Recombinant bacillus calmette guerin vaccine with long-term protection for preventing tuberculosis as well as preparation method and application of recombinant bacillus calmette guerin vaccine

Abstract

The invention relates to a recombinant bacillus calmette-guerin vaccine with long-term protection for preventing tuberculosis as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the recombinant shuttle plasmid disclosed by the invention, an Rv1813 gene sequence, an Rv2660c gene sequence, an Ag85B gene sequence, an Rv2623 gene sequence and an HspX gene sequence are inserted into an escherichia coli-mycobacterium shuttle plasmid. According to the recombinant bacillus calmette-guerin vaccine, preferably, a gene (Rv1813-Rv2660c-Ag85B-Rv2623-HspX) of a fusion protein A1D4 is cloned into the bacillus calmette-guerin vaccine, overexpression is carried out in the bacillus calmette-guerin vaccine, and the recombinant bacillus calmette-guerin vaccine rBCG:: A1D4 is formed. The rBCG:: A1D4 recombinant vaccine for expressing the fusion protein A1D4 has a good protection effect, the anti-tuberculosis primary infection protection property of the rBCG:: A1D4 recombinant vaccine is similar to that of a BCG vaccine in a short time, and the protection property is not weakened along with time along with the prolonging of immune time.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a recombinant BCG vaccine for tuberculosis prevention with long-term protection, a preparation method and an application, that is, the gene of the fusion protein A1D4 protein (Rv1813-Rv2660c-Ag85B-Rv2623-HspX) is cloned into the BCG vaccine, overexpressed in the BCG vaccine, and formed a recombinant BCG vaccine rBCG::A1D4. Background Art

[0002] Tuberculosis (TB) is one of the important public health issues that seriously affects human health worldwide. It is an infectious disease caused by infection with Mycobacterium tuberculosis (MTB) and spreads among the population mainly through the respiratory tract.

[0003] According to the World Health Organization, an estimated 10.6 million people will be newly diagnosed with TB in 2022. The global TB incidence has increased for two consecutive years, which means that the TB incidence in 2022 will return to the level of 2019. Globally, the net reduction rate of TB incidence from 2015 to 2022 was 8.7%, far below the WHO TB strategy's plan to reduce it by 50% by 2025.

[0004] Bacillus Calmette-Guerin (BCG) is the only preventive TB vaccine approved for use in the world. The immunization strategy of vaccinating newborns with BCG to prevent TB is implemented in more than 150 countries and regions around the world. The BCG coverage rate of newborns in my country exceeds 99.9%. It is generally believed that BCG immunization of newborns can effectively prevent severe TB in infants and young children. However, the protection period of BCG is only 10-15 years, and it lacks the protective power to prevent TB in adolescents and adults.

[0005] Therefore, research on more effective TB vaccines to replace the existing BCG is an important direction in the field of TB prevention research. The types of vaccines currently being studied include subunit vaccines, live attenuated vaccines, recombinant BCG (rBCG) vaccines, and DNA vaccines. In addition to developing recombinant BCG based on BCG itself, other types of candidate vaccines can hardly obtain the same protection as BCG immunity or exceed its effect in animal experiments. Therefore, there is currently no vaccine that can replace BCG for neonatal immunization.

[0006] Recombinant BCG is made by integrating the antigen gene obtained through screening into BCG. After being inoculated into the body, the modified BCG can reproduce itself and continuously express its own protein, maintaining the advantages of the parent BCG. At the same time, it will express the recombinant protein integrated into rBCG, continuously stimulating the body to produce specific immune responses, so as to achieve the purpose of extending the protection period of BCG after vaccination in the neonatal stage. VPM1002 is a recombinant BCG vaccine that is urease C-deficient and expresses listeriolysin. It was jointly developed by VPM and SSI. Currently, VPM1002 has entered Phase III clinical trials and its safety has been verified. It has been verified that the idea of ​​developing recombinant BCG is safe and effective.

