T cell receptor for specifically recognizing mycobacterium tuberculosis antigen peptide as well as preparation method and application of T cell receptor

Abstract

The invention discloses a T cell receptor for specifically recognizing mycobacterium tuberculosis antigen peptide as well as a preparation method and application of the T cell receptor. According to the invention, HLA-A * 0201 positive pulmonary tuberculosis patient PBMC is respectively stimulated by tuberculosis antigens CFP10 and Ag85B, CD8 + T cells are subjected to flow sorting, TCR alpha / beta chains are paired, corresponding HLA-A * 0201 restrictive TCR protein is successfully obtained, and eukaryotic expression and purification are carried out. The TCR protein can significantly inhibit proliferation of mycobacteria in THP-1 macrophages, shows that CFU is reduced and high content fluorescence intensity is weakened, and significantly improves expression of inflammatory factors such as IL-1beta, TNF-alpha, IL-6, IL-12 and the like. The tuberculosis antigen-specific TCR protein is proved to effectively inhibit intracellular mycobacterium tuberculosis replication by regulating the polarization state of macrophages and an inflammatory signal network for the first time, and a new strategy is provided for TCR-based tuberculosis immunotherapy.

Description

Technical Field

[0001] This invention relates to a T-cell receptor that specifically recognizes Mycobacterium tuberculosis antigen peptides, its preparation method, and its application, belonging to the field of biomedical technology. Background Technology

[0002] Pulmonary tuberculosis (TB) is a serious infectious disease caused by Mycobacterium tuberculosis (MTB) and remains a leading cause of death worldwide. Despite the availability of effective anti-tuberculosis drugs and vaccines, relapse and drug resistance remain significant challenges. Recent studies have revealed that T-cell receptors (TCRs) play a crucial role in regulating immune responses, particularly in the immune response to TB. The diversity and specificity of TCRs make them key to studying the immune response to TB.

[0003] CFP10 and Ag85B are both important antigens secreted by Mycobacterium tuberculosis (MTB). CFP10 belongs to the secretory protein family and has high immunogenicity, capable of activating the host's immune response. Its structural features include a β-sheet-rich core region, which allows it to bind to host MHC molecules and induce a strong T cell-mediated immune response. Ag85B belongs to the Ag85 complex and has important biological functions. Its main function is to promote cell wall synthesis and secretion, participating in the survival and pathogenicity of Mycobacterium tuberculosis. Ag85B can activate host macrophages, enhancing their ability to phagocytose and kill bacteria, and also influence the nature of the immune response by regulating cytokine secretion. Studies have shown that the proliferation and activation of specific T cells after MTB infection are key to combating tuberculosis. During tuberculosis infection, macrophages, as the host's main immune cells, play a crucial role. Macrophages are not only responsible for phagocytizing and eliminating pathogens but also regulate the immune response by secreting cytokines, promoting inflammation and tissue repair.

[0004] The function of TCRs primarily depends on their ability to bind to antigens. TCRs can recognize specific antigenic peptides presented by antigen-presenting cells (APCs), thereby activating T cells and initiating an immune response. Studies have shown that the diversity of TCRs in tuberculosis patients is closely related to disease severity and immune status. TCRs mediate cellular immune responses by recognizing MTB-associated antigens, promoting T cell activation and proliferation. In tuberculosis patients, the expansion of specific TCR clones is closely related to the establishment of protective immune responses. For example, CD4+... + T cells and CD8 +T cell aggregation in the lungs is significantly associated with MTB control; these cells suppress bacterial growth by secreting interferon-γ (IFN-γ) and other cytokines. Furthermore, the diversity and specificity of TCRs are also related to an individual's resistance to tuberculosis infection. Certain specific TCR clones are more abundant in individuals resistant to MTB infection, suggesting that these clones may play a key role in immune surveillance. TCRs on T cells consist of two heterodimer chains, α and β, each containing a constant region (C region) and a variable region (V region). The variable region consists of a backbone region and three hypervariable regions: CDR1, CDR2, and CDR3. CDR1 and CDR2 are generally considered to bind MHC molecules, while CDR3 binds to MHC-presented antigenic peptides. MHC-1 molecules exhibit polymorphism in the population, with one of the most prevalent MHC-1 types being HLA-A*0201, accounting for approximately 40% of the population.

