Preparation and application of novel mycobacterium tuberculosis subunit vaccine LK35

Abstract

The invention discloses preparation and application of a novel mycobacterium tuberculosis subunit vaccine LK35. The mycobacterium tuberculosis subunit vaccine is composed of fusion protein LK35 and a DP adjuvant. The fusion protein LK35 is prepared by sequentially connecting EsxL, EsxK and Rv2656c with one another. The invention also provides a preparation method of the fusion protein. The subunit vaccine can be used as a preventive vaccine for diseases related to mycobacterium tuberculosis / environmental mycobacterium, and can also be used as a therapeutic vaccine after mycobacterium tuberculosis / environmental mycobacterium infection. The vaccine immunized mice can provide good antigen-specific immune response, and the vaccine combined with fusion proteins LT20, LT57 and LT28 can provide effective long-term protective efficacy against mycobacterium bovis. Therefore, the subunit vaccine involved in the invention is expected to become a novel tuberculosis vaccine, and has good application and development prospects.

Description

Technical Field

[0001] This invention belongs to the field of vaccines and relates to Mycobacterium tuberculosis fusion protein LK35 and its subunit vaccines. Specifically, it relates to a vaccine containing paired secretory antigens and latent infection-associated antigens of Mycobacterium tuberculosis, and its application in the prevention and treatment of Mycobacterium tuberculosis / environmental mycobacterium infection. Background Technology

[0002] Tuberculosis (TB) is a highly contagious respiratory disease caused by Mycobacterium tuberculosis. Because the bacterium has coexisted with humans for centuries, it has evolved various self-protective mechanisms, cleverly evading the body's immune system while utilizing nutrients within host cells to maintain its long-term survival. Although BCG is the only TB vaccine approved for clinical use in many countries and offers excellent protection against severe TB conditions such as miliary tuberculosis in infants, its protection in adults remains limited. Therefore, there is an urgent need for new TB vaccines to overcome the current limitations of BCG, including its short duration of protection and significant differences in effectiveness across geographical distribution and population groups.

[0003] In recent years, significant progress has been made in the development of tuberculosis vaccines, with several different types of vaccines entering clinical trials. These mainly include inactivated vaccines, live attenuated vaccines, viral vector vaccines, and subunit vaccines. Currently available subunit vaccine candidates primarily utilize early-secreting antigens of Mycobacterium tuberculosis such as ESAT-6, CFP-10, and Ag85B. However, these vaccines lack antigens related to latent tuberculosis infection, resulting in poor efficacy in preventing latent tuberculosis infection (LTBI). Tuberculosis vaccine design should broaden the antigenic spectrum, adding antigens from different growth stages of Mycobacterium tuberculosis to enhance vaccine protection.

[0004] As research into the infection routes and mechanisms of Mycobacterium tuberculosis (MBTB) deepens, it has become clear that latent infection-associated antigens (LASIKs) are crucial for understanding the survival mechanisms of MBTB in vivo and their importance as candidate vaccine antigens. Currently, several multi-stage subunit vaccines containing LASIKs, such as ID93 / GLA-SE and H56:IC31, have entered clinical trials. Screening for novel LASIKs to construct subunit vaccines can significantly improve vaccine efficacy and holds great promise for clinical application. Summary of the Invention

[0005] The technical problem to be solved by this invention is:

[0006] We screened and evaluated highly immunogenic Mycobacterium tuberculosis latent infection-related antigens using BCG-immunized mouse models, Mycobacterium bovis-infected cattle, and Mycobacterium tuberculosis-infected humans. On the other hand, we constructed a fusion protein by using paired secreted Mycobacterium tuberculosis antigens and latent infection-related antigens, purified the fusion protein, and then immunized mice with DP adjuvant to evaluate its immunogenicity and protective effect.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A fusion protein comprising paired secretory antigens of Mycobacterium tuberculosis and latent infection-associated antigens of Mycobacterium tuberculosis was constructed and prepared. The fusion protein includes EsxL, EsxK, and Rv2656c antigens. EsxL (Rv1198) and EsxK (Rv1197) are putative ESAT-6 family proteins, belonging to ESX-5 in the type VII secretory system, and are encoded by the esxKL operon. EsxK and EsxL can form a dimeric complex with an α-helix structure, similar in structure to the ESAT6-CFP-10 complex. Previous studies have found that EsxL is present in the culture filtrate of Mycobacterium tuberculosis H37Rv, and the transcriptional level of Rv1198 is significantly increased in the sputum of patients diagnosed with tuberculosis, and it is highly expressed during active pulmonary tuberculosis infection in humans. Studies have shown that EsxL can induce the production of pro-inflammatory factors such as TNF-α by activating the NF-κB and MAPK signaling pathways through TLR2.