[0007] The key issue is how to develop recombinant BCG so that the new generation of BCG has better protection. A1D4 is a fusion protein designed and constructed by us. Its encoding genes are Rv1813, Rv2660c, Ag85B, Rv2623 and HspX genes connected in series in the order of Rv1813-Rv2660c-Ag85B-Rv2623-HspX. Antigens such as Ag85B are antigens expressed by Mycobacterium tuberculosis during the rapid growth and reproduction stage in vitro, and Rv1813, Rv2660c, Rv2623 and HspX are antigens expressed by Mycobacterium tuberculosis in a persistent or latent state, and are usually expressed at low levels or not expressed in BCG.

[0008] In previous studies, it has been confirmed that the A1D4 antigen has good immunogenicity and low vaccination risk, and a patent "A tuberculosis subunit vaccine containing fusion protein A1D4" (publication number CN104225586A) has been applied for. We prepared the recombinant protein A1D4 in the Escherichia coli protein expression and purification system, and mixed it with the oil-in-water adjuvant MTO to prepare the recombinant protein adjuvant vaccine A1D4 / MTO. The adjuvant MTO contains monophospholipid lipid A (MPL), trehalose 6,6' mycolic acid analog (TDB) and squalene-based oil-in-water adjuvant MF59. The recombinant protein adjuvant vaccine A1D4 / MTO was used to immunize SPF-grade 6-8-week-old C57BL / 6J mice twice and then challenged with Mycobacterium tuberculosis. Compared with the PBS group, the bacterial load in the lungs and spleens of mice in the A1D4 / MTO vaccine immunization group was significantly reduced, indicating that the recombinant protein adjuvant vaccine A1D4 / MTO can inhibit the proliferation of Mycobacterium tuberculosis in the lungs and spleen and has protective properties against Mycobacterium tuberculosis infection.

[0009] However, compared with BCG, the recombinant protein adjuvant vaccine A1D4 / MTO group had a higher bacterial load in the lungs and spleens of mice, and the protection against M.tb infection produced was weaker than that of BCG immunization, and it cannot replace BCG as a new generation of TB vaccine. At the same time, protein adjuvant vaccines require repeated immunizations, and have many preparation steps and are limited by protein purification processes and adjuvant preparation technologies. Recombinant BCG expresses immune antigens through live BCG bacteria themselves, which eliminates related technical barriers. Subsequent vaccine preparation only requires related steps such as culturing recombinant BCG live bacteria. If recombinant BCG is used to replace BCG, only one immunization is required.

[0010] Similarly, in the invention patent "A fusion protein CMFO and its application" with publication number CN105037561A, we designed a fusion protein CMFO, which was mixed with the liposome adjuvant DMT to prepare the recombinant protein adjuvant vaccine CMFO / DMT. The adjuvant DMT contains monophospholipid lipid A (MPL), trehalose 6,6' dimycolic acid analog (TDB) and cationic dimethyldioctadecyl ammonium (DDA). The recombinant protein adjuvant vaccine CMFO / DMT immunized SPF-grade 6-8-week C57BL / 6J mice twice and then attacked and infected with Mycobacterium tuberculosis. Compared with the PBS group, the bacterial load in the lungs and spleens of mice immunized with the CMFO / DMT vaccine was significantly reduced, which was comparable to the protectiveness of BCG, indicating that the recombinant protein adjuvant vaccine CMFO / DMT can inhibit the proliferation of Mycobacterium tuberculosis in the lungs and spleen, and has protectiveness against Mycobacterium tuberculosis infection. However, when a recombinant vaccine rBCG::CMFO overexpressing CMFO in BCG was constructed and mice were immunized and infected with the Mycobacterium tuberculosis strain H37Rv, the bacterial load in the spleen of the mice increased over time, indicating that the protective effect of the rBCG::CMFO vaccine decreased over time and its application value was reduced. Summary of the invention

[0011] The present invention provides a recombinant BCG vaccine for tuberculosis prevention with long-term protection and its preparation, which is obtained by transforming a recombinant shuttle plasmid into a BCG vaccine, wherein the recombinant shuttle plasmid is an Escherichia coli-Mycobacterium shuttle plasmid into which an Rv1813 gene sequence, an Rv2660c gene sequence, an Ag85B gene sequence, an Rv2623 gene sequence and an HspX gene sequence are inserted. The recombinant BCG vaccine in the present invention can significantly increase the immune protection period, thereby solving the technical problem of a short protection period of the BCG vaccine in the prior art.

