CN117683141A - Mycobacterium tuberculosis multi-antigen fusion protein and encoding genes and applications - Google Patents

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a Mycobacterium tuberculosis multi-antigen fusion protein, encoding genes and applications. The Mycobacterium tuberculosis multi-antigen fusion protein of the present invention consists of Rv3875 protein antigen, Rv3874 protein antigen, Rv0934-555 protein antigen and Rv2029c protein antigen. The fusion protein provided by the invention can induce the body to produce a strong protective immune response and has high safety. It can be used as a new tuberculosis vaccine candidate and has important application value.

Description

Mycobacterium tuberculosis multi-antigen fusion protein and encoding genes and applications

Technical field

The present invention relates to the field of biomedicine technology, and in particular to a Mycobacterium tuberculosis multi-antigen fusion protein, encoding genes and applications.

Background technique

Tuberculosis (TB) is a chronic infectious disease mainly caused by infection with Mycobacterium tuberculosis complex (MTBC). It is one of the main causes of death in the world. In recent years, the number of patients infected with HIV and Mycobacterium tuberculosis has increased, making TB increasingly a serious public health problem that endangers human health.

Vaccination is the most effective means to prevent and control infectious diseases. Bacillus Calmette Guerin (BCG) is currently the only vaccine approved to prevent human tuberculosis. The protective effect of BCG on newborns can reach up to 80% (Fourth Report to the Medical Research Council by its TubercμLosisVaccines Clinical Trials Committee, 1972, BμLl World Health Organ, 46(3):371-85.), but as the vaccine recipient As age increases, the protective power of BCG gradually decreases, and it cannot effectively prevent the occurrence of adult tuberculosis. In addition, BCG, as a live attenuated vaccine, carries the risk of infection when administered to people with immune deficiencies, and the problem of loss of protective antigens also occurs after multiple passages. Therefore, there is an urgent need to develop new tuberculosis vaccines as replacement or booster vaccines for BCG.

New tuberculosis vaccines undergoing clinical trials are mainly divided into the following three categories: protein subunit vaccines and viral vector vaccines, recombinant BCG and attenuated/inactivated vaccines, and DNA vaccines. Among them, the fastest-growing genetically modified BCG vaccine rBCGΔureChly+ (VPM1002) is currently It has entered Phase III clinical trials to evaluate the safety and protective effects of the vaccine in different types of test groups.

Genetically recombinant BCG is currently a hot topic in the field of new tuberculosis vaccine research. This type of vaccine uses BCG strains as engineered bacteria and uses genetic engineering technology to introduce foreign genes into BCG so that it can express immunologically advantageous foreign protein antigens. Recombinant BCG relies on the replication of bacteria in host cells and the expression of foreign genes to achieve the purpose of continuously stimulating the body's anti-tuberculosis immune response and thereby protecting the body from tuberculosis infection for a long time (Marques et al., 2021, Expert RevVaccines, 20 (8):1001-1011). At present, recombinant BCG is mainly expected to be used to replace traditional BCG as a primary vaccination vaccine and as a therapeutic vaccine to protect patients with active tuberculosis and latent tuberculosis infection. The body's immune resistance against Mycobacterium tuberculosis infection is a complex process. There are obvious differences in the expression of protective antigens of Mycobacterium tuberculosis in patients with active tuberculosis and patients with latent tuberculosis infection. Therefore, the current main research and development strategy for genetically recombinant BCG is It is to construct a genetically recombinant BCG strain that can express the protective antigen of tuberculosis bacteria. Through the continuous replication of the bacteria in the host cells and the expression of foreign genes, it can continuously stimulate the body to produce BCG-vaccinated people, active tuberculosis patients and latent tuberculosis infections. Protective immune responses against tuberculosis in a broad population of patients. Among them, selecting an effective protective Mycobacterium tuberculosis-related antigen combination is the key to developing new recombinant BCG vaccines.

In view of this, the present invention is proposed.

Contents of the invention

In view of the technical problems existing in the background technology, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a Mycobacterium tuberculosis multi-antigen fusion protein, which is composed of Rv3875 protein antigen, Rv3874 protein antigen, Rv0934-555 protein antigen and Rv2029c protein antigen, so The amino acid sequence of the Rv0934-555 protein antigen is shown in SEQ ID NO.5.

Tuberculosis vaccine candidates currently undergoing clinical trials generally use full-length antigens as vaccine components. Only limited epitopes in the full-length antigen can exert immune effects, and the remaining redundant fragments cannot enhance the intensity of the protective immune response. Based on the Rv0934 antigen, the present invention predicts and optimizes T cell epitopes and eliminates peptide segments with low epitope density to obtain the Rv0934-555 protein antigen (SEQ ID NO. 5), which has stronger immunogenicity.

From the perspective of the immune interaction between tuberculosis bacteria and the body, an important reason for the insufficient protective ability of traditional BCG is the lack of dominant protein antigens with good immune protection, and the inability to fully stimulate the body to produce sufficient specific cellular immune responses. To solve the above problems, the present invention provides a multi-antigen fusion protein, which is composed of Rv3875 protein antigen, Rv3874 protein antigen, Rv0934-555 protein antigen and Rv2029c protein antigen. Rv3875 protein antigen, Rv3874 protein antigen, Rv0934-555 protein antigen and Rv2029c protein antigen are all dominant antigens of Mycobacterium tuberculosis. The fusion protein obtained from these four protein antigens can induce more powerful and extensive protective immunity. reaction.

