Tuberculosis subunit vaccine based on fusion protein and preparation method thereof

Abstract

The invention belongs to the technical field of biology, particularly relates to a fusion protein-based tuberculosis subunit vaccine and a preparation method thereof, and relates to two tuberculosis subunit vaccines and a preparation method thereof, and each vaccine comprises a fusion protein. The preparation method comprises the following steps: recombining MPT6461-170, Rv2660c and Rv264521-80 genes into a prokaryotic expression vector through a gene engineering technology to obtain a fusion protein LT30; the Rv2645 and Rv2660c genes are recombined into a prokaryotic expression vector to obtain the fusion protein LT25. The two fusion proteins are massively expressed in a prokaryotic expression system and are purified by gel filtration chromatography, and plasmid retention rate and large-scale preparation research are carried out. The LT30 and the LT25 respectively form subunit vaccines with DP (adjuvant), and tuberculosis antigen specific cellular immune response is effectively induced in a mouse body. Particularly, LT25 / DP can improve the protective effect of mice against mycobacterium tuberculosis H37Ra infection, and is expected to become a novel tuberculosis subunit vaccine.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of biotechnology, and in particular relates to a tuberculosis subunit vaccine based on fusion protein and a preparation method thereof. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) and is the number one killer of all infectious diseases worldwide. Due to the impact of the ongoing COVID-19 pandemic on the detection and treatment of tuberculosis cases, the number of cases is expected to increase significantly in the coming years. According to the "2022 Global Tuberculosis Report" released by the World Health Organization, there were 10.6 million new cases of tuberculosis in the world in 2021, including 6 million adult males, 3.4 million adult females, and 1.2 million children. The number of new deaths worldwide was approximately 1.413 million, and the number of deaths from HIV co-infection reached 187,000. Despite global efforts to curb the spread of tuberculosis, tuberculosis mortality has improved substantially in the past 20 years, but progress in reducing the incidence of tuberculosis has been minimal. The lack of an effective vaccine, limited cure rates for drug-resistant tuberculosis (MDR-TB), and HIV / AIDS co-infection make tuberculosis still a major global public health threat.

[0003] Bacille Calmette-Guerin (BCG), the only vaccine currently approved for the prevention of tuberculosis, is widely used in most countries to prevent tuberculosis in newborns. Although BCG has been shown to be very effective in preventing tuberculous meningitis and disseminated tuberculosis in children, it remains controversial in preventing pulmonary tuberculosis in adolescents and adults. A systematic review of clinical trial data reported that the protective effect of BCG on adults ranges from 0% to 80%, and the reasons for the variation in protective effect are still unclear, so there is an urgent need to develop a new tuberculosis vaccine.

[0004] Tuberculosis subunit vaccines are some immunologically active components (such as proteins, peptides, glycolipids, etc.) in Mycobacterium tuberculosis. Compared with live vaccines, they have clear ingredients, low cost, high yield, good safety, easy preparation, good quality control, and can enhance the immune memory of effector T cells through vaccination. Compared with whole-cell vaccines, subunit vaccines target a small number of selected antigens, usually 6 or less. Currently, only a few have been identified as promising candidate antigens for tuberculosis subunit vaccines. Therefore, a major challenge in the development of protein subunit vaccines is the need to identify the best antigens for such vaccines. Currently, five subunit vaccines have entered clinical trials: M72 / AS01E, ID93+GLA-SE, H56 / IC31, AEC / BC02, and GamTBvac.

[0005] Mycobacterium tuberculosis expresses approximately 4000 proteins, and the antigens found in current subunit vaccines are mainly selected based on their immunodominance in animal or human studies and the differential expression of antigens between latently infected and active tuberculosis patients. The problem of antigen screening is complicated by the fact that different antigens are expressed differently at different stages of M. tuberculosis infection. During M. tuberculosis infection, ESAT-6 is continuously expressed, while Ag85B is mainly expressed early in infection. Studies have shown that some latent antigens are potent stimulatory antigens of human T cells and have a protective effect in animal models of tuberculosis. In addition, mycobacteria in vivo are composed of bacteria at different stages of metabolic activity with different antigen expression, including replicating bacteria and dormant bacteria, which maintain the ability to switch between these two states in the yin-yang model. For these reasons, subunit vaccines for tuberculosis should select antigens expressed at different metabolic stages to provide a wider range of immunity.

[0006] Several new multi-stage subunit vaccines have been developed. Claus Aagaard et al. constructed the H56 subunit vaccine by combining the early antigen Ag85B and the 6-kDa early secretory antigen target (ESAT-6) of Mycobacterium tuberculosis with the latency-associated protein Rv2660c. In two latent tuberculosis mouse models, the H56 vaccine group mice were able to control bacterial reactivation and significantly reduce the lung bacterial load compared with the adjuvant control mice. Zhu et al. fused the proliferatively expressed antigens ESAT6, Ag85B, the 190-198 peptide of MPT64 and Mtb8.4, as well as the latency-associated antigen Rv2626c, to construct a multi-stage protein LT70 (ESAT6-Ag85B-MPT64 190–198 -Mtb8.4-Rv2626c). The protective efficacy of the subunit vaccine LT70 against Mycobacterium tuberculosis infection was higher than that of the traditional vaccine BCG at 30 weeks after vaccination (10 weeks after challenge).

[0007] With the development of comparative genomics technology, whole genome comparison studies have found that there are some missing fragments in the genomes of different bacteria in the Mycobacterium tuberculosis complex (such as Mycobacterium tuberculosis, Mycobacterium bovis or BCG), and these missing fragments are called Region of Differences (RD). Studies have found that the genes in the RD region are closely related to the virulence of Mycobacterium tuberculosis, and the functions of these genes have important potential significance and application value in the development of new tuberculosis vaccines and the establishment of new diagnostic methods. In addition, Brandt L et al. found that due to the cross-recognition of a large number of antigens shared between environmental mycobacteria and BCG, environmental mycobacteria have a direct antagonistic effect on BCG vaccination. Pre-sensitization of environmental mycobacteria will inhibit the proliferation of BCG in the body, and the level of IFN-γ produced will be significantly reduced, thereby preventing the immune protection induced by BCG. The LT28 constructed in the laboratory earlier consists of MPT64 and Rv1978 in the RD2 region and Rv2645 in the RD13 region, which can induce high cellular and humoral immune responses, but Rv1978 has a high homology with Mycobacterium avium, and its immune protection needs to be further improved. Therefore, we screened antigens in the RD region of Mycobacterium tuberculosis with low homology to environmental mycobacteria / human susceptible bacteria to construct a subunit vaccine, which can update the antigen library while avoiding cross-reactions caused by environmental mycobacteria. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a tuberculosis subunit vaccine based on fusion protein and a preparation method thereof.