[0012] According to a first aspect of the present invention, a recombinant shuttle plasmid is provided, wherein the recombinant shuttle plasmid is an Escherichia coli-Mycobacterium shuttle plasmid in which an Rv1813 gene sequence, an Rv2660c gene sequence, an Ag85B gene sequence, an Rv2623 gene sequence and an HspX gene sequence are inserted, wherein the Rv1813 gene sequence is SEQ ID NO: 1, the Rv2660c gene sequence is SEQ ID NO: 2, the Ag85B gene sequence is SEQ ID NO: 3, the Rv2623 gene sequence is SEQ ID NO: 4, and the HspX gene sequence is SEQ ID NO: 5.

[0013] Preferably, the Rv1813 gene sequence, Rv2660c gene sequence, Ag85B gene sequence, Rv2623 gene sequence and HspX gene sequence are connected in series according to Rv1813-Rv2660c-Ag85B-Rv2623-HspX.

[0014] Preferably, the E. coli-Mycobacterium shuttle plasmid is pSMT3, pMV206, pMV261, pMV306 or pMV361.

[0015] According to another aspect of the present invention, a recombinant BCG vaccine is provided, which is obtained by transforming the recombinant shuttle plasmid into BCG vaccine.

[0016] Preferably, the BCG vaccine is BCG Chinese strain, BCG Pasteur strain, BCG Danish strain, BCG Copenhagen strain, BCG Japanese strain, BCG Tice strain or BCG Russian strain.

[0017] Preferably, the conversion is electroconversion.

[0018] According to another aspect of the present invention, there is provided the use of the recombinant BCG vaccine for preparing a vaccine with a prolonged protection period against tuberculosis.

[0019] In general, the recombinant BCG rBCG::A1D4 of the present invention has the following technical advantages compared with the prior art:

[0020] (1) The present invention uses the A1D4 gene to construct a recombinant BCG that overexpresses A1D4, providing a preventive live vaccine with simpler vaccine preparation technology, lower technical cost, better immune effect, and long-term anti-tuberculosis infection ability that significantly exceeds the protective ability of BCG.

[0021] (2) The present invention has good safety. Compared with wild-type BCG, the safety of the overexpressed fusion protein A1D4 of the present invention has been verified in small animals.

[0022] (3) The present invention is conducive to clinical application. The technology of modifying BCG is mature, stable, simple to prepare, and free of material technology bottlenecks, which is conducive to further production and application. Compared with the recombinant protein subunit vaccines A1D4 / MTO or A1D4 / DMT, which require repeated immunization, the recombinant BCG rBCG::A1D4 only requires one immunization, which is conducive to clinical promotion and use.

[0023] (4) The present invention has a good protective effect. Compared with recombinant protein adjuvant vaccines, the synergistic effect of recombinant BCG expressing antigen A1D4, including the advantages of BCG itself and overexpression of antigen A1D4, makes rBCG::A1D4 vaccine more protective against primary tuberculosis infection.

[0024] (5) The present invention can provide long-term protection. The protective effect of the recombinant BCG rBCG::A1D4 against primary tuberculosis infection does not weaken over time as the immunization time increases, thus solving the defects of the wild-type BCG. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a structural schematic diagram of the recombinant plasmid pM-A1D4 of the present invention.

[0026] Figure 2 The genomic DNA of recombinant BCG rBCG::A1D4 was extracted and PCR detection was performed to confirm that the A1D4 fusion sequence was introduced into BCG.

[0027] Figure 3 Western blotting was used to detect A1D4 fusion protein in rBCG::A1D4 cell lysates.

[0028] Figure 4 This is the bacterial load in the organs of C57BL / 6 mice after they were immunized with the A1D4 / MTO recombinant protein subunit vaccine and then infected with the Mycobacterium tuberculosis strain H37Rv.

[0029] Figure 5 This is the bacterial load in the organs of C57BL / 6 mice after they were immunized with CMFO / MTO and CMFO / DMT recombinant protein subunit vaccines and then infected with Mycobacterium tuberculosis strain H37Rv. Figure 6 This is the bacterial load in the organs of C57BL / 6 mice after they were immunized with A1D4 / DMT and CMFO / DMT recombinant protein subunit vaccines and then infected with Mycobacterium tuberculosis strain H37Rv.

