Mycobacterium tuberculosis tandem DNA vaccine W543 as well as preparation method and application thereof

Abstract

The invention discloses a mycobacterium tuberculosis tandem DNA vaccine W543 as well as a preparation method and application thereof. The DNA vaccine W543 provided by the invention is a novel mycobacterium tuberculosis DNA vaccine with multiple antigen immunodominance epitopes, which is constructed by connecting polypeptides or epitope coding genes of a mycobacterium tuberculosis proliferation period antigen and a latent related antigen in series through a linker by virtue of genetic engineering and inserting the polypeptides or epitope coding genes into an eukaryotic expression vector pVAX1. Experiments prove that the mycobacterium tuberculosis specific tandem DNA vaccine W543 disclosed by the invention is used as an immunomodulatory preparation, mainly stimulates immune response of Th1 type, and can significantly enhance the specific cellular immune function of mice; as a preventive treatment preparation, the bacterial count of lung and liver tissues can be reduced, the lesion range of the lung tissues is remarkably reduced, the lesion is relieved, and the treatment effect is slightly superior to that of an existing commercial tuberculosis latent infection prevention and treatment preparation, namely, microkalidium vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of tuberculosis medical immunology prevention and treatment, and specifically to a Mycobacterium tuberculosis specific tandem DNA vaccine W543 and a preparation method and application thereof, and particularly to a novel Mycobacterium tuberculosis specific tandem DNA vaccine W543 prepared by genetic engineering technology and an application thereof. Background Art

[0002] Tuberculosis (TB) is a chronic respiratory infectious disease that is full of challenges in terms of infection, immunity, prevention and treatment. According to the World Health Organization (WHO) report, there will be 10.6 million new TB patients in the world in 2022, and about 1.3 million patients will die from TB. Mycobacterium tuberculosis (MTB) is a facultative intracellular parasite that can interfere with the normal function of host cells to escape immune surveillance and clearance, and survive in host cells for a long time. About a quarter of the world's population is latently infected with tuberculosis (LTBI). The LTBI population in my country accounts for about 20% of the population, and the LTBI population is the main source of new TB (accounting for 85%-90%). When their immunity is reduced, such as combined with AIDS or diabetes, it is easy to develop active TB. Reasonable and regular chemotherapy can generally kill most of the tuberculosis bacteria in the lesions within 1 to 2 months, but there are still a small number of bacteria remaining, especially non-replicating tuberculosis bacteria retained in macrophages are not easy to kill, and treatment needs to be continued for 3 to 4 months, or even longer. At present, more than a dozen anti-tuberculosis drugs have been used clinically for decades. The chemotherapy regimen for the initial treatment of tuberculosis usually requires 3-4 drugs for 6 months, while retreatment or drug-resistant tuberculosis usually requires 5-6 drugs for more than 6-9 months. In addition to the toxic side effects of the drugs, second-line drugs are expensive and some patients find it difficult to stick to them. The development of new anti-tuberculosis drugs is not only more difficult than the development of new therapeutic vaccines, but also more difficult than the development of new therapeutic vaccines. New drugs may also quickly develop drug resistance. Vaccine treatment only requires a few injections in a few months, which is much more convenient than taking medicine every day, with fewer side effects and low cost. Anti-tuberculosis immunity is mainly a cell-mediated immune response. Vaccine treatment can achieve the purpose of treating the disease by regulating or selectively inducing the potential of the immune system of tuberculosis patients. Therefore, in recent years, the prevention and treatment of tuberculosis through immune regulation has become one of the research hotspots, and the research and development of new tuberculosis vaccines has become a very important research direction.