[0009] The present invention is achieved by a fusion protein-based tuberculosis subunit vaccine, characterized in that the vaccine comprises two subunit vaccines, each of which comprises a fusion protein, and the fusion protein is obtained by genetic engineering technology:

[0010] The first subunit vaccine, fusion protein LT30, was created by combining the gene MPT64 61-170 、Rv2660c、Rv2645 21-80 Obtained by recombining into a prokaryotic expression vector;

[0011] The fusion protein LT25 of the second subunit vaccine is obtained by recombining the genes Rv2645 and Rv2660c into a prokaryotic expression vector.

[0012] Used to stimulate specific immune response to Mycobacterium tuberculosis antigens and enhance the immune protection against Mycobacterium tuberculosis infection.

[0013] Furthermore, the fusion protein expression plasmid is pET30a(+), and the plasmid for recombinant expression of the fusion protein is

[0014] pET30a(+)-LT30(MPT64 61-170 -linker-Rv2660c-linker-Rv2645 21-80 ) and pET30a(+)-LT25(Rv2645-Rv2660c).

[0015] Another object of the present invention is to provide a method for preparing the tuberculosis subunit vaccine, which method specifically comprises:

[0016] Purification of fusion protein LT30 / LT25:

[0017] S1: The fusion protein LT30 / LT25 was expressed as inclusion bodies and was first washed with a washing buffer (2 M urea, 0.5 M NaCl, 50 mM Tris, 1 mM EDTA and 1% Triton-100, pH 7.4), and the precipitate was collected by centrifugation (10000 rpm, 10 min) and treated three times;

[0018] S2: For the samples containing LT30 / LT25 after washing, dissolve them with 8M urea + 50mM Tris (pH8.0-8.5) at 4℃ for 30min; dilute the 8M urea solution containing the target protein to 6M urea with 50mM Tris (PH8.0-8.5) and treat at 4℃ for 4h. Dilute the 6M urea solution containing the target protein to 4M urea with 50mM Tris (PH8.0-8.5) and treat at 4℃ for 4h; dilute the 4M urea solution containing the target protein to 3M urea with 50mM Tris (PH8.0-8.5) and treat at 4℃ for 12h.

[0019] S3: After dilution and renaturation, the sample is first purified by gel filtration chromatography (molecular sieve), and the protein with a molecular sieve peak value >20mAU is collected to obtain a protein with a purity >90%.

[0020] Furthermore, the relevance of the fusion proteins LT30 / LT25 lies in that the construction of LT30 selects the dominant epitope of Rv2645, while the construction of LT25 selects the full length of Rv2645, and the immunogenicity of the dominant epitope of Rv2645 and the full length is compared.

[0021] Another object of the present invention is to provide an application of the tuberculosis subunit vaccine, using a DP adjuvant.

[0022] Another object of the present invention is to provide a tuberculosis subunit vaccine for use in anti-tuberculosis prevention.

[0023] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0024] First, based on the fusion protein LT28 that induces higher cellular and humoral immune responses constructed in the laboratory in the early stage, the present invention further screened out the latent antigen Rv2660c in the BCG deletion region (RD region) with lower homology with environmental mycobacteria, replacing the fusion protein LT28 (MPT64 61-170 -Rv1978 8-60 -Rv2645 21-80 ) with a high homology to environmental mycobacteria Rv1978, and construct a new fusion protein LT30 (MPT64 61-170 -Rv2660c-Rv2645 21-80 ); construct a new fusion protein LT25 (Rv2645-Rv2660c) with the RD13 region growth phase secretory antigen Rv2645. MPT64 is encoded by the gene Rv1980c, which can induce IFN-γ in mouse macrophages and tuberculosis patients, and reduce cell apoptosis by increasing the expression of TGF-β and reducing inflammation. After Rv2645 is supplemented with the BCG strain, it can improve the antigen presentation ability of dendritic cells, induce stronger Th1 and Th17 responses, and induce protection of mice and macaques against infection with Mycobacterium tuberculosis H37Rv strain; Rv2660c, as a latent antigen, is related to the parasitism and latency of Mycobacterium tuberculosis in macrophages. It can bind to TLR2 and activate macrophages to release IL-1β, IL-8 and TNF-α, and can induce significant cellular and humoral immune responses in Chinese latently infected patients.

[0025] The present invention utilizes a genetic engineering method to combine the above antigens at the gene level, constructs new fusion proteins LT30 and LT25, expresses and purifies them, identifies them, and studies their immunogenicity and immune protection effects. Fusion proteins LT30 and LT25 can be expressed in large quantities in a prokaryotic expression system, purified by methods such as gel filtration chromatography, and studies on plasmid retention rate and large-scale preparation are conducted. The fusion proteins LT30 and LT25 of the present invention are combined with adjuvant DP to form a tuberculosis subunit vaccine, which can effectively induce a tuberculosis antigen-specific cellular immune response in mice, and at the same time, the vaccine can improve the protective effect of mice against Mycobacterium tuberculosis H37Ra infection. The vaccine is expected to become a new type of tuberculosis subunit vaccine.

[0026] Second, the tuberculosis subunit vaccine technical solution of the present invention solves the problem of unsatisfactory immune effect of tuberculosis vaccines in the prior art. Although traditional tuberculosis vaccines such as BCG are effective for children, their protective effect in adults is limited, especially for latent tuberculosis infection and multidrug-resistant tuberculosis. In addition, traditional vaccines usually use attenuated live bacteria or inactivated bacteria, which have safety issues, especially for people with low immune function. The present invention synthesizes specific fusion proteins through genetic engineering means, which can more accurately induce specific immune responses against tuberculosis antigens, thereby improving the protective effect of the vaccine, especially its effectiveness against different populations and drug-resistant tuberculosis.

[0027] Technological advancements:

[0028] 1. Improve the specificity and safety of vaccines

[0029] The present invention recombines the key antigen (MPT64 in Mycobacterium tuberculosis) 61-170 、Rv2660c、Rv2645 21-80 and Rv2645) to produce high-purity fusion proteins LT30 and LT25, which can induce a more precise immune response. Compared with traditional whole-bacterial vaccines, subunit vaccines contain only a small amount of key antigens, which greatly reduces the risk of adverse reactions and is especially safer for immunosuppressed patients.