[0005] HLA-A*0201 is a frequently expressed HLA allele in populations worldwide. By discovering and screening HLA-A*0201-restricted tuberculosis antigen CFP10 and Ag85B-specific T-cell receptor TCR, and obtaining their encoding genes and expressing them as soluble TCR proteins, these proteins are imbued with the HLA-A*0201-restricted pattern, enhancing their ability to clear intracellular Mycobacterium tuberculosis. Due to the high frequency of HLA-A*0201 expression, this method can be applied to a wide range of populations and has high practical value. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a T-cell receptor that specifically recognizes Mycobacterium tuberculosis antigen peptides, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a T-cell receptor that specifically recognizes a tuberculosis branch of the spp. antigen peptide, comprising an α-chain variable region and a β-chain variable region, wherein the T-cell receptor is selected from the group consisting of:

[0009] i) The T-cell receptor is a T-cell receptor that specifically recognizes the Mycobacterium tuberculosis CFP10 antigen peptide, and its α-chain variable region contains an α-chain complementarity-determining region ACDR3 composed of an amino acid sequence as shown in SEQ ID No. 3, and its β-chain variable region contains a β-chain complementarity-determining region BCDR3 composed of an amino acid sequence as shown in SEQ ID No. 6.

[0010] ii) The T-cell receptor is a T-cell receptor that specifically recognizes the Mycobacterium tuberculosis Ag85B antigen peptide. Its α-chain variable region contains an α-chain complementarity-determining region ACDR3 composed of an amino acid sequence as shown in SEQ ID No. 20, and its β-chain variable region contains a β-chain complementarity-determining region BCDR3 composed of an amino acid sequence as shown in SEQ ID No. 23.

[0011] In some embodiments of the present invention, the T cell receptor in i) is:

[0012] The α-chain variable region contains an α-chain complementarity-determining region ACDR1 composed of an amino acid sequence as shown in SEQ ID No. 1 and an α-chain complementarity-determining region ACDR2 composed of an amino acid sequence as shown in SEQ ID No. 2;

[0013] And / or, the β-chain variable region contains a β-chain complementarity-determining region BCDR1 consisting of an amino acid sequence as shown in SEQ ID No. 4 and a β-chain complementarity-determining region BCDR2 consisting of an amino acid sequence as shown in SEQ ID No. 5.

[0014] In some embodiments of the present invention, the T cell receptor in ii) is:

[0015] The α-chain variable region contains an α-chain complementarity-determining region ACDR1 composed of an amino acid sequence as shown in SEQ ID No. 18 and an α-chain complementarity-determining region ACDR2 composed of an amino acid sequence as shown in SEQ ID No. 19.

[0016] And / or, the β-chain variable region contains a β-chain complementarity-determining region BCDR1 consisting of an amino acid sequence as shown in SEQ ID No. 21 and a β-chain complementarity-determining region BCDR2 consisting of an amino acid sequence as shown in SEQ ID No. 22.

[0017] In some embodiments of the present invention, the T cell receptor in i) is:

[0018] The α-chain variable region contains an amino acid sequence as shown in SEQ ID No. 7, or contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 7;

[0019] And / or, the β-chain variable region contains an amino acid sequence as shown in SEQ ID No. 8, or contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 8;

[0020] In some embodiments of the present invention, the T cell receptor in ii) is:

[0021] The α-chain variable region contains an amino acid sequence as shown in SEQ ID No. 24, or contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 24;

[0022] And / or, the β-chain variable region contains an amino acid sequence as shown in SEQ ID No. 25, or contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 25.

[0023] In some embodiments of the present invention, the T cell receptor in i) has the following nucleotide sequences: the nucleotide sequence encoding ACDR1 is shown in SEQ ID No. 9, the nucleotide sequence encoding ACDR2 is shown in SEQ ID No. 10, the nucleotide sequence encoding ACDR3 is shown in SEQ ID No. 11, the nucleotide sequence encoding BCDR1 is shown in SEQ ID No. 12, the nucleotide sequence encoding BCDR2 is shown in SEQ ID No. 13, and the nucleotide sequence encoding BCDR3 is shown in SEQ ID No. 14.