The Mycobacterium tuberculosis multi-antigen fusion protein of the present invention consists of Rv3875 protein antigen, Rv3874 protein antigen, Rv0934-555 protein antigen and Rv2029c protein antigen. All four protein antigens can induce a protective immune response against Mycobacterium tuberculosis, and the Rv0934-555 protein antigen and Rv2029c protein antigen are tuberculosis latency antigens and can play a role in the latent infection process of tuberculosis; the Rv3874 protein antigen and Rv3875 protein are Important virulence factors of Mycobacterium tuberculosis are abundantly expressed in the early stages of bacterial invasion of the body; the fusion protein composed of four protein antigens can play an important immune role in new infections or reactivation of latent infections by Mycobacterium tuberculosis.

Preferably, the amino acid sequence of the Mycobacterium tuberculosis multi-antigen fusion protein of the present invention is shown in SEQ ID NO. 2.

In a second aspect, the present invention provides a gene encoding the Mycobacterium tuberculosis multi-antigen fusion protein, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

In a third aspect, the present invention provides biological materials for expressing the Mycobacterium tuberculosis multi-antigen fusion protein. The biological materials are selected from expression vectors and engineering bacteria. The biological materials include nucleotide sequences such as SEQ ID NO. The genes shown in .1.

Preferably, the biological material is recombinant BCG vaccine. The recombinant BCG vaccine uses BCG as a vector to express relevant antigens, thus retaining the original advantages of BCG, such as: BCG can be safely vaccinated in infants and young children; BCG is highly protective against invasive tuberculosis in infants; BCG can induce persistent antigens Stimulation; BCG intrinsic adjuvant activity; BCG is easy to produce and low cost, etc. Therefore, the development of new recombinant BCG vaccines has broad prospects.

In the present invention, the construction method of the recombinant BCG vaccine preferably includes:

S1. Connect the gene to the shuttle vector to obtain a recombinant plasmid carrying the fusion gene;

S2. Transform the recombinant plasmid described in step S1 into the BCG vaccine, and screen to obtain the recombinant BCG vaccine that stably expresses the mycobacterial multi-antigen fusion protein.

Preferably, the shuttle vector is pMV361; and/or the BCG vaccine is BCG-China strain.

In a more specific embodiment of the present invention, the construction method of the recombinant BCG vaccine is as follows:

Rv3875, Rv3874, Rv0934-555 and Rv2029c were connected sequentially from the 5' end to the 3' end, using GGTGGTTCTGGCGGT (the corresponding amino acid sequence is Gly-Gly-Ser-Gly-Gly) as a flexible connecting peptide to connect each antigen. Then, gene synthesis technology was used to synthesize the fusion antigen gene with EcoRI and HindIII restriction sites at both ends, and then it was double-digested and connected to the pMV361 integration plasmid to construct the pMV361-ECPP007 recombinant plasmid. The pMV361-ECPP007 recombinant plasmid was transformed into BCG competent cells by electroporation to construct a genetically recombinant BCG vaccine rBCG-ECPP007 capable of expressing the fusion antigen ECPP007. Among them, the conditions for electrical conversion are preferably: 20-30kV, 20-30uF, 900-1100Ω; more preferably, they are 25kV, 25uF, 1000Ω.

The present invention selects four immunodominant antigens of Mycobacterium tuberculosis, Rv3875, Rv3874, Rv0934-555 and Rv2029c, designs the Mycobacterium tuberculosis fusion antigen gene ECPP007, uses an integration plasmid to integrate the gene into the BCG genome, and constructs ECPP007 capable of expressing the fusion antigen Genetic recombination BCG.

The sequence of the ECPP007 fusion antigen gene from the 5' end to the 3' end is Rv3875, Rv3874, Rv0934-555 and Rv2029c. Each antigen component is connected with a flexible connecting arm. The final gene sequence is shown in SEQ ID NO: 1; The amino acid sequence of ECPP007 fusion antigen is shown in SEQ ID NO:2.

The fusion antigen provided by the present invention, which contains four immunodominant antigens of Mycobacterium tuberculosis, has insufficient immunogenicity due to the limited number of single protein epitopes and is difficult to induce the body to produce, including BCG vaccinated people, active tuberculosis patients and patients with latent tuberculosis infection. To overcome the shortcomings of producing effective protective immune responses in a wide range of populations, four immunodominant antigens of Mycobacterium tuberculosis, Rv3875, Rv3874, Rv0934-555 and Rv2029c, were selected, which can induce the body to produce a strong and extensive protective immune response. At the same time, the fusion antigen provided by the present invention containing four Mycobacterium tuberculosis immunodominant antigens is aimed at the presence of complex sequences with low T cell epitope content in the Rv0934 full-length protein antigen. The original Rv0934 gene sequence is used to predict T cell epitopes. According to the analysis results After deleting sequences with low T cell epitope content, a new epitope peptide Rv0934-555 was obtained as an antigen component of the ECPP007 fusion antigen.

Furthermore, the present invention integrates the ECPP007 fusion antigen gene into the BCG genome through the pMV361 integration plasmid, enabling it to stably express the ECPP007 fusion antigen, constructs a successful genetically recombinant BCG rBCG-ECPP007 subcutaneously immunized mouse, and conducts immunological evaluation and safety evaluate.