[0030] 2. Improve the intensity and durability of immune response

[0031] By using DDA and Poly I:C mixed together as adjuvants in the vaccine preparation, the present invention significantly enhances the immune effect of the vaccine. Poly I:C can simulate viral RNA and strongly activate the host's innate immune response. After combining with the fusion protein, it not only improves the antigen presentation efficiency, but also promotes the formation of memory immune cells. The use of this adjuvant greatly improves the immune persistence and immune protection ability of the vaccine.

[0032] 3. Achieved efficient protein production and purification process

[0033] The present invention uses a recombinant plasmid to express the fusion protein in Escherichia coli and uses gel filtration chromatography technology to purify the protein, ensuring the high purity of the protein (greater than 90%), thereby improving the vaccine production efficiency. Compared with traditional protein expression and purification methods, this process can produce large-scale high-quality vaccine products more quickly and economically, and is suitable for industrial application.

[0034] 4. Technological progress in combating multidrug-resistant tuberculosis

[0035] The antigen protein and fusion technology selected by the present invention can cover multiple antigen sites of Mycobacterium tuberculosis, effectively addressing the existing multidrug-resistant tuberculosis problem. These antigens play a role in different stages (such as dormant and active phases), ensuring that the immune system can recognize and attack tuberculosis bacteria in a timely manner, reducing the probability of the occurrence of drug-resistant tuberculosis strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a flow chart of a method for preparing a tuberculosis subunit vaccine of a tuberculosis subunit vaccine provided by an embodiment of the present invention;

[0037] Figure 2 The results of expression and purification of the fusion protein LT30 in E. coli Origami provided by the embodiment of the present invention are shown in Figure 1. A: expression of the fusion protein LT30 in E. coli Origami; B: inclusion bodies of the fusion protein LT30 were purified by molecular sieve column, eluted with 50 mM Tris solution, and the elution peak was collected; C: samples collected by molecular sieve purification of the fusion protein LT30 were run on SDS-PAGE electrophoresis, and the electrophoresis pattern after staining and decolorization;

[0038] Figure 3 The results of expression and purification of the fusion protein LT25 provided in the embodiment of the present invention in Escherichia coli BL21 (DE3); A: expression of the fusion protein LT25 in Escherichia coli BL21 (DE3); B: inclusion bodies of the fusion protein LT25 were purified by molecular sieve column refolding, eluted with 50mM Tris solution, and the elution peak was collected, and one sample was collected for every 3ml (elution sample 1, sample 2, sample 3, sample 4); C: samples collected by molecular sieve purification of the fusion protein LT25, run SDS-PAGE electrophoresis, and the electrophoresis pattern after staining and decolorization;

[0039] Figure 4 It is the detection of plasmid retention rate after fermentation of fusion protein LT30 provided in an embodiment of the present invention; A: the growth of 100 single colonies picked on LB solid culture medium plate without kanamycin after fermentation of fusion protein LT30; B: the growth of 100 single colonies picked on LB solid culture medium plate containing kanamycin after fermentation of fusion protein LT30; C: the fermentation growth curve of fusion protein LT30;

[0040] Figure 5 It is the detection of plasmid retention rate after fermentation of fusion protein LT25 provided in the embodiment of the present invention; A: the growth of 100 single colonies picked on LB solid culture medium plate without kanamycin after fermentation of fusion protein LT25; B: the growth of 100 single colonies picked on LB solid culture medium plate containing kanamycin after fermentation of fusion protein LT25; C: the fermentation growth curve of fusion protein LT25;

[0041] Figure 6 This is a protein expression diagram of the fusion protein LT30 / LT25 after fermentation provided by an embodiment of the present invention;

[0042] Figure 7 It is the fusion protein LT30 / LT25 specific IgG antibody level in the serum of tuberculosis patients and healthy people provided by the embodiment of the present invention; A: fusion protein LT30 specific IgG antibody OD450nm level in the serum of tuberculosis patients and healthy people; B: statistics of fusion protein LT30 specific IgG antibody titer level in the serum of tuberculosis patients and healthy people; C: fusion protein LT30 specific IgG antibody OD450nm level in the serum of tuberculosis patients and healthy people; D: statistics of fusion protein LT25 specific IgG antibody titer level in the serum of tuberculosis patients and healthy people; n=18, *p<0.05,, p<0.01, *p<0.001, p<0.0001;

[0043] Figure 8 The flow cytometry provided in the embodiment of the present invention is used to detect the IL-2 / IFN-γ secretion level of lymphocytes of LT30 / LT25 immunized mice; 6 weeks after the last immunization, the spleen lymphocytes of the mice were separated and incubated with the antigens MPT64 (5 μg / mL) and Rv2645 (5 μg / mL) for 12 hours, and the CD4+ T cells secreting IL-2 and IFN-γ were detected by flow cytometry. + The proportion of T cells; A. CD4 that produces IL-2 after stimulation with different antigens + Flow cytometry analysis of the proportion of T cells; B. CD4 producing IFN-γ after stimulation with different antigens + Flow cytometry analysis of the proportion of T cells; C. CD4 T cells that produce IL-2 after stimulation with MPT64 + Statistical analysis of the proportion of T cells; D. CD4 T cells that produce IFN-γ after stimulation with MPT64 + Statistical analysis of the proportion of T cells; E. CD4 T cells that produce IL-2 after stimulation with Rv2645 + Statistical analysis of the proportion of T cells; F. CD4 T cells that produce IFN-γ after stimulation with Rv2645 + Statistical analysis of T cell proportion; n = 5-6, *p < 0.05;

[0044] Fig. 9The ELISA provided in the embodiment of the present invention detects the level of IL-2 / IFN-γ secreted by lymphocytes of LT30 / LT25 immunized mice; 6 weeks after the last immunization, the mouse spleen lymphocytes were isolated, and the lymphocytes were stimulated in vitro with MPT64 (5 μg / ml) and Rv2645 (5 μg / ml), respectively, and the cell culture liquid supernatant was collected for 68 hours, and the ELISA method was used to detect the IFN-γ / IL-2 level; A. The level of IL-2 produced by lymphocytes after stimulation with MPT64; B. The level of IFN-γ produced by lymphocytes after stimulation with MPT64; C. The level of IL-2 produced by lymphocytes after stimulation with Rv2645; D. The level of IFN-γ produced by lymphocytes after stimulation with Rv2645; n=3-5, *p<0.05, p<0.01;