[0024] In some embodiments of the present invention, the T cell receptor in ii) has the following nucleotide sequences: the nucleotide sequence encoding ACDR1 is shown in SEQ ID No. 26, the nucleotide sequence encoding ACDR2 is shown in SEQ ID No. 27, the nucleotide sequence encoding ACDR3 is shown in SEQ ID No. 28, the nucleotide sequence encoding BCDR1 is shown in SEQ ID No. 29, the nucleotide sequence encoding BCDR2 is shown in SEQ ID No. 30, and the nucleotide sequence encoding BCDR3 is shown in SEQ ID No. 31.

[0025] In some embodiments of the present invention, the T cell receptor in i) encodes the nucleotide sequence of the α-chain variable region as shown in SEQ ID No. 15, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 15, and the nucleotide sequence encodes the β-chain variable region as shown in SEQ ID No. 16, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 16;

[0026] In some embodiments of the present invention, the T cell receptor in ii) has the following sequence: the nucleotide sequence encoding the α-chain variable region is as shown in SEQ ID No. 32, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 32; and the nucleotide sequence encoding the β-chain variable region is as shown in SEQ ID No. 33, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 33.

[0027] In a second aspect, the present invention provides a multivalent TCR complex comprising at least three TCR molecules, wherein at least one of the TCR molecules is a T cell receptor as described in the first aspect of the present invention.

[0028] A third aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the TCR described in the first aspect of the present invention or its complementary sequence, or a codon-optimized nucleotide sequence corresponding to the amino acid sequence of the TCR described in the first aspect of the present invention.

[0029] In a fourth aspect, the present invention provides a carrier containing the nucleic acid molecule described in the third aspect of the present invention.

[0030] In some embodiments of the present invention, the vector is a plasmid, a granule, a bacteriophage, or a viral vector; preferably, the vector is a lentiviral vector and / or a retroviral vector; in some more preferred embodiments, the vector is a lentiviral vector.

[0031] In a fifth aspect, the present invention provides a host cell containing an exogenous nucleic acid molecule as described in the third aspect of the present invention integrated into a vector or chromosome as described in the fourth aspect of the present invention.

[0032] Preferably, the host cell is an immune cell, such as a T cell or a stem cell; more preferably, the cell is a primary T cell or stem cell from the subject.

[0033] A sixth aspect of the present invention provides a pharmaceutical composition or kit comprising: i) a TCR as described in the first aspect of the present invention, a TCR complex as described in the second aspect, a nucleic acid molecule as described in the third aspect, a carrier as described in the fourth aspect, and / or a host cell as described in the fifth aspect, and ii) a pharmaceutically acceptable carrier or excipient.

[0034] A seventh aspect of the present invention provides the use of the T-cell receptor as described in the first aspect, the TCR complex as described in the second aspect, the nucleic acid molecule as described in the third aspect, the carrier as described in the fourth aspect, the host cell as described in the fifth aspect, and / or the pharmaceutical composition or kit as described in the seventh aspect, said use including: i) for the preparation of reagents or kits for the detection of Mycobacterium tuberculosis; and / or ii) for the preparation of medicaments for the prevention or treatment of pulmonary tuberculosis.

[0035] Preferably, the pulmonary tuberculosis is of type HLA-A*02; more preferably, the pulmonary tuberculosis is of type HLA-A*0201.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention involves stimulating PBMCs of HLA-A*0201-positive pulmonary tuberculosis patients with tuberculosis antigens (CFP10, Ag85B) to screen for antigen-specific CD8 antibodies. + T-cell TCR: The TCR protein screened through cell model verification has the effect of inhibiting the growth of intracellular Mtb (including Mycobacterium smegmatis and H37Rv strain). This invention is the first to demonstrate that the tuberculosis antigen-specific TCR protein effectively inhibits the replication of intracellular Mycobacterium tuberculosis by regulating macrophage polarization state and inflammatory signaling network, providing a new strategy for TCR-based tuberculosis immunotherapy. Attached Figure Description

