WO2010127492A1 - Recombinant bcg vaccine rbcg::xb - Google Patents

Abstract

Recombinant BCG vaccine rBCG::XB capable of overexpressing HspX and Ag85B simultaneously is prepared by the method comprising following steps: amplifying the whole genes of antigen HspX (acr,Rv2031c) and Ag85B (fbpB,Rv1886c), and then constructing a recombinant Escherichia coli-Mycobacterium shuttle expression plasmid containing these genes by subcloning, followed by transforming BCG vaccine with the shuttle expression plasmid. Compared with the traditional BCG vaccine, recombinant BCG vaccine rBCG::XB enables overexpression of HspX in the vaccine's bacterial lysate at high abundance and overexpression of Ag85B in the vaccine's bacterial lysate and culture filtrate at high abundance. After immunizing animals with rBCG::XB, significant cellular immunoreactions against proteins HspX and Asg85B can be induced individually, thus stable and long time protection from infection are formed.

Description

 Recombinant BCG RBCG: :XB

Technical field

 The invention belongs to the field of genetic engineering vaccines and tuberculosis vaccines. Specifically, the present invention provides a novel recombinant vaccine against Mycobacterium tuberculosis, which is a gene for HspX (acr, Rv2031c) and Ag85B (fbpB, Rvl886c) of Mycobacterium tuberculosis and/or BCG, simultaneously cloned into BCG, and Overexpression in BCG, formation of recombinant BCG rBCG: :XB.

Background technique

 Tuberculosis (TB) is one of the major public health issues that seriously affects human health worldwide. In the past decade or so, the incidence of TB has continued to rise, with approximately 2 million people dying each year due to TB. The number of people infected with Mycobacterium tuberculosis in China reached 550 million, and the number of patients with tuberculosis and the number of deaths were the highest among infectious diseases.

 B. m. BCG (BCG) is the only vaccine used clinically to prevent tuberculosis in infants and young children. It was attenuated in 1921 by repeated subculture of M. tuberculosis (M. ^νώ) in vitro. Live vaccines are currently used in 161 countries and territories around the world, with 90% of infants vaccinated every year worldwide. Clinical studies have confirmed that BCG is safe, inexpensive, and can prevent infants and young children from severe tuberculosis; but BCG has a short protection period of only 5-10 years; BCG has an unstable prevention effect on adult tuberculosis, the effect is between 0-80 Between %; can not prevent the recurrence of latent bacteria in the body. Therefore, researching more effective vaccines to replace existing BCG is an important and priority research direction in the field of tuberculosis control. The development of recombinant BCG is the most important and practical development strategy.

At present, strategies for the development of recombinant BCG vaccine at home and abroad mainly include: overexpression of cytokines such as IL-2, IL-4, IL-6, IL-10, IL-11, IL-13, IFN-γ, GM-CSF and TNF. -a et al; express importance Immunodominant antigens, such as Ag85A, Ag85B, 38kDa, 19kDa, ESAT-6, Ag85B-ESAT-6, ESAT-6-CFP10 (or RD1 region), Ag85B-MPT64-MTB8.4, etc.; expression of cytolysin; or expression Cytokine and antigen chimeric proteins, such as IL-2-ESAT-6, Ag85B-ESAT-6-IFN-y and the like. It has been confirmed that BCG expression of Ag85B (rBCG30, University of California, USA) and BCG expression cytolysin (Marne Blanche Institute of Infectious Diseases, Berlin, Germany) are more protective than BCG in immunized animals and entered I in 2005 and 2007 respectively. Clinical trials. However, there have been no reports or patent applications for the overexpression of HspX and Ag85B proteins of Mycobacterium tuberculosis and/or BCG in BCG.

Summary of the invention

 One of the objects of the present invention is to provide a novel recombinant plasmid;

 A second object of the present invention is to provide a vaccine prepared from the above novel recombinant plasmid; and a third object of the present invention is to provide a novel vaccine preparation method.

 The recombinant plasmid provided by the present invention replaces the promoter in the Escherichia coli-mycobacterial shuttle plasmid with a gene sequence encoding the HspX protein, and inserts the coding into the multiple cloning site in the Escherichia coli-mycobacterial shuttle plasmid. The genetic sequence of the Ag85B protein.