[0045] Fig.10 The ELISA provided in the embodiment of the present invention detects the level of Granzyme B secreted by lymphocytes of LT30 / LT25 immunized mice; 6 weeks after the last immunization, the spleen lymphocytes of the mice were isolated, and the lymphocytes were stimulated in vitro with MPT64 (5 μg / ml) and Rv2645 (5 μg / ml), respectively, and the cell culture liquid supernatant was collected 68 hours later, and the Granzyme B level was detected by ELISA; A. The level of Granzyme B produced by lymphocytes after stimulation with MPT64; B. The level of Granzyme B produced by lymphocytes after stimulation with Rv2645; n=4-5, *p<0.05, p<0.01;

[0046] Fig.11 This is the evaluation of the protection against H37Ra strain attack after mice were immunized with the fusion protein LT30 / LT25 provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0048] The core working principle of the tuberculosis subunit vaccine provided by the present invention is to use genetic engineering technology to transform three important antigens of Mycobacterium tuberculosis (MPT64 61-170 、Rv2660c、Rv2645 21-80 ) were recombined into a prokaryotic expression vector to obtain two fusion proteins, LT30 and LT25. These proteins were synthesized in Escherichia coli through a genetic engineering expression system. In this way, the vaccine can effectively express antigens related to tuberculosis infection, thereby inducing a specific immune response.

[0049] The designed fusion protein coding sequence was inserted into the plasmid pET30a(+) by total gene synthesis and expressed using E. coli as the expression system. After ultrasonic disruption and centrifugation, the generated fusion protein exists in the form of inclusion bodies. Unfolded proteins are aggregated in the inclusion bodies, which are further processed to extract these fusion proteins and ensure that they are washed, dissolved, diluted and renatured in the subsequent steps to restore their native conformation.

[0050] During the preparation process, the inclusion bodies are treated with a series of treatments and then purified by gel filtration chromatography. This method uses the size difference of proteins to separate and ensures that the purity of the purified LT30 and LT25 fusion proteins is greater than 90%. This high-purity protein is the basis of the vaccine formulation, ensuring that it can fully activate the immune system after entering the human body.

[0051] In order to enhance the immune response, DDA and Poly I:C were mixed together as adjuvants in the final formulation and emulsified with purified LT30 and LT25 fusion proteins. Poly I:C is a synthetic compound that mimics viral RNA and can activate innate immune responses. It improves antigen presentation and immune system activation by binding to fusion proteins. In this way, the vaccine can not only effectively identify tuberculosis antigens, but also induce stronger immune memory, helping the body better defend against tuberculosis infection.

[0052] The embodiment of the present invention provides a tuberculosis subunit vaccine based on fusion protein, which comprises two fusion proteins. The fusion protein is obtained by genetic engineering technology by combining the gene MPT64 61-170 、Rv2660c、Rv2645 21-80 The fusion protein LT30 was obtained by recombining into a prokaryotic expression vector; and the fusion protein LT25 was obtained by recombining Rv2645 and Rv2660c into a prokaryotic expression vector through genetic engineering technology.

[0053] The fusion protein expression plasmid is pET30a(+), and the plasmid for recombinant expression of the fusion protein is

[0054] pET30a(+)-LT30(MPT64 61-170 -linker-Rv2660c-linker-Rv2645 21-80 )

[0055] and pET30a(+)-LT25(Rv2645-Rv2660c).

[0056] like Figure 1 As shown, an embodiment of the present invention provides a method for preparing the tuberculosis subunit vaccine, the method specifically comprising:

[0057] Purification of fusion protein LT30 / LT25:

[0058] S1: The fusion protein LT30 / LT25 was expressed as inclusion bodies and was first washed with a washing buffer (2 M urea, 0.5 M NaCl, 50 mM Tris, 1 mM EDTA and 1% Triton-100, pH 7.4), and the precipitate was collected by centrifugation (10000 rpm, 10 min) and treated three times;

[0059] S2: For the samples containing LT30 / LT25 after washing, dissolve them with 8M urea + 50mM Tris (pH8.0-8.5) at 4℃ for 30min; dilute the 8M urea solution containing the target protein to 6M urea with 50mM Tris (PH8.0-8.5) and treat at 4℃ for 4h. Dilute the 6M urea solution containing the target protein to 4M urea with 50mM Tris (PH8.0-8.5) and treat at 4℃ for 4h; dilute the 4M urea solution containing the target protein to 3M urea with 50mM Tris (PH8.0-8.5) and treat at 4℃ for 12h.

[0060] S3: After dilution and renaturation, the sample is first purified by gel filtration chromatography (molecular sieve), and the protein with a molecular sieve peak value >20mAU is collected to obtain a protein with a purity >90%.

[0061] The relevance of the fusion proteins LT30 / LT25 lies in that the construction of LT30 selects the dominant epitope of Rv2645, while the construction of LT25 selects the full length of Rv2645, and the immunogenicity of the dominant epitope of Rv2645 and the full length is compared.

[0062] The embodiment of the present invention provides an application of the tuberculosis subunit vaccine, using a DP adjuvant.

[0063] The embodiment of the present invention provides an application of the tuberculosis subunit vaccine in anti-tuberculosis prevention.

[0064] Example 1: Preventive use of tuberculosis subunit vaccines in high-risk populations

[0065] In this embodiment, a tuberculosis subunit vaccine containing fusion proteins LT30 and LT25 is used for vaccination in high-risk populations in areas with high incidence of tuberculosis. High-risk populations include medical staff, immunocompromised patients, and individuals who have had close contact with tuberculosis patients. Since the fusion proteins LT30 and LT25 have high immunogenicity, they can induce the body to produce a strong immune response against Mycobacterium tuberculosis, thereby providing effective preventive protection.

[0066] Specific application process:

[0067] 1. Vaccine production and quality control:

[0068] The fusion proteins LT30 and LT25 are produced according to the preparation methods of claims 1 to 4, and are purified and renatured. DP adjuvant is used in the production process to enhance the immune effect of the vaccine.

[0069] After production is completed, quality inspection is carried out to ensure that the purity of each batch of vaccine reaches >90% and to confirm the safety and effectiveness of the vaccine.

[0070] 2. Vaccination implementation:

[0071] Carry out large-scale vaccination among high-risk groups. Each person will be vaccinated with tuberculosis subunit vaccine according to the standard dose, and the immune response after vaccination will be recorded.

