Recombinant rMS:: MPT64 and application thereof

Abstract

The invention discloses a recombinant rMS:: MPT64 and an application thereof. The recombinant rMS:: MPT64 is prepared by transfecting mycobacterium smegmatis by a recombinant vector for expressing an MPT64 gene. According to the present invention, the MPT64 gene is integrated into Mycobacterium smegmatis (MS) to construct the recombinant MS (rMS:: MPT64) for expressing the MPT64, and the recombinant MS (rMS:: MPT64) has excellent immunogenicity and excellent anti-tuberculosis effect when being used in the tuberculosis vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccines, and in particular to a recombinant rMS::MPT64 and an application thereof. Background Art

[0002] Currently, the only vaccine approved to prevent tuberculosis is Bacille Calmette-Guérin (BCG), which was first given orally to a child in Paris by Dr. Benjamin Weill-Halle in 1921. Although BCG vaccination of infants has a good effect in preventing severe extrapulmonary tuberculosis in young children, it has been shown in multiple clinical trials that BCG does not protect adults from pulmonary tuberculosis. Coupled with the increasing incidence of multidrug-resistant Mycobacterium tuberculosis strains, tuberculosis is one of the biggest challenges facing global health, and only the adoption of new and more effective vaccines can end the tuberculosis epidemic.

[0003] In its early stages, BCG was a live attenuated vaccine obtained by serial subculture of Mycobacterium bovis and distributed throughout the world. Continued subculture leads to the accumulation of genetic mutations, resulting in substrains with genetic and phenotypic differences. Due to the attenuation of BCG, 129 open reading frames (ORFs) were deleted, and these deleted genes can be divided into 16 genomic difference regions (named RD1-RD16). In each BCG substrain, not all of these regions are completely missing. The lack of the RD1 region containing ESAT-6 and CFP-10 in BCG was used to develop the interferon gamma release assay (IGRA) for the diagnosis of Mycobacterium tuberculosis infection. The length of time that an individual is protected after BCG vaccination also varies from study to study, with reports ranging from 10-60 years. Some studies have shown that the incidence of tuberculosis decreases after 10-15 years, which corresponds to the increase in the incidence of tuberculosis in early adulthood.

[0004] In the past few years, the use of mycobacterial models such as Mycobacterium smegmatis (MS) and BCG has greatly contributed to our current understanding of the biology and environmental adaptation of M. tuberculosis. BCG is an attenuated bovine tuberculosis bacillus obtained by serial passage in the laboratory; however, this mycobacterium grows slowly, similar to the growth rate of M. tuberculosis. M. smegmatis, first discovered in 1884, is a fast-growing saprophyte that shares more than 2000 homologous genes and a unique cell wall structure with M. tuberculosis. In humans, M. smegmatis is a powerful cellular immune adjuvant that is non-pathogenic and can coexist with the host. Unlike M. tuberculosis, cells infected with M. smegmatis rapidly destroy phagosomal proteases in phagosomes, but are unable to prevent the maturation of phagolysosomes and evade killing within the cell. In addition, compared with BCG, M. smegmatis can induce higher levels of cytokines through macrophages, activate maturation of dendritic cells (DCs) by upregulating major MHC class I molecules, and present mycobacterial antigens more efficiently through the MHC class I pathway. Because it can activate DCs and induce CD8-mediated immune responses, M. smegmatis has been used as a vaccine vector. In a previous study, researchers successfully prepared a cell-free M. smegmatis vaccine and produced protective effects in guinea pigs infected with Mycobacterium tuberculosis. To evaluate the safety, tolerability, and PPD skin reactions of the vaccine, a phase I clinical study was conducted in 55 healthy volunteers in China. The results showed that mild side effects were observed in 14 volunteers, but all volunteers tolerated the vaccine well, and the skin reactions showed strong positive PPD. In addition, M. smegmatis has been used as a vaccine vector because it can activate dendritic cells and induce CD8-mediated immune responses, in addition to its other functions. The study found that MS ESX-3 gene deletion can serve as a new vaccine vector with enhanced innate immune activation properties. When engineered to express M.tb ESX-3, the vector was found to be an effective TB vaccine, able to provide levels of protection superior to BCG when injected intravenously. Thus, recombinant MS could play an immunotherapeutic role in M. tuberculosis infection.