[0030] Figure 7 This is the bacterial load and changes in the organs of C57BL / 6 mice after they were immunized with recombinant BCG and then infected with the Mycobacterium tuberculosis strain H37Rv via aerosol.

[0031] Figure 8These are the results of HE and AF staining of lung pathological sections of C57BL / 6 mice immunized with recombinant BCG for 10 weeks and then infected with Mycobacterium tuberculosis strain H37Rv by aerosol.

[0032] Fig. 9 These are the results of HE and AF staining of lung pathological sections of C57BL / 6 mice immunized with recombinant BCG for 20 weeks and then infected with Mycobacterium tuberculosis strain H37Rv by aerosol.

[0033] Fig.10 After C57BL / 6 mice were immunized with recombinant BCG, the levels of antigen-specific T cells in the lungs of mice were analyzed by flow cytometry.

[0034] Fig.11 After C57BL / 6 mice were immunized with recombinant BCG, the levels of antigen-specific T cells in the spleen of mice were analyzed by flow cytometry.

[0035] Fig.12 After C57BL / 6 mice were immunized with recombinant BCG, the number of antigen-specific IFN-γ T cell spots in the spleen of mice was analyzed by ELISPOT. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] One object of the present invention is to provide a new recombinant shuttle plasmid. The recombinant shuttle plasmid is an Escherichia coli-Mycobacterium shuttle plasmid into which the Rv1813 gene sequence, the Rv2660c gene sequence, the Ag85B gene sequence, the Rv2623 gene sequence and the HspX gene sequence are inserted, wherein the Rv1813 gene sequence is SEQ ID NO: 1, the Rv2660c gene sequence is SEQ ID NO: 2, the Ag85B gene sequence is SEQ ID NO: 3, the Rv2623 gene sequence is SEQ ID NO: 4, and the HspX gene sequence is SEQ ID NO: 5.

[0038] Preferably, the connection sequence of the Rv1813 gene sequence, Rv2660c gene sequence, Ag85B gene sequence, Rv2623 gene sequence and HspX gene is Rv1813-Rv2660c-Ag85B-Rv2623-HspX, as shown in SEQ ID NO:6 according to the connection sequence.

[0039] The following principles should be followed when constructing fusion sequences:

[0040] (1) If there is a signal peptide, remove it, including the original start codon, and remove the stop codon (TAA is added after the last gene sequence spliced);

[0041] (2) For gene sequences without signal peptides, the start codon sequence is retained. If the start codon is GTG, since its original expression product is Met rather than Val, GTG should be expressed as Met when used as a non-start code, so GTG needs to be corrected to ATG.

[0042] Another object of the present invention is to provide a vaccine prepared by the above recombinant plasmid.

[0043] A novel recombinant plasmid in the present invention preferably inserts the tandem gene sequence of the fusion protein (i.e., A1D4 fusion protein) expressed by the Rv1813-Rv2660c-Ag85B-Rv2623-HspX sequence into the Escherichia coli-Mycobacterium tuberculosis shuttle plasmid sequence to form a recombinant shuttle plasmid. The amino acid sequence of the fusion protein expressed by the Rv1813-Rv2660c-Ag85B-Rv2623-HspX sequence is shown in SEQ ID NO:7.

[0044] In the present invention, the gene of A1D4 fusion protein comes from Mycobacterium tuberculosis (H37Rv). The gene sequence is derived from the US NIH GenBank public database (NC_000962.3). The fusion gene sequence is obtained by artificial gene synthesis.

[0045] The gene sequence of the A1D4 fusion protein is a tandem gene of Rv1813-Rv2660c-Ag85B-Rv2623-HspX, or an amino acid sequence encoding a protein with the same function that has 80% to 100% homology to the amino acid sequence translated after transcription of the sequence, or a derivative protein with equivalent activity produced by replacing any component or multiple components in the fusion protein in sequence, or a derivative protein with equivalent activity after adding, deleting or replacing one or more amino acids in the sequence.

[0046] In the process of preparing the recombinant plasmid of the present invention, the Escherichia coli-Mycobacterium shuttle plasmid that can be used can be one of pSMT3, pMV206, pMV261, pMV306 and pMV361, but is not limited thereto.

