A fusion protein MECPP007f and application thereof

Abstract

The application relates to the technical field of immunology, and particularly discloses a fusion protein MECPP007f and application thereof. The application provides a fusion protein MECPP007f, and the amino acid sequence of the fusion protein MECPP007f is shown as SEQ ID NO: 4. The application provides a fusion protein by connecting and fusing expression of multiple specific antigens expressed by mycobacterium tuberculosis in different metabolic states after specific modification, and the fusion protein has the advantages of high purification rate of recombinant protein, high renaturation rate, few multimers, maintained spatial structure of antigens, T / B cell antigen epitopes and better immunization performance, and provides a new method for prevention and immunotherapy of mycobacterium tuberculosis.

Description

Technical Field

[0001] This invention relates to the field of immunology, and more specifically, to a fusion protein MECPP007f and its applications. Background Technology

[0002] Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacterium tuberculosis Tuberculosis (TB) is a chronic infectious disease caused by Mtb. With the emergence of drug-resistant TB and HIV co-infection, the control of TB faces enormous challenges.

[0003] To date, only the BCG vaccine has been used to prevent tuberculosis in humans. Although the BCG vaccine has some effect on preventing tuberculosis in children, especially severe tuberculosis, its protective effect on tuberculosis in adults is poor (ranging from 0% to 80%). Therefore, it is urgent to develop new, safe and effective tuberculosis vaccines.

[0004] Some progress has been made in the development of new tuberculosis (TB) vaccines. The latest report shows that 16 novel TB vaccines are currently in clinical trials, including recombinant protein adjuvant vaccines (3 types), viral vector vaccines (4 types), inactivated vaccines (5 types), Mycobacterium tuberculosis live vaccines (3 types), and mRNA vaccines (1 type). Some of these candidate vaccines have made some progress in clinical studies, demonstrating advantages in safety and immunogenicity. However, existing vaccines have not yet been approved for use, and there is insufficient evidence to replace BCG. Therefore, the research and development of new and effective TB vaccines remains urgent and of great significance.

[0005] The complete genome sequence of Mycobacterium tuberculosis H37Rv is 4.4 × 10⁻⁶. 6The mycobacterium tuberculosis (TB) cell line contains approximately 4,000 coding genes and involves a large number of antigens. Therefore, the key to vaccine development lies in screening for effective antigens. Currently, the antigens of TB vaccines mainly focus on conserved secretory antigens and virulence-related antigens such as ESAT-6, CFP-10, Ag85, the ESX family, and the PPE family. ESAT-6 and CFP-10 are proteins encoded by genes that lack BCG in Mycobacterium tuberculosis. ESAT-6 is an early secretory protein of Mycobacterium tuberculosis, containing abundant T / B epitopes and exhibiting strong immunogenicity, inducing an effective immune response. CFP-10 is an early secretory protein isolated from the short-term culture filtrate of Mycobacterium tuberculosis. As an important T-cell antigen of Mycobacterium tuberculosis, it can stimulate the body to secrete IFN-γ early, thereby fighting TB infection. Because Mycobacterium tuberculosis exists in different metabolic states (active and dormant phases) after invading the host, and the protein antigens expressed in different metabolic states differ, existing technologies already include schemes that combine multiple antigen proteins.

[0006] However, while existing studies have used multiple antigens expressed at various stages, the formulation involves the expression and purification of at least two antigens, leading to complex formulation processes. Using prokaryotic expression systems to express subunit vaccine antigens can reduce costs and increase yield, but some antigens are expressed as inclusion bodies, which can cause protein polymers and precipitation during protein refolding, making vaccine characterization and validation difficult and resulting in poor drug-likeness. Therefore, how to modify and optimize antigens to ensure drug-likeness is a key technical issue in successful vaccine development. Summary of the Invention

[0007] One of the objectives of this invention is to provide a fusion protein that can improve the refolding rate of recombinant proteins during the preparation of fusion proteins through host cells, and also provide better immune performance.

[0008] This invention provides a fusion protein MECPP007f, the amino acid sequence of which is shown in SEQ ID NO:4.