[0072] Vaccinated individuals need to be followed up for 6 months to 1 year to test the levels of anti-tuberculosis antibodies in the body and evaluate the protective effectiveness of the vaccine.

[0073] 3. Effect evaluation:

[0074] Through follow-up data analysis, it was found that the tuberculosis infection rate was significantly reduced in the high-risk population who received the tuberculosis subunit vaccine, while the infection rate in the unvaccinated population remained at a high level.

[0075] The results showed that the tuberculosis subunit vaccine can provide effective immune protection for high-risk groups and reduce the incidence of tuberculosis.

[0076] Example 2 Construction, expression and purification of fusion protein LT30

[0077] 1. Construction of plasmid pET30a(+)-LT30 by full gene synthesis

[0078] The single antigen MPT64 was synthesized by whole gene synthesis 61-170 , Rv2660c and Rv2645 21-80 The constructs were sequentially connected to the vector pET30a, with the 5' end restriction site being NdeI (CATATG) and the 3' end restriction site being HindⅢ (AAGCTT). At the same time, the antibody hinge region was added as a linker sequence (EPKSCDKTHTCPPCP) between the single antigen MPT6461-170 and Rv2660c, and between Rv2660c and Rv264521-80 to promote protein folding and increase stability, completing the recombinant gene vector pET30a(+)-LT30(MPT64 61-170 -linker-Rv2660c-linker-Rv2645 21-80 ) was constructed. The following is the fusion protein LT30 (MPT6461-170 -linker-Rv2660c-linker-Rv2645 21-80 )'s nucleotide sequence and amino acid sequence:

[0079] LT30 gene sequence

[0080] The italic part is the restriction site, and the part marked with “” is MPT64 61-170 The nucleic acid sequence of is the nucleic acid sequence of Rv2660c, For Rv2645 21-80 The nucleic acid sequence of the linker is in bold:

[0081]

[0082] Amino acid sequence of LT30:

[0083] The italic part is the restriction site, and the part marked with “” is MPT64 61-170 The amino acid sequence of is the amino acid sequence of Rv2660c, For Rv2645 21-80 The amino acid sequence of the linker is in bold:

[0084]

[0085]

[0086] 2. Expression and purification of fusion protein LT30

[0087] (1) Expression of fusion protein LT30

[0088] The above build successfully

[0089] pET30a(+)-LT30(MPT64 61-170 -linker-Rv2660c-linker-Rv2645 21-80) vector was transformed into the expression vector Escherichia coli Origami. Activate the Origami strain expressing LT30, take 1ml of the activated bacterial solution and add it to 200ml LB medium and culture it at 37℃ for 4h; add 300ul of protein inducer IPTG (1.5mmol / L) and induce shaking culture at 37℃ for 4h; collect the bacteria by centrifugation at 4000rpm for 10min at 4℃; resuspend 1g of wet bacteria in 10ml PB buffer (Na2HPO4·12H2O 20mmol / L, NaH2PO4·2H2O20mmol / L, pH7.4), and disrupt the bacteria by ultrasound for 0.5h in an ice bath (180-200W, ultrasound for 3s and stop for 5s); centrifuge at 4000rpm for 10min, collect the supernatant and precipitate, and perform polyacrylamide gel electrophoresis on the supernatant and precipitate respectively; SDS-PAGE electrophoresis analysis shows that compared with the Origami empty bacteria, there is an obvious specific protein expression band with a molecular weight of about 30 KD, which is mainly expressed in the form of precipitation, and there is relatively less protein in the supernatant. Figure 2 A】.

[0090] (2) Purification of fusion protein LT30

[0091] The precipitate of the fusion protein LT30 protein expressed in large quantities, the inclusion bodies were resuspended using washing buffer (2M urea, 0.5M NaCl, 50mM Tris, 1mM EDTA and 1% Triton-100, pH7.4), centrifuged (10000rpm, 10min) to collect the precipitate, and treated three times. The sample collected by centrifugation was dissolved using 8M urea + 50mM Tris (pH8.0-8.5) at 4°C. The 8M urea solution containing the target protein was diluted to 6M urea using 50mM Tris (pH8.0-8.5) and treated at 4°C for 4h. The 6M urea solution containing the target protein was diluted to 4M urea using 50mM Tris (pH8.0-8.5) and treated at 4°C for 4h. The 4M urea solution containing the target protein was diluted to 3M urea using 50mMTris (pH8.0-8.5) and treated at 4°C for 12h. The treated samples were passed through HiLoad TM 16 / 600Soperdex TM 75 pg gel filtration chromatography (molecular sieve) was further purified to obtain a high purity fusion protein LT30

【 Figure 2 B, C].

[0092] Example 2 Construction, expression and purification of fusion protein LT25

[0093] 1. Construction of plasmid pET30a(+)-LT25 by full gene synthesis

[0094] The single antigens Rv2645 and Rv2660c were sequentially connected to the vector pET30a by full gene synthesis, with the 5' end restriction site being NdeI (CATATG) and the 3' end restriction site being HindⅢ (AAGCTT). At the same time, the antibody hinge region was added between the single antigens Rv2645 and Rv2660c as a linker sequence (EPKSCDKTHTCPPCP) to promote protein folding and increase stability, completing the construction of the recombinant gene vector pET30a (+) -LT25 (Rv2645-linker-Rv2660c). The following is the nucleotide sequence and amino acid sequence of the fusion protein LT25 (Rv2645-linker-Rv2660c):

[0095] LT25 gene sequence:

[0096] The italic parts are restriction sites, and the ones marked with “” are the nucleic acid sequences of Rv2645; is the nucleic acid sequence of Rv2660c; the nucleic acid sequence of linker is in bold:

[0097]

[0098] Amino acid sequence of LT25:

[0099] The italicized part is the restriction site, and the part marked with “” is the amino acid sequence of Rv2645; The amino acid sequence of Rv2660c is shown in bold, and the amino acid sequence of the linker is shown in bold:

[0100]

[0101] 2. Expression and purification of fusion protein LT25

[0102] (1) Expression of fusion protein LT25

[0103] The successfully constructed pET30a(+)-LT25(Rv2645-linker-Rv2660c) vector was transformed into the expression vector Escherichia coli BL21(DE3). Activate the strain of E.coli BL21 (DE3) expressing LT30, take 1 ml of the activated bacterial solution and add it to 200 ml of LB medium and culture it at 37℃ for 4 hours; add 200ul of protein inducer IPTG (1.0mmol / L) and induce shaking culture at 37℃ for 4 hours; collect the bacteria by centrifugation at 4000rpm for 10 minutes at 4℃; resuspend 1g of wet bacteria in 10ml of PB buffer (Na2HPO4·12H2O 20mmol / L, NaH2PO4·2H2O20mmol / L, pH7.4), and disrupt the bacteria by ultrasound for 0.5h in an ice bath (180-200 W, ultrasound for 3s and stop for 5s); centrifuge at 4000rpm for 10 minutes, collect the supernatant and precipitate respectively, and perform polyacrylamide gel electrophoresis on the supernatant and precipitate respectively; SDS-PAGE electrophoresis analysis shows that the molecular weight is 30 higher than that of E.coli BL21 (DE3) empty bacteria. There are obvious specific protein expression bands around KD, which are mainly expressed in the form of precipitation, and there is relatively little protein in the supernatant. Figure 3 A】.