[0047] The function of the E. coli-Mycobacterium shuttle plasmid is to carry the fusion gene sequence into the BCG vaccine, and further utilize the ability of the E. coli-Mycobacterium shuttle plasmid to replicate in the BCG vaccine or integrate into the BCG genome, and finally achieve the expression of A1D4 in the BCG vaccine. In one embodiment of the present invention, the E. coli-Mycobacterium shuttle plasmid used is pMV261.

[0048] On the other hand, the present invention provides a novel recombinant tuberculosis vaccine, that is, the recombinant plasmid containing the gene of the A1D4 fusion protein is transformed into BCG to obtain the rBCG::A1D4 recombinant vaccine.

[0049] The recombinant BCG rBCG::A1D4 against tuberculosis provided by the present invention is obtained by transforming the recombinant plasmid containing the gene of A1D4 fusion protein into BCG to obtain the recombinant BCG rBCG::A1D4.

[0050] The preparation method of the recombinant BCG rBCG::A1D4 provided by the present invention comprises the following steps:

[0051] (1) Amplifying or artificially synthesizing the gene sequence of A1D4 fusion protein;

[0052] (2) inserting the gene sequence encoding the A1D4 fusion protein into the sequence of the Escherichia coli-Mycobacterium shuttle plasmid to construct a recombinant Escherichia coli-Mycobacterium shuttle plasmid containing the gene encoding the A1D4 fusion protein;

[0053] (3) The recombinant Escherichia coli-Mycobacterium tumefaciens shuttle plasmid containing the gene encoding the A1D4 fusion protein is transformed into BCG to obtain the recombinant BCG rBCG::A1D4.

[0054] The BCG strain transformed by the recombinant plasmid of the present invention can be any BCG strain currently used for clinical immunization, such as BCG Chinese strain, BCG Pasteur strain, BCG Danish strain, BCG Copenhagen strain, BCG Japanese strain, BCG Tice strain, BCG Russian strain, etc. In one embodiment of the present invention, the BCG strain used is BCG Pasteur strain.

[0055] In previous studies, antigens Rv1813, Rv2660c, Ag85B, Rv2623, HspX, and A1D4 were screened by whole blood interferon release assay (WBIA) to stimulate the production of high levels of IFN-γ in the serum of tuberculosis patients, and the stimulation effect of fusion protein A1D4 was significantly improved compared with that of the individual components, confirming that A1D4 has good immunogenicity.

[0056] The following are specific embodiments

[0057] Example 1: Construction and identification of recombinant plasmid pM-A1D4

[0058] (1) Construction of recombinant plasmid of A1D4 fusion protein

[0059] The gene sequence of A1D4 fusion protein is Rv1813-Rv2660c-Ag85B-Rv2623-HspX tandem gene. BamHⅠ and HindⅢ were selected as restriction sites, and non-specific restriction sites were checked in the target sequence. The triplet code of HSP60 Promotor was supplemented at the restriction site to prevent frameshift mutation. A1D4 fusion protein gene with BamHⅠ at the 5' end and HindⅢ restriction sites at the 3' end was synthesized by gene synthesis technology. The fusion protein gene and pMV261 vector were double-digested by BamHⅠ and HindⅢ restriction endonucleases (37℃, 2h). The digestion products were identified by agarose gel electrophoresis and gel recovery, and then connected with T4 DNA ligase. The connection conditions were 16℃, 30min.

[0060] Double enzyme digestion system:

[0061]

[0062]

[0063] Connection system:

[0064]

[0065] 16℃,30min.

[0066] (2) Transformation of ligation products into E. coli DH5α competent cells

[0067] Take out 50 μL of frozen E. coli DH5α competent cells and place them on ice to dissolve. Add 5 μL of ligation product and mix well. Place in ice bath for 30 minutes. Heat shock at 42°C for 90 seconds. Place in ice bath for 2 minutes. Add 945 μL of LB liquid medium without resistance in a biosafety cabinet. Place in a shaker at 37°C and culture at 180 rpm for 1 hour. After the culture is completed, centrifuge at 4000 rpm for 1 minute, discard 800 μL of supernatant, gently blow and mix the bacterial precipitate, and take the remaining bacterial liquid to apply on LB solid plate containing kanamycin (50 ug / mL), and culture at 37°C inverted for 16 hours.