[0005] In view of this, the present invention is proposed, in which we integrate the preferred target gene MPT64 into Mycobacterium smegmatis, construct a recombinant Mycobacterium smegmatis expressing the target gene, immunize mice by subcutaneous injection, evaluate the immune effect stimulated by the recombinant vaccine, and create an infection model of mice after immunization by nasal drops of Mycobacterium tuberculosis. The lung bacterial load and lung pathology are used to evaluate the protective efficacy of the optimized heavy vaccine strain against tuberculosis infection, providing an experimental basis for the development of tuberculosis vaccines. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant rMS::MPT64 and its application. The present invention integrates the MPT64 gene into Mycobacterium smegmatis (MS) to construct a recombinant MS (rMS::MPT64) expressing MPT64, which has excellent immunogenicity and anti-tuberculosis effect when used in tuberculosis vaccines.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] In a first aspect, the present invention provides a recombinant rMS::MPT64, which is prepared by transfecting Mycobacterium smegmatis with a recombinant vector expressing the MPT64 gene.

[0009] Preferably, the recombinant vector expressing the MPT64 gene is constructed from the MPT64 gene and an expression vector.

[0010] Preferably, the expression vector comprises pMV361 and pET32a.

[0011] The second aspect of the present invention provides a use of the above-mentioned recombinant rMS::MPT64 in the preparation of tuberculosis vaccine.

[0012] The third aspect of the present invention provides a recombinant rMS::MPT64 tuberculosis vaccine, which comprises recombinant rMS::MPT64, a culture medium, and an immune adjuvant.

[0013] Preferably, the culture medium is beef extract peptone medium.

[0014] Preferably, the immune adjuvant is Mycobacterium smegmatis.

[0015] Preferably, the concentration of the recombinant rMS::MPT64 is 5×10 6 CFU / 100μL.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least:

[0017] The present invention integrates the MPT64 gene into the fast-growing MS to construct rMS::MPT64, which can efficiently express and purify the target protein and is used in the preparation of tuberculosis vaccines, thereby significantly improving the preparation efficiency and protein purity of the tuberculosis vaccine. In addition, the present invention uses optimized plasmids and electroporation technology to ensure the high stability of recombinant rMS::MPT64.

[0018] After mice were immunized with the recombinant rMS::MPT64 tuberculosis vaccine of the present invention, the rMS::MPT64 group showed a significantly enhanced cellular immune response compared with the traditional MS group, including: a significant increase in proinflammatory factors (such as IL-2, IL-17, IFN-γ and TNF-α) in CD4+ and CD8+ T cells; an increase in the proportion and activity of memory T cells, especially a significant increase in the proportion of effector memory T cells; enhanced antibody production (significantly increased levels of IgA, IgG1 and IgG2a), indicating that it induced a strong humoral immune response; improved the proportion of regulatory T cells (Treg) and B cells (Breg), reduced the influence of immunosuppressive cells, and helped to enhance the host's resistance to tuberculosis infection.

[0019] The MS in the present invention grows faster than traditional tuberculosis vaccine vectors such as BCG, which significantly shortens the vaccine production cycle; in addition, the efficient recombinant plasmid construction and target gene expression technology reduce the use of complex steps and expensive reagents in production.

[0020] The present invention adopts electrotransformation and plasmid expression system, and the standardized preparation process reduces the complexity of operation; in addition, rMS::MPT64 is based on non-pathogenic bacteria MS, which reduces biosafety risks and environmental pollution compared with traditional pathogenic vectors.

[0021] In a mouse experiment, the recombinant rMS::MPT64 tuberculosis vaccine of the present invention significantly reduced the bacterial load in the lungs and spleen after immunization, without the presence of acid-fast bacilli, and histology showed that the integrity of the organ structure was significantly improved; this result indicates that the vaccine has a good protective effect in the prevention and treatment of tuberculosis infection in the lungs and spleen. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0023] Figure 1 This is a gel image of the gene fragment of the RD region in the Mycobacterium tuberculosis genome amplified by PCR technology in Example 1 of the present invention;

[0024] Figure 2 This is a gel run after double enzyme digestion of pET32a-RDs in Example 2 of the present invention;

[0025] Figure 3 The protein expression of pET32a-RDs in Example 2 of the present invention;

[0026] Figure 4A and Figure 4B The peptide fingerprint of the amino acid sequence of the MPT64 recombinant protein peptide in Example 2 of the present invention;