Specifically, the present invention will successfully construct the genetically recombinant BCG rBCG-ECPP007 to immunize BALB/c mice, and use four experimental technologies to detect rBCG: enzyme-linked immunosorbent assay (ELISA), Luminex technology, fluorescence quantitative PCR technology and flow cytometry technology. -Specific antibody titer of mice after ECPP007 immunization, spleen and lung cells express IL-2 (interleukin 2), IL-4 (interleukin 4), IL-6 (interleukin 6), IL-10 (interleukin 10), IL-12 (interleukin 12), IL-17 (interleukin 17), IFN-γ (interferon-γ), TNF-α (tumor necrosis factor α), GM-CSF (granulocyte-macrophage colony-stimulating factor) and iNOS (inducible nitric oxide synthase) and other important inflammatory factors, as well as the grouping of mouse spleen lymphocytes after stimulation with corresponding antigens and PPD, to evaluate the immunogenicity of the vaccine; conventional paraffin sections and HE staining were used to analyze rBCG- The degree of pathological damage to the liver, spleen and lungs of mice after ECPP007 immunization was used to evaluate the safety of the vaccine. The results show that rBCG-ECPP007 can induce the body to produce a strong protective immune response and has high safety. It can be used as a new tuberculosis vaccine candidate and has important application value. In addition, the present invention uses the BCG-China strain as the mother strain for preparing the genetically recombinant BCG vaccine rBCG-ECPP007, which is the same as the BCG strain stipulated in the domestic vaccination policy, which is beneficial to the late-stage clinical trials of the rBCG-ECPP007 vaccine candidate in the country and its nationwide promotion.

In a fourth aspect, the present invention provides the use of the Mycobacterium tuberculosis multi-antigen fusion protein, or the encoding gene, or the biological material in preparing a Mycobacterium tuberculosis vaccine.

In a fifth aspect, the present invention provides a Mycobacterium tuberculosis vaccine, the active ingredients of which include the Mycobacterium tuberculosis multi-antigen fusion protein; or are composed of the encoding gene, or the biological material Express the prepared multi-antigen fusion protein of Mycobacterium tuberculosis.

In the sixth aspect, the present invention provides the Mycobacterium tuberculosis multi-antigen fusion protein, or the encoding gene, or the biological material in the preparation of Mycobacterium tuberculosis detection reagents or drugs for treating Mycobacterium tuberculosis diseases. Applications.

Beneficial effects:

The invention provides a Mycobacterium tuberculosis multi-antigen fusion protein, which is composed of Rv3875 protein antigen, Rv3874 protein antigen, Rv0934-555 protein antigen and Rv2029c protein antigen. The fusion protein provided by the invention can induce the body to produce a strong protective immune response and has high safety. It can be used as a new tuberculosis vaccine candidate and has important application value.

Description of the drawings

In order to more clearly illustrate the present invention or the technical solutions in the prior art, the following will describe the accompanying drawings that are needed in the description of the embodiments or the prior art.

Figure 1 is a schematic structural diagram of the pMV361-ECPP007 recombinant plasmid in Example 1 of the present invention.

Figure 2 is the verification result of the integration of foreign genes in Example 2 of the present invention. Among them: M is the standard DNA molecular weight marker, and samples 1, 2, 3, and 4 are rBCG-ECPP007 single clone ①, rBCG-ECPP007 single clone ②, rBCG-pMV361 empty vector, and BCG-China respectively.

Figure 3 is the verification result of exogenous gene expression in Example 2 of the present invention. Among them: M is the standard DNA molecular weight marker, and samples 1 and 2 are rBCG-ECPP007 monoclonal ① and rBCG-ECPP007 monoclonal ② respectively.

Figure 4 is the detection result of serum IgG antibody titer in Example 3 of the present invention.

Figure 5 is the detection results of the levels of nine cytokines secreted by mouse spleen lymphocytes after stimulation with different antigens in Example 3 of the present invention. Among them: A, B, C, D, E, F, G, H, I respectively represent the IL-2, IL-4, IL-6, IL-10, IL- Measurements of 12p70, IL-17a, IFN-γ, TNF-α and GM-CSF, * and ** represent statistical significance of differences <0.05 and 0.01, respectively.

Figure 6 is the detection result of the levels of nine cytokines secreted by mouse spleen lymphocytes after PPD stimulation in Example 3 of the present invention. Among them: A, B, C, D, E, F, G, H, I respectively represent IL-2, IL-4, IL-6, IL-10, IL-12p70 in the supernatant of spleen lymphocytes after stimulation with PPD. , measured values of IL-17a, IFN-γ, TNF-α and GM-CSF, * and ** represent statistical significance of differences <0.05 and 0.01, respectively.

Figure 7 is the detection result of the levels of multiple inflammatory factors expressed in mouse lung cells after immunization in Example 3 of the present invention. Among them: the ordinate represents the expression levels of various inflammatory factors in the lung cells of mice in the rBCG-ECP001 group and BCG group compared with the PBS negative control group. *Represents statistical significance of difference <0.05.

Figure 8 shows the grouping of mouse spleen lymphocytes after stimulation with corresponding antigens detected by flow cytometry in Example 3 of the present invention. Among them: Pictures A, B and C respectively represent the secretion of IFN-γ and TNF-α by antigen-specific CD3 ⁺ CD4 ⁺ T cells and CD3 ⁺ CD8 ⁺ T cells in the spleen lymphocytes of mice in the PBS group, rBCG-ECPP007 group and BCG group. and IL-2 ratio of three cytokines.

Figure 9 is a flow cytometric analysis of the splenic lymphocyte clustering of mice after PPD stimulation in Example 3 of the present invention. Among them: A, B and C respectively represent the secretion of IFN-γ, TNF-α and T cells by antigen-specific CD3 ⁺ CD4 ⁺ T cells and CD3 ⁺ CD8 ⁺ T cells in the spleen lymphocytes of mice in the PBS group, rBCG-ECPP007 group and BCG group. The ratio of three IL-2 cytokines.

