An antigen composition and use thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to an antigen composition and application thereof. The antigen composition comprises ESAT-6, CFP-10, Mpt83 and Ag85B, and a vaccine prepared by using the antigen composition can be used to solve the defects that the tuberculosis candidate vaccines in the prior art can only induce cellular immune response of the body, and protective immune response of mutual balance of humoral immunity and cellular immunity is generated.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antigen composition and its application. Background Technology

[0002] Tuberculosis is a chronic infectious disease mainly caused by infection with the Mycobacterium tuberculosis complex (MTBC), and it is increasingly becoming an important public health problem.

[0003] Vaccination is the most effective means of preventing and controlling infectious diseases. Bacillus Calmette-Guerin (BCG) is currently the only approved vaccine for the prevention of human tuberculosis. BCG offers up to 80% protection in newborns, but its protective efficacy gradually declines with age, and it is no longer effective in preventing adult pulmonary tuberculosis. Furthermore, as a live attenuated vaccine, BCG poses a risk of infection in immunocompromised individuals, and it also suffers from the loss of protective antigens after multiple passages. Therefore, there is an urgent need to develop novel tuberculosis vaccines as alternatives to or booster versions of BCG.

[0004] Adjuvants, as non-specific immune enhancers, have been widely used in vaccine formulation. The combined use of adjuvants with subunit vaccines can maximize the immunogenicity of the vaccine, resulting in better protective efficacy. Aluminum salt adjuvants, with good safety and stability, are widely used in various vaccines, including the DPT vaccine, human papillomavirus vaccine, H5N1 avian influenza vaccine, and inactivated polio vaccine.

[0005] The novel tuberculosis vaccines currently undergoing clinical trials are mainly divided into the following three categories: subunit vaccines and viral vector vaccines, recombinant BCG and attenuated / inactivated vaccines, and DNA vaccines. Among them, subunit vaccines such as ID93 / GLA-SE, GamTBvac, and M72 / AS01E have entered the phase II clinical trial stage.

[0006] Recombinant protein subunit vaccines with adjuvants have become a hot topic in vaccine development due to their good safety profile and well-defined composition. These vaccines contain a large number of human T / B cell epitopes, which can be recognized by human MHC class I and MHC class II molecules, stimulating a significant protective immune response. They induce the activation and differentiation of T and B lymphocytes into memory lymphocytes, providing long-term protection against corresponding pathogens. Currently, tuberculosis subunit vaccines are mainly used for booster immunization after BCG vaccination and as therapeutic vaccines to protect patients with active tuberculosis and those with latent tuberculosis infection.

[0007] The immune response to Mycobacterium tuberculosis infection is a complex process. There are significant differences in the expression of protective antigens of Mycobacterium tuberculosis in patients with active tuberculosis and those with latent tuberculosis infection. Therefore, the common strategy for developing tuberculosis subunit vaccines is to select and combine multiple immunodominant antigens to induce a highly efficient and specific protective immune response against a broad population, including those vaccinated with BCG, patients with active tuberculosis, and patients with latent tuberculosis infection. Commonly used candidate antigens for tuberculosis vaccines are mainly Mycobacterium tuberculosis secreted proteins and cell membrane / cell wall proteins. ESAT-6 (Rv3875), CFP-10 (Rv3874), Mpt83 (Rv2873), and Ag85B (Rv1886c) are important immunodominant antigens of Mycobacterium tuberculosis, expressed at high levels at different stages of Mycobacterium tuberculosis infection and each playing an important role. ESAT-6 and CFP-10 belong to the Esx family of Mycobacterium tuberculosis virulence factors and are important early secretory antigens of Mycobacterium tuberculosis; Mpt83 is a glycolipid protein secreted by Mycobacterium tuberculosis and is associated with the adhesion and dissemination of Mycobacterium tuberculosis; Ag85B is mainly expressed in the logarithmic growth phase of Mycobacterium tuberculosis and is an important virulence factor for Mycobacterium tuberculosis to invade host cells.