[0027] Figure 5 The ELISA analysis results of IFN-γ levels in spleen cells of mice immunized with pET32a-RDs at different times in Example 3 of the present invention;

[0028] Figure 6 The plasmid structure diagram of pMV361, MPT64 identification and the colony morphology of rMS::MPT64 in Example 4 of the present invention are shown;

[0029] Figure 7 The verification results of MPT64 in rMS::MPT64 in Example 4 of the present invention and the growth conditions of rMS::MPT64 and MS in culture;

[0030] Fig. 8A and Figure 8B The levels of IFN-γ, TNF-α, IL-2 and IL-17 in CD4+ cells in the spleen and lymph nodes of the immunized mice in Example 5 of the present invention;

[0031] Fig. 9 CD8 + Intracellular levels of GzmB and Perforin;

[0032] Fig.10 The CD8 + Intracellular levels of Tregs and Breg cells;

[0033] Fig.11 The ratio of DC cells in the spleen of the immunized mice and the ratio of DC cells secreting IL-10 and IL-12 cytokines in Example 5 of the present invention;

[0034] Fig.12 is the proportion of memory T cells in the spleen and lymph nodes of the immunized mice in Example 5 of the present invention;

[0035] Fig.13 is the cell proliferation level in the spleen of the immunized mice in Example 5 of the present invention;

[0036] Fig.14 is the level of specific antibodies IgA, IgG1 and IgG2a in the serum of the immunized mice in Example 5 of the present invention;

[0037] Fig.15 The colony counts in the lungs and spleens of mice in the PBS, MS and rMS groups after immunization and infection in Example 6 of the present invention;

[0038] Fig.16 Pathological tissue sections of the lungs and spleens of mice in the PBS, MS and rMS groups after immunization and infection in Example 6 of the present invention. DETAILED DESCRIPTION

[0039] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0040] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0041] The embodiment of the present invention provides a recombinant rMS::MPT64, wherein the recombinant rMS::MPT64 is prepared by transfecting Mycobacterium smegmatis with a recombinant vector expressing the MPT64 gene.

[0042] The invention integrates the MPT64 gene into the fast-growing MS to construct rMS::MPT64, which can efficiently express and purify the target protein. In addition, rMS::MPT64 is based on non-pathogenic bacteria MS, which reduces biosafety risks and environmental pollution compared to traditional pathogenic vectors.

[0043] In one embodiment, the recombinant vector expressing the MPT64 gene is constructed from the MPT64 gene and an expression vector.

[0044] In one embodiment, the expression vector comprises pMV361 and pET32a.

[0045] Another embodiment of the present invention provides a use of the above-mentioned recombinant rMS::MPT64 in the preparation of tuberculosis vaccine.

[0046] The present invention uses recombinant rMS::MPT64 in the preparation of tuberculosis vaccines, which significantly improves the preparation efficiency and protein purity of tuberculosis vaccines; the growth rate of MS in the present invention is faster than that of traditional tuberculosis vaccine vectors such as BCG, which significantly shortens the vaccine production cycle; in addition, efficient recombinant plasmid construction and target gene expression technology reduce the use of complex steps and expensive reagents in production.

[0047] Another embodiment of the present invention provides a recombinant rMS::MPT64 tuberculosis vaccine, wherein the recombinant rMS::MPT64 tuberculosis vaccine comprises recombinant rMS::MPT64, culture medium, and immune adjuvant.

[0048] After mice were immunized with the recombinant rMS::MPT64 tuberculosis vaccine of the present invention, the rMS::MPT64 group showed a significantly enhanced cellular immune response compared with the traditional MS group, including: a significant increase in proinflammatory factors (such as IL-2, IL-17, IFN-γ and TNF-α) in CD4+ and CD8+ T cells; an increase in the proportion and activity of memory T cells, especially a significant increase in the proportion of effector memory T cells; enhanced antibody production (significantly increased levels of IgA, IgG1 and IgG2a), indicating that it induced a strong humoral immune response; improved the proportion of regulatory T cells (Treg) and B cells (Breg), reduced the influence of immunosuppressive cells, and helped to enhance the host's resistance to tuberculosis infection; in addition, the bacterial load in the lungs and spleen was significantly reduced after immunization, no acid-fast bacilli were present, and histology showed that the integrity of the organ structure was significantly improved; this result shows that the vaccine has a good protective effect, especially in the prevention and treatment of tuberculosis infection in the lungs and spleen.

