CN116606356A - Preparation and application of a Mycobacterium tuberculosis antigen composition EPRHP014 - Google Patents

Abstract

The invention relates to the technical field of biological medicine, in particular to preparation and application of a mycobacterium tuberculosis antigen composition EPRHP 014. The antigen composition provided by the invention consists of EsxH, nPPE18, rv2628, hspX and nPstS1, and can induce organisms to generate specific immune response, thereby having good application potential. The vaccine prepared by the antigen composition can overcome the defect of single antigen type used by subunit vaccines in the prior art.

Description

Preparation and application of a Mycobacterium tuberculosis antigen composition EPRHP014

technical field

The invention relates to the technical field of biomedicine, in particular to the preparation and application of a Mycobacterium tuberculosis antigen composition EPRH014.

Background technique

Tuberculosis is widespread in the world, and Mycobacterium tuberculosis has become the second largest cause of death from a single source of infection after the new coronavirus pneumonia, seriously threatening human health. Since the 1990s, due to factors such as the increase of tuberculosis-AIDS co-infection cases, the spread of drug-resistant tuberculosis strains, and the frequent movement of the population around the world, the disease burden of tuberculosis has risen sharply. Achieving WHO's End TB Strategy is critical.

The current research and development strategies for tuberculosis vaccines can be divided into subunit protein vaccines, viral vector vaccines, DNA vaccines, mRNA vaccines, whole-bacteria inactivated vaccines, and recombinant BCG vaccines. Among them, protein subunit vaccines can efficiently induce protective immune responses and have good efficacy. Safety is one of the more ideal vaccine forms.

So far, most of the proteins or polypeptides used as vaccines are screened from immunodominant antigens, such as ESAT-6 family proteins, Ag85 complex, MTB39 and MTB32, etc. Among them, the EsxH antigen of the ESAT-6 gene family can stimulate the body to produce specific cytotoxic T lymphocytes, induce a high level of CD4+ T cell response and IFN-γ secretion, and is a candidate dominant antigen for vaccines; PPE18 protein can induce the production of macrophages High levels of IL-10, and conducive to Th2-type immune response. Secreted proteins in the growth phase have become research hotspots because they are related to the strong pathogenicity of Mycobacterium tuberculosis and can induce specific immune responses in the body. For example, the DosR regulatory protein Rv2628 and HspX expressed in latent infection can induce specific pro-inflammatory cells. Factor secretion, while stimulating the production of protective cytokines such as IL-2 and IL-17, can effectively induce Th1 type immune response and humoral response, and has the potential to be developed into a tuberculosis subunit vaccine. Phosphate-specific transport substrate-binding protein-1 (pstS1) encoded by the Rv0934 gene is an antigen involved in the active transport of inorganic phosphate across membranes, and has been shown to induce activation of mouse CD8+ T cells and generate Th1 and Th17 immune protection responses.

Conventional vaccines, mostly employing a single antigen, are insufficient to induce adequate immune protective responses. Therefore, the mixed or fusion expression of multiple antigens has become an effective measure to improve the immune protection effect of vaccines. Studies have shown that fusion proteins are more effective at inducing immune protection than multiple antigen mixtures. Compared with other types of vaccines, protein subunit vaccines prepared by fusion or mixing of multiple antigens have great application prospects due to the advantages of multiple effective antigens, good specificity, high purity, and clear components.

Bacillus Calmette-Guerin (BCG), as the only vaccine approved so far for the prevention of tuberculosis, part of its protective antigens are lost during repeated passaging and storage, resulting in large differences in its immune effects in different populations, and it cannot fully simulate the distribution of tuberculosis in nature. The real process of mycobacterium infection in the human body and the prevention of the invasion of Mycobacterium tuberculosis. Therefore, the research and development of safe, efficient and new anti-tuberculosis vaccines is imminent. At present, tuberculosis vaccines in different stages of clinical trials can be divided into three categories, subunit vaccines, viral vector vaccines and DNA vaccines. Compared with Mycobacterium tuberculosis protein subunit vaccines, although DNA vaccines can produce stronger IFN-γ secretion and cytotoxic CD8+ T cell responses, they are generally less immunogenic to large mammals, while viral vector vaccines The disadvantage is that the time for inducing the body to produce immune memory is short, and it cannot establish long-lasting immune protection, and the vectors used are mostly vaccinia virus and adenovirus. Antibodies against the vector may already exist in the human body before vaccination, which limits its vaccination. immune effect. Studies have shown that recombinant protein subunit vaccines are safe, have clear ingredients, can provide long-term immune protection effects, and have good application and development prospects.