[0003] At present, the new tuberculosis candidate vaccines being studied at home and abroad mainly fall into the following five categories: inactivated vaccines, subunit vaccines, viral vector vaccines, live attenuated mycobacterium vaccines and nucleic acid vaccines. DNA vaccine is a new vaccine technology developed in the 1990s and is the third revolution in the history of vaccine development. DNA vaccine is to directly introduce foreign genes encoding a certain antigen protein into the body's cells, synthesize the antigen protein through the expression system of the host cell, and induce the host to produce an immune response to the antigen protein to achieve the purpose of preventing and treating the disease. The number of research reports on the use of DNA vaccines to prevent or treat human and animal diseases has gradually increased, and it has become one of the hot spots in the field of vaccine research. It can be used as a booster vaccine for BCG or as a therapeutic vaccine. Compared with other types of vaccines, DNA vaccines have the following advantages: (1) They can induce a comprehensive immune response: they can activate both humoral and cellular immune responses, especially cytotoxic T lymphocyte (CTL) responses, which can identify, kill, and destroy infected cells and eliminate pathogens within cells. This is an effective elimination pathway for killing Mycobacterium tuberculosis parasitic in macrophages, which can make up for the weak CTL response induced by BCG, which is unmatched by recombinant protein subunit vaccines and inactivated tuberculosis vaccines; (2) They can overcome the problem of protein subunit vaccines being prone to misfolding and incomplete glycosylation; (3) The immune effect of vaccines can be improved by mixing plasmids expressing multiple antigens or constructing complex plasmids that can simultaneously express multiple antigen epitopes to prepare multivalent vaccines. (4) A small amount of DNA can produce a long-lasting immune response after several inoculations. (5) DNA does not have the infectivity and replication of bacteria, and is safer than live attenuated vaccines. In addition, DNA vaccines only encode beneficial proteins, which can avoid harmful antigens and reduce the occurrence of side effects. (6) It is easy to prepare and can produce high-purity and stable DNA vaccines on a large scale with low production costs. (7) The plasmid DNA of DNA vaccines is stable, easy to monitor quality, does not require refrigeration, and is easy to store and transport. In addition, DNA vaccines are simple to prepare and use, do not require any chemical carriers, and are safer than BCG for immunocompromised people.

[0004] Although a variety of DNA vaccines for the preventive treatment of latent tuberculosis infection have been reported, their protective efficacy and therapeutic effects still need to be further improved. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to induce rapid and effective cellular immune response to different stages of Mycobacterium tuberculosis infection, inhibit endogenous recurrence of Mycobacterium tuberculosis, and inhibit or eliminate Mycobacterium tuberculosis in the body.

[0006] In order to solve the above technical problems, the present invention provides a Mycobacterium tuberculosis-specific tandem DNA vaccine W543 for preventive treatment of latent tuberculosis infection and its related biomaterials and applications.

[0007] In a first aspect, the present invention claims a Mycobacterium tuberculosis DNA vaccine.

[0008] The active ingredient of the Mycobacterium tuberculosis DNA vaccine claimed in the present invention is a recombinant eukaryotic cell expression vector, which includes a gene sequence encoding an antigenic epitope A, a gene sequence encoding an antigenic epitope B, a gene sequence encoding an antigenic epitope C, a gene sequence encoding an antigenic epitope D, a gene sequence encoding an antigenic epitope E, a gene sequence encoding an antigenic epitope F, a gene sequence encoding an antigenic epitope G, a gene sequence encoding an antigenic epitope H, a gene sequence encoding an antigenic epitope I, a gene sequence encoding an antigenic epitope J, a gene sequence encoding an antigenic epitope K, a gene sequence encoding an antigenic epitope L, a gene sequence encoding an antigenic epitope M, a gene sequence encoding an antigenic epitope N, a gene sequence encoding an antigenic epitope O, and a gene sequence encoding an antigenic epitope P;

[0009] The amino acid sequence of the antigen epitope A is shown in sequence 1, positions 3-291 in the sequence listing;

[0010] The amino acid sequence of the antigenic epitope B is shown in sequence 1 at positions 299-313 in the sequence listing;

[0011] The amino acid sequence of the antigen epitope C is shown in sequence 1 at positions 319-327 in the sequence list;

[0012] The amino acid sequence of the antigen epitope D is shown in sequence 1 at positions 333-368 in the sequence list;

[0013] The amino acid sequence of the antigenic epitope E is shown in sequence 1 at positions 374-393 in the sequence list;

[0014] The amino acid sequence of the antigen epitope F is shown in sequence 1 at positions 399-407 in the sequence list;

[0015] The amino acid sequence of the antigen epitope G is shown in sequence 1, positions 413-426 in the sequence list;