 When the E. coli-mycobacterial shuttle plasmid is PMV261, the gene sequence encoding the HspX protein replaces the promoter PHsp60 in PMV261, and the gene sequence encoding the Ag85B protein is located at the cleavage sites BamlU and Hindm. between.

The recombinant BCG rBCG _{: :} XB provided by the present invention is composed of the above recombinant plasmid and transformed into BCG. The BCG vaccine may be any BCG strain currently used for clinical immunization, such as BCG Pasteur, BCG, Danish BCG, BCG, Bacillus Ticensis, BCG, and BCG.

The preparation method of the recombinant BCG rBCG _{: :} XB provided by the invention comprises the following steps: (1) amplifying or artificially synthesizing a gene encoding an HspX protein and a gene encoding an Ag85B protein;

 (2) constructing a recombinant Escherichia coli-mycobacterial shuttle plasmid comprising a gene encoding a HspX protein and a gene encoding an Ag85B protein;

(3) The recombinant Escherichia coli-mycobacterial shuttle plasmid constructed in the step (2) is transformed into BCG to obtain the recombinant BCG vaccine of the present invention! "BCG _{: :} XB.

 The BCG vaccine used in the above method may be any BCG strain currently used for clinical immunization, such as BCG Pasteur, BCG, Danish BCG, BCG, Bacillus Tice, and BCG.

 The gene encoding the HspX protein and the gene encoding the Ag85B protein of the present invention are derived from different strains of Mycobacterium tuberculosis, Bacillus subtilis strains or obtained by artificial synthesis.

 The E. coli-mycobacterial shuttle plasmid used in the present invention may include, but is not limited to, pSMT3, pMV206, pMV261, pMV306, pMV361. The role of the E. coli-Mycobacterium shuttle plasmid is to carry the genes of exogenous HspX and Ag85B proteins into BCG, and further utilize the ability of E. coli-mycobacterial shuttle plasmid to replicate in BCG, ultimately benefiting HspX and Ag85B. The protein is also overexpressed at high abundance in BCG.

 One of the objects of the present invention is to provide a novel recombinant plasmid in which the genes of HspX and Ag85B proteins are cloned into the same Escherichia coli-mycobacterial shuttle plasmid to construct a new recombinant plasmid.

The recombinant plasmid provided by the present invention is characterized in that the gene sequence encoding the HspX protein comprises a HspX self-promoter sequence, a coding sequence and a regulatory sequence, which can direct the overexpression of the HspX protein; the gene sequence encoding the Ag85B protein comprises the Ag85B self-promoter. Sequence, secretion signal peptide, The coding sequence and regulatory sequences can direct the overexpression of Ag85B protein. Overexpression of HspX and Ag85B in BCG is not affected by the promoter and regulatory sequences carried by the E. coli-Mycobacterium shuttle plasmid due to the promoter and regulatory sequences of the respective proteins themselves; at the same time overexpressing HspX and Ag85B The nature of the two proteins is identical to the properties of the two proteins expressed by BCG itself. The genes inserted by HspX and Ag85B are not oriented in the construction of recombinant plasmids.

 In one embodiment of the present invention, the full-length sequences of the genes encoding the Mycobacterium tuberculosis HspX and Ag85B proteins were cloned, inserted into the E. coli-mycobacterial shuttle plasmid PMV261, and replaced with heat shock protein promoter in pMV261. Sub Hsp60. Since the heat shock protein promoter Hsp60 in pMV261 was replaced, it was verified that the genes of these two proteins could be non-directionally inserted, have self-promoter and self-regulated expression.

 The gene reference sequence of the gene encoding the HspX and Ag85B proteins in the recombinant plasmid of the present invention is derived from the NIH GenBank public database of the United States. Through the Blast homology comparison, the coding genes of Mycobacterium tuberculosis HspX and Ag85B proteins are identical to the coding sequences of the corresponding genes of BCG. Therefore, the strategy for the source of the gene encoding the HspX and Ag85B proteins can be amplified from the genome of the following strains by designing primers, which can be different strains of Mycobacterium tuberculosis, such as H37Rv, H37Ra, Beijing strain or Clinical isolates, etc., or different strains of Mycobacterium tuberculosis and BCG. The genes encoding the HspX and Ag85B proteins derived from these strains are identical. The genes encoding the HspX and Ag85B proteins can also be referred to by the GenBank public database by artificial gene synthesis. In one embodiment of the invention, it is obtained from genomic amplification of the Mycobacterium tuberculosis H37Rv strain.