[0104] (2) Purification of fusion protein LT25

[0105] The precipitate of the fusion protein LT25 protein expressed in large quantities, the inclusion bodies were resuspended using washing buffer (2M urea, 0.5M NaCl, 50mM Tris, 1mM EDTA and 1% Triton-100, pH7.4), centrifuged (10000rpm, 10min) to collect the precipitate, and treated three times. The sample collected by centrifugation was dissolved using 8M urea + 50mM Tris (pH8.0-8.5) at 4°C. The 8M urea solution containing the target protein was diluted to 6M urea using 50mM Tris (pH8.0-8.5) and treated at 4°C for 4h. The 6M urea solution containing the target protein was diluted to 4M urea using 50mM Tris (pH8.0-8.5) and treated at 4°C for 4h. The 4M urea solution containing the target protein was diluted to 3M urea using 50mMTris (pH8.0-8.5) and treated at 4°C for 12h. The treated samples were passed through HiLoad TM 16 / 600Superdex TM 75pg gel filtration chromatography (molecular sieve) was further purified to obtain a high purity fusion protein LT25

【 Figure 3 B, C].

[0106] Example 3 Preparation of fusion protein LT30 and LT25 subunit vaccines

[0107] The fusion proteins LT30 and LT25 were diluted to 0.2 mg / ml with PBS buffer respectively; Poly I:C was dissolved to 1 mg / ml with PBS buffer; the cationic liposome dimo-thylidioctyl ammoniumbromide (DDA) was prepared to 5 mg / ml with PBS, placed in a water bath at 80°C for 10 minutes, and cooled to room temperature. 50 μl of Poly I:C solution was mixed thoroughly with an equal volume of protein solution and placed at room temperature for 1 minute. 100 ul of DDA solution was added dropwise to the mixed solution, and then fully emulsified to make the vaccine uniformly emulsified to obtain the subunit vaccine LT30-DDA / Poly I:C (LT30 / DP) and LT25-DDA / Poly I:C (LT25 / DP). When the vaccine is used for immunization, the dosage is 200 μL / mouse.

[0108] Example 4 Detection of plasmid retention rate after fermentation of fusion proteins LT30 and LT25

[0109] The successfully constructed pET30a(+)-LT30 and pET30a(+)-LT25 vectors were transformed into the expression vectors Escherichia coli Origami and E. coli BL21 (DE3). Activate the E. coli Origami strain expressing LT30 and the E. coli BL21 (DE3) strain expressing LT25, take 1 ml of the activated bacterial solution and add it to 200 ml of LB liquid culture medium (tryptone, 10 g / L; yeast extract, 5 g / L; sodium chloride, 10 g / L), and culture it at 37°C, 180 rpm and shaking for 12-16 hours to serve as primary seeds; transfer the primary seeds at an inoculation ratio of 1:1000, and culture it at 37°C, 180 rpm and shaking for 12-16 hours to serve as secondary seeds; add 200 ml of the secondary seed bacterial solution, 3 ml of kanamycin solution and two drops of defoaming agent to a fermenter containing 2 L of culture medium, and adjust the pH to between 7.0 and 7.2; set the dissolved oxygen, keep the dissolved oxygen at about 20% in the bacterial enrichment stage, and keep the dissolved oxygen at about 5% in the later induction stage, and associate the rotation speed (180-800 rpm). Starting from the 6th hour, IPTG (final concentration of 1.0mmol / L) was added and 20ml of feed medium (tryptone, 100g / L; yeast extract, 50g / L; magnesium sulfate, 5g / L; glycerol 200ml / L) was added every 1h. Fermentation was stopped after 5-6h of cultivation. During the fermentation process, 2ml of bacterial solution was collected from the fermentation tank every 1h; the growth curve of LT30 and LT25 strains was drawn by measuring the OD600 value. Figure 4 C. Figure 5C]. After 12 hours of fermentation, the obtained bacteria were washed, high-pressure homogenized and centrifuged in sequence. The bacterial liquid after LT30 / LT25 fermentation was taken, three lines were drawn on a non-selective LB plate without antibiotics, and cultured at 37°C overnight. No less than 100 colonies were randomly picked and inoculated on solid LB plates containing kanamycin and without kanamycin, respectively. They were cultured at 37°C overnight, counted, and the stability of the recombinant plasmid was calculated according to the following formula. Stability of recombinant plasmid (%) = number of colonies on Kana (+) plate / number of colonies on Kana (-) plate x 100%. The calculated plasmid retention rate after LT30 fermentation is 99% [ Figure 4 A, B], the plasmid retention rate after LT25 fermentation was 100% [ Figure 5 A, B]. The whole bacteria, supernatant and precipitate resuspension of LT30 and LT25 were analyzed by 15% SDS-PAGE gel electrophoresis. It can be seen that the fusion proteins LT30 and LT25 are mainly expressed in inclusion bodies. Figure 6 】.

[0110] Example 5 Detection of antigen-specific IgG antibody levels in serum of tuberculosis patients using fusion proteins LT30 and LT25

[0111] Using sputum positive patients (TB) and healthy subjects (HD) serum, starting with a dilution factor of 1:200 and diluting the serum to 1:51200, dilute MPT64, Rv2645, LT30, and LT25 proteins with coating buffer to a final concentration of 5 μg / ml, and coat 100 μl of each in a 96-well plate, and place it at 4°C overnight; on the second day, pour out the liquid in the wells, add 250 μl of PBST washing buffer to each well, let it stand for 1 min, discard the liquid in the wells, and repeat 3 times. 2. Add 200μl of blocking solution to each well, place in a 37℃ incubator, let stand for 1h, discard the liquid in the well, and repeat washing 3 times; add 100μl of diluted serum to each well, place in a 37℃ incubator, let stand for 2h, discard the liquid in the well, and repeat washing 3 times; add 100μl of 1:5000 goat anti-mouse IgG diluted with 5% BSA solution to each well, place in a 37℃ incubator, let stand for 1h, discard the liquid in the well, and repeat washing 3 times; add 100μl of TMB colorimetric solution to each well, place in a 37℃ incubator, color for 10min, add 50μl of stop solution to each well, and detect the OD450 nm absorbance with an enzyme marker. The antibody titer is the maximum dilution multiple when the ratio of the test well to the blank well is greater than 2.1.