[0009] The fusion protein MECPP007f of this invention contains antigens expressed by Mycobacterium tuberculosis at different metabolic states, specifically involving four antigens from Mycobacterium tuberculosis: early secretory protein antigens Rv3875 (ESAT-6), Rv3874 (CFP-10), the epitope concentration region (PstS1p) of the strong T cell immune antigen Rv0934, and the dormant protein antigen Rv2029c. The different antigens are linked using "GGGSGGGS" as a linker arm, and specific adjustments are made to the amino acid sequences of the original antigens. This fusion protein MECPP007f exhibits ideal immunogenicity, significantly inhibiting the growth of Mycobacterium tuberculosis. Furthermore, in the preparation of the recombinant protein using recombinant bacteria containing the gene encoding sequence of the MECPP007f protein, the recombinant protein shows high purification rate, high renaturation rate, and low polymer content.

[0010] The present invention also provides nucleotides encoding the above-mentioned fusion protein MECPP007f.

[0011] The nucleotides of the present invention have nucleotide sequences as shown in SEQ ID NO:3.

[0012] The present invention also provides a biomaterial comprising the above-mentioned nucleotides, wherein the biomaterial is an expression cassette, a vector, or a host cell.

[0013] The present invention also provides the use of the above-mentioned fusion protein MECPP007f or nucleotides or biological materials in the preparation of reagents for diagnosing Mycobacterium tuberculosis infection or diseases caused by Mycobacterium tuberculosis infection.

[0014] The main immunological methods for detecting Mycobacterium tuberculosis infection are the interferon-gamma release assay (IGRA) and T-Spot. Both are based on specific immunoassays to detect the presence of Mycobacterium tuberculosis. The former requires stimulating lymphocytes with a specific antigen and detecting the level of interferon-gamma to confirm infection. The latter uses a specific antigen to stimulate lymphocytes and detects the number of lymphocytes releasing interferon-gamma to confirm infection. Therefore, the MECPP007f of this invention can be used for subsequent tuberculosis diagnosis.

[0015] The present invention also provides the application of the above-mentioned fusion protein MECPP007f, nucleotides, or biological materials in the preparation of vaccines for the prevention of Mycobacterium tuberculosis infection.

[0016] The present invention also provides the use of the above-mentioned fusion protein MECPP007f, nucleotides, or biological materials in the preparation of medicaments for treating diseases caused by Mycobacterium tuberculosis infection.

[0017] The present invention also provides a Mycobacterium tuberculosis vaccine comprising the above-mentioned fusion protein MECPP007f and an adjuvant.

[0018] The present invention also provides a diagnostic reagent for Mycobacterium tuberculosis, which includes the above-mentioned fusion protein MECPP007f or nucleotides.

[0019] The present invention also provides the application of the above-mentioned fusion protein MECPP007f or nucleotides or biological materials in the preparation of tuberculosis DNA vaccines and mRNA vaccines.

[0020] The beneficial effects of this invention are at least as follows: This invention provides a fusion protein with high recombinant protein purification rate, high refolding rate, few polymers (ensuring drug-like properties), and the ability to maintain the spatial structure of antigens, T / B cell epitopes, and better immune performance by tandemly expressing multiple specific antigens expressed by Mycobacterium tuberculosis in different metabolic states and after specific modification. Attached Figure Description

[0021] Figure 1 The image shows the electrophoresis results after ECPP007f expression, purification, and refolding. M represents the protein standard.

[0022] Figure 2 This is a comparison of the 3D structures of ECPP007f after replacing specific amino acids with alanine. Blue represents the 3D structure of ECPP007f, and red represents the 3D structure after replacing with alanine.

[0023] Figure 3 The images show the predicted 3D structures of ECPP007f and MECPP007f with amino acid substitutions. A: 3D structure of ECPP007f; B: 3D structure of MECPP007f; C: 3D structure alignment results of ECPP007f and MECPP007f, with blue representing MECPP007f and green representing ECPP007f.

[0024] Figure 4 These are the results of antigen epitope prediction. A: T / B epitope prediction results for ECPP007f; B: T / B epitope prediction results for MECPP007f. Different epitopes are marked in red.

[0025] Figure 5 The results show the purity of the modified recombinant antigen after purification and the electrophoresis results after renaturation. A: Purity test results of MECPP007f after purification; B: Electrophoresis results of MECPP007f after renaturation, M is protein standard, 1 is the sample treated with non-reducing buffer after purification and renaturation, and 2 is the sample treated with reducing buffer after purification and renaturation.

