ No.51, SEQ No.52, SEQ No.53, SEQ No.61, SEQ No.65, SEQ No.69, SEQ No.75, SEQ No.84, SEQ No.106, SEQ No.107, SEQ No.112, SEQ No.138, SEQ No.147, SEQ No.150, SEQ No.169, SEQ No.170, SEQ No.178 and SEQ No.188 (SEQ ID No.1-SEQ ID No. 30) can produce more cytokines after stimulating T cells.

EXAMPLE 2 ELISPOT detection of level of gamma interferon release in tuberculosis patients

Dividing the screened polypeptide fragments into two groups, wherein one group comprises SEQ NO.2, SEQ NO.9, SEQ NO.15, SEQ NO.18, SEQ NO.19, SEQ NO.23, SEQ NO.25, SEQ NO.36, SEQ NO.37, SEQ NO.38, SEQ NO.42, SEQ NO.43, SEQ NO.51, SEQ NO.52 and SEQ NO.53 encoded by the Rv3878 gene and is named as antigen series 1; another group comprises SEQ No.61, SEQ No.65, SEQ No.69, SEQ No.75, SEQ No.84, SEQ No.106, SEQ No.107, SEQ No.112, SEQ No.138, SEQ No.147, SEQ No.150, SEQ No.169, SEQ No.170, SEQ No.178, SEQ No.188, encoded by the Rv3879c gene, designated as antigen series 2.

The two groups of polypeptides are used for diagnosing tuberculosis, and the specific process is as follows:

100 samples were selected, 30 of which were active tuberculosis samples, 30 of which were latent tuberculosis samples, and 40 of which were negative samples. 4 tests were performed for each sample, including: blank control, antigen series 1 stimulation, antigen series 2 stimulation, positive control stimulation. Wherein the blank control is to add culture medium into T cells, the antigen series 1 stimulation is to add antigen series 1 into T cells, the antigen series 2 stimulation is to add antigen series 2 into T cells, and the positive control is to add positive quality control PHA into T cells. Other positive controls may also be added, such as one of staphylococcal enterotoxin (seph-coccal enterotoxin B, SEB), phytohemagglutinin (PHA), CD4 monoclonal antibody, CEF peptide library or a mixture of PMA and ionomycin (or a 23187). Wherein CEF is a mixture of epitope peptides of MHC class I molecules of T cells derived from 32 Cytomegalovirus (CMV), epstein-Barr virus (EBV) and influenza virus (InflLlenzavirus), the stimulatory response is a specific response similar to the stimulatory effect of in vitro administration of specific peptide fragments or antigens, and the response to CEF is different in different humans. PHA was chosen as the positive quality control in the examples of the present application.

The specific diagnostic procedure of the embodiment of the present application is as follows:

PBMCs isolation: 100 tuberculosis patients were selected from whole blood samples, 30 active tuberculosis samples, 30 latent tuberculosis samples, and 40 negative samples. By Ficoll-Paque TM ^Plus+ PBMCs were isolated according to the protocol, layers of white, hazy PBMCs were pipetted and transferred to 15 mL-bottomed centrifuge tubes, 10mL of medium was added, and 600g was centrifuged for 7 minutes. The supernatant was discarded and the cells were resuspended in 1mL of medium. The medium was added to 10mL and centrifuged at 350g for 7 min. The supernatant was discarded and the cells were resuspended in 0.7mL of medium.

And (3) detection: the culture plate bar is taken out and restored to room temperature in the embedded culture plate frame. 100 samples were tested using 400 holes. Each sample operates as follows: 50. Mu.L of medium was added to well 1 as a negative control, 50. Mu.L of antigen series 1 was added to well 2, 50. Mu.L of antigen series 2 was added to well 3, and 50. Mu.L of PHA was added to well 4 as a positive control. Then 100. Mu.L of a solution containing 2.5X10 s was added to 1-4 wells ⁶ Samples of individual/mL PBMCs were placed in a 5% CO solution ₂ Is cultured in an incubator of (a).

Spot count: the plates were removed from the incubator, the medium was discarded, and washed 3 times with PBS buffer. 50. Mu.L of labeled antibody was added to each well and incubated at 2-8deg.C for 1 hour. The labeled antibody was discarded and washed 3 times with PBS buffer. mu.L of substrate chromogenic solution was added to each well and incubated at room temperature for 7 minutes. The reaction was stopped by discarding the substrate developing solution, thoroughly washing the plate with distilled water, and drying the plate. The number of spots generated and the average spot area were counted using a spot counter.