[0009] Rv2656c was identified as a PhiRv2 phage protein belonging to the RD13 region. It was highly expressed in a nutrient starvation model and was predicted to have a dominant CTL epitope. Previously, Wu Xueqiong's team constructed a multi-epitope vaccine, PP19128R, containing the dominant epitope of Rv2656c, which exhibited good antigenicity and immunogenicity. This dominant epitope was then incorporated into the multi-epitope-based diagnostic biomarker HP16118P. This biomarker demonstrated strong antigenicity and immunogenicity, inducing the production of cytokines such as CM-CSF, IL-23, and IL-5, and showed potential for differentiating between LTBI and ATB.

[0010] This invention utilizes genetic engineering methods to horizontally conjugate the genes of three antigens, EsxL, EsxK, and Rv2656c, and clones them into the pET30a(+) vector for expression. The protein is expressed in large quantities as inclusion bodies in E. coli BL21(DE3) strain, and the fusion protein is subsequently purified through inclusion body washing and renaturation. The subunit vaccine constructed from this fusion protein and adjuvant holds promise for the prevention and treatment of Mycobacterium tuberculosis / environmental mycobacteria infections.

[0011] Preferably, the amino acid sequence of the fusion protein LK35 is listed in SEQ ID No. 2;

[0012] As a preferred method, the purification method of the fusion protein LK35 is as follows: first, LK35 cells are disrupted by ultrasound, the precipitate after cell disruption is collected, the precipitate is washed with inclusion body washing solution, and then denatured with 8M urea. Subsequently, the purified LK35 fusion protein is obtained by gradient dialysis refolding.

[0013] Preferably, the adjuvant is composed of PolyI:C and DDA; the weight ratio of PolyI:C to DDA is 1:5.

[0014] This invention provides a method for preparing an LK35 tuberculosis subunit vaccine. The method involves diluting the fusion protein LK35, PolyI:C, and DDA separately using sterile PBS. After dilution, DDA is dissolved in an 80°C water bath for 10 minutes. After dissolution, the solution is allowed to return to room temperature. The PolyI:C solution is mixed with the protein dilution solution, and then the DDA solution is added, followed by thorough emulsification.

[0015] The present invention also provides the application of the above-mentioned tuberculosis subunit vaccine in the prevention and treatment of Mycobacterium tuberculosis / environmental mycobacterium infection.

[0016] The present invention also provides a gene sequence encoding the above-mentioned fusion protein LK35;

[0017] Preferably, the gene sequence encoding the fusion protein LK35 is the sequence listed in SEQ ID No. 1;

[0018] The present invention also provides an expression vector containing the above-mentioned genes; preferably, the expression vector is a prokaryotic expression vector; more preferably, the prokaryotic expression vector is pET30a(+).

[0019] The present invention also provides a host cell comprising the above-described vector; preferably, the host cell is E. coli BL21(DE3).

[0020] This invention utilizes genetic engineering technology to successfully construct, express, and purify the tag-free Mycobacterium tuberculosis fusion protein LK35. The purified LK35 fusion protein can be obtained using inclusion body washing and dialysis refolding. The LK35 fusion protein expression is stable and can be scaled up. Using the LK35 fusion protein provided by this invention, an LK35 / DP subunit vaccine was constructed in combination with a laboratory DP adjuvant. Immunization of mice induced a good antigen-specific cellular and humoral immune response, and the vaccine also provided effective protection against Mycobacterium bovis. This vaccine shows promising development potential in the prevention and treatment of Mycobacterium tuberculosis / environmental mycobacteria infections. Attached Figure Description

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings:

[0022] Figure 1 Screening for antigens related to latent tuberculosis infection.

[0023] (A) Peripheral blood of bovine herds infected with Mycobacterium bovis was stimulated with a single latent-associated antigen, and IFN-γ secretion levels were measured. (B) Peripheral blood of bovine herds infected with Mycobacterium bovis was stimulated with Rv2656c, and IFN-γ secretion levels were measured. (C) Peripheral blood of human subjects was stimulated with Rv2656c, and IFN-γ secretion levels were measured.

[0024] Figure 2 This study aims to provide a map of the construction of the fusion protein LK35 in the pET30a(+) plasmid, the three-dimensional protein structure of LK35, the expression and purification of LK35, and its Western Blot validation.

[0025] Figure 3 An immunization strategy diagram for detecting the immunogenicity of the LK35 fusion protein in mice.