At present, the antigens used in subunit vaccines that have entered the clinical stage mainly include ESAT-6 family proteins, Ag85 complexes, PPE families, and latency proteins. Most of them use one or two antigens/peptides as immunogens. A single type of problem cannot induce a comprehensive immune protective response.

Contents of the invention

The invention provides the preparation and application of a Mycobacterium tuberculosis antigen composition EPRHP014, which is used to solve the defect of single antigen type used in subunit vaccines in the prior art.

The present invention provides a Mycobacterium tuberculosis antigen composition named EPRHP014, said antigen composition is composed of EsxH, nPPE18, Rv2628, HspX and nPstS1,

The amino acid sequence of the EsxH is shown in SEQ ID NO.6;

The amino acid sequence of nPPE18 is shown in SEQ ID NO.7;

The amino acid sequence of the Rv2628 is shown in SEQ ID NO.8;

The amino acid sequence of the HspX is shown in SEQ ID NO.9;

The amino acid sequence of nPstS1 is shown in SEQ ID NO.10.

The present invention selects the antigen composition EsxH, nPPE18, Rv2628, HspX and nPstS1 as potential candidate components for constructing the EPRHP014 subunit vaccine.

The antigen composition EPRH014 provided by the invention can induce a specific immune response in the body, and has good application potential.

The present invention provides DNA molecules encoding said Mycobacterium tuberculosis antigen composition.

Preferably, the DNA molecule includes one or more of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.

The invention provides a recombinant bacterium, a recombinant expression vector or a transgenic cell line, including the DNA molecule.

The invention provides a tuberculosis vaccine or tuberculosis infection detection and diagnostic kit, comprising the Mycobacterium tuberculosis antigen composition;

Preferably, the antigens EsxH, nPPE18, Rv2628, HspX and nPstS1 exist independently to form the mixed antigen EPRHPO14m.

Alternatively, the antigens EsxH, nPPE18, Rv2628, HspX and nPstS1 exist in the form of fusion proteins to constitute the antigen EPRHP014f.

During the research and screening of potential effective antigens for Mycobacterium tuberculosis, it was found that different application methods of EPRHP014m and EPRHP014f, the antigen composition formed by EsxH, nPPE18, Rv2628, HspX and nPstS1, can induce protective immune responses.

The antigen combination method of the present invention includes a composition formed by the antigens of each component in any ratio and/or in any order, and also includes a fusion protein expressed after the genes Rv0288, nRv1196, Rv2628, Rv2031c, and nRv0934 are linked, optimized, and modified in any order.

Preferably, the gene fragments expressing the antigens EsxH, nPPE18, Rv2628, HspX and nPstS1 are sequentially connected after splicing and optimization, and the fusion expresses the antigen EPRHP014f.

According to the tuberculosis vaccine or tuberculosis infection detection and diagnosis kit of the present invention, it also includes an adjuvant.

Preferably, the adjuvant is aluminum hydroxide adjuvant, and its components are aluminum hydroxide and magnesium hydroxide.

According to the preparation method of the tuberculosis vaccine or tuberculosis infection detection and diagnostic kit, the construction of the fusion protein EPRHP014f comprises the following steps:

The five gene fragments corresponding to EsxH, nPPE18, Rv2628, HspX, and nPstS1 were sequentially connected by linker and codon-optimized, and then connected to pET43.1a to construct a recombinant plasmid of fusion gene, which was expressed and purified in Escherichia coli.

The fusion protein EPDPA015f comprises Rv0288, nRv1196, Rv2628, Rv2031c and nRv0934 combined genes connected by a linker, the corresponding nucleotide sequence is shown in the sequence listing SEQ ID NO: 11, and the amino acid sequence of the encoded protein EPDPA015f is shown in the sequence listing SEQ ID NO: 12 shown.