[0049] In one embodiment, the culture medium is beef extract peptone medium.

[0050] In one embodiment, the immunoadjuvant is Mycobacterium smegmatis.

[0051] In one embodiment, the recombinant rMS::MPT64 concentration is 5×10 6 CFU / 100μL.

[0052] The technical solution of the present invention is further described in detail below through specific embodiments.

[0053] Example 1

[0054] This embodiment is a method for constructing a recombinant vector expressing the MPT64 gene, and the construction method comprises the following steps:

[0055] Using the genomic DNA of the MTBH37Rv standard strain as a template, PCR was used to amplify the gene fragment of the RD region in the Mycobacterium tuberculosis genome (e.g. Figure 1 As shown, Figure 1The PCR amplification products (1-7) of some genes in the RD region correspond to the RD region genes Rv3874, Rv3875, Rv1773, MPT63, MPT83, MPT64 and CLE, respectively, and their sizes are approximately 282bp, 270bp, 607bp, 390bp, 697bp, 506bp and 620bp; M represents marker; MPT64 is approximately 506bp in size. The pMV361 (KANR) plasmid was digested with restriction enzyme sites (EcoRI, HindIII), and the PCR-amplified MPT64 gene fragment was inserted into the plasmid through a ligation reaction to obtain a recombinant vector expressing the MPT64 gene, recorded as pMV361-MPT64.

[0056] Identification of the above pMV361-MPT64:

[0057] pMV361-MPT64 was transformed into E. coli DH5α competent cells, and positive clones were screened. After plasmid extraction, gel electrophoresis was used to confirm that the size of the inserted fragment was consistent with the vector and the target gene. The recombinant plasmid was sent to Shanghai Bioengineering for sequence determination, and sequence analysis confirmed that the target gene was accurately cloned into the pMV361 plasmid.

[0058] Example 2

[0059] This embodiment is a method for constructing a recombinant vector expressing RD region genes, and the construction method comprises the following steps:

[0060] Using the genomic DNA of the MTBH37Rv standard strain as a template, PCR was used to amplify the gene fragment of the RD region in the Mycobacterium tuberculosis genome (e.g. Figure 1 As shown, Figure 1 The PCR amplification products of some genes in the RD region (1-7) correspond to the RD region genes Rv3874, Rv3875, Rv1773, MPT63, MPT83, MPT64 and CLE, respectively, with sizes of approximately 282bp, 270bp, 607bp, 390bp, 697bp, 506bp and 620bp; M represents marker). Through restriction enzyme digestion and ligation reactions, the above gene fragments were respectively inserted into the pET32a prokaryotic expression vector to construct a recombinant vector, recorded as pET32a-RDs.

[0061] Identification of the above pET32a-RDs:

[0062] The pET32a-RDs were double-digested and the digested products were run on agarose gel. The results of the run were as follows: Figure 2 As shown; Figure 2Double enzyme digestion was used to identify some RD region genes of the recombinant pET32a plasmid (1-7 correspond to RD region genes CLE, MPT83, MPT64, MPT63, Rv1773, Rv3875, and Rv3874, respectively; the size of the pET32a plasmid is about 5000 bp, and M represents a marker);

[0063] Depend on Figure 2 It can be seen that the double enzyme digestion of the recombinant vector was successful.

[0064] Positive clones were identified by PCR and restriction digestion. The results of gel running after restriction digestion were as follows: Figure 2 As shown by Figure 2 It can be seen that when double enzyme digestion identification is performed using EcoRⅠ, HindⅢ or BamHⅠ according to the restriction site, specific bands are seen at the corresponding positions, and the double enzyme digestion identification is correct.

[0065] The constructed expression strain was induced by IPTG, and then the protein was purified using a Ni-NTA agarose column, and then the target protein was identified by SDS-PAGE; the identification results are shown in Figure 3 As shown by Figure 3 It can be seen that protein bands of corresponding molecular weight appeared after induction of recombinant bacteria, indicating that the purification and identification were correct.

[0066] In order to further confirm the correctness of the purified recombinant MPT64 protein, a protein band was screened out by binding the recombinant MPT64 protein to a Ni column; the protein band was then cut and sent to Shanghai Bioengineering for mass spectrometry analysis. Figure 4A and Figure 4B A to G in the middle represent the peptide fingerprint of the amino acid sequence of the MPT64 recombinant protein peptide;

[0067] Depend on Figure 4A and Figure 4B It can be seen that the recombinant MPT64 protein was purified successfully.