[0026] Figure 4 Flow cytometry results and statistical graphs of LK35 / DP in a mouse model for cellular immunoassay.

[0027] (A) LK stimulation of mouse spleen lymphocytes CD4 + IFN-γ, CD8 + IFN-γ, CD4 + IL-2 and CD8 + Flow cytometry results of IL-2 secretion levels. (B) LK stimulation of mouse spleen lymphocytes CD4 + IFN-γ, CD8 + IFN-γ, CD4 + IL-2 and CD8 + Flow cytometry results of IL-2 secretion levels. (C) Rv2656c stimulation of mouse spleen lymphocytes CD4 + IFN-γ, CD8 + IFN-γ, CD4 + IL-2 and CD8 + Flow cytometry results of IL-2 secretion levels. (D) Rv2656c stimulation of mouse spleen lymphocytes CD4 + IFN-γ, CD8 + IFN-γ, CD4 + IL-2 and CD8 +Statistical analysis of IL-2 secretion levels by flow cytometry. (E) Statistical analysis of granzyme B production by mouse splenic lymphocytes stimulated by LK. (F) Statistical analysis of granzyme B production by mouse splenic lymphocytes stimulated by Rv2656c.

[0028] Figure 5 Figure showing the humoral immunity detection results of LK35 / DP in a mouse model.

[0029] (A) Rectangular and line graphs of anti-LK IgG antibody titers. (B) Rectangular and line graphs of anti-Rv2656c IgG antibody titers. (C) Rectangular and line graphs of anti-LK IgG1 antibody titers. (D) Rectangular and line graphs of anti-Rv2656c IgG1 antibody titers. (E) Rectangular and line graphs of anti-LK IgG2c antibody titers. (F) Rectangular and line graphs of anti-Rv2656c IgG2c antibody titers.

[0030] Figure 6 Flow cytometry results and statistical graphs of LK35 / DP-induced long-term survival memory T cell levels in a mouse model.

[0031] (A) LK stimulation of mouse spleen lymphocytes CD4 + TIFN-γ + and CD4 + T IL-2 + The flow cytometry results. (B) LK stimulation of mouse spleen lymphocytes CD4 + TIFN-γ + and CD4 + T IL-2 + Statistical analysis of flow cytometry results. (C) Rv2656c stimulation of mouse spleen lymphocytes CD4 + TIFN-γ + and CD4 + T IL-2 + Flow cytometry results. (D) Rv2656c stimulation of mouse spleen lymphocytes CD4 + TIFN-γ + and CD4 + T IL-2 + Statistical analysis of flow cytometry results. (E)LK and Rv2656c stimulated CD4+ in mouse lymphocytes. + Flow cytometry results of T cell proliferation capacity. (F) LK and Rv2656c stimulation of CD4+ in mouse lymphocytes. + Statistical graph of flow cytometry results for T cell proliferation capacity.

[0032] Figure 7Immunization strategy diagram for evaluating the long-term protective effect of LT20 / DP, LT28 / DP, LK35 / DP, LT57 / DP and the vaccine combination L4 / DP against Mycobacterium bovis intranasal challenge mice.

[0033] Figure 8 To evaluate the long-term protective effects of LT20 / DP, LT28 / DP, LK35 / DP, LT57 / DP and the vaccine combination L4 / DP against Mycobacterium bovis intranasal challenge in mice. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0036] Example 1: Screening for antigens related to latent infection with Mycobacterium tuberculosis

[0037] This study successfully obtained a screened antigen related to latent tuberculosis infection via Ni column purification. The purified protein (10 μg / ml) was then used to stimulate peripheral blood from bovine herds infected with *Mycobacterium tuberculosis*, and the mixture was incubated at 37°C for 20 hours. The IFN-γ secretion level in the blood supernatant was subsequently detected using ELISA.

[0038] Purified protein (Rv2656c, 10 μg / ml) was used to stimulate peripheral blood of bovine herds infected with Mycobacterium bovis and human peripheral blood infected with Mycobacterium tuberculosis, and incubated at 37°C for 20 hours. The IFN-γ secretion level in the blood supernatant was subsequently detected using ELISA. Figure 1 )

[0039] in Figure 1 (A) Peripheral blood of bovine herds infected with Mycobacterium bovis was stimulated with a single latent-associated antigen, and IFN-γ secretion levels were measured. (B) Peripheral blood of bovine herds infected with Mycobacterium bovis was stimulated with Rv2656c, and IFN-γ secretion levels were measured. (C) Peripheral blood of human subjects was stimulated with Rv2656c, and IFN-γ secretion levels were measured.