[0008] Currently, all novel tuberculosis vaccines suffer from the drawback of inducing a relatively singular protective immune response. Traditionally, cellular immunity is considered the main pathway for the body to resist Mycobacterium tuberculosis infection. However, the latest research results increasingly suggest that humoral immunity and cellular immunity also play irreplaceable roles in the body's anti-tuberculosis immune response. Most of the more than ten tuberculosis vaccine candidates currently undergoing clinical trials can only induce a cellular immune response and cannot induce a comprehensive immune response against Mycobacterium tuberculosis. Therefore, the protective effect of the vaccines is relatively limited, making it difficult to induce a highly efficient and specific protective immune response against a wide range of populations, including those who have received BCG vaccination, patients with active tuberculosis, and patients with latent tuberculosis infection. Summary of the Invention

[0009] This invention provides an antigen composition and its application, which addresses the deficiency of existing tuberculosis candidate vaccines, which can only induce cellular immune responses in the body, and generates a protective immune response that balances humoral and cellular immunity.

[0010] This invention provides an antigen composition comprising ESAT-6, CFP-10, Mpt83, and Ag85B;

[0011] The amino acid sequence of the ESAT-6 is shown in SEQ ID NO.5;

[0012] The amino acid sequence of CFP-10 is shown in SEQ ID NO.6;

[0013] The amino acid sequence of Mpt83 is shown in SEQ ID NO.7;

[0014] The amino acid sequence of Ag85B is shown in SEQ ID NO.8.

[0015] This invention selects four Mycobacterium tuberculosis immunogenic antigens, ESAT-6, CFP-10, Mpt83, and Ag85B, to construct two multi-component subunit vaccines, ECMA019m and ECMA019f, which can overcome the deficiency that most candidate vaccines in the prior art can only induce the body to produce a cellular immune response.

[0016] ECMA019m contains four components: ESAT-6, CFP-10, Mpt83, and Ag85B. The amino acid sequences of each component are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.

[0017] The components of the ECMA019f fusion protein, from N-terminus to C-terminus, are ESAT-6, CFP-10, Mpt83, and Ag85B. The components are connected by flexible linkers. The codon-optimized gene sequence of the ECMA019f fusion protein is shown in SEQ ID NO.9, and the amino acid sequence of the ECMA019f fusion protein is shown in SEQ ID NO.10.

[0018] The antigen combination method of the present invention includes a composition of the component antigens formed in any proportion and / or in any order, and the fusion protein formed by linking ESAT-6, CFP-10, Mpt83 and Ag85B in any order is also within the protection scope of the present invention.

[0019] Preferably, according to the antigen composition of the present invention, the four antigens ESAT-6, CFP-10, Mpt83 and Ag85B are directly mixed as individual antigens and then used.

[0020] Preferably, the flexible connecting arms are sequentially connected from the N end to the C end in the order of ESAT-6, CFP-10, Mpt83 and Ag85B.

[0021] This invention provides a method for preparing an antigen composition, including the expression, purification, refolding, and harvesting of the target product of four separate antigen components of ECMA019m and the ECMA019f fusion protein.

[0022] This invention provides a method for constructing the fusion protein ECMA019m, wherein gene fragments corresponding to antigens ESAT-6, CFP-10, Mpt83 and Ag85B are respectively ligated into the pET-32a plasmid to construct recombinant plasmids of ESAT-6, CFP-10, Mpt83 and Ag85B and transform them into Escherichia coli DH5α competent cells.

[0023] Preferably, the four engineered bacteria ESAT-6, CFP-10, Mpt83 and Ag85B that were successfully constructed were enriched in LB liquid medium and then induced to express recombinant proteins using isopropyl β-d-thiogalactoside. The target proteins with physiological conformations were then purified by Ni affinity chromatography and DEAE ion exchange chromatography.

[0024] In some embodiments of the present invention, the acquisition of each antigenic component of ECMA019m includes the following steps: using the genome of Mycobacterium tuberculosis strain H37Rv as a template, the gene sequences of ESAT-6, CFP-10, Mpt83, and Ag85B with EcoRI and HindIII double restriction sites at both ends are amplified by PCR. Using molecular cloning technology, the ESAT-6, CFP-10, Mpt83, and Ag85B gene fragments are double-digested and ligated into the pET-32a plasmid to construct recombinant plasmids of ESAT-6, CFP-10, Mpt83, and Ag85B. These plasmids are then transformed into Escherichia coli DH5α competent cells. After plasmid extraction and sequencing verification, they are transformed into Escherichia coli BL21(DE3) competent cells. The four successfully constructed engineered bacteria are enriched in LB liquid medium and then induced to express recombinant proteins using isopropyl β-d-thiogalactoside (IPTG). The target proteins with physiological conformations are purified by Ni affinity chromatography and DEAE ion exchange chromatography.