Escherichia coli-mycobacterial shuttle plasmid for selection of recombinant plasmid of the present invention Such as pSMT3, pMV206, pMV261, pMV306, pMV361 and so on. The E. coli-Mycobacterium shuttle plasmid functions to carry the gene encoding the exogenous HspX and Ag85B proteins into BCG, and further utilizes the ability of the E. coli-mycobacterial shuttle plasmid to replicate in BCG, ultimately benefiting HspX and Ag85B. At the same time over-expression in BCG. In one embodiment of the present project, the E. coli-mycobacterial shuttle plasmid used is pMV261.

Another object of the present invention is to provide a novel vaccine against Mycobacterium tuberculosis, which converts the above recombinant plasmid containing HspX and Ag85B into BCG, thereby obtaining recombinant BCG.

 Mycobacterium tuberculosis Ag85B (βρΒ), the full length of the gene (including promoter, secretion signal peptide, coding sequence and regulatory sequence) is 1.5 kb, belonging to one of the Mycobacterium tuberculosis antigen Ag85 complex components. The anti-Ag85 complex (molecular weight 30-32kDa) is a large amount of protein (culture filtrate protein) secreted by M. tuberculosis during its growth and reproduction, and is a key component that stimulates the body to produce an anti-tuberculosis protective immune response. The antigen Ag85 complex belongs to the cellulose-binding protein family and consists of three closely related proteins: AgS5 A (βρΑ, Rv3804c, 32 kDa), Ag85B (bpB, Rvl 886c, 30 kDa) and Ag85C ifbpCl, Rv3803c, 32.5 kDa). It has mycobacterial acid transferase activity and is involved in the synthesis of cell wall and cord-like factors in bacteria. Proportion of extracellular secretion to the bacteria Ag85A: Ag85B: Ag85C is 3:2: 1. We and other domestic and foreign studies have confirmed that the establishment of recombinant BCG by Ag85B has obtained stronger protection than BCG alone. Therefore, Ag85B protein is one of the most important target antigens for new tuberculosis vaccine research.

Mycobacterium tuberculosis HspX, also known as heat shock protein Hspl6.3, Rv2031c, Acr, alpha-crystallin. Mycobacterium tuberculosis or BCG is low in expression under normal aerobic conditions, and even difficult to detect; a protein that significantly increases expression during the dormancy of Mycobacterium tuberculosis or BCG Quality, while Mycobacterium tuberculosis or BCG is thickened in the process of cell wall formation, which is conducive to the parasitism and long-term latency of bacteria in the body macrophages. Therefore, the HspX protein is considered to be one of the hallmark proteins of Mycobacterium tuberculosis dormancy. The HspX protein consists of 144 amino acids with a molecular weight of 16.3 KD. The HspX protein is highly immunogenic, but does not produce a cellular immune response against HspX after neonatal immunization with BCG. In chronic patients, HspX protein can stimulate the body to produce strong humoral and cellular immune responses. Thl CD4 and CD8 T cells in tuberculosis patients can recognize HspX protein, and after effective treatment, the cellular immune response against HspX protein is changed from ThO to Th1. Mice immunized with the HspX gene can induce a strong Th1 cellular immune response, and the immunized guinea pig can provide infection against Mycobacterium tuberculosis. Therefore, HspX protein is an important T cell immune antigen of Mycobacterium tuberculosis and an important target antigen for the development of anti-tuberculosis vaccine. It is also an important target antigen for the development of recombinant BCG vaccine against tuberculosis. It is difficult to obtain a large amount of HspX protein. In the preliminary work, we established an E. coli genetic engineering system and purification technology that overexpressed HspX protein, and applied for a national invention patent (National Invention Patent Application No. 200710051915.5). At the same time, we established recombinant BCG rBCG _{: :} X, which overexpressed HspX protein alone, and confirmed the significantly enhanced immunoprotection compared with the original BCG. These work laid the foundation for the patent vaccine application.