[0040] Example 2: Construction and validation of an immunization combination containing Mycobacterium tuberculosis paired secretory antigens EsxL-EsxK and latent infection-associated antigen Rv2656c

[0041] This invention first fuses the paired secretory antigens EsxL and EsxK from Mycobacterium tuberculosis at the gene level. By predicting the protein's spatial structure and considering its impact on stability, the (GGGS)3Linker is used to link the paired proteins, effectively preserving their spatial structure. The spatial structure of the Rv2656c protein shows a flexible fragment at its N-terminus; this flexible fragment is used as the natural linker for the fusion protein. The three-gene linker is named LK35 (sequence No. 1), and the linking order is as follows: Figure 1 As shown in Figure A, the gene and protein sequences of LK35 are as shown in Sequence No. 1 of the sequence listing. The LK35 gene was constructed by using a (GGGS)3Linker to link the DNA sequences encoding EsxL and EsxK, directly linking the Rv2656c sequence, and then constructing the recombinant plasmid pET30a(+)-LK35(EsxL-linker-EsxK-Rv2656c) using a standard cloning procedure.

[0042] The three-dimensional structure of the LK35 protein was predicted using the alpha-Fold3 server by uploading the amino acid sequence encoded by the LK35 gene.

[0043] The pET30a(+)-LK35 recombinant plasmid was transformed into *E. coli* BL21(DE3) for LK35 fusion protein expression. Finally, based on the protein's characteristics, the fusion protein was successfully purified using inclusion body washing and dialysis refolding. The specific steps are as follows:

[0044] 1. The plasmid pET30a(+)-LK35(EsxL-linker-EsxK-Rv2656c) was constructed by synthesizing the entire genome.

[0045] The single antigens EsxL, EsxK, and Rv2656c were sequentially ligated into the vector pET30a(+) using a whole-genome synthesis method. The 5' restriction enzyme site was NdeI (CATATG), and the 3' restriction enzyme site was Hind III (AAGCTT). Simultaneously, a (GGGS)3-linker was added between the single antigens EsxL and EsxK to promote protein structural stability. The recombinant gene vector pET30a(+)-LK35(EsxL-linker-EsxK-Rv2656c) was constructed. The following are the nucleotide and amino acid sequences of the fusion protein LK35(EsxL-linker-EsxK-Rv2656c):

[0046] LK35 nucleotide sequence

[0047] The italics indicate enzyme cleavage sites; "—" indicates the nucleotide sequence of antigen EsxL; "–––" indicates the nucleotide sequence of antigen EsxK; "~~~" indicates the nucleotide sequence of antigen Rv2656c; the lowercase nucleotide sequence is the linker (GGGGS)3.

[0048]

[0049]

[0050] The amino acid sequence of LK35

[0051] Italics indicate enzyme cleavage sites; "—" indicates the nucleotide sequence of antigen EsxL; "–––" indicates the nucleotide sequence of antigen EsxK; "~~~" indicates the nucleotide sequence of antigen Rv2656c; bold indicates the amino acid sequence of linker (GGGGS)3.

[0052]

[0053] 2. Three-dimensional protein structure of fusion protein LK35

[0054] The three-dimensional structure of the LK35 protein was predicted using alpha-Fold3 software after uploading its amino acid sequence. The predicted structure was displayed using PyMol. Figure 2 )

[0055] 3. Expression of the fusion protein LK35

[0056] The successfully constructed pET30a(+)-LK35(EsxL-linker-EsxK-Rv2656c) vector was transformed into *E. coli* BL21(DE3). The *E. coli* BL21(DE3) strain expressing LK35 was activated, and a single colony was placed in 5 ml of sterile LB broth. A final concentration of 50 μg / ml kanamycin was added, and the culture was incubated overnight at 37°C with shaking at 180 rpm. Take 2 ml of overnight culture and add it to 200 ml of LB liquid medium. Add kanamycin to a final concentration of 50 μg / ml and incubate at 37°C with shaking at 180 rpm for 4 hours. Add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 1 mmol / L and incubate at 37°C with shaking at 180 rpm for 4 hours. Collect the bacterial cells by centrifugation at 8000 rpm for 10 min at 4°C. Wash the bacterial cells three times with PB buffer (Na2HPO4·12H2O 20 mmol / L, NaH2PO4·2H2O 20 mmol / L, pH 7.4) and resuspend the bacterial cells. The resuspended bacterial cells were placed on ice and sonicated (180-200W, 3s sonication, 3s pause); then centrifuged at 8000rpm for 10min at 4℃, and the supernatant and precipitate were collected separately. The supernatant and precipitate were subjected to polyacrylamide gel electrophoresis. SDS-PAGE analysis showed that, compared with empty *E. coli* BL21(DE3), there were obvious specific protein expression bands at approximately 35KD, with the protein mainly expressed in inclusion body form, and less expression in the supernatant. Figure 2 ).