[0068] Example 3

[0069] This example is the screening of RD proteins that highly express IFN-γ:

[0070] In order to screen for RD proteins that highly express IFN-γ, endotoxin-free RD proteins were used to stimulate mouse spleen cells pre-immunized with inactivated H37Rv (iH37Rv) for 24, 48, and 72 hours. Subsequently, the IFN-γ levels induced by each RD protein were determined by ELISA; the results are shown in Figure 5 As shown by Figure 5It can be seen that MPT64 protein can stimulate mouse macrophages to express IFN-γ, and among the seven RD proteins, MPT64 protein induced the strongest IFN-γ production level (P < 0.001).

[0071] Example 4

[0072] This embodiment is a method for constructing a recombinant rMS::MPT64, which comprises:

[0073] The pMV361-MPT64 of Example 1 was electroporated into Mycobacterium smegmatis, and the recombinant Mycobacterium smegmatis rMS:MPT64 was identified by PCR and Western blot.

[0074] The structure of the above pMV361-MPT64 is shown in Figure 6 As shown in A, pMV361-MPT64 was digested with double enzymes and run on gel. Figure 6 As shown in B, lane 1: double enzyme digestion identification of pMV361-MPT64; lane 2: PCR product band of MPT64; colony morphology of rMS::MPT64 after electroporation and subculture is as shown in Figure 6 As shown in C;

[0075] Depend on Figure 6 It can be seen that the pMV361-MPT64 recombinant plasmid was successfully constructed.

[0076] Verification of MPT64 expression in rMS::MPT64:

[0077] PCR was used to verify whether the MPT64 gene existed in the rMS::MPT64 bacterial solution. The results were as follows: Figure 7 As shown in A; Western blotting was used to verify the expression of MPT64 protein. Figure 7 As shown in B; rMS::MPT64 and MS were cultured, and the growth of the bacteria was as follows Figure 7 As shown in C;

[0078] Depend on Figure 7 It can be seen that:

[0079] After the rMS::MPT64 colony was cultured for 48 hours, the bacterial solution was used for PCR amplification of MPT64, H37Rv was used as a positive control, and MS was used as a negative control. The agarose gel electrophoresis results showed that the bands amplified by rMS::MPT64 and H37Rv were consistent, while MS did not amplify the bands, indicating that rMS::MPT64 was successfully constructed ( Figure 7 Western blot results confirmed that both rMS::MPT64 and H37Rv expressed MPT64, but MS did not ( Figure 7Middle B). The growth rate of rMS::MPT64 was slightly slower than that of MS, but there was no significant difference ( Figure 7 C).

[0080] Example 5

[0081] PBS, MS, and rMS:MPT64 were added to the culture medium containing immune adjuvant to obtain vaccines, and mice were immunized for 30 days. Flow cytometry was used to analyze the cytokine and antibody levels of immunized mice, including CD4 + 、CD8 + Changes in T cells, memory T cells, Treg, Breg, DC cells and spleen cell proliferation; ELISA was used to detect the levels of specific antibodies IgA, IgG1 and IgG2a in mouse serum.

[0082] The experimental results are as follows Fig. 8A , 8B as well as Figures 9 to 14 As shown, Fig. 8A and 8B To detect the levels of IFN-γ, TNF-α, IL-2, and IL-17 in CD4+ cells in the spleen and lymph nodes of immunized mice, Figure 8 shows the levels of cytokines IFN-γ (A), TNF-α (B), IL-2 (C), and IL-17 (D) in CD4+ cells in the spleen (upper) and lymph nodes (lower) of immunized mice, and representative images are shown. The gated area represents CD4+ cells; E: Statistical data of flow cytometry results, showing the cytokine levels of IFN-γ, TNF-α, IL-2, and IL-17 in CD4+ cells in the spleen; F: Statistical data of flow cytometry results, showing the cytokine levels of IFN-γ, TNF-α, IL-2, and IL-17 in CD4+ cells in the lymph nodes.