The present invention provides the preparation method of described tuberculosis vaccine or tuberculosis infection detection and diagnostic kit, the construction of mixed protein EPRHP014m comprises the following steps:

For the genes Rv0288, Rv2628 and Rv2031c encoding the three antigens of EsxH, Rv2628 and HspX, using the Mycobacterium tuberculosis strain H37Rv genome as a template, the gene fragments of each component were connected to the pET32a vector to construct a recombinant plasmid;

For the two antigens nPPE18 and nPstS1, the genes nRv1196 and nRv0934 of the above two proteins were linked to the PMD19T plasmid and then linked to the pET32a vector to construct a recombinant plasmid.

Based on bioinformatics analysis, the present invention predicts, designs and optimizes Rv1196 and Rv0934 genes and their products, constructs gene fragments nRv1196 and nRv0934 that can efficiently express T cell epitope clusters, and expresses subunit protein nPPE18 in vitro through genetic engineering technology and nPstS1.

Preferably, the present invention uses bioinformatics analysis platforms such as NetMHCIIpan3.2 Server, SYFPEITHI, TEpredict, and IEDB to perform T cell epitope prediction on Mycobacterium tuberculosis Rv1196 and Rv0934 genes, and select the T cell epitope concentration region of Rv1196 and Rv0934 , the nucleotides corresponding to the epitope region were respectively spliced and optimized to form epitope-enriched region genes nRv1196 and nRv0934 in series, and the encoded proteins were nPPE18 and nPstS1. EsxH, nPPE18, Rv2628, HspX and nPstS1 were combined with adjuvants to construct multicomponent subunit vaccines EPRHP014f/adjuvant and EPRHP014m/adjuvant.

Preferably, for the two antigens nPPE18 and nPstS1 obtained by epitope screening, use gene synthesis technology to synthesize the genes nRv1196 and nRv0934 encoding the above two proteins and connect them to the PMD19T plasmid and then connect them to the pET32a vector after double enzyme digestion to construct recombinant plasmid. Finally, the above plasmids were transformed into Escherichia coli competent cells BL21 (DE3), expressed and purified, and then mixed in an equimolar ratio.

The invention provides the application of the Mycobacterium tuberculosis antigen composition in preparing tuberculosis vaccine.

The invention provides the application of the Mycobacterium tuberculosis antigen composition in the preparation of medicines for preventing or treating Mycobacterium tuberculosis infection or diseases caused by Mycobacterium tuberculosis infection.

The invention provides the application of the Mycobacterium tuberculosis antigen composition in the preparation of reagents for diagnosing Mycobacterium tuberculosis infection or diseases caused by Mycobacterium tuberculosis infection.

The present invention selects Mycobacterium tuberculosis active phase antigens EsxH, PPE18, latent phase antigens Rv2628, HspX and a transport protein PstS1 with good immunogenicity to prepare mixed protein EPRHP014m and fusion protein EPRHP014f, and then combine with adjuvant to construct two tuberculosis subtypes The unit vaccine was evaluated by mouse model for immunological effect. Specifically, the constructed protein was mixed with an adjuvant to immunize BALB/C mice, BCG was used as a positive control group, PBS and adjuvant were used as a blank control group and a negative control group, and enzyme-linked immunosorbent assay (ELISA), Multiple microsphere Luminex technology, enzyme-linked immunospot (Elispot), in vitro mycobacterial growth inhibition assay (MGIA) and other means were used to evaluate and analyze its immune protection effect.

After the mice were immunized with EPRHP014m/adjuvant and EPRHP014f/adjuvant, the level of specific antibody IgG was the same as that of the BCG group, indicating that the mixed protein EEPRHP014m and the fusion protein EPRHP014f used to prepare protein subunit vaccines could induce the body's production not lower than that of the BCG group. The level of humoral immunity, and IgG2a/IgG1 results also showed that the two groups induced Th2 type immune response. The levels of GM-CSF, IL-12, and IL-6 produced by EPRHP014m/adjuvant stimulation and induction were significantly higher than those in the BCG control group. IL-12 and IL-6, as key factors for inducing T lymphocyte differentiation, can stimulate CD4 ⁺ T cells and CD8 ⁺ T cells produce cytokines such as GM-CSF and IFN-γ, and activate macrophages to exert stronger antigen presentation ability, indicating that EPRHP014m/adjuvant can stimulate the body to produce a stronger immune response than BCG. EPRHP014f/adjuvant stimulated and induced IFN-γ, IL-4, IL-10 levels were significantly higher than BCG control group, indicating that EPRHP014f/adjuvant can not only induce CD4+ T cells to secrete IFN-γ, but also stimulate the body to produce stronger It can promote the proliferation and differentiation of B cells by enhancing the secretion of IL-4 and IL-10, and play a role in assisting humoral immunity. Therefore, the use of EPRHP014m and EPRHP014f as subunit antigens in tuberculosis vaccines can induce more abundant cytokines and stimulate the body to produce a more comprehensive immune protective response.