[0057] 4. Purification of fusion protein LK35

[0058] The fusion protein LK35 was expressed in large quantities. Bacterial cells were collected, resuspended in 20 mM PB buffer, placed on ice, and sonicated to disrupt the cells. The precipitate containing the LK35 fusion protein was collected by centrifugation at 8000 rpm for 10 min at 4°C. Subsequently, the inclusion bodies were resuspended in inclusion body washing buffer (5.85 g NaCl, 30 g urea, 0.186 g EDTA, 5 ml Triton-100, 20 mM PB buffer to a final volume of 500 ml, pH 7.4), centrifuged at 10000 rpm for 5 min at 4°C, and the precipitate was collected. This process was repeated 5 times. The protein collected by the final centrifugation was resuspended in 8 M urea + 50 mM Tris (pH 7.4) and incubated overnight at 4°C to dissolve. After overnight incubation, the dissolved protein was placed in a 3KD dialysis bag and treated in 6M urea + 50mM Tris (pH=7.4) dialysis buffer at 4°C for 12 hours; then in 4M urea + 50mM Tris (pH=7.4) dialysis buffer at 4°C for 12 hours; then in 2M urea + 50mM Tris (pH=7.4) dialysis buffer at 4°C for 12 hours; then in 1M urea + 50mM Tris (pH=7.4) dialysis buffer at 4°C for 12 hours; and finally in 50mM Tris (pH=7.4) dialysis buffer at 4°C for 12 hours. The treated samples were collected and subjected to polyacrylamide gel electrophoresis; SDS-PAGE analysis showed a protein band with 90% purity at approximately 35KD. Figure 2 )

[0059] Figure 2 The following data are presented: Construction method of the LK35 fusion protein gene; alpha-Fold prediction of the three-dimensional structure of the LK35 fusion protein; expression and purification results of the LK35 fusion protein in *E. coli* BL21(DE3) and Western blot validation. (A) Construction method of the LK35 fusion protein gene. (B) Alpha-Fold prediction of the three-dimensional structure of the LK35 fusion protein. (C) Expression and purification results of the LK35 fusion protein. M, Marker; 1, Empty BL21 bacteria; 2, Supernatant after LK35 lysis; 3, Precipitate after LK35 lysis; 4, Purified LK35 protein. (D) Western blot validation using LK and Rv2656c mouse polyclonal antibodies. M, Marker; 1, Purified LK35 protein; 2, Blank control; 3, LB28 (no cross-antigen with LK35).

[0060] Depend on Figure 2 It can be seen that the LK35 protein is mainly expressed in the form of inclusion bodies in E. coli BL21 strain, and can be purified by washing and dialysis refolding of inclusion bodies.

[0061] Example 3: Formulation of LK35 fusion protein subunit vaccine

[0062] The LK35 fusion protein was diluted to 0.1 mg / ml using sterile PBS; PolyI:C was dissolved to 1 mg / ml using sterile PBS buffer; cationic liposome dimethyl hexadecylamine (DDA) was prepared to 5 mg / ml using sterile PBS and incubated at 80°C for 10 min to dissolve the DDA, followed by cooling to room temperature. The protein dilution was then mixed with PolyI:C at a 1:1 ratio and incubated at room temperature for 1 min. Two volumes of DDA solution were added dropwise to the mixture to achieve a protein solution:PolyI:C:DDA ratio of 1:1:2. The mixture was then thoroughly emulsified to a homogeneous emulsion, which constituted the LK35 / DP subunit vaccine. The dosage for immunization was 200 μl per mouse.

[0063] Example 4: Immunogenicity evaluation of the subunit vaccine LK35 / DP

[0064] 1. Materials used: Subunit vaccine LK35 / DP; BCG; Phosphate-buffered saline (PBS).

[0065] 2. Experimental animals: 6-8 week old female C57BL / 6 mice.

[0066] 3. Immune indicators and their measurement methods

[0067] (1) Flow cytometry was used to detect the secretion levels of IFN-γ and IL-2 in mouse spleen lymphocytes.