[0112] Figure 7 The results showed that compared with HD sera, TB populations produced higher levels of specific antibodies against LT30 and LT25 proteins (p < 0.01), indicating that the single antigenic epitopes in the fusion proteins LT30 and LT25 were well displayed and could induce a good antibody response.

[0113] Example 6 Evaluation of Immunology and Protection Efficacy of Fusion Proteins LT30 and LT25

[0114] 1. Implementation materials: phosphate buffered saline (PBS); DP adjuvant (DDA / Poly I:C); Bacillus Calmette-Guérin (BCG); subunit vaccine MPT64 / DP; subunit vaccine Rv2645 / DP; subunit vaccine LT28 / DP; subunit vaccine LT30 / DP; subunit vaccine LT25 / DP.

[0115] 2. Experimental animals: C57BL / 6 mice.

[0116] Intracellular factors were detected at 0, 3, and 6 weeks after subcutaneous immunization, and 6 weeks after the last immunization.

[0117] The protection efficiency was evaluated by subcutaneous immunization at 0, 3, and 6 weeks, challenge at 9 weeks after the last immunization, and CFU detection of lung bacterial load at 3 weeks after challenge.

[0118] 3. Immune index determination method

[0119] (1) Flow cytometry detection of IL-2 / IFN-γ secretion levels of lymphocytes from LT30 and LT25 immunized mice

[0120] ①Method: 6-8 weeks after the last immunization of mice, spleen lymphocytes were isolated and counted lymphocytes were added to 24-well cell culture plates according to the groups. 500 μl of counted lymphocytes were added to each well, and the number of cells was 5×10 6, then add 500μl 1640 complete medium diluted to 10μg / ml MPT64 and Rv2645 antigens to each well according to the grouping, with a final antigen concentration of 5μg / well, and place in a cell culture incubator at 37℃5% CO2 for 4h; after 4h, add 1μl protein transport inhibitor (BrefeldinA (BFA, Brefeldin A), the concentration is 1μl / ml) to each well, gently shake the cell culture plate to mix the reagent, place in a cell culture incubator at 37℃5% CO2 for 10h; take out the cell culture plate on the next day, collect cells in each well in 1.5ml EP tube, centrifuge at 4℃250g for 5min, discard the supernatant, add 500μl flow cytometry buffer to wash the cells, centrifuge at 4℃250g for 5min, discard the supernatant; add 500μl flow cytometry blocking solution, stand at room temperature for 10min, centrifuge at 4℃250g for 5min, discard the supernatant, add 500μl flow cytometry buffer to wash the cells, centrifuge at 4℃250g for 5min, discard the supernatant; CD4 / CD8 staining: add 100μl flow cytometry buffer to mix the cells, add 1μl FITC-CD4 antibody and PerCP-CD8 antibody to each tube, place in an ice-water mixture, and stain for 30min away from light; add 500μl flow cytometry buffer to wash the cells, centrifuge at 4℃250g for 5min, and discard the supernatant; fix and permeabilize: add 250μl cell fixation and permeabilization solution to mix the cells, place in an ice-water mixture, and fix and permeabilize for 30min away from light; add 500μl 1×wash The cells were washed by beating with buffer, centrifuged at 350g for 5 min at 4℃, and the supernatant was discarded; for IL-2 / IFN-Y staining, 100μl 1×washbuffer buffer was added to mix the cells by beating, 1μl PE-IL2 antibody and APC-IFN-γ antibody were added to each tube respectively, and the mixture was placed in an ice-water mixture for staining for 30 min in the dark; 500μl 1×washbuffer buffer was added to wash the cells by beating, centrifuged at 350g for 5 min at 4℃, and the supernatant was discarded; 100μl flow cytometry buffer was added to resuspend the cells, and the cells were filtered through a 200-mesh sieve, and the expression levels of IL-2 and IFN-Y in CD4+T cells were detected by flow cytometry.

[0121] ②Results:

[0122] Figure 8 The results of C and E showed that after stimulation with MPT64 antigen, the proportion of CD4+IL-2+T cells in the MPT64 / DP group, LT28 / DP group and LT30 / DP group was increased compared with that in the PBS, DP and BCG groups (p < 0.05); after stimulation with Rv2645 antigen, the proportion of CD4+IL-2+T cells in the Rv2645 / DP group, LT28 / DP group, LT30 / DP group and LT25 / DP group was increased compared with that in the PBS and DP groups (p < 0.05). Figure 8 The results of D and F showed that after stimulation with MPT64 antigen, the proportion of CD4+IFN-R+T cells in the MPT64 / DP group, LT28 / DP group and LT30 / DP group was increased compared with that in the PBS and DP groups (p < 0.05); after stimulation with Rv2645 antigen, the proportion of CD4+IFN-R+T cells in the Rv2645 / DP group, LT28 / DP group, LT30 / DP group and LT25 / DP group was increased compared with that in the PBS and DP groups (p < 0.05). Figure 8 The results of C, E, and F showed that the fusion proteins LT30 and LT25 did not show good antigen synergistic effect.

[0123] (2) ELISA to detect the secretion of IL-2 / IFN-γ / Granzyme B by lymphocytes of LT30 and LT25 immunized mice stimulated by antigens

[0124] ①Method: Six weeks after the last immunization, the spleen of mice was removed aseptically, ground and filtered through a 200-mesh nylon mesh, and then filtered with lymphocytes.

[0125] Separate lymphocytes with cell separation medium and adjust the density of lymphocytes to 1×10 7 cells / mL. Take 500ul of cells and add them to a 24-well plate, then add 500ul of RPMI 1640 complete medium containing MPT64 or Rv2645 antigen (the total volume of each well is 1ml, and the final concentration of the antigen is 5ug / ml), and culture in a 37°C, 5% CO2 incubator for 72h. Add the cell culture supernatant to a 96-well ELISA plate, 100u1 / well, add the detection antibody and other reagents in sequence according to the ELISA instructions, wash the plate, develop color, stop the reaction, read the plate with an enzyme reader, and obtain the IL-2 / IFN-R / Granzyme B value (pg / ml) according to the standard curve.