 Therefore, the antigens HspX and Ag85B are important protective antigens for Mycobacterium tuberculosis. The new recombinant BCG vaccine we have established is based on both HspX and Ag85B proteins.

 The Bacillus strains transformed with the novel recombinant plasmid may be any BCG strains currently used for clinical immunization, such as BCG Pasteur, BCG, Danish BCG, BCG, Bacillus Tice, BCG, and many more.

A novel recombinant BCG rBCG::XB that overexpresses HspX and Ag85B proteins established by the present invention is used to immunize C57BL/6 mice. BCG rBCG::261 (BCG contains empty plasmid PMV261) and PBS served as positive and negative controls. The results show that the novel recombinant BCG rBCG _{: :} XB can provide long-term and stable protection against Mycobacterium tuberculosis infection in immunized mice, and the short-term and long-term protection types are stronger than the original BCG. This enhanced protection has been shown to be associated with increased expression of HspX and Ag85B proteins, respectively, as well as for Bacillus Calmette-Guerin, and for induction of cellular immune responses against HspX and Ag85B proteins.

Still another object of the present invention is to provide a method for preparing the novel recombinant BCG rCGG _{: :} XB described above, which comprises the steps of:

 (1) amplifying or artificially synthesizing the genes of HspX and Ag85B;

 (2) constructing a recombinant E. coli-mycobacterial shuttle plasmid comprising a gene encoding a HspX protein and a gene encoding an Ag85B protein;

(3) The recombinant Escherichia coli-mycobacterial shuttle plasmid constructed in the step (2) is transformed into BCG to obtain the recombinant BCG vaccine of the present invention! "BCG _{: :} XB.

The novel recombinant BCG rBCG _{: :} XB provided by the present invention has the following advantages:

1. Safety is unchanged. Compared with the original BCG, HspX and Ag85B proteins are also proteins secreted by BCG, so the safety of recombinant BCG overexpressing HspX and Ag85B proteins will not change.

 2. The properties of the overexpressed HspX and Ag85B proteins are unchanged. The expression strategies of HspX and Ag85B proteins in recombinant BCG are achieved by using HspX and Ag85B's own promoters and regulatory sequences, respectively, so that the expression of HspX and Ag85B proteins can be fully realized without additional temperature or other specific conditions. The HspX and Ag85B proteins of BCG itself are identical in structure and function.

3. Conducive to human body applications. The expression strategy of HspX and Ag85B proteins in recombinant BCG is By utilizing its own promoter and regulatory sequences, it does not require additional temperature or other specific conditions to induce expression, which is beneficial to the human body application of recombinant BCG.

 4. Expression stability. In the presence of kanamycin antibiotics and the absence of kanamycin antibiotics, rBCG::XB stably expressed HspX and Ag85B proteins, see Figures 2 and 3.

5. The protection effect is strong. At the same time, the novel recombinant BCG rBCG _{: :} XB overexpressing HspX and Ag85B proteins was significantly stronger than the original BCG.

DRAWINGS

 Figure 1 is a structural schematic diagram of the recombinant plasmid pMXAg85B, pMHspX, pMAg85B and the original plasmid pMV261 of the present invention.

 Figure 2 shows the high-expression HspX protein (anti-Rv2031c as a primary antibody) in recombinant BCG raffectin rBCG::XB cell lysates by Western blotting. 1, rBCG:: 261 (BCG empty vector pMV261); 2, rBCG::85B (BCG contains recombinant expression Ag85B plasmid-pMAg85B); 3, rBCG: :X (BCG contains recombinant expression HspX plasmid-pMHspX); The present invention is rBCG::XB (BCG contains the recombinant plasmid pMXAg85B:). Gl is the first generation culture; G4 is the fourth generation culture. The results showed that the rBCG::XB (BCG containing recombinant plasmid pMXAg85B) and rBCG:: compared to rBCG::261 (BCG empty vector pMV261) and rBCG::85B (BCG containing recombinant expression Ag85B plasmid-pMAg85B) X (BCG contains recombinant expression HspX plasmid-pMHspX) The expression level of HspX protein was significantly increased in both bacterial lysate and culture filtrate.