[0038] Figure 1 The figures show the results of TCR clone screening and protein expression experiments in Examples 1 and 2 of this invention. In Example A: CD8+ of Mycobacterium tuberculosis-specific TCRs was sorted by flow cytometry. + A: Results of T cell amplification; B: Results of TCR Vα and Vβ gene amplification by PCR; C: Schematic diagram of TCR variable region gene confirmation by comparison; D: Results of TCR Vα and Vβ gene amplification by PCR;

[0039] Figure 2 The figures show the experimental results of TCR protein inhibiting intracellular Mycobacterium smegmatis obtained in Examples 1 and 2; where A: cell fluorescence image; B: bacterial count result image; C: average bacterial intensity at time points of 24h TCR treatment and 24h TCR removal.

[0040] Figure 3 The figure shows the experimental results of TCR protein obtained in Examples 1 and 2 inhibiting Mycobacterium tuberculosis H37Rv.

[0041] Figure 4The figures show the experimental results of TCR protein inducing the release of inflammatory cytokines from THP-1 cells obtained in Examples 1 and 2; where A: ELISA results of inflammatory cytokines detection; B: qPCR results of inflammatory cytokines detection. Detailed Implementation

[0042] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0043] The scientific and technical terms used in this invention have the meanings commonly understood by those skilled in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the Mycobacterium tuberculosis, materials, reagents, etc., used in the following examples are commercially available reagents and materials.

[0044] Example 1: Preparation of TCRs specific to tuberculosis antigen CFP10 protein

[0045] Patients with HLA-A*0201-positive pulmonary tuberculosis (positive T-SPOT results) were screened, and peripheral blood was obtained. Mononuclear lymphocytes were separated by density gradient centrifugation using Ficoll reagent. The cells were then cultured for 7 to 10 days with 5 μg / ml of the AQEAGNFERISGDLK (SEQ ID No. 17) peptide derived from the CFP10 protein at positions 12-26, along with 10 ng / ml of recombinant IL-2. The HLA-A*0201 tetramer of the fluorescently labeled conjugated peptide was stained, and the CD8+ of the Mycobacterium tuberculosis-specific TCR was sorted by flow cytometry. + T cells ( Figure 1 A). Primers were designed based on the conserved regions at both ends of the variable regions of the TCRα and β chains to amplify the Vα and Vβ genes using polymerase chain reaction (PCR). Figure 1 B), the gene sequence was obtained through sequencing and confirmed as a TCR variable region gene by comparison on the website www.imgt.org / . Figure 1 C). Two TCR clones targeting the CFP10 protein epitope peptide of tuberculosis antigen were obtained, each containing its own Vα and Vβ variable region sequences. The paired Vα and Vβ genes from these clones were ligated using a GGGGS linker sequence and constructed into a His-tagged pcDNA3.1-IL12 eukaryotic expression vector. HEK293 cell lines were transfected with Transfection reagent. Three days later, HEK293 cell supernatant was collected, affinity purified, and a soluble protein containing TCR Vα and Vβ (TCR-C) was obtained. Figure 1 D).

[0046] The TCR clone α-chain variable region and β-chain variable region sequences of the TCR clone that recognizes the complex of the tuberculosis antigen CFP10 protein TCR epitope peptide and HLA-A*0201 (Table 1) involved in this invention were compared and analyzed on the website www.imgt.org / . The amino acid sequences of the complementarity-determining regions ACDR1, ACDR2, and ACDR3 in the α-chain variable region were confirmed as shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively, and the amino acid sequence of the α-chain variable region was confirmed as shown in SEQ ID No. 7. The amino acid sequences of the complementarity-determining regions BCDR1, BCDR2, and BCDR3 in the β-chain variable region were confirmed as shown in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively, and the amino acid sequence of the β-chain variable region was confirmed as shown in SEQ ID No. 8.