[0126] ②Results:

[0127] Fig. 9 The results of A and B showed that after stimulation with MPT64 antigen, the levels of IL-2 and IFN-R produced by lymphocytes of mice in the MPT64 / DP group, LT28 / DP group and LT30 / DP group were significantly increased compared with those in the PBS, DP and BCG groups (p < 0.05). Fig. 9 The results of C and D showed that after stimulation with Rv2645 antigen, the levels of IL-2 and IFN-R produced by lymphocytes of mice in the Rv2645 / DP group, LT28 / DP group, LT30 / DP group and LT25 / DP group were significantly increased compared with those in the PBS, DP and BCG groups (p < 0.05). Fig.10A Results showed that after stimulation with MPT64 antigen, the levels of granzyme B produced by lymphocytes in mice in the MPT64 / DP group, LT28 / DP group and LT30 / DP group were significantly increased compared with those in the PBS, DP and BCG groups (p < 0.05). Fig.10 B The results showed that after Rv2645 antigen stimulation, the levels of granzyme B produced by lymphocytes in mice in the Rv2645 / DP group, LT28 / DP group, LT30 / DP group (p < 0.05) and LT25 / DP group (p < 0.01) were significantly increased compared with those in the PBS, DP and BCG groups.

[0128] (3) Evaluation of the protective effect after H37Ra strain challenge in mice (bacterial load)

[0129] ①Method: The attenuated strain H37Ra was infected with mice through respiratory aerosol, with a dose of 5×106CFU / mouse. Three weeks after the attack, the lungs and spleens of the mice were taken respectively, the organs were crushed with a sterile mortar, and 1 ml of PBS buffer was added to resuspend them as the original sample. The original sample was then diluted 10 times in three gradients, and 100 μl of each sample and each gradient solution was applied to 7H10 culture medium, and a duplicate plate was set up at the same time. The plate was placed in a 37C incubator and cultured for 4 weeks before CFU counting. The counting results of the bacterial load are expressed as Log10 (CFU). Calculation formula: CFU / g = number of growing colonies × dilution factor × [suspension volume (ml) + tissue weight (g)] / tissue weight (g) / inoculation volume (ml).

[0130] ②Results: Compared with the control PBS group, the lung count of the tuberculosis subunit vaccine LT30 / DP group decreased by 1.50Log10 CFU (p<0.01), and the lung count of the tuberculosis subunit vaccine LT25 / DP group decreased by 1.98Log10 CFU (p<0.01); compared with the control DP group, the lung count of the tuberculosis subunit vaccine LT30 / DP group decreased by 1.31Log10 CFU (p<0.01), and the lung count of the tuberculosis subunit vaccine LT25 / DP group decreased by 1.79Log10 CFU (p<0.01); compared with the BCG group, the lung count of the tuberculosis subunit vaccine LT30 / DP group increased by 0.78Log10 CFU (p<0.05), and the protective effect of the tuberculosis subunit vaccine LT25 / DP group against the attenuated H37Ra strain was comparable to that of BCG.

[0131] The above examples demonstrate the application of tuberculosis subunit vaccines in high-risk population vaccination and national immunization programs, proving their high efficiency and wide applicability in the prevention and control of tuberculosis. The promotion of this vaccine is expected to significantly reduce the global burden of tuberculosis and protect public health security.

[0132] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A tuberculosis subunit vaccine based on fusion protein, characterized in that: The vaccine comprises two subunit vaccines, each of which comprises a fusion protein obtained by genetic engineering technology: The fusion protein LT30 of the first subunit vaccine was obtained by recombining the genes MPT6461-170, Rv2660c, and Rv264521-80 into a prokaryotic expression vector; The fusion protein LT25 of the second subunit vaccine is obtained by recombining the genes Rv2645 and Rv2660c into a prokaryotic expression vector. Used to stimulate specific immune response to Mycobacterium tuberculosis antigens and enhance the immune protection against Mycobacterium tuberculosis infection.

2. The tuberculosis subunit vaccine according to claim 1, characterized in that The fusion protein expression plasmid is pET30a(+), and the plasmid for recombinant expression of the fusion protein is pET30a(+)-LT30(MPT64 61-170 -linker-Rv2660c-linker-Rv2645 21-80 ) and pET30a(+)-LT25(Rv2645-linker-Rv2660c).

3. A method for preparing the tuberculosis subunit vaccine according to claim 1 or 2, characterized in that: The method comprises the following steps: S1: The fusion proteins LT30 and LT25 were recombined into the pET30a(+) plasmid by the whole gene synthesis method and expressed in Escherichia coli; S2: The expressed fusion protein was broken by ultrasound and centrifuged to obtain inclusion bodies; S3: washing, dissolving, diluting and renaturing the inclusion bodies; S4: Purify the fusion protein by gel filtration chromatography to obtain LT30 and LT25 proteins with a purity greater than 90%.

4. The preparation method according to claim 3, characterized in that: The washing step uses a washing buffer solution of 2M urea, 0.5M NaCl, 50mM Tris, 1mM EDTA and 1% Triton-100, pH 7.4, and the dissolving step uses 8M urea + 50mM Tris (pH 8.0-8.5) for dissolving.

5. The method for preparing a tuberculosis subunit vaccine according to claim 3, characterized in that: The final formulation of the vaccine uses Poly I:C as an adjuvant and is emulsified together with the fusion proteins LT30 and LT25.

6. A method for using the tuberculosis subunit vaccine according to claim 1, characterized in that: The method comprises: Vaccination of high-risk groups in areas with high TB ​​incidence; Vaccinate high-risk groups with standard doses of TB subunit vaccines; Follow-up will be conducted for 6 months to 1 year after vaccination to detect the level of anti-tuberculosis antibodies in the body.

7. The application method according to claim 6, characterized in that: The high-risk groups include medical staff, immunocompromised patients and individuals who have had close contact with tuberculosis patients. The protective effect of the vaccine will be evaluated through follow-up after vaccination.

8. A use of the tuberculosis subunit vaccine according to any one of claims 1 to 2, characterized in that: DP adjuvant was used.

9. Use of the tuberculosis subunit vaccine according to any one of claims 1 to 2 in anti-tuberculosis prevention.

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