Figure 10 is the histopathological test results in Example 3 of the present invention. Among them: A, B and C represent the HE staining results (40×) of the liver, spleen and lung pathological sections of mice in the PBS group, rBCG-ECPP007 group and BCG group respectively.

Detailed ways

The invention provides a fusion antigen of Mycobacterium tuberculosis ECPP007, which is composed of Rv3875 protein antigen, Rv3874 protein antigen, Rv0934-555 protein antigen and Rv2029c protein antigen. Specifically, the ECPP007 fusion antigen is formed by sequentially connecting four antigen components: Rv3875, Rv3874, Rv0934-555 and Rv2029c through flexible connecting arms.

Among them, the amino acid sequence of Rv3875 is:

The amino acid sequence of Rv3874 is:

The amino acid sequence of Rv0934-555 is:

The amino acid sequence of Rv2029c is:

The nucleotide sequence encoding the recombinant protein ECPP007 is:

The amino acid sequence of its encoded protein is:

The present invention selects Rv2029c, Rv3874, Rv3875 full-length antigen and Rv0934 antigen epitope peptide enriched region Rv0934-555, a total of four tuberculosis protective antigens to construct a fusion antigen gene, and uses genetic engineering technology to prepare recombinant Bacillus Calmette-Guérin rBCG that can express the above-mentioned fusion antigen. -ECPP007 and evaluate its immunogenicity and safety. The results show that rBCG-ECPP007 can induce the body to produce a strong protective immune response and has high safety. It can be used as a new tuberculosis vaccine candidate and has important application value. In addition, the present invention uses the BCG-China strain as the mother strain for preparing the genetically recombinant BCG vaccine rBCG-ECPP007, which is the same as the BCG strain stipulated in the domestic vaccination policy, which is beneficial to the late-stage clinical trials of the rBCG-ECPP007 vaccine candidate in the country and its nationwide promotion.

The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be understood as limiting the protection scope of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used can be obtained from commercial sources.

Example 1 Construction of pMV361-ECPP007 recombinant plasmid

(1) Design of ECPP007 fusion antigen gene

Query the gene sequences corresponding to Rv3875, Rv3874 and Rv2029c on the NCBI (National Center for Biotechnology Information) website. Rv0934-555 is the Rv0934 antigen gene after T cell epitope prediction and optimization, and the elimination of peptides with low epitope density. Antigenic epitope peptides. Rv3875, Rv3874, Rv0934-555 and Rv2029c were connected sequentially from the 5' end to the 3' end, and GGTGGTTCTGGCGGT (amino acid sequence: Gly-Gly-Ser-Gly-Gly) was used as a flexible connecting peptide to connect each antigen to design a fusion antigen gene. ECPP007.

(2) Construction of pMV361-ECPP007 recombinant plasmid

Gene synthesis technology (Beijing Qingke) was used to synthesize the ECPP007 fusion antigen gene with EcoRⅠ at the 5' end and HindⅢ restriction site at the 3' end, and EcoRⅠ and HindⅢ restriction endonucleases (NEB, USA) were used to cleave the fusion antigen gene. Perform double enzyme digestion (37°C, 25min) with pMV361 vector. The enzyme digestion system is shown in Table 1. The digested products were identified by agarose gel electrophoresis and recovered using T4 DNA ligase (NEB, USA). The ligation conditions were 25°C and 30 min. The ligation system is shown in Table 2.

Table 1 Enzyme digestion system

Table 2 Connection system

Components Volume(μL) empty vector Add to 0.03pmol destination segment Add to 0.3pmol T4 DNA ligase 2 10×digestion buffer 2.5 ddH2O Make up to 25μL

The ligation product is transformed into E. coli DH5α competent cells. The steps are:

Take out 100 μL of frozen Escherichia coli DH5α competent cells (Beijing Quanshijin Biotechnology) and place it on ice to dissolve, add 10 μL of ligation product, mix well, and then stand in an ice bath for 30 minutes, heat shock at 42°C for 90 seconds, and let stand in an ice bath for 2 minutes. Add 800 μL of non-resistant LB liquid culture medium to the biological safety cabinet and place it on a shaker at 37°C and 140 rpm for 1 hour. After the culture is completed, centrifuge at 4000 rpm for 1 min, discard 400 μl of the supernatant, mix the bacterial sediment by gently pipetting, and apply 200 μl of the bacterial solution on an LB solid plate containing kanamycin, and incubate upside down at 37°C for 12 to 16 hours. Single colonies with good growth status were picked and cultured in LB liquid medium containing kanamycin, and the bacterial liquid was extracted for plasmid minipreparation and then identified by EcoRⅠ and HindⅢ double enzyme digestion. The digested products were identified by agarose gel electrophoresis as expected and sent to the company for sequencing. The sequencing results were compared with BLAST and the bacterial samples were completely correct. They were enriched and cultured in LB liquid medium containing kanamycin, and the plasmid was extracted by alkaline lysis. And stored at -20℃. The structural diagram of pMV361-ECPP007 recombinant plasmid is shown in Figure 1.