[0025] In some embodiments of the present invention, obtaining the ECMA019f fusion protein antigen includes the following steps: synthesizing an ECMA019f fusion protein gene sequence with NdeI and XhoI restriction sites at both ends using gene synthesis technology; double digesting the sequence and ligating it into the pET-43.1a plasmid; transforming the sequence into *E. coli* DH5α competent cells; extracting the plasmid and verifying its sequence by sequencing; and then transforming the plasmid into *E. coli* BL21(DE3) competent cells. After successful engineering, the engineered bacteria are enriched in LB liquid medium and recombinant protein expression is induced using isopropyl β-d-thiogalactoside (IPTG). The target protein with a physiological conformation is purified using Ni affinity chromatography and DEAE ion exchange chromatography.

[0026] This invention utilizes an Escherichia coli expression system to express and purify exogenous proteins, which has advantages such as high expression efficiency, mature fermentation process, and low production cost, making it suitable for large-scale commercial production.

[0027] The present invention provides a DNA molecule encoding the antigen composition, including one or more of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.

[0028] The present invention provides a recombinant bacterium, a recombinant expression vector or a transgenic cell line, comprising the DNA molecule.

[0029] This invention provides a tuberculosis vaccine or a tuberculosis infection detection and diagnostic kit, utilizing antigens ESAT-6, CFP-10, Mpt83, and Ag85B.

[0030] The tuberculosis vaccine or tuberculosis infection detection and diagnostic kit provided according to the present invention also includes an adjuvant.

[0031] In the tuberculosis vaccine or tuberculosis infection detection and diagnostic kit according to the present invention, the adjuvant is aluminum hydroxide.

[0032] This invention provides a method for preparing a tuberculosis vaccine or a tuberculosis infection detection and diagnostic kit, comprising expressing recombinant proteins of each component of ECMA019m or ECMA019f fusion protein through an Escherichia coli expression system, and obtaining the target protein by purification and refolding.

[0033] The present invention provides the use of the antigen composition in the preparation of vaccines for the prevention of Mycobacterium tuberculosis infection or medicaments for the treatment of diseases caused by Mycobacterium tuberculosis infection.

[0034] The present invention provides the use of the antigen composition in the preparation of reagents for diagnosing Mycobacterium tuberculosis infection or diseases caused by Mycobacterium tuberculosis infection.

[0035] The recombinant proteins of each component of ECMA019m and the fusion protein of ECMA019f obtained by purification in this invention were mixed with aluminum hydroxide adjuvant and then used to immunize BALB / c mice. The specific antibody titers of the immunized mice were detected using four experimental techniques: enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISpot), Luminex technology, and mycobacterial growth inhibition assay (MGIA). The immunogenicity and protective efficacy of the vaccine were evaluated by assessing the inhibitory effects of eight cytokines (IFN-γ, IL-4, IL-2, IL-6, IL-10, IL-17, TNF-α, and GM-CSF) and splenic lymphocytes on mycobacterial growth. The results showed that:

[0036] The two subunit vaccines ECMA019m and ECMA019f provided by this invention contain four Mycobacterium tuberculosis immunodominant antigens. Addressing the shortcomings of insufficient immunogenicity due to the limited number of single protein antigenic epitopes, and the difficulty in inducing effective protective immune responses in a broad population including BCG-vaccinated individuals, active tuberculosis patients, and patients with latent tuberculosis infection, ECMA019m and ECMA019f select four Mycobacterium tuberculosis immunodominant antigens—ESAT-6, CFP-10, Mpt83, and Ag85B—to induce a strong and widespread protective immune response.