Figure 3 shows Western blotting detection of Ag85B protein (anti-Ag85B Ab as primary antibody:) in recombinant BCG rBCG::XB cell lysate and cell culture filtrate. 1, rBCG:: 261 (BCG empty vector pMV261); 2, rBCG::85B (BCG contains recombinant expression Ag85B plasmid-pMAg85B); 3, rBCG::X (BCG contains recombinant expression HspX plasmid-pMHspX); rBCG::XB (BCG contains recombinant plasmid pMXAg85B). Gl is the first generation culture; G4 is the fourth generation culture. The results showed that the rBCG::XB (BCG contains recombinant plasmid pMXAg85B) and rBCG::85B (BCG contains recombinant expression Ag85B plasmid-pMAg85B), rBCG:: compared to rBCG::261 (BCG empty vector pMV261) X (BCG contains recombinant expression HspX plasmid-pMHspX) significantly increased the expression level of Ag85B protein in the culture filtrate.

Figure 4 shows the immunization of C57BL/6 mice with four vaccines (rBCG::261, rBCG::85B _; rBCG::X; rBCG::XB) for 6 weeks (short-term:), ELISPOT analysis of secreted antigen-specific IFN - γ changes in the number of spleen cells.

Figure 5 shows the immunization of C57BL/6 mice with four vaccines (rBCG::261, rBCG::85B _; rBCG::X; rBCG::XB) for 24 weeks (long-term), ELISPOT analysis of secreted antigen-specific IFN- The number of gamma splenocytes changes.

Figure 6 shows that the BALB/c and C57BL/6 mice were immunized with four vaccines (rBCG:: 261, rBCG::85B _; rBCG::X; rBCG::XB) for 6 weeks, and the tail vein was infected with 10 ⁶ CFU of nodules. After 4 weeks, the bacterial load of the lungs of different mice was observed. * P < 0.05 vs. rBCG: :261; * * P < 0.05 vs. rBCG: :XB.

Figure 7 shows that the BALB/c and C57BL/6 mice were immunized with four vaccines (rBCG::261, rBCG::85B _; rBCG::X; rBCG::XB) for 6 weeks, and the tail vein was infected with 10 ⁶ CFU of nodules. After 4 weeks, the bacterial load of the spleen of different kinds of mice was observed. * P < 0.05 vs. rBCG: :261; * * P < 0.05 vs. rBCG: :XB.

Figure 8 uses four vaccines (rBCG:: 261, rBCG::85B _; rBCG::X; rBCG::XB) for 6 weeks after immunization of C57BL/6 mice, and 10 ⁶ CFU of Mycobacterium tuberculosis in the tail vein for 4 weeks. Changes in bacterial loads in the lungs of mice at different time points after 10 and 18 weeks. Figure 9 uses four vaccines (rBCG:: 261, rBCG::85B; rBCG::X; rBCG::XB) to immunize C57BL/6 mice for 6 weeks, and the tail vein is infected with 10 ⁶ CFU of Mycobacterium tuberculosis for 4 weeks. Changes in bacterial loads of spleens of mice at different time points after 10 and 18 weeks. detailed description

 Example 1

 Construction and identification of recombinant plasmid pMXAg85B

Molecular biology techniques were routinely performed: 0.8 kb of HspX (acr, Rv2031c) and 1.5 kb of Ag85B (fbpB, Rvl886c) genes, which were amplified from the genome of Mycobacterium tuberculosis H37Rv by PCR, respectively. The amplification conditions were: 95 °C for 5 min, then 94 °C for 45 s, 60 °C for 45 s, 72 °C for 50 s, 30 cycles, and finally 72 °C for 5 min; 95 °C for 5 min, then 94 °C for lmin, 60 ° C lmin, 72 ° C for 2 min, 30 cycles, and finally 72 ° C for 10 min. The PCR product was recovered using the AxyPrep PCR Product Recovery Kit (Axygen). The 0.8 kb acr gene was digested with BamHI and Xbal, and the 1.5 kb fbpB gene was digested with BamHI and Hindlll and recovered using the AxyPrep DNA Gel Recovery Kit (Axygen). Then the acr gene was ligated with pcDNA3.10 which was similarly digested; the fbpB gene was ligated with pcDNA3.1(+) which was similarly digested; acr and fbpB were subcloned into the same Escherichia coli-mycobacterial shuttle plasmid pMV261 alone or sequentially. , recombinant plasmids pMHspX and pMAg85B, pMXAg85B were formed, respectively. Further restriction enzyme digestion and sequence analysis confirmed that the constructed recombinant expression plasmid was completely correct. The sequencing results showed that the cloned acr and fbpB were identical to the coding sequences of the corresponding genes in the genome-wide sequence of Mycobacterium tuberculosis and Mycobacterium bovis, respectively, published in NIH GenBanK. The E. coli-mycobacterial shuttle recombinant plasmid pMXAg85B expressing both Ag85B and HspX, the empty vector pMV261 and the plasmid -pMAg85B expressing Ag85B alone, The structural pattern of the plasmid-pMHspX expressing HspX is shown in Figure 1.