Example 2 Preparation of rBCG-ECPP007 gene recombinant BCG

(1) Preparation of BCG competent state

Use an inoculation loop to take a small amount of BCG-China culture from Roche's culture medium, inoculate it into 7H9 liquid culture medium, and culture it statically at 37°C until the logarithmic growth phase (about 20 days). After the culture is completed, ice-bath the bacterial solution for 1 hour. , centrifuge at 4000 rpm and 4°C for 15 min, discard the supernatant, and collect the bacteria. For every 50 mL of bacterial solution, add 50 mL of autoclaved 10% glycerol (pre-cooled on ice) to each 50 mL of bacterial solution. Gently mix with a pipette to fully suspend the bacterial cells. Centrifuge at 4000 rpm and 4°C for 15 min. Discard the supernatant. Wash, collect the cells, and repeat washing three times. After the last wash, add 1000 μL of 10% glycerol to resuspend the bacteria. Dispense the prepared BCG competent cells into EP tubes at 200 μL per tube and store them at -80°C for later use.

(2) Transformation of BCG competent cells by electroporation

Take out the prepared BCG competent cells from the refrigerator and let them stand on ice to thaw. Pipette 10 μL of the recombinant plasmid successfully constructed in Example 1 and add it to 200 μL of BCG competent cells. After standing in an ice bath for 10 minutes, transfer all to an electroporation cup for electroporation transformation (American Biorad, parameters are 25kV, 25uF, 1000Ω). The electroporation is completed. The subsequent bacterial solution was transferred to a 1.5 mL EP tube, 800 μL of 7H9 liquid culture medium was added, and the culture was revived and cultured in a shaker at 37°C and 180 rpm for 24 hours. Centrifuge the recovered bacterial solution at 4000 rpm for 10 minutes at room temperature, discard 400 μL of the supernatant, resuspend the precipitate, and apply 200 μL of the precipitate to 7H10 solid medium containing kanamycin at a final concentration of 25 μg/mL, and culture at 37°C until the medium length of the plate is reached. A single colony emerged.

(3) Verification of rBCG-ECPP007 recombinant BCG

A) Verification of foreign gene integration: Query the host genome integration site (attB site) of the pMV361 vector in the whole genome of the BCG strain provided on the NCBI website and design integration verification primers, where the upstream primer is located at the BCG genome attB site The upstream is about 700 bp, and the downstream primer is about 150 bp downstream of the pMV361 vector restriction site. The primer sequences are shown in Table 3. Pick the recombinant BCG single clone in TE buffer, heat and lyse it at 100°C for 10 minutes to release the DNA, use 1 μl of the lysis supernatant as a template to amplify the target fragment by PCR, and confirm that the amplified product band meets the expected length by agarose gel electrophoresis. , send the sample for Sanger sequencing verification, and the agarose gel electrophoresis results are shown in Figure 2. The length of pMV361-ECPP007 recombinant plasmid is 6641bp, and the length of pMV361 empty vector is 4445bp. The electrophoresis results showed that the pMV361-ECPP007 recombinant plasmid was successfully integrated into the genome of the host BCG.

B) Verification of foreign gene expression: Inoculate the recombinant BCG strain with completely correct BLAST comparison into 7H9 liquid culture medium containing kanamycin at a final concentration of 25 μg/mL, and culture it statically at 37°C for 20 days. After the culture, a small amount of bacterial liquid was taken and subjected to heat shock at 45°C for 60 minutes to induce the expression of integrated genes. After the induction, the total RNA was extracted using the one-step method of Trizol (Invitrogen, USA) and verified by RT-PCR. The specific method was: centrifuge the collected bacteria. Add 1ml of Trizol solution and grinding beads, shake in an ice bath to crush, add 300μl of chloroform, shake and mix, let stand at room temperature for 3 minutes, centrifuge at 4°C and 12000g for 15 minutes, carefully draw 400μl of the top colorless water phase into a clean 1.5ml centrifuge tube , add an equal volume of isopropyl alcohol and mix well, freeze at -80°C overnight, centrifuge at 4°C and 12000g for 15 min, discard the supernatant, add 1 ml of 75% ethanol and gently pipet to wash the precipitate, centrifuge at 4°C and 12000g for 15 min and discard. Evaporate the ethanol to dryness, add 50 μL DEPC water to dissolve the precipitate, and measure the purity and concentration of the RNA sample. Reverse-transcribe RNA samples with qualified purity and concentration, and use the reverse-transcription product cDNA as a template to design expression verification primers to perform PCR amplification of the target gene and conduct agarose gel electrophoresis and DNA sequencing for verification. The primer sequences are shown in Table 3. Agar The results of sugar gel electrophoresis are shown in Figure 3. Experimental results showed that both rBCG-ECPP007 monoclonal strains could express foreign proteins.

Table 3 Primers for verification of recombinant BCG

primer Primer sequence Integration verification primer F CGGCTTATCAACTAGATCGGCGCAG Integrated verification primer R GACGTCAGGTGGCTAGCTGATCA Expression verification primer F CTGCGGCATGTTCTGGATTT Expression verification primer R GGTCTCAACCCCCAGCTAGT

Example 3 Immunological evaluation and safety evaluation of rBCG-ECPP007 gene recombinant BCG

(1) Preparation of rBCG-ECPP007 vaccine

Centrifuge the rBCG-ECPP007 and BCG-China strains cultured in 7H9 liquid culture medium at 4°C and 4000 rpm to collect the cells. Wash the cells once with freshly prepared antibiotic-free 7H9 culture medium. Finally, suspend the cells in sterile PBS and mix them. Adjust the bacterial concentration to 5×10 ⁶ CFU/mL.

(2) Mouse immunization process

6-8 weeks old SPF grade female BALB/c mice (Beijing Vitong Lever) were used for immune experiments. The mice were randomly divided into 3 groups: PBS group, rBCG-ECPP007 group and BCG group, with 6 mice in each group. The process is shown in Table 4. Mice were sacrificed 30 days after immunization, and various immunological tests were performed to evaluate the immunogenicity and safety of rBCG-ECPP007.