[0016] The amino acid sequence of the antigenic epitope H is shown in sequence 1, positions 432-449 in the sequence listing;

[0017] The amino acid sequence of the antigenic epitope I is shown in sequence 1, positions 455-469 in the sequence listing;

[0018] The amino acid sequence of the antigen epitope J is shown in sequence 1, positions 475-510 in the sequence listing;

[0019] The amino acid sequence of the antigen epitope K is shown in sequence 1 at positions 518-641 in the sequence list;

[0020] The amino acid sequence of the antigen epitope L is shown in sequence 1, positions 649-662 in the sequence list;

[0021] The amino acid sequence of the antigen epitope M is shown in sequence 1 at positions 668-680 in the sequence list;

[0022] The amino acid sequence of the antigen epitope N is shown in sequence 1, positions 686-700 in the sequence listing;

[0023] The amino acid sequence of the antigenic epitope O is shown in positions 706-717 of sequence 1 in the sequence list;

[0024] The amino acid sequence of the antigen epitope P is shown in the sequence 1 at positions 723-742 in the sequence listing.

[0025] Furthermore, the coding gene sequence of the antigen epitope A is shown in sequence 2, positions 22-888 in the sequence list;

[0026] The coding gene sequence of the antigen epitope B is shown in sequence 2, positions 910-954 in the sequence list;

[0027] The coding gene sequence of the antigen epitope C is shown in sequence 2, positions 970-996 in the sequence list;

[0028] The coding gene sequence of the antigen epitope D is shown in sequence 2, positions 1012-1119 in the sequence list;

[0029] The coding gene sequence of the antigenic epitope E is shown in sequence 2, positions 1135-1194 in the sequence list;

[0030] The coding gene sequence of the antigen epitope F is shown in sequence 2, positions 1210-1236 in the sequence list;

[0031] The coding gene sequence of the antigen epitope G is shown in sequence 2, positions 1252-1293 in the sequence list;

[0032] The coding gene sequence of the antigen epitope H is shown in sequence 2, positions 1309-1362 in the sequence list;

[0033] The coding gene sequence of the antigen epitope I is shown in sequence 2, positions 1378-1422 in the sequence list;

[0034] The coding gene sequence of the antigen epitope J is shown in sequence 2, positions 1438-1545 in the sequence list;

[0035] The coding gene sequence of the antigen epitope K is shown in sequence 2, positions 1567-1938 in the sequence list;

[0036] The coding gene sequence of the antigen epitope L is shown in sequence 2, positions 1960-2001 in the sequence list;

[0037] The coding gene sequence of the antigen epitope M is shown in sequence 2, positions 2017-2055 in the sequence list;

[0038] The coding gene sequence of the antigen epitope N is shown in sequence 2, positions 2071-2115 in the sequence list;

[0039] The coding gene sequence of the antigenic epitope O is shown in sequence 2, positions 2131-2166 in the sequence list;

[0040] The coding gene sequence of the antigen epitope P is shown in sequence 2, positions 2182-2241 in the sequence list.

[0041] Furthermore, the recombinant eukaryotic cell expression vector expresses a fusion protein; the amino acid sequence of the fusion protein is any one of the following P1) or P2):

[0042] P1) The amino acid sequence is the protein shown in SEQ ID NO:1;

[0043] P2) A fusion protein having the same function is obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1.

[0044] Furthermore, the recombinant eukaryotic cell expression vector is a vector obtained by inserting the DNA molecule shown in positions 7-2244 of sequence 2 in the sequence list into the restriction site of the eukaryotic expression vector.

[0045] In a specific embodiment of the present invention, the recombinant eukaryotic cell expression vector is a vector obtained by inserting the DNA molecule shown in positions 7-2244 of Sequence 2 in the sequence listing into the Nhe I and EcoR I restriction sites of the eukaryotic expression vector pVAX1.

[0046] In a second aspect, the present invention claims protection for any of the following biological materials:

[0047] Q1) the above fusion protein;

[0048] Q2) a nucleic acid molecule encoding the above fusion protein;

[0049] Q3) an expression cassette containing a nucleic acid molecule encoding the above fusion protein;

[0050] Q4) A recombinant vector containing a nucleic acid molecule encoding the above fusion protein.