The results of Mycobacterium tuberculosis growth inhibition assay (MGIA) in vitro showed that both EPRHP014m/adjuvant and EPRHP014f/adjuvant exhibited the same ability to inhibit the growth of mycobacteria as the BCG group. That is, when Mycobacterium tuberculosis enters or lurks in the body, the immune protection response induced by it can inhibit the proliferation of Mycobacterium tuberculosis or promote its clearance, which plays an important role in the prevention of tuberculosis. At the same time, the combined application of various antigenic components can exert their respective antigenic effects, and is also suitable for the treatment of patients with latent Mycobacterium tuberculosis infection.

The multi-component protein subunit vaccine constructed by the present invention combines the protective immunogen components expressed by Mycobacterium tuberculosis in different infection stages, which has immunological pertinence, can reach or even exceed the immune protection effect of BCG, and can also be applied to latent tuberculosis The treatment of infection, which assists the body to eliminate latent infection bacteria and has the advantages of low cost and good safety, is expected to become a candidate vaccine for tuberculosis prevention and treatment.

Description of drawings

In order to more clearly illustrate the present invention or the technical solutions in the prior art, the accompanying drawings that need to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings in the following description are the present invention. For some embodiments of the invention, those skilled in the art can also obtain other drawings based on these drawings without creative effort.

Fig. 1 is the SDS-PAGE result graph of purified EsxH, nPPE18, Rv2628, HspX and nPstS1 and fusion protein EPRHPO14f in Example 3 of the present invention;

Note: 1 is EPRHP014f fusion protein, 2 is EsxH, 3 is nPPE18, 4 is Rv2628, 5 is HspX, 6 is nPstS1, and their theoretical molecular weights are 70.1kD, 31.07kD, 31.4kD, 33.71kD, 36.35kD, 40.75 kD.

Fig. 2 is the detection result of the serum antibody titer of each group in Example 4 of the present invention.

Fig. 3 is a graph of IgG2a/IgG1 ratio of mouse serum after immunization in EPRHP014m group and EPRHP014f group provided by Example 4 of the present invention.

In Fig. 4, a is the IFN-γ result graph of ELISPOT of Example 4 of the present invention, and b is the result graph of IL-4 of Example 4 of the present invention.

Fig. 5 is a graph showing the levels of four cytokines, GM-CSF, IL-12, IL-6 and IL-10 in Example 4 of the present invention.

Fig. 6 is a graph showing the number of colonies grown in the in vitro growth inhibition test of Mycobacterium tuberculosis in Example 5 of the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention , but not all examples. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

Example 1 Prediction of T cell epitopes and selection of rich T cell epitope sequences

Search the Mycobacterium tuberculosis Rv1196 (PPE18), Rv0934 (PstS1) gene sequences (Gene ID: 886073; Gene ID: 885724) in the National Center for Biotechnology Information (NCBI) database. Online bioinformatics analysis tools such as NetMHCIIpan3.2 Server, SYFPEITHI, TEpredict and IEDB were used to predict CD4+ and CD8+ T cell epitopes of PPE18 and PstS1, and HLA-II molecules (HLA-DRB1*0101, DRB1*0301, DRB1 *0401, DRB1*0701, DRB1*0802, DRB1*0901, DRB1*1101, DRB1*1302, DRB1*1501), MHC type selection HLA-class I molecule HLA-A*0201, *0202, *0203, *0206 As a limiting condition for prediction, epitope peptides with better binding to HLA-class I molecules and HLA-II class molecules were screened to obtain the concentrated region of dominant antigenic epitope peptides. The 201-300 amino acid sequence of PPE18, the 57-135 amino acid sequence and the 268-373 amino acid sequence of PstS1 were respectively spliced and optimized to construct epitope-enriched region gene fragments nPPE18 and nPstS1.