Table 4 Immunization process

Group Immunity dose/only immune pathways PBS group 200μl PBS Subcutaneous immunization once rBCG-ECPP007 group 1×106CFU (total 200μl) Subcutaneous immunization once BCG group 1×106CFU (total 200μl) Subcutaneous immunization once

(3)Humoral immunity evaluation

Detection of serum specific antibody titers using enzyme-linked immunosorbent assay (ELISA)

A) Serum separation: Remove the eyeballs of the immunized mice before killing, collect the blood, let it stand at room temperature for 2 hours, and centrifuge at 4000 rpm for 10 minutes to collect the serum.

B) ELISA method to detect serum antibody titer: ultrasonic lyse the whole bacteria of rBCG-ECPP007 and BCG-China, use the BCA method to measure the protein concentration of the whole bacteria, and use the coating buffer (50mM carbonate buffer, pH 9.0) to adjust the corresponding protein The concentration is 2 μg/mL, and 100 μL per well is added to the 96-well plate, and coated at 37°C for 2 hours. After coating, wash 5 times with PBST (PBS containing 0.5‰ Tween-20) and remove the residual liquid in the wells. Add 200 μL PBST containing 5% skim milk powder to each well, block at 37°C for 1 hour, and use PBST after blocking. Wash 5 times and completely remove residual liquid in the wells. Dilute the serum of each group with PBS, add 100 μL of diluted serum to each well, and incubate at 4°C for 12 hours. After the incubation, wash 5 times with PBST and completely remove the residual liquid in the well. Then dilute the HRP-labeled goat anti-mouse IgG antibody (Suzhou Boaolong) at 1:5000 with PBS. Add 100 μL of the diluted antibody to each well and incubate at 37°C for 1 hour. After the incubation, wash 5 times with PBST and remove the contents of the wells. Residual liquid is completely removed. Add 100 μL of TMB chromogenic substrate solution to each well, and after 15 minutes of color development at 37°C, add 100 μL of 2M sulfuric acid to each well to terminate the reaction. Use a microplate reader to detect the absorbance value at 450 nm.

The criterion for judging antibody titer is: if the OD value of the 2nd ⁿ -fold diluted serum/the OD value of the PBS group is ≥2.1 and the OD value of the 2nd ⁿ⁺¹ -fold diluted serum/the OD value of the PBS group is <2.1, then ²ⁿ is the serum. The antibody titer corresponding to the sample. The experimental results are shown in Figure 4. The results showed that the serum specific antibody titer test results showed that the serum specific antibody titer of mice after rBCG-ECPP007 immunization increased significantly, and the difference was statistically significant compared with the BCG group.

(4) Cellular immunity evaluation:

A) Isolation of mouse spleen lymphocytes: The mice were sacrificed by cervical dislocation after blood collection, and the spleen was isolated in a biological safety cabinet after soaking in 75% alcohol. Soak the spleen in mouse lymphocyte separation solution (purchased from Beijing Dawei Biotechnology Co., Ltd.), grind the spleen using a 75 μm filter and a grinding rod, and perform density gradient centrifugation to separate spleen lymphocytes. The experimental operation is performed according to the instructions. Finally, spleen lymphocytes were resuspended in RPMI 1640 medium (Gibco, USA), and the cells were adjusted to a final concentration of 1×10 ⁶ /mL for subsequent experiments.

B) Luminex method detects nine cytokines: IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17a, IFN-γ, TNF-α and GM-CSF.

The Luminex Multiplex Cytokine Detection Kit was purchased from R&D Systems. The specific operation is as follows: Add 100 μL of adjusted concentration of spleen lymphocytes to each well of a 96-well cell culture plate, and then add 2 μg of the corresponding stimulant (ECPP007 whole bacterial lysis product, BCG whole cell lysate or PPD). Each group has a negative control well stimulated by sterile PBS and a positive control well stimulated by 5 μg/mL concanavalin. Cover the plate and culture it in a 37°C, 5% CO2 incubator for 16 to 24 hours. After the culture, centrifuge the 96-well cell plate at 4000 rpm for 10 minutes, and take the supernatant for Luminex multiple cytokine detection. The detection steps are carried out according to the instructions.

The results of the Luminex test stimulated by specific antigens are shown in Figure 5, and the results of the Luminex test stimulated by PPD are shown in Figure 6. Among them, IL-2, IL-12p70, IFN-γ, and TNF-α are Th1-type cytokines that stimulate cellular immune responses; IL-4, IL-6, and IL-10 are Th2-type cytokines that stimulate humoral immune responses; IL -17a and GM-CSF are innate immune-related cytokines and play a role in the body's innate immune response.

The experimental results showed that after antigen stimulation, the secretion levels of IL-2, IFN-γ, TNF-α, GM-CSF and IL-17a were higher in the spleen lymphocytes of mice in the rBCG-ECPP007 immunized group compared with the BCG group, while IL There was no significant difference in the secretion of -12p70, IL-4, IL-6 and IL-10; compared with the BCG group, the spleen lymphocytes of mice in the rBCG-ECPP007 immunized group stimulated by PPD had higher levels of IFN-γ, TNF-α, The secretion levels of IL-6, IL-10, GM-CSF and IL-17a were higher, but there was no significant difference in the secretion of IL-2, IL-12p70 and IL-4. The experimental results suggest that rBCG-ECPP007 can induce the body to produce strong immune responses related to humoral immunity, cellular immunity and innate immunity.