[0047] Table 1 Sequence Information Table

[0048]

[0049]

[0050] Example 2: Preparation of TCRs specific to tuberculosis antigen Ag85B protein

[0051] Patients with HLA-A*0201-positive pulmonary tuberculosis (positive T-SPOT results) were screened, and peripheral blood was obtained. Mononuclear lymphocytes were separated by density gradient centrifugation using Ficoll reagent. In vitro, ALLDPSQGM (SEQ ID No. 34) peptide at positions 191-199, derived from the tuberculosis antigen Ag85B protein, was added at 5 μg / ml, followed by stimulation with 10 ng / ml recombinant IL-2 for 7 to 10 days. HLA-A*0201 tetramer incubation with the fluorescently labeled conjugated peptide was used for staining, and CD8+ of the Mycobacterium tuberculosis-specific TCR was sorted by flow cytometry. + T cells ( Figure 1 A). Primers were designed based on the conserved regions at both ends of the variable regions of the TCRα and β chains to amplify the Vα and Vβ genes using polymerase chain reaction (PCR). Figure 1 B), the gene sequence was obtained through sequencing and confirmed as a TCR variable region gene by comparison on the website www.imgt.org / . Figure 1C). Two TCR clones targeting the Ag85B protein epitope peptide of tuberculosis antigen were obtained, each containing its own Vα and Vβ variable region sequences. The paired Vα and Vβ genes from these clones were ligated using a GGGGS linker sequence and constructed into a His-tagged pcDNA3.1-IL12 eukaryotic expression vector. HEK293 cell lines were transfected with Transfection reagent. Three days later, HEK293 cell supernatant was collected, affinity purified, and a soluble protein containing TCR Vα and Vβ (TCR-A) was obtained. Figure 1 D).

[0052] The TCR clone α-chain variable region and β-chain variable region sequences of the TCR epitope peptide of tuberculosis antigen Ag85B protein and HLA-A*0201 complex involved in this invention (Table 2) were compared and analyzed on www.imgt.org / . The amino acid sequences of the complementarity-determining regions ACDR1, ACDR2, and ACDR3 in the α-chain variable region were confirmed as shown in SEQ ID No. 18, SEQ ID No. 19, and SEQ ID No. 20, respectively, and the amino acid sequence of the α-chain variable region was confirmed as shown in SEQ ID No. 24. The amino acid sequences of the complementarity-determining regions BCDR1, BCDR2, and BCDR3 in the β-chain variable region were confirmed as shown in SEQ ID No. 21, SEQ ID No. 22, and SEQ ID No. 23, respectively, and the amino acid sequence of the β-chain variable region was confirmed as shown in SEQ ID No. 25.

[0053] Table 2 Sequence Information Table

[0054]

[0055]

[0056] Example 3: Inhibition of intracellular Mycobacterium smegmatis survival

[0057] Recombinant Mycobacterium smegmatis CFP10 (rM. Smegmatis) and Ag85B recombinant Mycobacterium smegmatis (rM. Smegmatis) were constructed and used to infect THP1 cells stably transfected with human HLA-A*0201 for 4 h. Cells were then treated with TCR protein (TCR-C) from Example 1 and TCR protein (TCR-A) obtained from Example 2, respectively. Two TCR protein treatment methods were set up: one group was treated with TCR protein (10 μg / mL) for 24 h, and the other group was cultured for another 24 h after TCR treatment with fresh medium. Using the plain scan function of a high-content fluorescence microscope stage, cells treated with TCR protein for 24 h were stained with DAPI, and cell fluorescence images were captured. Figure 2A) After 24 hours of TCR removal treatment, the intracellular fluorescence intensity of TCR-treated cells was downregulated to varying degrees compared with the control group. Cells were collected at 24 hours and 24 hours after TCR removal, plated according to dilution gradients, and CFU counts were performed 3-4 days later. The results showed that the number of intracellular bacteria in the treatment group was significantly reduced ( Figure 2 B). Furthermore, calculations of fluorescence intensity at the same level revealed a significant decrease in bacterial count in the treatment group. Measurements of the average fluorescence intensity of cells showed a significant decrease in the average fluorescence intensity of recombinant bacteria within the treatment group (TCR protein treatment group). Figure 2 C). The above results indicate that both 24h treatment with TCR protein and 24h removal of TCR protein significantly inhibited the proliferation of the corresponding recombinant rM. smegmatis in macrophages.