[0037] Furthermore, based on ECMA019m, the ECMA019f fusion protein was constructed using gene synthesis technology. The four components ESAT-6, CFP-10, Mpt83, and Ag85B were sequentially linked using a flexible linker arm. Only one fusion protein needs to be expressed and purified to obtain the above four antigen components, which simplifies the steps of recombinant protein expression and purification and facilitates the standardization of production procedures.

[0038] Immunological evaluation was performed on mice immunized with two subunit vaccines, ECMA019m and ECMA019f. The results showed that both ECMA019f and ECMA019m could induce a strong protective immune response and inhibit the growth of mycobacteria, making them potential candidates for novel tuberculosis vaccines with significant application value. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0040] Figure 1 This is the SDS-PAGE identification result of the recombinant protein in Example 2 of this invention;

[0041] Note: 1, 2, 3, 4, and 5 correspond to the purified ECMA019f, ESAT-6, CFP-10, Mpt83, and Ag85B, respectively, with corresponding molecular weights of 77.8 kD, 27 kD, 28.8 kD, 40.1 kD, and 52.6 kD.

[0042] Figure 2 This is a graph showing the serum antibody titer detection results of Example 3 of the present invention;

[0043] Note: A is the serum IgG / IgG1 / IgG2a antibody titer, and B is the IgG1 / IgG2a ratio.

[0044] Figure 3 This is the result of ELISpot cytokine detection in Example 3 of this invention;

[0045] Note: Antigen-specific IFN-γ (A) and IL-4 (B) secretion levels were measured in SFCs, spot-forming cells, and spleen lymphocytes from the adjuvant and PBS groups were used as negative controls.

[0046] Figure 4 This is the result of Luminex multiple cytokine detection in Example 3 of this invention;

[0047] Note: A, B, C, D, E, and F represent the measured values ​​of IL-2, IL-6, IL-17, TNF-α, IL-10, and GM-CSF in the supernatant after co-culturing splenic lymphocytes with the corresponding stimulants, respectively.

[0048] Figure 5 This is the result of the mycobacterial growth inhibition test in Example 3 of this invention;

[0049] Note: CFU, Colony Forming Unit. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0051] Example 1 Construction of recombinant plasmids of ECMA019m components and ECMA019f fusion protein (1) Primer design

[0052] Using the gene sequences encoding antigens ESAT-6, CFP-10, Mpt83, and Ag85B from the genome of Mycobacterium tuberculosis strain H37Rv in the NCBI database as templates, primers were designed using Primer Premier 5.0 software. Primer information is shown in Table 1.

[0053] Table 1 Primer Information

[0054]

[0055] Note: __ indicates the EcoRI and HindIII restriction sites.

[0056] (2) Obtaining the target gene

[0057] The gene sequences of ESAT-6, CFP-10, Mpt83 and Ag85B were amplified using PCR technology. The PCR amplification system and reaction procedure are shown in Tables 2 and 3.

[0058] Table 2 PCR amplification system

[0059]

[0060]

[0061] Table 3 PCR reaction procedure

[0062]

[0063] (3) Construction of recombinant plasmids

[0064] The target gene and the pET32a / pET43.1a vector were double-digested using EcoRI / NdeI and HindIII / XhoI restriction endonucleases (37℃, 25 min). The digestion system is shown in Table 3. The digestion products were recovered. The recovered target gene and the vector were ligated using T4 DNA ligase at 25℃ for 30 min. The ligation system is shown in Table 4.

[0065] Table 3. PCR products and plasmid digestion system

[0066]

[0067]

[0068] Table 4 Connection System

[0069]

[0070] The ligation product was transformed into *E. coli* DH5α competent cells. The procedure was as follows: 10 μL of the ligation product was mixed with *E. coli* DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, incubated on ice for 2 min, and then 800 μL of antibiotic-free LB liquid medium was added. The cells were then incubated on a shaker at 37℃ and 140 rpm for 1 h. After incubation, the cells were centrifuged at 4000 rpm for 1 min, 400 μL of supernatant was discarded, and the bacterial pellet was remixed by pipetting. 200 μL of the bacterial suspension was spread onto LB agar plates containing ampicillin and incubated upside down at 37℃ for 12–16 h. Single colonies were picked and enriched in LB liquid medium containing ampicillin. The bacterial suspension was then used for PCR identification. The PCR system and procedure are shown in Tables 2 and 3. After identification, the bacterial samples were enriched in LB liquid medium containing ampicillin, and plasmids were extracted by alkaline lysis and stored at -20℃.