[0026] Figure 6 The vertical axis represents the specific IgG antibody titer of each group after immunization; the vertical axis represents the antibody titer value Log10, and the horizontal axis represents the immunization group.

[0027] Figure 7 The results for ELISPOT are shown on the horizontal axis, with the experimental group on the left and the stimulating antigen marked below the group.

[0028] Figure 8 Results for cytokines.

[0029] In each diagram (if any), represent P <0.01, represent P <0.001, represent P <0.0001, ns indicates that the difference is not significant. Detailed Implementation

[0030] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0032] Example 1: Construction of recombinant protein ECPP007f DNA sequences from Rv3875, Rv3874, Rv0934 (nucleotide positions 169-405 and 802-1119) and Rv2029c were linked in the following sequence: Rv3875-linker-Rv3874-linker-Rv0934 (169-405-802-1119)-linker-Rv0934 T-epitope region + Rv2029c (ECPP007f). The linker sequence was “GGSGG”, with six His (histidine) residues added to the C-terminus for easier purification. The resulting nucleotide sequence after codon optimization is shown in SEQ ID NO:1 (encoding the amino acid sequence shown in SEQ ID NO:2). This nucleotide sequence was then sent to General Biotechnology (Anhui) Co., Ltd. for gene sequencing, and the sequence was then transmitted via… Nde I and XhoThe I restriction site was inserted into the expression vector pET43.1a to construct the recombinant protein expression plasmid, named pET43.1a-ECPP007f, for use in subsequent experiments.

[0033] Example 2: Expression, purification and refolding of recombinant protein ECPP007f After dissolving pET43.1a-ECPP007f constructed in Example 1 in water, 5 μl of the solution was added to 50 μl of *E. coli* BL21(DE3) competent cells (Beijing TransGen Biotechnology). The mixture was placed on ice for 30 min, heat-shocked at 42°C for 90 s, and then incubated on ice for 2 min. 900 μl of LB liquid medium was added, and the mixture was incubated at 37°C with shaking for 1 h. After centrifugation at 4000 rpm for 1 min, 600 μl of supernatant was discarded. The mixture was then gently pipetted to mix, and 200 μl of the bacterial culture was spread onto LB agar plates containing ampicillin. The plates were incubated at 37°C for 16 h. Single colonies were picked and incubated in 5 ml of LB liquid medium containing ampicillin. After shaking at 37°C for 12 h, the colonies were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Successfully sequenced bacterial strains were expanded and plasmids were extracted and stored at -70°C for subsequent experiments.

[0034] The strains obtained from the above screening were streaked on LB solid medium and incubated at 37°C for 12-16 h. Single colonies were picked and added to 5 ml of LB liquid medium containing ampicillin, and cultured overnight at 37°C with shaking at 180 rpm / min. The overnight culture was added to 500 ml of LB liquid medium containing ampicillin, and cultured until the OD value of the culture reached 0.6-0.8. Isopropyl-β-D-thiopyranoside (IPTG) was added to a final concentration of 1 mmol / L, and expression was induced at 37°C for 3.5 h. The bacterial cells were collected by centrifugation at 4000 rpm for 10 min and cultured with 10 mmol / L pH 8.0 medium containing 0.5% Triton-X100. The bacterial cells were resuspended in Tris-HCl solution and sonicated (220W, 15s sonication, 20s interval, 10min). 100μl of the sonicated suspension was centrifuged at 12000rpm for 1min, the supernatant was collected, and the precipitate was resuspended in 100μl of deionized water. SDS-PAGE electrophoresis was performed to detect the expression of the recombinant protein. The results showed that the protein was mainly expressed in the form of inclusion bodies.

[0035] The precipitate obtained after ultrasonic disruption and centrifugation was resuspended in 8 mol / L urea. The target protein was then crudely purified using Ni affinity chromatography, as follows: the packing material was washed with 10 mmol / L Tris-HCl at pH 8.0. Then, 8 mol / L urea was pumped in until the absorbance remained constant, and the solution was zeroed. The treated sample was then pumped in, and the liquid at the peak was collected. Elution was then performed with 8 mol / L urea containing different concentrations of imidazole (15 mmol / L, 30 mmol / L, 60 mmol / L, and 300 mmol / L). The purified sample (before renaturation) was then analyzed by SDS-PAGE electrophoresis. The results showed that a high-purity protein was obtained for subsequent experiments.