Example 2 Mycobacterium tuberculosis EsxH, nPPE18, Rv2628, HspX and nPstS1 and fusion protein EPRHP014f recombinant plasmid construction

Specific primers were designed for Mycobacterium tuberculosis antigens EsxH, Rv2628, and HspX (see Table 1 for primer information), and the target genes were amplified by PCR using the Mycobacterium tuberculosis H37Rv genome as a template. The coding sequences of the two antigens nPPE18 and nPstS1 obtained by epitope screening were synthesized by gene synthesis technology and connected to the PMD19T plasmid to obtain recombinant plasmids PMD19T-nRv1196 and PMD19T-nRv0934. The pET32a vector, PCR amplification products, and recombinant plasmids PMD19T-nRv1196p and PMD19T-nRv0934 were double digested with restriction endonucleases EcoRI/HindIII or BamHI/HindIII, and the digested products were ligated with the target gene fragment using T4 ligase. pET32a expression vector (reaction condition: 16°C 14h).

The fusion protein EPRHP014f was constructed based on the above five antigens including EsxH, nPPE18, Rv2628, HspX and nPstS1. [linker]-Rv2031c-[linker]-nRv0934 was sequentially synthesized in tandem and cloned into the pET43.1a vector. The restriction sites connected at both ends of the sequence were NdeI and XhoI. Linker was composed of GGTGGTTCTGGCGGT (SEQID NO.13) gene sequence, corresponding to The amino acid is GGSGG (SEQ ID NO.14).

The synthetic fusion protein EPRHP014f plasmid and the above ligation products pET32a-Rv0288, pET32a-nRv1196, pET32a-R2628, pET32a-Rv2031c, pET32a-nRv0934 were respectively transformed into E. coli DH5α competent cells. After mixing with competent cells, ice-bath for 30min, heat shock at 42°C for 90s, ice-bath for 2min, add 800μl LB liquid medium, shake culture at 37°C for 1h, centrifuge at 4000rpm for 1min, discard 600μl supernatant, resuspend the remaining medium, take 200μl The bacterial solution was spread on LB solid plates containing ampicillin, and cultured at 37°C for 16h. Finally, a single colony was picked and placed in 5 ml LB liquid medium containing ampicillin, cultured with shaking at 37°C for 12 hours, and then sequenced for verification.

Table 1

Example 3 Expression, purification and preparation of Mycobacterium tuberculosis EsxH, nPPE18, Rv2628, HspX and nPstS1 and fusion protein EPRHPO14f.

Transform the successfully constructed recombinant plasmid in Example 2 into Escherichia coli BL21 (DE3) competent cells. The transformation method is the same as Example 2. Pick a single colony and inoculate it into 2ml of ampicillin-containing LB liquid medium at 37°C and culture with shaking at 180rpm After 15 hours, 1ml was transferred to 300mL LB medium containing 100μg/mL ampicillin, cultured until the A ₆₀₀ value was 0.6-0.8, and 1mM final concentration of isopropyl β-d-thiogalactoside ( IPTG) was cultured for a certain period of time under appropriate temperature conditions to induce the expression of the target protein. (Protein induction expression conditions are shown in Table 2). After the induction, centrifuge at 4°C and 4000rpm for 10min to collect the bacterial pellet, add it to 20mmol/L Tris-HC1 buffer (pH8.0) at a volume ratio of 10:1, sonicate the bacteria, and analyze the protein expression by SDS-PAGE electrophoresis . For the four proteins nPPE18, Rv2628, nPstS1 and EPRHP014f expressed in the form of inclusion bodies, use 8M urea to denature and dissolve them. For the soluble forms, EsxH and HspX are dissolved in the lysate (20mM Tris-HC1) during ultrasonic lysis, and Ni affinity The target protein was purified by chromatography and DEAE ion exchange chromatography.