 Example 2

 Recombination of BCG to establish rBCG::XB

Recombinant BCG rBCG _{: :} XB was prepared as follows. First, the competent state of BCG was prepared. The BCG strain lml in logarithmic growth phase was aseptically inoculated into 50 ml of 7H9 liquid medium, and cultured at 37 ° C for 2 weeks. After the medium was ice bathed for 2 hours, the bacteria were collected by centrifugation at 4 °C. Resuspend with 1 ml of 10% ice-cold glycerin, and disperse the bacteria by a stick grinder. After that, it was washed 3 times with the volume of 1/2, 1/10 and 1/50 of the original culture volume, and finally resuspended with 1 ml of ice-cold glycerin, and dispensed into ΙΟΟμΙ each tube, and stored at -80 °C for use. Followed by the electrical conversion of BCG. The ΙΟΟμΙ competent BCG strain was firstly added to a pre-cooled 2mm Bio-Rad electroporation cup, and the purified recombinant plasmid pMXAg85B (less than 5μ1) was added and mixed, and the ice bath was removed for 10 minutes to remove the bubbles in the cup and the water outside the cup. Beads, electrically pierced with a Bio-Rad electroporator. The electrical wear parameters are: voltage 2kv, 25 F, 1000 Ω conversion. The time constant is between 15-25ms. Immediately after the completion of the transformation, the bacteria were transferred to 10 ml of 7H9 liquid medium, and shaken overnight at 37 ° C; the bacteria were collected by centrifugation the next day, and plated on a 7H11 solid plate containing 25 g/ml kanamycin. After 4 weeks of culture at 37 °C, the resistant growth clones were picked and inoculated into 7H9 liquid medium (containing 25 g/ml kanamycin:). After expanding for 4 weeks at 37 °C, the bacteria and supernatant were collected by centrifugation. Western Blotting was performed. The primary antibody was an anti-Rv2031c murine monoclonal antibody (ab64786, Abeam) and a rabbit anti-Ag85B polyclonal antibody (ab43019, Abeam), which were developed by chemiluminescence. The results confirmed that the bacterial lysate had 16kDa protein (HspX) expressed in recombinant BCG (results shown in Figure 2); while in the bacterial lysate and supernatant, there were protein-specific expression of molecular weight of about 30kDa (Ag85B), but Ag85B protein was mainly Secreted expression in bacterial culture supernatant (results shown in Figure 3); and recombinant vaccine rBCG _{: :} XB simultaneously overexpressed The amount of HspX and Ag85B protein was significantly greater than the amount of the two proteins expressed by the original BCG. At the same time, subculture was carried out four times without antibiotics, and the results were compared with the first generation products to evaluate the stability of expression. Recombinant BCG containing empty plasmid pMV261 (rBCG::261), expressing HspX (rBCG::X) alone, and expressing Ag85B (rBCG::85B) alone was prepared as above and used as an experimental control.

 Example 3

 Cellular immune characteristics of recombinant BCG vaccine rBCG::XB

C57BL/6 mice were immunized with 10 ⁶ CFU recombinant vaccine rBCG::XB, rBCG::85B, rBCG::X, rBCG::261, and PBS was used as a control. Six weeks and 24 weeks after immunization of the mice, the spleen lymphocytes of the immunized mice were aseptically isolated, and the secretion number of spleen lymphocyte antigen-specific IFN-γ cells was detected by ELISPOT technique. Antigens include PPD, HspX and Ag85B proteins with an antigen concentration of 2 g/ml and a cell count of 10 ⁶ . The recombinant vaccine rBCG::XB induced an increase in the secretion of IFN-γ cells specific for HspX and Ag85B proteins in the short-term (see Figure 4) and long-term (see Figure 5) spleen lymphocytes.