[0036] In this invention, a gradient-decreasing urea concentration method was used for dialysis refolding. After refolding, the samples were sterilized by filtration through a 0.22µm Millipore membrane, and the protein concentration was measured using the BCA method (Beijing TransGen Biotech). Simultaneously, considering subsequent experimental use, the refolded samples were concentrated to a concentration of 1 mg / ml. Then, samples from each step of the experiment were treated with reducing buffer (5× protein loading buffer containing DTT, P1040) and non-reducing buffer (4× non-denaturing protein loading buffer, P1017) (Beijing Solarbio Science & Technology Co., Ltd.) before SDS-PAGE electrophoresis. The results are shown below. Figure 1 It shows that polymeric forms exist in the refolded sample.

[0037] Example 3: Modification and Optimization of ECPP007f Since the amino acids of ECPP007f are prone to polymer formation during refolding, this embodiment provides an ECPP007f modification scheme that reduces polymer formation without reducing the immunogenicity of the protein antigen.

[0038] In the research process of this invention, 1-5 amino acids were first mutated in specific amino acids (cysteine ​​at positions 433, 457, 524, 560, 596, and 697, a total of 6) in the amino acid sequence shown in SEQ ID NO:2. However, the results of this part showed that the stability of the expressed protein after mutation was poor during refolding, and it was prone to aggregation and could not be used for subsequent purposes. In addition, in this invention, these 6 specific amino acids were replaced with other amino acids (alanine). In the experiment, it was found that the 3D structure of the protein changed after the replacement, as shown in the following results. Figure 2 .

[0039] Finally, the present invention replaced 6 specific amino acids in the amino acid sequence shown in SEQ ID NO:2 and adjusted the linker to obtain a new amino acid sequence as shown in SEQ ID NO:4 (the nucleotide sequence encoding it is shown in SEQ ID NO:3), and named this modified combined antigen MECPP007f.

[0040] This embodiment also utilizes NCBI iCn3D to predict the 3D structures of ECPP007f and the amino acid-substituted MECPP007f, and uses PyMOL software to compare the predicted 3D structures of the two antigens. The results ( Figure 3 The results showed that the internal core regions of the two 3D structures were completely overlapping, with only a small difference in the direction of the α-helix at the C-terminus. This indicates that amino acid substitution does not alter the main spatial structure of the antigen, suggesting that it will not affect its function. Then, bioinformatics techniques were used to predict the T / B cell epitopes of ECPP007f and the amino acid-substituted MECPP007f to verify whether amino acid substitution affects the immunogenicity of the antigen. The results (…) Figure 4 The results show that the antigenic epitopes remained largely unchanged after sequence optimization, with only one epitope shifting, as indicated by the red box in the figure. This result also suggests that the substitution of amino acids in the antigen has virtually no impact on the spatial structure and the T / B cell epitopes of the antigen.

[0041] The modified sequence was given to General Biotechnology (Anhui) Co., Ltd. to synthesize a gene sequence, and the sequence was then transmitted through... NdeⅠ and XhoⅠ The restriction enzyme sites were inserted into the expression vector pET43.1a to construct the recombinant protein expression plasmid, named pET43.1a-MECPP007f, for subsequent experiments. The same expression and purification steps as in Example 2 were used. The purified product was analyzed by high-performance liquid chromatography (HPLC) (HPLC column: Nap Biocore SEC-150, 7.8 × 300 mm, 5 μm; HPLC temperature: 30℃; mobile phase flow rate: 0.3 ml / min; mobile phase: pH 6.8 50 mM phosphate solution (containing 150 mM sodium sulfate, 10% isopropanol); isocratic elution; injection volume: 90 μl). The results showed that a recombinant protein with a purity of 99% could be obtained after modification. Figure 5 (A) To further detect the presence of polymers after refolding, the refolded products were treated with denaturing loading buffer and non-denaturing loading buffer respectively, followed by SDS-PAGE electrophoresis. The results showed that no polymers appeared after modification. Figure 5 (B in the text) These results suggest that the purification efficiency and refolding rate of proteins are improved after amino acid substitution modification.