[0058] Example 4: Inhibition of intracellular Mycobacterium tuberculosis survival

[0059] The standard strain of Mycobacterium tuberculosis H37Rv was inoculated into 7H9 medium (containing 10% OADC) for resuscitation, and then passaged twice until the logarithmic growth phase with an OD600 of 0.4–0.75 for single-bacterial preparation. THP-1 cells were then cultured at a concentration of 1 × 10⁻⁶ cells. 5 (48 holes) or 5×10 5 (12 wells) were seeded in medium containing 10% FBS 1640, and cells were stimulated with 100 nM PMA for 48 h to differentiate into macrophages. H37Rv was used to infect cells at an MOI of 1:1. After 24 h of treatment with TCR protein (10 μg / mL), cell lysates (ddH2O lysis), cell supernatant, and RNA samples (Trizol lysed cells) were collected. Cell supernatant was collected and cells were lysed, serially diluted, and plated. Plates were removed after 13–18 days for CFU counting. The results showed that both the TCR protein (TCR-C) obtained in Example 1 and the TCR protein (TCR-A) obtained in Example 2 significantly inhibited the survival of H37Rv cells. Figure 3 As shown.

[0060] Example 5: TCR protein induces THP-1 cells to release cytokines

[0061] The inflammatory factors TNF-α, IL-1β, IL-6, and IL-12 (p40) induced by TCR protein (TCR-C) obtained in Example 1 and TCR protein (TCR-A) obtained in Example 2, respectively, were detected by ELISA and qPCR. Cell supernatants were collected for ELISA experiments to detect the protein levels of the corresponding cytokines. The Xinbosheng ELISA kit catalog numbers were: Cat#: EHC103a, Cat#: EHC002b, Cat#: EHC007(H), and Cat#: EHC152. The results are as follows: Figure 4 As shown in Figure A. The specific steps for detecting mRNA expression using qPCR were as follows: Total RNA was extracted from cells using the Trizol method, and reverse transcribed from RNA to cDNA using HiScript IIIRT SuperMix for qPCR (+gDNA wiper) (Vazyme). The steps included PCR at 42°C for 2 min to remove genomic DNA, and PCR at 37°C for 15 min; followed by 85°C for 5 s to reverse transcribe into cDNA. The qPCR reaction was performed using ChamQ UniversalSYBR qPCR Master Mix (Vazyme), with HPRT1 as the internal reference gene. -△△Ct The method for quantifying target genes yielded the following results: Figure 4 As shown in B.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A T-cell receptor that specifically recognizes Mycobacterium tuberculosis antigenic peptides, comprising an α-chain variable region and a β-chain variable region, characterized in that, The T cell receptors are selected from the following group: i) The T-cell receptor is a T-cell receptor that specifically recognizes the Mycobacterium tuberculosis CFP10 antigen peptide, and its α-chain variable region contains an α-chain complementarity-determining region ACDR3 composed of an amino acid sequence as shown in SEQ ID No. 3, and its β-chain variable region contains a β-chain complementarity-determining region BCDR3 composed of an amino acid sequence as shown in SEQ ID No.

6. ii) The T-cell receptor is a T-cell receptor that specifically recognizes the Mycobacterium tuberculosis Ag85B antigen peptide. Its α-chain variable region contains an α-chain complementarity-determining region ACDR3 composed of an amino acid sequence as shown in SEQ ID No. 20, and its β-chain variable region contains a β-chain complementarity-determining region BCDR3 composed of an amino acid sequence as shown in SEQ ID No.

23.