Example 4: Recombinant BCG rBCG _{: :} Short-term and long-term protection of XB

In order to evaluate the effect of the murine line on the immune effect, 10 ⁶ CFU recombinant vaccine will be given separately! "BCG _{: :} XB, rBCG::85B, rBCG::X and rBCG::261, intradermally immunized BALB/c and C57BL/6 mice, using PBS as control. After 6 weeks of immunization, use 10 ⁶ CFU of Mycobacterium tuberculosis immunized intravenously infected mice, after 4 weeks, the different mice lungs (see FIG. 6) and the number of bacterial load in spleen (see FIG. 7) of the (bacterial loads) ₀ recombinant vaccine _rBCG:: XB are generated in both mice The strong protection of the lungs and spleen, its enhanced protection from the mouse strain, its protection is the strongest among the four vaccines.

To further evaluate the short-term and long-term protective effects of recombinant BCG rBCG _{: :} XB, 10 ⁶ CFU recombinant vaccine rBCG::XB, rBCG::85B, rBCG::X and rBCG::261 C57BL/6 mice were treated with PBS as a control. After 6 weeks of immunization, the immunized mice were infected with 10 ⁶ CFU of Mycobacterium tuberculosis tail vein, and the bacterial load of the mouse lung (see Figure 8) and the spleen (see Figure 9) after 4 weeks, 10 weeks and 18 weeks (bacterial loads) ). Recombinant BCG rBCG::XB is short-term protective against the lungs of immunized mice, and provides long-term protection against the lungs, which is the most protective of the four vaccines.

Claims

Claim  A recombinant plasmid characterized in that it replaces a promoter in an Escherichia coli-mycobacterial shuttle plasmid with a gene sequence encoding a HspX protein, and a multiple cloning site in the Escherichia coli-mycobacterial shuttle plasmid The gene sequence encoding the Ag85B protein was inserted.  The recombinant plasmid according to claim 1, wherein the Escherichia coli-mycobacterial shuttle plasmid is pMV261, and the gene sequence encoding the HspX protein replaces the promoter PHsp60 in PMV261, The gene sequence encoding the Ag85B protein is located between the cleavage sites BamlU and Hindm.  A recombinant BCG rBCG::XB, which is characterized in that it is transformed into BCG by the recombinant plasmid of claim 1 or 2. 4. The recombinant BCG rBCG _:: XB according to claim 3, wherein the BCG vaccine can be any BCG strain currently used for clinical immunization.  The recombinant plasmid according to claim 1 or 2, wherein the gene encoding the HspX protein and the gene encoding the Ag85B protein are derived from different strains of Mycobacterium tuberculosis, Bacillus Calmette-Guerin strains or obtained by artificial synthesis.  The recombinant plasmid according to claim 1, wherein the Escherichia coli-mycobacterial shuttle plasmid used may include, but is not limited to, pSMT3, pMV206, pMV261, pMV306, pMV361 o The recombinant plasmid according to claim 1 or 2, wherein the gene sequence encoding the HspX protein comprises a HspX self-promoter sequence, a coding sequence and a regulatory sequence; and the gene sequence encoding the Ag85B protein comprises an Ag85B self-promoter sequence, Secretion of signal peptides, coding sequences and regulatory sequences. 8. A method for preparing a recombinant BCG rBCG _{: :} XB, comprising the steps of:  (1). Amplification or artificial synthesis of a gene encoding the HspX protein and a gene encoding the Ag85B protein (2) Constructing a recombinant Enterobacteriaceae-mycobacterial shuttle plasmid comprising a gene encoding a HspX protein and a gene encoding an Ag85B protein; (3) The recombinant Escherichia coli-mycobacterial shuttle plasmid constructed in the step (2) is transformed into a BCG to obtain the recombinant BCG rBCG _{: :} XB of the present invention.  9. The preparation method according to claim 8, wherein the carcass used in the method is any BCG strain currently used for clinical immunization.