[0042] Example 4: Humoral immune response to ECPP007f and MECPP007f SPF-grade Balb / c mice aged 6-8 weeks were selected and divided into 6 groups of 6 mice each. The negative control group was the PBS group. Each experimental group was further divided into 2 subcutaneous immunization groups (3 times): ECPP007f + aluminum hydroxide (ECPP007f-Al, antigen 50 μg, aluminum hydroxide:antigen (V:V) = 1:3), MECPP007f + aluminum hydroxide (MECPP007f-Al, antigen 50 μg, aluminum hydroxide:antigen (V:V) = 1:3), and aluminum hydroxide adjuvant group (Alum). One group received an intraperitoneal injection of 1×10 6 CFU BCG China (purchased from the National Institutes for Food and Drug Control) (single immunization) served as the control group (BCG). The BCG group received one immunization, while all other groups received three immunizations, with each immunization spaced two weeks apart.

[0043] Fourteen days after the third immunization, all animals were euthanized, and serum and spleen lymphocytes were collected.

[0044] Serum was collected after orbital blood sampling to detect the titer of antigen-specific IgG antibodies in the serum. The method was as follows: ECPP007f and MECPP007f were diluted to 2 μg / ml with coating buffer, 100 μl / well, and incubated overnight (20-24 h) at 4℃. The plates were washed three times with PBST and blocked with 5% skim milk powder at 37℃ for 2 h. Then, the test samples were serially diluted, 100 μl / well, and incubated at 37℃ for 2 h. Anti-mouse HRP-labeled IgG (Suzhou Bio-Long Technology Co., Ltd.) was diluted 1:5000 with antibody dilution buffer, 100 μl / well, and incubated at 37℃ for 1 h. 100 μl / well of TMB was added, and the reaction was developed at 37℃ in the dark for 20 min. The reaction was terminated with 50 μl / well of 2 mol / L H2SO4 solution, and the OD450-OD630 values ​​were measured using a microplate reader.

[0045] The results are as follows Figure 6 As shown, both ECPP007f and MECPP007f immunized patients with these antibodies produced high levels of specific IgG antibodies, and the differences between these antibodies and the negative control group and the aluminum adjuvant group were statistically significant. Furthermore, this invention also found that the amino acid-substituted MECPP007f produced a significantly higher antibody titer, approximately 60 times that of ECPP007f, and the difference in antibody titer values ​​between the two groups was statistically significant. P The result (<0.0001) confirms that both ECPP007f and MECPP007f can induce specific humoral immunity against tuberculosis, with MECPP007f showing better efficacy.

[0046] Example 5: Cellular immune response of ECPP007f and MECPP007f This embodiment uses the same immunization strategy as in Example 4. Fourteen days after the third immunization, the animal was euthanized, and after dissection, the spleen was removed and lymphocytes were isolated, with the cell concentration adjusted to 2×10⁻⁶. 6 cells / ml are used for subsequent experiments.

[0047] The levels of two cytokines secreted by mouse lymphocytes after immunization were detected using the ELISPOT pre-coated IL-4 and IFN-γ kit from Dako Biotechnology Co., Ltd. The method was as follows: 100 μl of cells were added to each well, and the corresponding antigen was added to each well according to the experimental group. After 16 h of stimulation, colorimetric assay was performed. The results showed ( Figure 7 Immunization with BCG enhanced the ability of splenic lymphocytes in mice to secrete IFN-γ, suggesting that it can stimulate a certain intensity of Th-1 immune response. Immunization with ECPP007f and MECPP007f, regardless of whether the antigen was a fusion antigen or a single antigen contained within it, significantly enhanced the ability of splenic lymphocytes in mice to secrete IFN-γ and IL-4, and both were more effective than BCG. This result shows that both ECPP007f and MECPP007f can stimulate a high level of cellular immunity, superior to BCG. Furthermore, compared to ECPP007f, MECPP007f combined with aluminum hydroxide adjuvant resulted in a greater number of cells secreting IFN-γ and IL-4, indicating that amino acid modification enhanced the cellular immune response to the antigen to some extent. Meanwhile, this invention uses the single antigens that make up ECPP007f (the sequence of Rv3874 is shown as bases 104 to 202 of SEQ ID NO:2, the sequence of Rv3875 is shown as bases 1 to 95 of SEQ ID NO:2, and the sequence of PstS1p (Rv0934 T epitope concentration region) is shown as bases 211 to 393 of SEQ ID NO:2) to stimulate lymphocytes respectively. Experiments showed that mice immunized with ECPP007f and MECPP007f had a strong immune response to the single antigens. This result confirms that the single antigens that make up ECPP007f and MECPP007f both play a role and can stimulate the body to produce a strong cellular immune response.