After the diagnosis, the number of generated spots of at least one of the active tuberculosis sample and the latent tuberculosis sample is more than or equal to 5 blank control spots after the active tuberculosis sample and the latent tuberculosis sample are stimulated by the antigen series 1 and the antigen series 2; the number of spots generated by the negative sample after being stimulated by the antigen series 1 and the antigen series 2 is less than 5 compared with the number of blank control spots. The specificity and sensitivity of the reagent for distinguishing tuberculosis (including active tuberculosis and latent tuberculosis) reach 100%. The number of spots produced by the antigen series 1 and the antigen series 2 stimulated PBMCs represents the number of PBMCs sensitive to mycobacterium tuberculosis in the sample to be tested, the average spot area produced by the antigen series 1 and the antigen series 2 stimulated PBMCs represents the intensity of the response of the PBMCs sensitive to mycobacterium tuberculosis to the mycobacterium tuberculosis, the number of spots produced by the positive control stimulated PBMCs represents the number of PBMCs with immunological activity in the sample to be tested, and the average spot area produced by the positive control stimulated PBMCs represents the intensity of the immunological activity of the PBMCs with immunological activity. From this, the number of antigen series 1 spots x the average spot area of antigen series 1 represents the overall intensity of response of PBMCs in the sample to antigen series 1 stimulation, defined as antigen series 1 efficacy; antigen series 2 spot number x antigen series 2 average spot area represents the overall intensity of response of PBMCs to antigen series 2 stimulation in the sample, defined as antigen series 2 efficacy; the number of positive control spots x the average positive control spot area represents the overall intensity of the response of the PBMCs in the sample to the positive control stimulus, defined as the overall efficacy; antigen series 1 potency + antigen series 2 potency is defined as mycobacterium tuberculosis antigen potency. The ratio of the antigen potency of Mycobacterium tuberculosis/total potency representing the antigen potency of Mycobacterium tuberculosis to the total potency in the sample is defined as the risk value of Mycobacterium tuberculosis infection. The greater the risk of a tuberculosis infection, the more reactive the sample is to the mycobacterium tuberculosis antigen, the more likely it is to be an active tuberculosis infection. The risk value of the mycobacterium tuberculosis infection of the active tuberculosis case and the latent tuberculosis case is analyzed, and found that the risk value of the mycobacterium tuberculosis infection of the active tuberculosis is generally larger than that of the latent tuberculosis. The ROC curve is drawn by using the data of active tuberculosis and latent tuberculosis as the diagnosis result of gold standard, the area AUC=0.883 under the curve shows that the infection risk value of the mycobacterium tuberculosis has good diagnosis effect, the optimal index threshold value is determined by Youden's index, the cut-off value is determined to be 0.689, the diagnosis of the active tuberculosis and the latent tuberculosis is carried out according to the cut-off value, the sensitivity is 86.7%, and the specificity is 93.3%. Detailed experimental results for the examples of the present application are shown in table 2, and ROC curves distinguishing active binding from latent binding are shown in fig. 1.

TABLE 2 detailed detection results of Mycobacterium tuberculosis cell-specific immunoreactions

/>

/>

In the embodiment of the application, the proteins synthesized by the Rv3878 and Rv3879c genes in the genome of the Mycobacterium tuberculosis H37Rv are split into a series of polypeptide fragments, polypeptide fragments SEQ NO.2, SEQ NO.9, SEQ NO.15, SEQ NO.18, SEQ NO.19, SEQ NO.23, SEQ NO.25, SEQ NO.36, SEQ NO.37, SEQ NO.38, SEQ NO.42, SEQ NO.43, SEQ NO.51, SEQ NO.52, SEQ NO.53, SEQ NO.61, SEQ NO.65, SEQ NO.69, SEQ NO.75, SEQ NO.84, SEQ NO.106, SEQ NO.107, SEQ NO.112, SEQ NO.138, SEQ NO.147, SEQ NO.150, SEQ NO.169, SEQ NO.170, SEQ NO.178 and SEQ NO.188 are screened, and the amino acid sequences of the 30 polypeptide fragments are sequentially numbered from SEQ ID NO.1 to 30. The 30 fragments are subjected to an ELISPOT experiment to diagnose tuberculosis, and the sensitivity and the specificity reach 100 percent; the number and average area of spots generated by ELISPOT experiments of active tuberculosis and latent tuberculosis cases are further analyzed, a cutoff value is determined, diagnosis of active tuberculosis and latent tuberculosis is carried out, the sensitivity reaches 86.7%, and the specificity reaches 93.3%.

The experimental results show that the mycobacterium T cell epitope polypeptide can be applied to a kit for detecting mycobacterium tuberculosis infection, and compared with the existing T-SPOT.TB kit, the sensitivity and the specificity of tuberculosis diagnosis are greatly improved, and active tuberculosis and latent tuberculosis can be distinguished.

The foregoing examples represent only a few embodiments of the present application, which are described in more detail and are not thereby to be construed as limiting the scope of the present application. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.

Claims

1. The T cell epitope polypeptide is characterized in that the amino acid sequence of the T cell epitope polypeptide is shown in SEQ ID NO.1-SEQ ID NO.30, and the T cell epitope polypeptide is derived from proteins coded by Rv3878 and Rv3879c genes of a mycobacterium tuberculosis H37Rv genome.

2. A kit for detecting a mycobacterium tuberculosis infection comprising the T cell epitope polypeptide of claim 1 and means for recognizing the T cell epitope polypeptide.

3. A kit for detecting a mycobacterium tuberculosis infection as in claim 2, wherein the means for recognition of the T cell epitope polypeptide comprises an IFN- γ antibody.

4. A method for detecting a mycobacterium tuberculosis infection, comprising the steps of:

contacting a T cell of a host of mycobacterium tuberculosis with the T cell epitope polypeptide of claim 1;

determining whether the T cell recognizes said T cell epitope polypeptide by detecting a cytokine secreted from the T cell.

5. The method for detecting a mycobacterium tuberculosis infection according to claim 4, wherein the detection method for detecting a cytokine secreted from a T cell is any one of ELISA, ELISPOT, intracellular cytokine staining, immunoblotting, T cell proliferation assay.

6. The method for detecting a mycobacterium tuberculosis infection according to claim 4, further comprising the steps of:

and for the T cells capable of recognizing the T cell epitope polypeptides, drawing a ROC curve according to the number and average spot area of generated spots, determining a cut-off value, and judging whether the T cells are infected with active tuberculosis or inactive tuberculosis according to the cut-off value.

7. The use of a mycobacterium tuberculosis T cell epitope polypeptide according to claim 1 for preparing a kit for detecting mycobacterium tuberculosis infection.