Purification steps: pump Loading Buffer (20mmol/LTris-HCl for soluble protein; 8M urea for inclusion body protein) into the installed nickel column, adjust to zero after the 280nm UV absorbance is stable. Pump the sample solution after ultrasonic treatment, and then add Wash Buffer (20mmol/L Tris-HCl for soluble protein; 8M urea for inclusion body protein) until the absorbance is stable, then pump in different concentrations of Elution Buffer (30mmol/L imidazole, 60mmol/L imidazole, 150mmol/L imidazole, 300mmol/L imidazole) elution. Collect samples at each stage for SDS-PAGE electrophoresis detection. For the two proteins EsxH and Rv2628 whose purity is not enough for nickel-affinity chromatography purification, use a dialysis bag with a molecular weight cut-off of 10kDa to desalt after dialysis and perform ion-exchange chromatography. EsxH is equilibrated with 20mmol/L Tris-HC1, and Rv2628 is equilibrated with 8M urea. After the column material, 100mM, 200mM, 400mM NaCl gradient elution was used, and samples at each stage were collected for SDS-PAGE electrophoresis detection. (See Table 3 for the expression forms and elution conditions of each protein.) After purification, put the target protein into a dialysis bag for gradient dialysis to remove urea, imidazole and other small molecule impurities, and finally place it in PBS for 14-16 hours at 4°C. Afterwards, concentrate by ultrafiltration and sterilize by filtration with a 0.22 μm filter, measure the protein concentration with a BCA protein assay kit, and store in -70°C. The results of SDS-PAGE of purified EsxH, nPPE18, Rv2628, HspX and nPstS1 and fusion protein EPRHP014f are shown in Fig. 1 .

Table 2 protein induction expression conditions

Table 3 protein expression form and purification conditions

Example 4 mixture EPRHP014m and fusion protein EPRHP014f immunogenicity evaluation

1. Animal grouping and immunization: 30 BALB/c mice were randomly divided into 5 groups, 6 mice in each group, respectively PBS group, aluminum hydroxide adjuvant group, BCG group, EPRHP014f group, EPRHP014m group. The BCG group was immunized with 1×10 ⁶ CFU live bacteria, and immunized once in total; the PBS group and the adjuvant group were immunized with PBS and adjuvant at an amount of 200 μl per mouse, the EPRHP014m antigen was mixed according to the equimolar ratio of each component protein, and the EPRHP014f group was immunized with the fusion protein EPRHP014f , with a dose of 50 μg/rat. After mixing the fusion protein antigen EPRHP014f and the mixed antigen EPRHP014m with aluminum hydroxide adjuvant at a volume ratio of 3:1, each mouse was subcutaneously immunized three times with an interval of 10 days.

2. Animal immune serum preparation

Orbital blood was collected before immunization, and blood was collected from eyeballs after the last immunization. Serum was separated and stored at –20°C for ELISA humoral immunity detection.

3. Humoral immunity test

Total IgG antibody level, IgG antibody subclasses IgG1, IgG2a were detected by indirect ELISA method to detect the titer of the corresponding antibody in mouse serum. Coat the 96-well ELISA plate with a concentration of 2 μg/ml, and place it at 4°C for 14-16 hours; wash the plate 5 times with 200 μl of PBS-T, block with 5% skimmed milk powder (200 μl/well), and stand at 37°C for 2 hours; Add 100 μl of 2-fold diluted serum to be tested in each well, let it stand at 37°C for 1 hour; dilute HRP-labeled goat anti-mouse IgG, IgG1, IgG2a according to 1:5000 times, wash the plate 5 times, add 100 μl/well, 37°C Let stand for 1 hour; wash the plate, add 100 μl/well of TMB substrate chromogenic solution, and then add 100 μl/well of stop solution (3mol/LH ₂ SO ₄ ) for 30 minutes at room temperature in the dark; stop the reaction; detect the absorbance value A at a wavelength of 450nm ₄₅₀ .

The detection results of serum antibody titers in each group are shown in Figure 2. The results showed that the IgG, IgG2a and IgG1 antibody titers in serum of mice in EPRHP014m group and EPRHP014f group after immunization were equivalent to those in BCG immunized mice, indicating that both EPRHP014m group and EPRHP014f group could induce humoral immunity comparable to that of BCG group Response, the ratio of IgG2a/IgG1 showed that the immune effects of the EPRHP014m group and the EPRHP014f group were biased towards the Th2 type immune response after stimulation, which could induce the body to produce an effective humoral immune response (Figure 3).