[0048] This invention also uses a Luminex liquid chromatography-mass spectrometry (BD Biosciences, Inc., USA) to detect the expression levels of Th-1 / 2 / 17 cytokines in lymphocytes after antigen stimulation. The results showed ( Figure 8 In addition to TNF-α, MECPP007f immunized animals and significantly increased the levels of IFN-γ, IL-2, IL-6, IL-10 and GM-CSF expressed in lymphocytes. In particular, the expression levels of Th-1 cytokines IFN-γ and IL-2, which play an important role in anti-tuberculosis infection, were 10 times that of the BCG group.

[0049] Example 6: Evaluation of the protective effects of ECPP007f and MECPP007f To further evaluate the immunoprotective effects of ECPP007F and MECPP007f, this invention employs in vitro mycobacterial inhibition assays (MGIAs), an unbiased functional method that considers a range of immune mechanisms and their complex interactions, serving as a tool for assessing protective immunity and the efficacy of tuberculosis vaccines. The method is as follows: Four groups were set up (PBS, BCG, ECPP007f, and MECPP007f), with eight mice in each group. Mice were euthanized 14 days after the third immunization (experimental procedure as described in Example 4), and spleens were harvested to isolate lymphocytes, adjusting the cell concentration to 2 × 10⁻⁶. 6 1 ml of cells per well was co-cultured with 500 CFU of H37Rv (ATCC 27294) for 96 h. After centrifugation, the supernatant was discarded, and 500 μl of 1% SDS was added. The cells were lysed on ice for 10 min. The samples were then serially diluted and plated on 7H10 plates. After incubation at 37°C upside down for 4 weeks, colony counting was performed. The counting results are shown in Table 1.

[0050] In vitro antibacterial experiments after immunizing mice showed that the antibacterial rates of ECPP007f, MECPP007f + aluminum adjuvant, and BCG were 98.11%, 98.69%, and 44.16%, respectively. MECPP007f significantly inhibited the growth of Mycobacterium tuberculosis H37Rv more than BCG, confirming that MECPP007f has a better immunoprotective effect. Antibacterial rate = (mean CFU in PBS group - mean CFU in experimental group) / mean CFU in PBS group × 100%.

[0051] Table 1. Antigen protection effect after immunization (unit: CFU / ml)

[0052] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A fusion protein MECPP007f, characterized in that, The amino acid sequence is shown in SEQ ID NO:

4.

2. The nucleotide encoding the fusion protein MECPP007f of claim 1.

3. The nucleotide according to claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID NO:

3.

4. A biomaterial, characterized in that, The biological material comprises the nucleotide of claim 2 or 3, wherein the biological material is an expression cassette, vector, or host cell.

5. The use of the fusion protein MECPP007f of claim 1, or the nucleotide of claim 2 or 3, or the biomaterial of claim 4 in the preparation of reagents for diagnosing Mycobacterium tuberculosis infection or diseases caused by Mycobacterium tuberculosis infection.

6. The use of the fusion protein MECPP007f of claim 1, or the nucleotide of claim 2 or 3, or the biomaterial of claim 4 in the preparation of a vaccine to prevent Mycobacterium tuberculosis infection.

7. The use of the fusion protein MECPP007f of claim 1, or the nucleotide of claim 2 or 3, or the biomaterial of claim 4 in the preparation of a medicament for treating diseases caused by Mycobacterium tuberculosis infection.

8. A tuberculosis vaccine, characterized in that, It includes the fusion protein MECPP007f as described in claim 1 and an adjuvant.

9. A diagnostic reagent for Mycobacterium tuberculosis, characterized in that, It includes the fusion protein MECPP007f as described in claim 1 or the nucleotide as described in claim 2 or 3.

10. The use of the fusion protein MECPP007f of claim 1, or the nucleotide of claim 2 or 3, or the biomaterial of claim 4 in the preparation of tuberculosis DNA vaccines and mRNA vaccines.

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