[0068] Six weeks after the last immunization of mice, spleen lymphocytes were isolated, counted, and then divided into groups of 5 × 10⁻⁶. 6 Cells were seeded into 24-well cell culture plates and stimulated in vitro with the corresponding single antigen (5 μg / ml) for 4 hours. Then, 1 μl of a protein transport inhibitor was added to each well, and after incubation for 16 hours, CD4 counts were detected by flow cytometry. + T cells and CD8 + The secretion levels of IFN-γ and IL-2 in T cells. Figure 4 )

[0069] (2) ELISA was used to detect the granzyme B secretion level of mouse spleen lymphocytes.

[0070] Six weeks after the last immunization of mice, spleen lymphocytes were isolated, counted, and then divided into groups of 5 × 10⁻⁶. 6Cells were seeded into 24-well cell culture plates and stimulated in vitro with the corresponding single antigen (5 μg / ml) for 72 hours. Cell supernatant was then collected, and CD8+ was detected using a granzyme B assay kit. + T cell secretion of granzyme B level ( Figure 4 ).

[0071] Figure 4 In this study, the BCG group was immunized once at week 0, while the LK35 / DP subunit tuberculosis vaccine group was immunized three times at weeks 0, 3, and 6. Six weeks after the last immunization, splenic lymphocytes were isolated from the mice and co-incubated with antigens EsxL-EsxK and Rv2656c (5 μg / ml) for 20 hours. CD4 counts were then performed using flow cytometry. + T cells and CD8 + The secretion levels of IFN-γ and IL-2 in T cells. Specifically, (A)LK stimulation of mouse splenic lymphocytes CD4... + IFN-γ, CD8 + IFN-γ, CD4 + IL-2 and CD8 + Flow cytometry results of IL-2 secretion levels. (B) LK stimulation of mouse spleen lymphocytes CD4 + IFN-γ, CD8 + IFN-γ, CD4 + IL-2 and CD8 + Flow cytometry results of IL-2 secretion levels. (C) Rv2656c stimulation of mouse spleen lymphocytes CD4 + IFN-γ, CD8 + IFN-γ, CD4 + IL-2 and CD8 + Flow cytometry results of IL-2 secretion levels. (D) Rv2656c stimulation of mouse spleen lymphocytes CD4 + IFN-γ, CD8 + IFN-γ, CD4 + IL-2 and CD8 + Statistical analysis of IL-2 secretion levels by flow cytometry. (E) Statistical analysis of granzyme B production in splenic lymph nodes of mice stimulated by LK using ELISA. (F) Statistical analysis of granzyme B production in splenic lymph nodes of mice stimulated by Rv2656c using ELISA. N = 5. *p < 0.05, **p < 0.01, ***p < 0.005.

[0072] Depend on Figure 4It can be seen that the LK immunized mouse group induced more antigen-specific T cells than the fusion protein LK35 group, while the Rv2656c immunized mouse group induced fewer antigen-specific T cells than the LK35 group. This indicates that the fusion protein composed of EsxL, EsxK and Rv2656c has good immunogenicity and displays single antigen T cell epitopes well.

[0073] (3) The levels of mouse serum-specific antibodies IgG, IgG1 and IgG2c were detected by ELISA.

[0074] Serum was collected from mice 6 weeks after their last immunization. Each individual antigen (5 μg / ml) was added to a 96-well ELISA plate, and the plates were incubated overnight at 4°C for protein pre-coating. The next day, the plate solution was discarded, and the plates were washed four times with 300 μl / well of PBST wash buffer. 100 μl of 5% BSA was added to each well for blocking, and the plates were incubated at 37°C for 1 hour. After repeating the washing steps, 100 μl of serially diluted serum sample was added to each well, and the plates were incubated at 37°C for 2 hours. After repeating the washing steps, 100 μl of a 1:10000 diluted goat anti-mouse IgG, IgG1, or IgG2c was added to each well, and the plates were incubated at 37°C for 1 hour. After repeating the washing steps, 100 μl of TMB chromogenic solution was added to each well, and the reaction was incubated at room temperature in the dark for 15 minutes. Finally, 50 μl of stop solution (2MH2SO4) was added to each well to terminate the reaction. The OD value was measured at 450 nm. Figure 5 )

[0075] Figure 5 The level of specific antibodies in the serum of mice immunized with the subunit vaccine LK35 / DP.