4. Detection of cellular immunity

(1) Preparation of spleen lymphocytes

Put the aseptically removed spleen of each mouse on the sieve, add the lymphocyte separation liquid dropwise and grind it, suck the grinding liquid into a 15ml centrifuge tube with a Pasteur tube, add 200-500 μl serum-free medium dropwise for low-speed gradient centrifugation, Centrifuge at 800×g for 30 min at 25°C, aspirate the lymphocyte layer, add 10 ml of serum-free medium, wash upside down, and collect cells by centrifugation at room temperature at 250×g for detection of cellular immunity.

(2) Detection of the number of spleen lymphocytes secreting IFN-γ and IL-4

After isolating mouse spleen lymphocytes, take a 96-well cell culture plate, add 100 μl of splenocytes at a concentration of 1× ¹⁰⁶ /ml to each well, and add 10 μl of EPRHP014f and EPRHP014m antigens to each well (final concentration is 2 μg/well) . Three replicate wells were made for each antigen stimulus, and a control well was set up separately. After incubation for 20 hours, reagents such as detection antibodies were added in turn, the plate was washed, color was developed, the number of spots was counted, and the results were analyzed.

The IFN-γ results of ELISPOT are shown in Figure 4 a, and the IL-4 results are shown in Figure 4 b. The results of IFN-γ showed that the release of IFN-γ in the EPRHP014f group was much higher than that in the EPRHP014m group and the BCG group, and the release level of IFN-γ in the EPRHP014m group was the same as that in the BCG group, that is, both EPRHP014f and EPRHP014m could induce Th1-type protective cellular immune responses . The results of IL-4 showed that both EPRHP014f group and EPRHP014m group could induce mouse splenocytes to produce higher IL-4 release level, and the IL-4 release amount of EPRHP014f group was significantly higher than that of BCG group and EPRHP014m group, that is, EPRHP014f group and EPRHP014m group EPRHP014m group can induce protective humoral immune response. Overall, compared with the BCG group, both EPRHP014f and EPRHP014m could induce a more balanced protective immune response after immunization.

(3) Lumminex method to detect the production level of spleen cytokines in immunized mice

Take 100 μL of splenocytes with the above-mentioned adjusted concentration in each group and place them in a 24-well culture plate, add 10 μg/well specific antigen, and incubate the splenocytes with the corresponding stimulators in a 37°C, 5% CO2 incubator for 72 hours. Cell supernatants were collected, and the levels of four cytokines, GM-CSF, IL-12, IL-6 and IL-10, were detected by Luminex multifactor detection technology.

The results are shown in Figure 5. The levels of the above four cytokines in the EPRHP014m group were significantly higher than those in the BCG group, suggesting that EPRHP014m can induce a more comprehensive immune protection effect than BCG after immunization. The secretion levels of GM-CSF, IL-12 and IL-6 in the EPRHP014f group were comparable to those in the BCG group, and the secretion level of IL-10 was significantly higher than that in the BCG group. The results showed that compared with the BCG group, the EPRHP014f group could produce a stronger immune protective effect after immunization. Example 5 subunit vaccine in vitro protective evaluation

Mycobacterial In Vitro Growth Inhibition Assay (MGIA)

1. Isolation of mouse spleen lymphocytes

The separation steps were the same as those in Example 4 above. After the separation of lymphocytes, the concentration of lymphocytes was measured and adjusted to 1×10 ⁶ cells/ml.

2. Take a 24-well cell culture plate, add 1ml of splenocytes at a concentration of 1× ¹⁰⁶ /ml to each well, take 3 mouse splenocytes in each group, make 2 duplicate holes, and inoculate 100 μl of Mycobacterium tuberculosis strain in each well H37Rv suspension (final concentration is 500CFU/ml), and 50CFU suspension was directly coated with 7H10 plate as a control. Incubate in a 5% _CO2 incubator at 37°C for 96 hours. After incubation, centrifuge the culture, discard the supernatant of the cell culture medium in each well, resuspend the cells in 500 μL of sterile tissue culture grade water and incubate at room temperature for 15 minutes to lyse the cells and vortex Spin and mix well, take 50 μl spread on the 7H10 plate, culture at 37°C for 2 weeks, and count the colonies on each group of plates. The experimental data is expressed as log ₁₀ CFU of each tube of sample, and compared with the growth ratio of the sample (CFU sample (96 hours)/CFU control on day 0).