[0071] Example 2: Prokaryotic expression and purification of ECMA019m components and ECMA019f fusion protein.

[0072] The recombinant plasmid successfully constructed in Example 1 was transformed into *E. coli* BL21(DE3) competent cells using a heat shock method. Single colonies were picked and inoculated into LB broth containing ampicillin, and cultured at 37°C and 180 rpm for enrichment. Isopropyl β-d-thiogalactopyranoside (IPTG) was added to a final concentration, and the cells were cultured under the same conditions for 3 hours to induce the expression of the target protein. After induction, the cells were collected by centrifugation at 4°C and 4000 rpm for 10 min. The cells were resuspended in PBS and then sonicated (ultrasonic parameters: 220 W, 15 s operation, 20 s interval, 15 min total). After sonication, the supernatant and precipitate were separated by centrifugation at 12000 rpm for 10 min. The expression level and form of the recombinant protein were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The target protein was purified using an Aktaavant 25 automated protein purification system via Ni affinity chromatography and DEAE ion exchange chromatography. The specific steps are as follows:

[0073] The four individual antigenic components of ECMA019f and ECMA019m were first subjected to Ni affinity chromatography:

[0074] (1) Pack Ni-IDA affinity chromatography packing material and pass in 3 column volumes of equilibration buffer (use PBS equilibration packing material with pH 8.0 for soluble recombinant proteins; use 8M urea equilibration packing material with pH 8.0 for inclusion body recombinant proteins), and zero the column after the 280nm UV absorbance value stabilizes.

[0075] (2) Pump in the sample and collect the flow-through solution, then pump in the equilibration buffer corresponding to the recombinant protein to rinse the packing material until the UV absorbance value at 280 nm is stable.

[0076] (3) Perform linear elution with 0-300mM imidazole, collect all elution peaks, identify the purity of the target protein in each sample by SDS-PAGE, and select the sample with the best purity for subsequent operations.

[0077] The Ni affinity chromatography purified products of the three recombinant proteins ESAT-6, Mpt83 and Ag85B had low purity. After dialysis with a dialysis bag with a molecular weight cutoff of 10 kDa to remove salt, they were subjected to DEAE ion exchange chromatography.

[0078] (4) Pack DEAE ion exchange packing material and pass in 3 column volumes of equilibration buffer (use 10 mM pH 8.0 Tris buffer to equilibrate the packing material for soluble recombinant proteins; use 8 M urea buffer at pH 8.0 to equilibrate the packing material for inclusion body recombinant proteins), and zero the packing material after the 280 nm UV absorbance value stabilizes.

[0079] (5) Pump in the sample and collect the flow-through solution, then pump in the equilibration buffer corresponding to the recombinant protein to rinse the packing material until the UV absorbance value at 280 nm is stable.

[0080] (6) Perform linear elution with 0-400mM sodium chloride, collect all elution peaks, identify the purity of the target protein in each sample by SDS-PAGE, and select the sample with the best purity for subsequent operations.

[0081] (7) After purification, the target protein was placed in a dialysis bag and subjected to gradient dialysis to remove urea, imidazole, and other small molecule impurities. Finally, it was dialyzed in PBS at pH 7.4 at 4°C for 2 hours. After dialysis, the protein was concentrated by ultrafiltration and sterilized by filtration through a 0.22 μm filter. After aliquoting, it was stored at -70°C. The SDS-PAGE identification results of the ECMA019f recombinant protein and each fraction of ECMA019m are shown in the figure. Figure 1 .

[0082] Example 3: Immunological evaluation of ECMA019m and ECMA019f multi-component subunit vaccines

[0083] 1. Preparation of ECMA019m and ECMA019f multi-component subunit vaccines

[0084] The antigen components of ECMA019m were mixed in equimolar ratios and then mixed with aluminum hydroxide adjuvant at a volume ratio of 3:1; the antigen components of ECMA019f were directly mixed with aluminum hydroxide adjuvant at a volume ratio of 3:1 to prepare the vaccine.