C) RT-qPCR method to detect the expression levels of inflammatory factors in lung cells

The total RNA from lung tissue was extracted using the Trizol one-step method (Invitrogen, USA) and the expression levels of each inflammatory factor were detected by qPCR. The steps were as follows: the mice were sacrificed by cervical dislocation, the lungs were isolated in a biosafety cabinet after soaking in 75% alcohol, and cut Take lungs with a wet weight of about 0.1g for extraction of total RNA. Add 1 ml Trizol solution and grinding beads to every 0.1 g lung sample, shake and crush in an ice bath, add 300 μl chloroform, shake and mix, let stand at room temperature for 3 min, and centrifuge at 12000g for 15 min at 4°C. Carefully transfer 400 μl of the top colorless aqueous phase into a clean 1.5 ml centrifuge tube, add an equal volume of isopropyl alcohol, mix well, freeze at -80°C overnight, and centrifuge at 12,000g for 15 min at 4°C. Discard the supernatant, add 1 ml of 75% ethanol (prepared with ribozyme-free water) and gently pipette to wash the precipitate. Be careful to avoid breaking up the white precipitate. Centrifuge at 12000g for 15 minutes at 4°C and discard the supernatant. After evaporating the ethanol, add 50 μL of ribozyme-free water to dissolve the RNA and measure the purity and concentration of the RNA sample. RNA samples with qualified purity and concentration are subjected to reverse transcription. The reverse transcription system is shown in Table 5. The specific operations are the same as mentioned above. Using the reverse transcription product as a template, specific primers were designed to perform qPCR detection of the target gene. The primer sequences are shown in Table 6. The experimental results are presented in the form of 2 ^-ΔΔCt .

Table 5 Reverse transcription system

Reverse transcription system components Volume (μL) RNA 300ng 2×Reverse Transcription Mix 25μL ddH2O Make up to 50μL

Table 6 Specific primer sequences

Gene forward primer backward primer IL-2 CTGCGGCATGTTCTGGATTT ATGTGTTGTCAGAGCCCTTTAGT IL-4 GGTCTCAACCCCCAGCTAGT GCCGATGATCTCTCTCAAGTGAT IL-6 TCCGGAGAGGAGACTTCACA TTGCATTGCACAACTCTTTTC IL-10 GCTGTCATCGATTTCTCCCCT ACACCTTGGTCTTGGAGCTTAT IL-12a TGTCAATCACGCTACCTCCTC TGGTCTTCAGCAGGTTTCGG GM-CSF TGCCTGTCACGTTGAATGAAG TGCCTGTCACGTTGAATGAAG IFN-γ TTGCCAAGTTTGAGGTCAACAA TTGCCAAGTTTGAGGTCAACAA iNOS GGAGTGACGGCAAACATGACT TCGATGCACAACTGGGTGAAC TNF-α CCACCACGCTCTTCTGTCTAC ATGAGAGGGAGGCCATTTGGG GAPDH AGGTCGGTGTGAACGGATTTG TGTAGACCATGTAGTTGAGGTCA

The experimental results are shown in Figure 7. The results show that compared with the BCG group, the expression levels of TNF-α in lung cells of mice in the rBCG-ECPP007 immunized group increased, and the expression levels of IL-2, IL-6, IL-10 and IL-12a decreased. , there is no statistical difference in the other results.

D) Detection of mouse spleen lymphocyte grouping by flow cytometry: First, prepare a stimulator and blocker mixture according to the number of samples: containing 20 μg of the corresponding stimulatory antigen (rBCG-ECPP007 whole bacterial lysate, BCG whole bacterial lysate, or PPD) /mL, CD28 antibody 2 μg/mL, CD49d antibody 2 μg/mL and BFA blocker (500-fold dilution) in RPMI1640 medium. Take 100 μL of cell suspension (a total of 1x10 ⁵ cells) and inoculate it into a 96-well culture plate. Add 100 μL of the prepared stimulation mixture. The final concentration of each component is: antigen stimulator 10 μg/mL, CD28 antibody 1 μg/mL, CD49d Antibody 1 μg/mL, diluted 1000 times with BFA blocker, mixed and placed in a 37°C, 5% CO2 cell incubator for stimulation and culture for 8 hours. After stimulation, collect the cells and transfer them to a flow tube, add 2 ml of PBS, mix well, and centrifuge at 300 g for 5 minutes to discard the supernatant. Add FVS510 Live-Dead dye prepared in PBS, mix well, and incubate at room temperature in the dark for 15 minutes. Add 2 mL of flow staining wash solution, mix well and centrifuge at 300g for 5 minutes, discard the supernatant. Resuspend the cells into 100 μL flow cytometry staining wash solution, add fluorescently labeled antibodies to cell surface antigens (BD, USA) (CD4, CD8, CD62L and CD44), mix and incubate at room temperature in the dark for 20 minutes. Add 2 ml of flow staining wash solution, mix well and centrifuge at 300g for 5 minutes, discard the supernatant. Add 0.5 mL of cell fixative to each tube, mix well, and incubate at room temperature in the dark for 20 minutes. Centrifuge at 300 g for 5 minutes and discard the supernatant. Dilute the 10× membrane rupture wash solution with deionized water to 1× working solution. Add 2 mL of membrane rupture working solution to each tube. Mix well and centrifuge at 300 g for 5 minutes. Discard the supernatant. Add 2 mL of membrane-breaking working solution to each tube again, mix and centrifuge at 300g for 5 minutes, discard the supernatant. Add fluorescently labeled antibodies to cytoplasmic antigens (BD, USA) (IL-2, IL-4 and IFN-y), mix and incubate at room temperature in the dark for 30 minutes. Add 2 mL of membrane-breaking working solution to each tube, mix and centrifuge at 350g for 5 minutes, discard the supernatant. Add 2 ml of flow staining wash solution to each tube, mix well and centrifuge at 300g for 5 minutes, discard the supernatant. Add 0.5 ml of cell staining wash solution to resuspend the cells and detect them on a flow cytometer.