[0083] Fig. 9 To detect CD8 + Intracellular levels of GzmB and Perforin; Fig. 9 In the figure, flow cytometry was used to evaluate the levels of GzmB (A) and Perforin (B) in CD8+ cells in the spleen (upper) and lymph nodes (lower) of immunized mice, and representative images are shown; the gated area indicates CD8+ cells; C: Statistical data of flow cytometry results, showing the expression of CD8+ in spleen + Cytokine levels of GzmB and Perforin in cells; D: Statistical data of flow cytometry results, showing the cytokine levels of GzmB and Perforin in lymph node CD8+ cells;

[0084] Fig.10 To detect CD8 +Intracellular levels of Tregs and Breg cells; Fig.10 A: Flow cytometry results and statistical data of the proportion of Tregs cells in immunized mice (PBS, MS and rMS groups). The gated area represents CD4 + A: Breg cells; B: Flow cytometry results and statistical data of Breg cell ratio in immunized mice (PBS, MS and rMS groups). The gated area represents CD19+ cells.

[0085] Fig.11 To detect the proportion of DC cells in the spleen of immunized mice and the proportion of DC cells that secrete IL-10 and IL-12 cytokines. A: Representative images and statistical data of flow cytometry of the proportion of DC cells in immunized mice (PBS, MS and rMS groups), the gated area represents CD11c+ cells. B: Representative images and statistical data of flow cytometry of the proportion of DC cells that secrete IL-10 cytokines in immunized mice (PBS, MS and rMS groups), the gated area represents CD11c + C: Representative flow cytometry images and statistical data of the proportion of DC cells secreting cytokine IL-12 in immunized mice (PBS, MS and rMS groups), and the gated area indicates CD11c + cell.

[0086] Fig.12 To detect the proportion of memory T cells in the spleen and lymph nodes of immunized mice. A: Representative images and statistical data of flow cytometry of the proportion of memory T cells in the spleen of immunized mice (PBS, MS and rMS groups), the gated area represents CD4+ cells. B: Representative images and statistical data of flow cytometry of the proportion of memory T cells in the lymph nodes of immunized mice (PBS, MS and rMS groups), the gated area represents CD4+ cells.

[0087] Fig.13 A: Cell proliferation in spleen of immunized mice (PBS, MS and rMS groups). B: Statistical diagram of flow cytometry results.

[0088] Fig.14 The levels of specific antibodies IgA, IgG1 and IgG2a in the sera of immunized mice. ELISA analysis shows the statistical graph of the levels of specific antibodies IgA (A), IgG1 (B) and IgG2a (C) in the sera of immunized mice (PBS, MS and rMS groups).

[0089] Depend on Fig. 8A , 8B as well as Figures 9 to 14 It can be seen that:

[0090] In the CD4+ cells of the mouse spleen and lymph nodes, 4 weeks after immunization, the expression levels of IL-2 and IL-17 in the rMS::MPT64 group were significantly higher than those in the MS group. This suggests that rMS::MPT64 may have a stronger stimulatory effect in local and systemic inflammatory responses by enhancing the expression of these pro-inflammatory factors in CD4+ cells. + In the cells, after immunization, the expression levels of GzmB and Perforin in the rMS group were significantly higher than those in the MS group. This indicates that the rMS immunization strategy can more effectively enhance the cytotoxic function of CD8+T cells and more effectively fight pathogen-infected cells and tumor cells by increasing the expression levels of GzmB and Perforin. Tregs (marked by FoxP3) and Bregs (mainly IL-10-secreting B10 cells, marked by IL-10, CD9 and CD19) play an important role in immune regulation. At 30 days after immunization, the proportion of Tregs and Bregs in mice in the rMS group was significantly lower than that in the MS group, but higher than that in the PBS group, indicating that rMS may have a certain regulatory effect on immunosuppressive cells. Dendritic cells (DCs) take up antigens through phagocytosis, pinocytosis and receptor-mediated endocytosis, and are the only antigen-presenting cells that can effectively trigger primary immune responses. DCs are divided into multiple subgroups according to phenotype, cytokine secretion, tissue distribution or function. The cytokines secreted by different subgroups (such as IL-12, IL-10 and IL-4) affect the differentiation of activated T cells. This study analyzed the proportion of DCs and DCs secreting IL-10 and IL-12 in the spleen of three groups of mice 30 days after immunization. Flow cytometry results showed that the proportion of DCs and the proportion of DCs secreting IL-12 in rMS group mice were significantly higher than those in MS group, while the proportion of DCs secreting IL-10 was significantly lower than that in MS group.