[0085] 2. Mouse immunization procedure

[0086] Six- to eight-week-old SPF-grade female BALB / c mice were used for the immunization experiment. Mice were randomly divided into five groups: PBS group, adjuvant group, ECMA019f-adjuvant group, ECMA019m-adjuvant group, and BCG group, with six mice in each group. The immunization procedure is shown in Table 5.

[0087] Table 5. Mouse immunization procedure

[0088]

[0089]

[0090] Mice in the PBS group, adjuvant group, ECMA019f-adjuvant group, and ECMA019m-adjuvant group were sacrificed 10 days after the third immunization. Mice in the BCG group were sacrificed 30 days after immunization. Various immunological tests were performed to evaluate the immunoprotective effects of the ECMA019m and ECMA019f multi-component subunit vaccines.

[0091] 3. Evaluation of humoral immunity

[0092] Serum specific antibody titers were detected using enzyme-linked immunosorbent assay (ELISA).

[0093] (1) Serum separation

[0094] Before euthanasia, blood was collected from the eyes of immunized mice. The blood was left to stand at room temperature for 2 hours and then centrifuged at 4000 rpm for 10 minutes to collect serum.

[0095] (2) ELISA method for detecting serum antibody titer

[0096] 1) Adjust the concentration of the target protein using coating buffer (50mM carbonate buffer, pH 9.0). Adjust each of the four components of ECMA019m to 400ng / mL and the recombinant protein of ECMA019f to 2μg / mL. Add 100μL to each well of a 96-well plate and coat at 4℃ for 12h.

[0097] 2) On the second day, wash 5 times with PBST (PBS containing 0.5‰ Tween-20), add 100μL of PBST containing 5% skim milk powder to each well, block at 37℃ for 2 hours, wash 5 times with PBST and completely remove the residual liquid in the well.

[0098] 3) Serially dilute each group of serum with PBS, add 100 μL of diluted serum to each well, incubate at 37°C for 2 hours, wash 5 times with PBST and completely remove any residual liquid in the wells.

[0099] 4) Dilute HRP-labeled goat anti-mouse IgG, IgG1 and IgG2a antibodies with PBS at a ratio of 1:5000. Add 100 μL of diluted antibody to each well, incubate at 37°C for 1 hour, wash 5 times with PBST and completely remove any residual liquid in the wells.

[0100] 5) Add 100 μL of TMB substrate solution, develop at 37°C for 15 minutes, then add 100 μL of 2M sulfuric acid to terminate the reaction.

[0101] 6) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value at 450 nm.

[0102] 7) Antibody titer judgment criteria: If the second... n The OD value of the diluted serum / PBS group was ≥2.1 and the second... n+1 If the OD value of the diluted serum / PBS group is <2.1, then 2 n That is, the antibody titer corresponding to the serum sample.

[0103] Serum antibody titer test results are shown in Figure 2The experimental results showed that the serum specific IgG, IgG1, and IgG2a antibody titers of mice immunized with ECMA019m and ECMA019f were significantly increased. Compared with the BCG group, there was no significant difference in serum IgG levels between the ECMA019m and ECMA019f groups, but the levels of IgG1 and IgG2a were higher in the ECMA019m and ECMA019f groups. The IgG1 and IgG2a subtypes indicate the levels of humoral and cellular immune responses, respectively. The IgG1 / IgG2a ratio is usually used to measure the strength of the humoral and cellular immune responses induced by the vaccine. The experimental results showed that the serum IgG1 / IgG2a ratios of the ECMA019m, ECMA019f, and BCG groups were all around 1.5, suggesting that both ECMA019m and ECMA019f subunit vaccines induce a protective immune response in which humoral and cellular immunity are balanced.

[0104] 4. Cellular immune evaluation:

[0105] 4.1 Isolation of mouse spleen lymphocytes

[0106] 1) Mice were euthanized by cervical dislocation after blood collection, and their spleens were separated after soaking in 75% alcohol.