[0051] In the above fusion protein, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0052] The above nucleic acid molecule is shown in the 7th to 2244th positions of sequence 2 in the sequence listing or in the 16th to 2244th positions of sequence 2 in the sequence listing.

[0053] The expression cassette may include a promoter, the nucleic acid molecule and a terminator. Further, the expression cassette may also include an enhancer sequence.

[0054] The above-mentioned vector refers to a eukaryotic cell expression vector that can carry the above-mentioned nucleic acid molecule into a host cell for amplification and expression. The recombinant vector refers to a recombinant DNA molecule constructed by connecting the above-mentioned nucleic acid molecule and the vector in vitro. Any existing eukaryotic cell expression vector can be used to construct a recombinant vector containing the above-mentioned nucleic acid molecule. In a specific embodiment of the present invention, the eukaryotic cell expression vector is a pVAX1 vector, and the recombinant vector is a vector obtained by inserting the DNA molecule shown in the 7th to 2244th position of the sequence 2 in the sequence table into the Nhe I and EcoR I restriction sites of the eukaryotic expression vector pVAX1.

[0055] In a third aspect, the present invention claims a preparation for preventing and treating latent tuberculosis infection.

[0056] The active ingredient of the preparation for preventing and treating latent tuberculosis infection claimed in the present invention is the above-mentioned DNA vaccine or the above-mentioned biological material.

[0057] When preparing the preparation for preventing and treating latent tuberculosis infection, auxiliary materials such as diluents, cosolvents, buffers, pH regulators, etc., such as physiological saline, may also be added.

[0058] The tuberculosis latent infection prevention and treatment preparation can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, or respiratory administration, including nasal spray, oral inhalation administration, etc.

[0059] When the tuberculosis latent infection prevention and treatment preparation is used to prevent and / or treat tuberculosis latent infection, an effective amount of the above DNA vaccine or the above biological material is administered to the subject.

[0060] In a fourth aspect, the present invention claims to protect new uses of the above-mentioned DNA vaccine or the above-mentioned biological material or the above-mentioned preparation for preventing and treating latent tuberculosis infection.

[0061] The present invention claims to protect the use of the above-mentioned DNA vaccine or the above-mentioned biological material or the above-mentioned tuberculosis latent infection prevention and treatment preparation in the preparation of a product for preventing and / or treating tuberculosis latent infection.

[0062] The present invention also claims to protect the use of the above DNA vaccine or the above biological material or the above tuberculosis latent infection prevention and treatment preparation in the preparation of products for inhibiting the growth and / or proliferation and / or endogenous recurrence of Mycobacterium tuberculosis.

[0063] The present invention also claims to protect the use of the above-mentioned DNA vaccine or the above-mentioned biological material or the above-mentioned tuberculosis latent infection prevention and treatment preparation in the preparation of products for improving or enhancing cellular immune function or cellular immune response level.

[0064] In any of the above applications, the improvement or enhancement of cellular immune function or cellular immune response level is embodied in the improvement or enhancement of Th1 type cellular immune response level.

[0065] In any of the above applications, the product may be a drug or a vaccine.

[0066] The beneficial effects of the present invention are as follows: the present invention clones, expresses and purifies a novel Mycobacterium tuberculosis tandem DNA vaccine W543 through genetic engineering technology, which can stimulate the body's humoral immunity and cellular immune response, inhibit the growth and endogenous recurrence of Mycobacterium tuberculosis in the body, and is thus used for preventive treatment of latent tuberculosis infection.