The results of the flow cytometry experiment stimulated by specific antigens are shown in Figure 8, and the results of the flow cytometry experiment stimulated by PPD are shown in Figure 9. The experimental results show that the spleen lymphocytes of mice in the rBCG-ECPP007 group and BCG group secrete IL-2 and secrete IFN- γ CD4 ⁺ and CD8 ⁺ T lymphocytes both account for the majority, but the proportion of T lymphocytes secreting both IFN-γ/IL-2 and IFN-γ/TNF-α in the spleen lymphocytes of mice after rBCG-ECPP007 immunization More. Experimental results show that compared with the PBS group and BCG group, the antigen-specific T lymphocytes induced by rBCG-ECPP007 have a stronger ability to secrete three cytokines, IFN-γ, TNF-α and IL-2, and secrete important cytokines. The T lymphocyte population is more diverse.

(5) Safety evaluation of rBCG-ECPP007 gene recombinant BCG

The mice were sacrificed by cervical dislocation. After soaking in 75% alcohol, the spleen, liver, spleen and lungs were separated in a biological safety cabinet and fixed in 10% formalin solution for 1 week. After the fixation, routine paraffin embedding and sectioning were performed. Hematoxylin-Eosin Staining (HE staining), and observe morphological changes under a microscope.

The experimental results are shown in Figure 10. The gross anatomy of the organs was observed with the naked eye: Compared with the control group, the organs of the mice in the rBCG-ECPP007 group had normal appearance, color, and size, and no obvious pathological changes such as proliferation, atrophy, and edema were found. . Microscopic observation showed that the liver cells had a clear structure, were tightly arranged, and had abundant cytoplasm. The nuclei were large, round, and centered. The liver cords were arranged neatly and orderly. There was no difference compared with the control group. The spleen tissue cells had normal morphology, which was similar to the control group. There was no difference compared with the control group; there were no abnormalities in the bronchi and alveolar structures of the lung tissue, and a small amount of inflammatory cell infiltration could be seen, but there was no significant difference compared with the control group. All organ structures were within the normal morphological range, and no obvious pathological changes such as cell edema and necrosis were found.

The above-mentioned embodiments only express several implementation modes of the present invention to facilitate a specific and detailed understanding of the technical solutions of the present invention, but they cannot be understood as limiting the scope of invention patent protection. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims (10)

1. The mycobacterium tuberculosis multi-antigen fusion protein is characterized by comprising an Rv3875 protein antigen, an Rv3874 protein antigen, an Rv0934-555 protein antigen and an Rv2029c protein antigen, wherein the amino acid sequence of the Rv0934-555 protein antigen is shown as SEQ ID NO. 5.

2. The mycobacterium tuberculosis multi-antigen fusion protein according to claim 1, wherein the amino acid sequence of the mycobacterium tuberculosis multi-antigen fusion protein is shown as SEQ ID No. 2.

3. A gene encoding the mycobacterium tuberculosis multi-antigen fusion protein according to claim 1 or 2, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 1.

4. A biological material for expressing the mycobacterium tuberculosis multi-antigen fusion protein of claim 1, the biological material being selected from the group consisting of expression vectors, engineering bacteria, characterized in that the biological material comprises the gene of claim 3.

5. The biomaterial of claim 4, wherein the biomaterial is recombinant bcg; the construction method of the recombinant BCG vaccine comprises the following steps:

s1, connecting the gene of claim 3 to a shuttle vector to obtain a recombinant plasmid carrying a fusion gene;

s2, transforming the recombinant plasmid in the step S1 into bacillus calmette-guerin vaccine, and screening to obtain the recombinant bacillus calmette-guerin vaccine which stably expresses the multi-antigen fusion protein combined with the mycobacterium.

6. The biomaterial of claim 5, wherein the shuttle vector is pMV361; and/or, the BCG vaccine is BCG-China strain.

7. The biomaterial of claim 5 or 6, wherein the transformation is performed by electrotransformation, and wherein the conditions of the electrotransformation are: 20-30kV,20-30uF,900-1100 omega.

8. Use of a mycobacterium tuberculosis multi-antigen fusion protein according to claim 1 or 2, or a coding gene according to claim 3, or a biomaterial according to any one of claims 4-7 in the preparation of a mycobacterium tuberculosis vaccine.

9. A mycobacterium tuberculosis vaccine, characterized in that the effective component of the mycobacterium tuberculosis vaccine comprises the mycobacterium tuberculosis multi-antigen fusion protein of claim 1 or 2; or the mycobacterium tuberculosis multi-antigen fusion protein prepared by the encoding gene of claim 3 or the expression of the biological material of any one of claims 4-7.

10. Use of a mycobacterium tuberculosis multi-antigen fusion protein according to claim 1 or 2, or a coding gene according to claim 3, or a biomaterial according to any one of claims 4-7, in the preparation of a mycobacterium tuberculosis detection reagent or a medicament for the treatment of a mycobacterium tuberculosis disease.