The results of MGIA are shown in Figure 6. Compared with the PBS control group and the adjuvant group, the splenocytes of mice immunized with the EPRHP014f group and the EPRHP014m group can better inhibit the growth of Mycobacterium tuberculosis, and the inhibitory effect is equivalent to that of BCG, that is, EPRHP014f and EPRHP014m When used to prepare protein subunit vaccines, it can produce an immune protection effect not lower than that of BCG.

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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it can still be Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims (10)

1. a Mycobacterium tuberculosis antigen composition, is characterized in that, described antigen composition is made up of EsxH, nPPE18, Rv2628, HspX and nPstS1, The amino acid sequence of the EsxH is shown in SEQ ID NO.6; The amino acid sequence of nPPE18 is shown in SEQ ID NO.7; The amino acid sequence of the Rv2628 is shown in SEQ ID NO.8; The amino acid sequence of the HspX is shown in SEQ ID NO.9; The amino acid sequence of nPstS1 is shown in SEQ ID NO.10. 2. the DNA molecule of the Mycobacterium tuberculosis antigen composition described in coding claim 1; Preferably, the DNA molecule includes one or more of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5. 3. A recombinant bacterium, recombinant expression vector or transgenic cell line, characterized in that it comprises the DNA molecule of claim 2. 4. A tuberculosis vaccine or tuberculosis infection detection and diagnostic kit, is characterized in that, comprises the Mycobacterium tuberculosis antigen composition described in claim 1; Preferably, the antigens EsxH, nPPE18, Rv2628, HspX and nPstS1 exist in an independent form to form the mixed antigen EPRHPO14m; Or, the antigens EsxH, nPPE18, Rv2628, HspX and nPstS1 exist in the form of fusion proteins to form the antigen EPRHP014f; Preferably, the gene fragments expressing the antigens EsxH, nPPE18, Rv2628, HspX and nPstS1 are sequentially connected after splicing and optimization, and the fusion expresses the antigen EPRHP014f. 5. tuberculosis vaccine or tuberculosis infection detection and diagnostic kit according to claim 4, is characterized in that, also comprises adjuvant; Preferably, the adjuvant is aluminum hydroxide adjuvant, and its components are aluminum hydroxide and magnesium hydroxide. 6. the preparation method of tuberculosis vaccine or tuberculosis infection detection and diagnostic kit described in claim 4 or 5, is characterized in that, the construction of fusion protein EPRHP014f comprises the following steps: The five gene fragments corresponding to EsxH, nPPE18, Rv2628, HspX, and nPstS1 were sequentially connected by linker and codon-optimized, and then connected to pET43.1a to construct a recombinant plasmid of fusion gene, which was expressed and purified in Escherichia coli. 7. the preparation method of tuberculosis vaccine or tuberculosis infection detection and diagnostic kit described in claim 4 or 5, is characterized in that, the construction of mixed protein EPRHP014m comprises the following steps: For the genes Rv0288, Rv2628 and Rv2031c encoding the three antigens of EsxH, Rv2628 and HspX, using the Mycobacterium tuberculosis strain H37Rv genome as a template, the gene fragments of each component were connected to the pET32a vector to construct a recombinant plasmid; For the two antigens nPPE18 and nPstS1, the genes nRv1196 and nRv0934 of the above two proteins were linked to the PMD19T plasmid and then linked to the pET32a vector to construct a recombinant plasmid. 8. The application of the Mycobacterium tuberculosis antigen composition described in claim 1 in the preparation of tuberculosis vaccine. 9. The application of the Mycobacterium tuberculosis antigen composition according to claim 1 in the preparation of medicines for preventing or treating Mycobacterium tuberculosis infection or diseases caused by Mycobacterium tuberculosis infection. 10. The use of the Mycobacterium tuberculosis antigen composition of claim 1 in the preparation of reagents for diagnosing Mycobacterium tuberculosis infection or diseases caused by Mycobacterium tuberculosis infection.