[0068] (3) Screening and identification of positive monoclones

[0069] Single colonies with good growth were picked and cultured in LB liquid medium containing kanamycin (50ug / mL). After plasmid mini-extraction using the bacterial liquid plasmid mini-extraction kit (Axygen), double enzyme digestion identification was performed. The enzyme digestion products were observed after 1% agarose gel electrophoresis. After the identification was in line with expectations, the candidate strains were sent to the company for sequencing. The bacterial samples with completely correct sequencing results after BLAST alignment were cultured in LB liquid medium containing kanamycin (50ug / mL), and the plasmids were extracted and stored in a -20℃ refrigerator. The structural diagram of the pM-A1D4 recombinant plasmid is shown in Figure 1 .

[0070] Example 2: Construction of recombinant BCG rBCG::A1D4

[0071] (1) Preparation of BCG competent cells

[0072] Inoculate BCG in 10mL 7H9 liquid culture medium and culture for 10 days. Collect bacteria by centrifugation, ice-bath with 10mL ice-cold 10% glycerol sterile aqueous solution for 2h, centrifuge and discard the supernatant, resuspend with 2mL ice-cold 10% glycerol sterile aqueous solution, centrifuge, discard the supernatant, resuspend with 500uL of 10% glycerol, and divide into EP tubes to make BCG competent cells, and freeze at -80℃ for later use.

[0073] (2) Electrotransformation of recombinant plasmid into BCG

[0074] Add 100uL BCG electrocompetent cells to 5ul recombinant plasmid pM-A1D4, mix well and transfer into an electroporation cup with a diameter of 0.20mm, place the electroporation cup in an ice bath for 10min, wipe off the moisture outside the electroporation cup, and use an electroporator to transform pM-A1D4 into BCG competent cells.

[0075] The electroporation conditions were: resistance 1,000Ω, voltage 2.5kV, capacitance 25uF, and transformation time 17ms. Immediately after electroporation, the bacteria were transferred into 10mL 7H9 liquid medium and cultured overnight with shaking. The cells were collected by centrifugation and immediately spread on 7H11 agar plates containing kanamycin (25μg / mL). Cultured at 37℃ for 3-4 weeks.

[0076] Example 3: Identification of the gene level of recombinant BCG rBCG::A1D4

[0077] Only rBCG transformed with the plasmid containing Kan resistance gene can grow on 7H11 medium containing Kan (25ug / mL). Randomly select colonies visible to the naked eye and inoculate them into 7H11 medium containing Kan (25ug / mL) for expansion culture.

[0078] The cells after expansion culture were picked and the genomic DNA of recombinant BCG was extracted using the Mycobacterium Genome Extraction Kit (Shanghai Jingnuo). The gene fragment of PCR fusion protein A1D4 was detected and the target band was observed after 1% agarose gel electrophoresis of the PCR product. The results showed that the band obtained by PCR amplification was consistent with the size of the A1D4 fusion protein gene band (such as Figure 2 ).

[0079] Example 4: Identification of protein levels of recombinant BCG rBCG::A1D4

[0080] Take the recombinant BCG that has been identified at the genetic level and inoculate it into 7H9 medium containing Kan (25ug / mL) for expansion culture. Collect the bacteria, remove the medium by centrifugation and resuspend with PBS. Add lysozyme 20ug / mL and digest at 37℃ for 2h. Ultrasonicate the digested bacterial solution, centrifuge at 2000rpm for 20min, and collect the supernatant. Western-blot results show that the obtained band is about 100KD, which is consistent with the predicted size. (such as Figure 3 ).