[0076] Figure 5 In this study, the BCG group was immunized once at week 0, while C57BL / 6 mice were immunized three times at weeks 0, 3, and 6 with the tuberculosis subunit vaccine LK35 / DP. Six weeks after the last immunization, mouse serum was collected to detect the titers of EsxL, EsxK, and Rv2656c antigen-specific IgG, IgG1, and IgG2c antibodies. The graphs show: (A) rectangular and line graphs of anti-LK IgG antibody titers; (B) rectangular and line graphs of anti-Rv2656c IgG antibody titers; (C) rectangular and line graphs of anti-LK IgG1 antibody titers; (D) rectangular and line graphs of anti-Rv2656c IgG1 antibody titers; (E) rectangular and line graphs of anti-LK IgG2c antibody titers; and (F) rectangular and line graphs of anti-Rv2656c IgG2c antibody titers. n = 5. *p<0.05, **p<0.01, ***p<0.005

[0077] Depend on Figure 5It can be seen that the individual antigens EsxL, EsxK and Rv2656c B cell epitopes that make up the fusion protein LK35 are well displayed and can induce a good antibody response.

[0078] Example 5: The ability of the subunit vaccine LK35 / DP to induce long-term surviving memory T cells

[0079] 1. Materials used: Subunit vaccine LK35 / DP; BCG; Phosphate-buffered saline (PBS).

[0080] 2. Experimental animals: C57BL / 6 mice.

[0081] 3. Methods for detecting immune markers

[0082] (1) Flow cytometry was used to detect the secretion levels of IFN-γ and IL-2 in mouse spleen lymphocytes.

[0083] Twelve weeks after the last immunization, mice were intraperitoneally injected with a mixture of antigens EsxL-EsxK (5 μg / mouse) and Rv2656c (5 μg / mouse). Three days later, splenic lymphocytes were isolated, counted, and then divided into groups at a ratio of 5 × 10⁻⁶ cells / mouse. 6 Cells were seeded into 24-well cell culture plates and stimulated in vitro with the corresponding single antigen (5 μg / ml) for 4 hours. A protein transport inhibitor was added to each well, and the plates were incubated for 16 hours. CD4 counts were then measured. + T cells and CD8 + The levels of IFN-γ and IL-2 secreted by T cells. Figure 6 )

[0084] (2) Flow cytometry detection of the proliferative capacity of long-term surviving memory T cells

[0085] Twelve weeks after their last immunization, mice were intraperitoneally injected with a mixture of antigens EsxL-EsxK (5 μg / mouse) and Rv2656c (5 μg / mouse). Three days later, splenic lymphocytes were isolated from the mice and stimulated in vitro with antigens LK and Rv2656c (5 μg / ml), respectively. After 4 hours of stimulation, EdU was added and co-incubated for 68 hours. Intracellular cytokine staining was performed, and EdU was detected by flow cytometry. + Cell count was used to analyze their proliferative capacity.

[0086] Figure 6 The ability of the subunit vaccine LK35 / DP to induce long-term survival memory T cells

[0087] Figure 6In this study, the BCG group was immunized once at week 0, while the LK35 / DP group (tuberculosis subunit vaccine) was immunized three times at weeks 0, 3, and 6. Twelve weeks after the last immunization, mice were intraperitoneally injected with a mixture of antigens EsxL-EsxK (5 μg / mouse) and Rv2656c (5 μg / mouse). Three days later, splenic lymphocytes were isolated from the mice and stimulated with antigens LK and Rv2656c (5 μg / ml), respectively. Twenty hours after stimulation, intracellular cytokine staining was performed, and the expression levels of IFN-γ and IL-2 were detected by flow cytometry. Specifically, (A) LK stimulation of mouse splenic lymphocytes showed increased CD4+ expression. + TIFN-γ + and CD4 + T IL-2 + (B) Flow cytometry results; LK stimulation of mouse spleen lymphocytes CD4 + TIFN-γ + and CD4 + T IL-2 + Statistical analysis of flow cytometry results; (C) Rv2656c stimulation of mouse spleen lymphocytes CD4 + TIFN-γ + and CD4 + T IL-2 + Flow cytometry results; (D) Rv2656c stimulation of mouse spleen lymphocytes CD4 + TIFN-γ + and CD4 + T IL-2 + Statistical analysis of flow cytometry results; (E)LK and Rv2656c stimulation of CD4+ in mouse lymphocytes + Flow cytometry results of T cell proliferation capacity. (F) LK and Rv2656c stimulation of CD4+ in mouse lymphocytes. + Statistical graph of flow cytometry results for T cell proliferation capacity.

[0088] Figure 6 It can be seen that 12 weeks after the last immunization, the LK35 fusion protein can induce mice to produce more long-lived T cells than the single antigen group, and its proliferation ability is better than that of the single antigen immunization group.

[0089] Example 5: Evaluation of the immunoprotective effect after Mycobacterium bovis nasal drop challenge

[0090] 1. Materials used: Subunit vaccines LK35 / DP, LT20 / DP, LT57 / DP, LT28 / DP, L4 / DP; BCG; Phosphate-buffered saline (PBS).