[0107] 2) The spleen was immersed in mouse lymphocyte separation medium (purchased from Beijing Dakewei Biotechnology Co., Ltd.) to separate lymphocytes, and the spleen lymphocytes were adjusted to a final concentration of 1×10⁻⁶. 6 / mL of solution is ready for use.

[0108] 4.2 Detection of Th1 cytokine IFN-γ and Th2 cytokine IL-4 by ELISA

[0109] 1) Add 200 μL of RPMI 1640 medium to the ELISpot detection wells pre-coated with IFN-γ and IL-4 antibodies, and let stand at room temperature for 10 min to activate the pre-coated plate.

[0110] 2) Add 100 μL of spleen lymphocyte solution to each well, and add 2 μg of the corresponding stimulant (ECMA019f antigen, ECMA019m antigen, or BCG whole cell lysis product). Set up one negative control well (PBS stimulation) and one positive control well (5 μg / mL concanavalin A). Incubate at 37°C and 5% CO2 for 16–24 hours.

[0111] 3) After the culture is complete, discard the liquid in the well, wash with PBST 5 times to completely remove the residual liquid in the well.

[0112] 4) Add biotin-labeled anti-IFN-γ and anti-IL-4 antibody working solution at a rate of 100 μL / well, incubate at 37°C for 1 h, discard the liquid in the well, wash 5 times with PBST to completely remove any remaining liquid in the well.

[0113] 5) Add streptavidin-labeled HRP working solution at a rate of 100 μL / well, incubate at 37°C for 1 h, discard the liquid in the well, wash 5 times with PBST to completely remove any residual liquid in the well.

[0114] 6) Add ACE colorimetric solution at a rate of 100 μL / well and incubate at 37°C in the dark. Once clear and obvious spots appear in each well, rinse each well with running water to stop the colorimetric reaction and invert the well to dry.

[0115] 7) Use an ELISpot plate reader to detect the SFCs (spot-forming cells) value of each well.

[0116] Test results are shown Figure 3 IFN-γ is a typical Th1 cytokine that plays an important role in cellular immunity, while IL-4 is a typical Th2 cytokine that plays an important role in humoral immunity. Experimental results showed that after immunization with ECMA019f and ECMA019m, the levels of IFN-γ and IL-4 secreted by splenic lymphocytes in mice were significantly increased upon stimulation with their respective stimuli. The ECMA019f group showed comparable IFN-γ and IL-4 secretion capacities in splenic lymphocytes, while the ECMA019m group showed the lowest IFN-γ secretion capacity but the highest IL-4 secretion capacity compared to the ECMA019f and BCG groups. This suggests that ECMA019f induces a strong protective immune response that balances humoral and cellular immunity, while ECMA019m induces a predominantly humoral immune response.

[0117] 4.3. Luminex assay for the detection of six cytokines: IL-2, IL-6, IL-10, IL-12, IL-17, and GM-CSF.

[0118] The Luminex Multiple Cytokine Assay Kit was purchased from R&D Systems. The specific procedure is as follows:

[0119] 1) Add 100 μL of spleen lymphocytes to each well of a 96-well cell culture plate, followed by 10 μg of the corresponding stimulant (ECMA019f antigen protein, ECMA019m antigen protein, or BCG whole-cell lysate). Set up one negative control well stimulated with sterile PBS and one positive control well stimulated with 5 μg / mL concanavalin A protein in each group. Cover the plates and incubate at 37°C in a 5% CO2 incubator for 16–24 hours.

[0120] 2) After the culture is completed, centrifuge the 96-well cell plate at 4000 rpm for 10 min, and take the supernatant for Luminex multiplex cytokine assay. The assay procedure is performed according to the instructions.

[0121] Test results are shown Figure 4 The experimental results showed that after immunization with ECMA019f and ECMA019m, mouse splenic lymphocytes secreted significantly increased levels of six cytokines: IL-2, IL-6, IL-10, IL-12, IL-17, and GM-CSF. IL-2 and IL-12 are Th1 cytokines that induce cellular immune responses; IL-6 and IL-10 are Th2 cytokines that induce humoral immune responses; and IL-17 and GM-CSF are innate immune-related cytokines that play a role in the body's innate immunity. The results indicate that compared to ECMA019f, ECMA019m can induce higher levels of Th1 and Th2 cytokines and has a stronger ability to induce protective immune responses. However, in terms of innate immunity, ECMA019f can induce more GM-CSF (granulocyte-macrophage colony-stimulating factor), which is beneficial for macrophage activation and phagocytosis of Mycobacterium tuberculosis.