[0081] Example 5: Short-term and long-term protection of recombinant BCG rBCG::A1D4

[0082] SPF grade C57BL / 6 female mice were used, female, 6-8 weeks old, 18-20g. They were divided into PBS control group, adjuvant control group, BCG immunization group, and adjuvant subunit candidate vaccine immunization group. Mice were subcutaneously immunized with 0.2 ml of vaccine three times, 3 weeks apart, and infected with Mycobacterium tuberculosis H37Rv 10 weeks after the last immunization. Four weeks after infection, the mice were killed and the spleen and lungs of the mice were aseptically removed. Add 2 mL of sterile PBS to each organ and grind the homogenate with a homogenizer. Take 100 μL of the homogenate, add it to 900 μL of sterile PBS for dilution, and make multiple dilutions in turn. Take 100 μL of the dilution and spread it on 7H11-ADC solid culture medium (add TCH to selectively inhibit the growth of residual BCG), and count the colonies after static culture at 37°C for 3-4 weeks. The number of bacteria in each organ of the mouse was estimated based on the plate count, and the bacterial load in the mouse lung and spleen and the pathological changes in the mouse lung were evaluated.

[0083] The recombinant protein subunit vaccine A1D4 / MTO was used to immunize C57BL / 6J mice, and the immunization method and dosage were the same as above. A control group of PBS, MTO and BCG groups were set up to evaluate the bacterial load in the mouse organs. Figure 4 ) Compared with the PBS group, A1D4 / MTO produced protection against TB infection and reduced the bacterial load in the lungs and spleen of mice. However, compared with the BCG group, the protection against M.tb infection produced by A1D4 / MTO was not enough to replace BCG.

[0084] The mice were immunized with the recombinant protein subunit vaccines CMFO / MTO and CMFO / DMT, and the immunization method and dosage were the same as above. A control group PBS group was set up. The bacterial load in the lungs and spleen of the mice was evaluated. (e.g. Figure 5 ) Compared with the CMFO / MTO group, the bacterial load in the lungs and spleens of the mice in the CMFO / DMT group was significantly reduced. Compared with the MTO adjuvant, the combination of DMT adjuvant and recombinant protein CMFO can produce better protection against M.tb infection.

[0085] C57BL / 6J mice were immunized with recombinant protein subunit vaccines CMFO / DMT and A1D4 / DMT, and the immunization method and dosage were the same as above. A control group PBS group, a DMT group, and a BCG group were set up to evaluate the bacterial load in the mouse organs. Figure 6 ) Compared with the PBS group, the bacterial load in the lungs and spleens of the CMFO / DMT group and the A1D4 / DMT group was significantly reduced. The effect of the CMFO / DMT group on the spleen was more significant. Compared with the BCG group, the CMFO / DMT group and the A1D4 / DMT group showed similar protection against M.tb infection. Therefore, both A1D4 and CMFO can be modified with recombinant BCG. In order to evaluate the protective effect of recombinant BCG on mice against primary infection with Mycobacterium tuberculosis, C57BL / 6J mice were subcutaneously immunized for 10 6 CFU of recombinant BCG vaccine, set up control group PBS group and BCG group. After 10 weeks (short-term) and 20 weeks (long-term) of immunization, mice were infected with Mycobacterium tuberculosis H37Rv through an aerosol generator via the respiratory route (lung bacterial load ≈ 400 CFU). Four weeks after infection, the bacterial load in the lungs and spleens of mice and the pathological changes in the lungs of mice were evaluated. (e.g. Figure 7 ) Compared with the PBS group, the bacterial load in the lungs and spleens of mice in rBCG::A1D4 and rBCG::CMFO groups decreased significantly at 10 and 20 weeks. Compared with the BCG group, at the 10-week time point, the bacterial load in the lungs and spleens of mice in the rBCG::A1D4 and rBCG::CMFO groups was similar to that of BCG. However, as the immunization time prolonged, the bacterial load in the lungs and spleens of mice in the BCG group increased over time. However, the bacterial load in the lungs and spleens of mice in the rBCG::A1D4 group further decreased over time, producing stronger protection than BCG at long-term time points. The bacterial load in the lungs of mice in the rBCG::CMFO group also showed a downward trend over time, but the bacterial load in the spleen increased significantly over time, exceeding the bacterial load in the spleens of mice in the BCG group during the same period.