[0091] 2. Experimental animals: C57BL / 6 mice.

[0092] 3. Methods for determining bacterial load index

[0093] Twenty-six weeks after the last immunization, all experimental mice were infected nasally with Mycobacterium bovis. Four weeks later, the mice were euthanized, and the middle lobe of their lungs was collected for HE staining. The remaining lung tissue was collected, ground, and spread onto 7H11+OADC plates and incubated at 37°C. The Mycobacterium bovis load was then detected by CFU counting. Figure 8 ).

[0094] Figure 8 Evaluation of the immunoprotective effect after nasal challenge with Mycobacterium bovis

[0095] Figure 8 In this study, the BCG group was immunized once at week 0, while the tuberculosis subunit vaccine group was immunized three times at weeks 0, 3, and 6. Twenty-six weeks after the last immunization, mice were infected nasally with Mycobacterium bovis at a dose of 2000 CFU / mouse. Four weeks after infection, the bacterial load in the lungs and spleen of mice in each group was measured. Each group consisted of 4-5 mice.

[0096] Depend on Figure 8 It can be seen that, due to the long post-immunization period of 26 weeks, neither the individual fusion protein nor BCG showed good protective effects against Mycobacterium bovis. However, the L4 / DP combination, consisting of LT20, LT28, LT57, and LK35, still exhibited good long-term protective efficiency, significantly higher than the BCG group. This indicates that the L4 / DP combination can induce long-term protective efficiency against Mycobacterium bovis in mice.

[0097] Subunit vaccines constructed based on latent tuberculosis infection-related antigens exhibit certain preventive and therapeutic effects against Mycobacterium tuberculosis. This invention discloses a tuberculosis subunit vaccine comprising a fusion protein LK35 combined with a DP adjuvant (a mixture of dioctadecyl dimethylammonium bromide and polyinosinic acid). The fusion protein LK35 is prepared by sequentially linking EsxL, EsxK, and Rv2656c. This invention also provides a method for preparing this fusion protein. This subunit vaccine can be used as a prophylactic vaccine against Mycobacterium tuberculosis / environmental mycobacteria-related diseases. Immunization of mice with the vaccine provides a good antigen-specific immune response, and its combination with fusion proteins LT20, LT57, and LT28 provides effective long-term protection against Mycobacterium bovis. Therefore, the subunit vaccine involved in this invention shows promise as a novel anti-tuberculosis subunit vaccine with good application and development prospects.

[0098] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that, It contains paired secreted antigens of Mycobacterium tuberculosis and latent infection-associated antigens, and the fusion protein antigens include: EsxL, EsxK and Rv2656c.

2. The fusion protein according to claim 1, characterized in that, The recombinant fusion protein was constructed using pET30a(+) vector, and the fusion protein was prepared by sequentially linking antigens EsxL, EsxK, and Rv2656c.

3. The fusion protein according to claim 2, characterized in that, The host cell of the vector is E. coli BL21(DE3) strain.

4. The fusion protein according to claim 1 or 2, characterized in that, The amino acid sequence of the fusion protein is SEQ ID No.

2.

5. A tuberculosis subunit vaccine comprising the fusion protein of claim 1 or 2.

6. The tuberculosis subunit vaccine according to claim 5, characterized in that, Its adjuvant consists of PolyI:C and DDA; the preparation ratio is PolyI:C:DDA = 1:

5.

7. The method for expressing and purifying the fusion protein according to claim 1 or 2, characterized in that, The following steps are involved: (1) The fusion protein was stably expressed in the form of inclusion bodies in E. coli BL21(DE3) strain; (2) Purification of fusion protein LK35 First, LK35 cells were disrupted by ultrasound, and the precipitate was collected. The precipitate was washed with inclusion body washing buffer and then denatured with 8M urea. The purified fusion protein was then obtained by gradient dialysis.

8. The method for preparing the tuberculosis subunit vaccine according to claim 5, characterized in that, The fusion proteins LK35, PolyI:C, and DDA were diluted separately using sterile PBS. After dilution, DDA was dissolved in an 80°C water bath for 10 minutes. After dissolution, the solution was allowed to stand and return to room temperature. The PolyI:C solution was mixed with the protein dilution solution, and then the DDA solution was added and emulsified thoroughly.

9. The tuberculosis subunit vaccine of claim 5 is used in the prevention / treatment of diseases related to Mycobacterium tuberculosis and environmental mycobacteria infection.

10. The application according to claim 9, characterized in that, The fusion protein can be used alone or in combination with other tuberculosis subunit vaccines.

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