2. The T cell receptor according to claim 1, characterized in that, In i), the α-chain variable region contains an α-chain complementarity-determining region ACDR1 composed of an amino acid sequence as shown in SEQ ID No. 1 and an α-chain complementarity-determining region ACDR2 composed of an amino acid sequence as shown in SEQ ID No. 2; and / or, the β-chain variable region contains a β-chain complementarity-determining region BCDR1 composed of an amino acid sequence as shown in SEQ ID No. 4 and a β-chain complementarity-determining region BCDR2 composed of an amino acid sequence as shown in SEQ ID No. 5; And / or, in ii) the α-chain variable region contains an α-chain complementarity-determining region ACDR1 composed of an amino acid sequence as shown in SEQ ID No. 18 and an α-chain complementarity-determining region ACDR2 composed of an amino acid sequence as shown in SEQ ID No. 19; and / or the β-chain variable region contains a β-chain complementarity-determining region BCDR1 composed of an amino acid sequence as shown in SEQ ID No. 21 and a β-chain complementarity-determining region BCDR2 composed of an amino acid sequence as shown in SEQ ID No.

22.

3. The T-cell receptor according to claim 1, characterized in that, In i), the α-chain variable region comprises an amino acid sequence as shown in SEQ ID No. 7, or comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 7; and / or, the β-chain variable region comprises an amino acid sequence as shown in SEQ ID No. 8, or comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 8; And / or, in ii) the α-chain variable region comprises an amino acid sequence as shown in SEQ ID No. 24, or comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 24; and / or the β-chain variable region comprises an amino acid sequence as shown in SEQ ID No. 25, or comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No.

25.

4. The T cell receptor according to claim 2, characterized in that, In i), the nucleotide sequence encoding ACDR1 is shown in SEQ ID No. 9, the nucleotide sequence encoding ACDR2 is shown in SEQ ID No. 10, the nucleotide sequence encoding ACDR3 is shown in SEQ ID No. 11, the nucleotide sequence encoding BCDR1 is shown in SEQ ID No. 12, the nucleotide sequence encoding BCDR2 is shown in SEQ ID No. 13, and the nucleotide sequence encoding BCDR3 is shown in SEQ ID No.

14. And / or, in ii): the nucleotide sequence encoding ACDR1 is shown in SEQ ID No. 26, the nucleotide sequence encoding ACDR2 is shown in SEQ ID No. 27, the nucleotide sequence encoding ACDR3 is shown in SEQ ID No. 28, the nucleotide sequence encoding BCDR1 is shown in SEQ ID No. 29, the nucleotide sequence encoding BCDR2 is shown in SEQ ID No. 30, and the nucleotide sequence encoding BCDR3 is shown in SEQ ID No.

31.

5. The T-cell receptor according to claim 3, characterized in that, In i), the nucleotide sequence encoding the α-chain variable region is as shown in SEQ ID No. 15, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 15; and the nucleotide sequence encoding the β-chain variable region is as shown in SEQ ID No. 16, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No.

16. And / or, in ii): the nucleotide sequence encoding the α-chain variable region is as shown in SEQ ID No. 32, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 32; and the nucleotide sequence encoding the β-chain variable region is as shown in SEQ ID No. 33, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No.

33.

6. A multivalent TCR complex, characterized in that, It contains at least three TCR molecules, and at least one of the TCR molecules is a T cell receptor as described in any one of claims 1-5.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding the TCR of any one of claims 1-5 or its complementary sequence, or a codon-optimized nucleotide sequence corresponding to the amino acid sequence of the TCR of any one of claims 1-5.

8. A carrier, characterized in that, The carrier contains the nucleic acid molecule as described in claim 7.

9. A host cell, characterized in that, The host cell contains the vector of claim 8 or the chromosome in which the exogenous nucleic acid molecule of claim 7 is integrated.

10. A pharmaceutical composition or kit, characterized in that, The composition or kit contains: i) any one of claims 1-5, the TCR complex of claim 6, the nucleic acid molecule of claim 7, the vector of claim 8, and / or the host cell of claim 9, and ii) a pharmaceutically acceptable carrier or excipient.

11. Use of the T cell receptor of any one of claims 1-5, the TCR complex of claim 6, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, and / or the pharmaceutical composition or kit of claim 10, said use comprising: i) Reagents or kits used to prepare for the detection of Mycobacterium tuberculosis; and / or ii) for the preparation of drugs for the prevention or treatment of pulmonary tuberculosis.

12. The use as described in claim 11, characterized in that, The tuberculosis is of type HLA-A*02.

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