[0086] At the same time (such as Figure 8 and Fig. 9) There were also significant differences in the lung pathological changes of mice. The lungs of mice in the PBS group showed severe multiple nodules and large-area inflammatory cell infiltration, and a large number of Mycobacterium tuberculosis growth was observed after acid-fast staining. The BCG group showed significant improvement compared with the PBS group. Compared with the BCG group, the lung inflammation response of mice in the rBCG::A1D4 group and the rBCG::CMFO group was alleviated to a certain extent, among which the rBCG::A1D4 group showed further relief, with normal alveolar structure and limited inflammation area. Therefore, among the candidate recombinant BCG vaccines, rBCG::A1D4 has better protection.

[0087] Example 6: Antigen-specific immunogenicity of recombinant BCG rBCG::A1D4

[0088] SPF grade C57BL / 6 female mice were used. They were 6-8 weeks old and weighed 18-20 g. They were divided into PBS control group, BCG control group, and candidate vaccine rBCG::A1D4 immunization group. C57BL / 6J mice were subcutaneously immunized for 10 6 CFU vaccine, 10 weeks (short-term) and 20 weeks (long-term) after immunization, the lungs and spleens of mice were taken to prepare single cell suspensions. Mouse cells were plated and stimulated with A1D4 for 12 hours, and the cells were surface stained and intracellularly stained using fluorescent antibodies. Flow cytometry was used to analyze the levels of antigen-specific T cells in the lungs and spleens of mice.

[0089] Mouse spleen cells were plated and stimulated with A1D4 for 12 hours before color development using an ELISPOT kit. The number of antigen-specific IFN-γT cell spots in the mouse spleen was analyzed using an ELISPOT spot counter.

[0090] Flow cytometry analysis of mouse lungs (eg Fig.10 ) and mouse spleen (e.g. Fig.11 ) antigen-specific T cell types and trends. In the mouse lungs, compared with the BCG group, the rBCG::A1D4 group had a significant advantage in most immune cells. Among them, lung IFN-γ + CD4, IL2 + CD4, TNF + CD4, IL2 + CD8, IFN-γ + CD8 T EM and IL2 + CD8 T CM cells, and IFN-γ in mouse spleen + CD4, IL2 + CD4 T CM , IFN-γ +CD8 showed a trend of further increase over time, reflecting that the recombinant BCG rBCG::A1D4 induced a stronger antigen-specific T cell response. This difference was further verified by ELISPOT spot counts of mouse spleen cells (e.g. Fig.12 The spleens of mice in the rBCG::A1D4 group produced more antigen-specific IFN-γT cell spots compared with those in the PBS and BCG groups, confirming that rBCG::A1D4 has great potential as a candidate vaccine to replace BCG.

[0091] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant shuttle plasmid, characterized in that: The recombinant shuttle plasmid is an Escherichia coli-Mycobacterium shuttle plasmid in which the Rv1813 gene sequence, Rv2660c gene sequence, Ag85B gene sequence, Rv2623 gene sequence and HspX gene sequence are inserted, wherein the Rv1813 gene sequence is SEQ ID NO: 1, the Rv2660c gene sequence is SEQ ID NO: 2, the Ag85B gene sequence is SEQ ID NO: 3, the Rv2623 gene sequence is SEQ ID NO: 4, and the HspX gene sequence is SEQ ID NO:

5.

2. The recombinant shuttle plasmid according to claim 1, characterized in that: The Rv1813 gene sequence, Rv2660c gene sequence, Ag85B gene sequence, Rv2623 gene sequence and HspX gene sequence are connected in series according to Rv1813-Rv2660c-Ag85B-Rv2623-HspX.

3. The recombinant shuttle plasmid according to claim 1, characterized in that: The E. coli-Mycobacterium shuttle plasmid is pSMT3, pMV206, pMV261, pMV306 or pMV361.

4. A recombinant BCG vaccine, characterized in that: The recombinant shuttle plasmid according to any one of claims 1 to 3 is transformed into BCG.

5. The recombinant BCG vaccine according to claim 4, characterized in that The BCG vaccine is a BCG Chinese strain, a BCG Pasteur strain, a BCG Danish strain, a BCG Copenhagen strain, a BCG Japanese strain, a BCG Tice strain or a BCG Russian strain.

6. The recombinant BCG vaccine according to claim 4 or 5, characterized in that The conversion is electro-conversion.

7. Use of the recombinant BCG vaccine as described in any one of claims 4 to 6 for preparing a vaccine with a longer protection period for immune prevention against tuberculosis.

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