[0091] To further explore the effect of rMS::MPT64 on memory T cells, the study used the expression levels of CD44 and CD62L to distinguish effector memory T cells from central memory T cells, and compared the ratios of the two types of memory T cells after different immune treatments. hi CD62L low ) were higher in the MS group, while central memory T cells (CD44 low CD62L hi) was lower in the rMS group than in the MS group. After immunization with rMS::MPT64, the proportion of Ki67+ cells increased. Ki67 is a cell cycle-related nuclear protein used to evaluate cell proliferation ability. The study evaluated the proliferation effect of rMS::MPT64 recombinant vaccine on T cells by cell proliferation experiment. The spleen cells of immunized mice were stimulated with specific antigen PPD for 72 hours, and the proliferation was evaluated using Ki67 detection kit. The results showed that the proportion of Ki67-positive cells in the rMS group was significantly higher than that in the MS group, indicating that the degree of cell proliferation in the rMS group was higher. Antibodies play a key role in connecting adaptive immune responses with the innate immune system. Among them, IgG and IgA are the most abundant antibodies in serum, accounting for 90% of circulating antibodies. In this study, the levels of specific antibodies IgA, IgG1, and IgG2a were quantitatively determined using ELISA technology. The results showed that the levels of specific IgA, IgG1, and IgG2a in the rMS group were significantly higher than those in the MS group. .

[0092] Example 6

[0093] PBS, MS and rMS:MPT64 were added to the culture medium containing immune adjuvant to obtain the vaccine. After 30 days of immunization, the mice were challenged with H37Ra to establish the mouse infection model. The mice were killed 4 weeks later, and the organs were aseptically extracted for colony count, acid-fast staining and HE staining to evaluate the anti-tuberculosis effect of the vaccine. The results are shown in Figures 15-16 As shown;

[0094] Fig.15 The colony counts in the lungs and spleens of mice in the PBS, MS, and rMS groups after immunization and infection were as follows: Fig.15 A: Statistical graph of colony counts in the lungs of mice in the PBS, MS and rMS groups. B: Statistical graph of colony counts in the spleens of mice in the PBS, MS and rMS groups.

[0095] Fig.16 Pathological tissue sections of the lungs and spleens of mice in the PBS, MS and rMS groups after immunization and infection. Fig.16 In the figure, the first row: H&E stained sections of the lungs of mice in the PBS, MS and rMS groups after immunization and infection. The second row: H&E stained sections of the spleens of mice in the PBS, MS and rMS groups after immunization and infection.

[0096] Depend on Figures 15-16 It can be seen that:

[0097] The number of bacteria in the lungs and spleens of mice in the rMS group was significantly lower than that in the MS group and PBS group, and the difference was statistically significant. Fig.16) showed that in the observation of lung tissue, granuloma formation and incomplete alveolar structure appeared in the PBS group, which was improved in the MS group, while the alveolar structure in the rMS group was more complete, and no obvious pathological changes were observed. HE staining of the spleen showed that the lymph nodes in the PBS group were morphologically destroyed and the boundaries were unclear, which was improved in the MS group, and the rMS group performed better. In addition, acid-fast staining did not find acid-fast bacilli.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A recombinant rMS::MPT64, characterized in that The recombinant rMS::MPT64 is prepared by transfecting Mycobacterium smegmatis with a recombinant vector expressing the MPT64 gene.

2. The recombinant rMS::MPT64 according to claim 1, characterized in that The recombinant vector expressing the MPT64 gene is constructed from the MPT64 gene and an expression vector.

3. The recombinant rMS::MPT64 according to claim 2, characterized in that The expression vector includes pMV361.

4. Use of the recombinant rMS::MPT64 according to any one of claims 1 to 3 in the preparation of tuberculosis vaccines.

5. A recombinant rMS::MPT64 tuberculosis vaccine, characterized in that The recombinant rMS::MPT64 tuberculosis vaccine comprises recombinant rMS::MPT64, culture medium and immune adjuvant.

6. The recombinant rMS::MPT64 tuberculosis vaccine according to claim 5, characterized in that The culture medium is beef extract peptone culture medium.

7. The recombinant rMS::MPT64 tuberculosis vaccine according to claim 5, characterized in that The immune adjuvant is Mycobacterium smegmatis.

8. The recombinant rMS::MPT64 tuberculosis vaccine according to claim 5, characterized in that The concentration of the recombinant rMS::MPT64 was 5×10 6 CFU / 100μL.

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