[0122] 5. Evaluation of the protective efficacy of ECMA019f / ECMA019m subunit vaccines

[0123] The inhibitory effect of mouse spleen lymphocytes on the growth of Mycobacterium tuberculosis was evaluated using the in vitro mycobacterial growth inhibition assay (MGIA).

[0124] 1) Add 1 ml of spleen lymphocytes of the correct concentration to each well of a 24-well plate, inoculate with 50 CFU (colony forming unit) of Mycobacterium tuberculosis strain H37Rv, mix well and incubate at 37°C and 5% CO2 for 4 days.

[0125] 2) After the culture is completed, transfer the co-culture to a centrifuge tube, centrifuge at 12,000 rpm for 10 minutes and discard 900 μL of supernatant.

[0126] 3) While centrifuging, add 500 μL of sterile water to each well of the 24-well plate, repeatedly pipette the bottom and sidewalls, let stand for 5 minutes, then transfer the liquid in the well to the corresponding centrifuge tube, vortex to completely lyse the cells and release the intracellular Mycobacterium tuberculosis.

[0127] 4) Spread 50 μL of the solution onto a 7H10 plate, and simultaneously spread 50 μL of H37Rv bacterial solution directly onto the plate as a blank control. Incubate the plates upside down at 37°C for 2–3 weeks, and then count the colonies.

[0128] See results Figure 5 Compared with the BCG group, the splenic lymphocytes of mice in the ECMA019m and ECMA019f groups had a weaker ability to inhibit the growth of Mycobacterium tuberculosis, but there was still a significant difference compared with the negative control group. The two multi-component subunit vaccines, ECMA019m and ECMA019f, had comparable inhibitory effects on the growth of Mycobacterium tuberculosis in mouse splenic lymphocytes. The experimental results show that both ECMA019m and ECMA019f can induce a strong protective immune response against Mycobacterium tuberculosis infection.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The fusion protein ECMA019f, characterized in that, The amino acid sequence of the ECMA019f fusion protein is shown in SEQ ID NO.

10.

2. The gene encoding the fusion protein ECMA019f, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

9.

3. The method for constructing the fusion protein ECMA019f according to claim 1, characterized in that, The gene sequence of ECMA019f fusion protein with NdeI and XhoI restriction sites at both ends was synthesized using gene synthesis technology. After double digestion, it was ligated into pET-43.1a plasmid and transformed into Escherichia coli DH5α competent cells. The plasmid was extracted and sequenced for verification before being transformed into Escherichia coli BL21(DE3) competent cells. After the successfully constructed engineered bacteria were enriched in LB liquid medium, recombinant protein expression was induced by isopropyl β-d-thiogalactoside (IPTG). The target protein with physiological conformation was obtained by purification using Ni affinity chromatography and DEAE ion exchange chromatography.

4. A recombinant bacterium, a recombinant expression vector, or a transgenic cell line, characterized in that, Includes the encoding gene as described in claim 2.

5. A tuberculosis vaccine or a tuberculosis infection detection and diagnostic kit, characterized in that, Using the encoding gene as described in claim 2.

6. The tuberculosis vaccine or tuberculosis infection detection and diagnostic kit according to claim 5, characterized in that, It also includes adjuvants.

7. The tuberculosis vaccine or tuberculosis infection detection and diagnostic kit according to claim 6, characterized in that, The adjuvant is aluminum hydroxide.

8. The use of the fusion protein ECMA019f of claim 1 in the preparation of a vaccine for the prevention of Mycobacterium tuberculosis infection or a medicament for the treatment of diseases caused by Mycobacterium tuberculosis infection.

9. The use of the fusion protein ECMA019f of claim 1 in the preparation of reagents for diagnosing Mycobacterium tuberculosis infection or diseases caused by Mycobacterium tuberculosis infection.

Images