[0067] The present invention provides a novel Mycobacterium tuberculosis tandem DNA vaccine W543 targeting different Mycobacterium tuberculosis infection stages. The vaccine is constructed by genetically engineering the polypeptide or epitope encoding genes of Mycobacterium tuberculosis proliferation phase antigens and latency-related antigens together through a linker and inserting them into a eukaryotic expression vector pVAX1 to obtain a novel Mycobacterium tuberculosis DNA vaccine with multiple antigen immunodominant epitopes. Experiments have shown that: the tuberculosis-specific tandem DNA vaccine W543 of the present invention can significantly increase the number of T lymphocyte spots that secrete interferon-γ when used as an immunomodulatory preparation to immunize mice, and the anti-W543 protein antibody and subtype levels in the plasma are slightly increased, showing a Th1 type cellular immune response, confirming that the vaccine mainly stimulates the Th1 type immune response to obtain a good immunomodulatory effect, and can be used to improve or enhance the cellular immune function or cellular immune response level; as a preventive treatment preparation, the tuberculosis latent infection mice can be immunized, and the number of bacteria in the lung and liver tissues can be reduced, the range of lung tissue lesions is significantly reduced, and the lesions are alleviated, and the therapeutic effect is slightly better than the therapeutic effect of the existing commercialized tuberculosis latent infection prevention and treatment preparation Microcard vaccine, and can be used for preventive treatment of tuberculosis latent infection. Therefore, the vaccine prepared by the present invention will have a broad application prospect in the preventive treatment of tuberculosis latent infection.

[0068] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation modes, but this does not mean to limit the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 To identify the recombinant plasmid pVAX1-W543 by restriction endonucleases NheI and EcoRI. M: DM10000 DNA molecular weight standard; W543: The recombinant plasmid pVAX1-W543 was digested by restriction endonucleases NheI and EcoRI.

[0070] Figure 2 The anti-W543 IgG antibody level in the plasma of each group of mice 3 weeks after the third immunization in Example 2 of the present invention. Note: Compared with the normal saline group: **P<0.01; compared with the microcard vaccine group: &P<0.05.

[0071] Figure 3 The anti-W543 IgG1 antibody levels in the plasma of mice in each group 3 weeks after the third immunization in Example 2 of the present invention. Note: Compared with the normal saline group: *P<0.05.

[0072] Figure 4The anti-W543 IgG2a antibody level in the plasma of each group of mice 3 weeks after the third immunization in Example 2 of the present invention. Notes: Compared with the normal saline group: ***P<0.0001; compared with the pVAX1 vector group: ##P<0.01; compared with the microcard vaccine group: &&P<0.01; compared with the ag85ab plasmid DNA group: $$P<0.01.

[0073] Figure 5 It is the number of T lymphocyte spots secreting IFN-γ in the spleen lymphocytes of each group of mice in Example 2 of the present invention. Notes: Compared with the normal saline group: **P<0.01, ***P<0.0001; compared with the pVAX1 vector group: ##P<0.01, ###P<0.0001; compared with the microcard vaccine group: &&P<0.01, &&&P<0.0001; compared with the ag85ab plasmid DNA group, $P<0.05.

[0074] Figure 6 It is the expression level of IFN-γ in the culture supernatant of mouse spleen cells in each group of the present invention in Example 2. Notes: Compared with the normal saline group: ***P<0.0001; compared with the pVAX1 vector group: ###P<0.0001; compared with the microcard vaccine group: &&&P<0.0001; compared with the ag85ab plasmid DNA group: $$P<0.01.

[0075] Figure 7 The expression levels of TNF in the culture supernatant of spleen cells of each group of mice in Example 2 of the present invention. Note: Compared with the pVAX1 vector group: ##P<0.01.

[0076] Figure 8 It is the IL-4 expression level in the culture supernatant of spleen cells of each group of mice in Example 2 of the present invention.

[0077] Fig. 9 The expression level of IL-6 in the culture supernatant of spleen cells of each group of mice in Example 2 of the present invention.

[0078] Fig.10 It is the number of T lymphocyte spots secreting IFN-γ in each group of mice in Example 3 of the present invention. Notes: Compared with the saline group: ***P<0.0001; compared with the pVAX1 vector group: ###P<0.0001; compared with the microcard vaccine group: &&P<0.01, &&&P<0.0001; compared with the ag85ab plasmid DNA group: $$P<0.01.

[0079] Fig.11 The CD4 + CD25 + FOXP3 +Cell percentage. Note: Compared with the microcard vaccine group: &&P<0.01.

[0080] Fig.12 The expression levels of IFN-γ in the culture supernatant of mouse spleen cells in each group in Example 3 of the present invention. Notes: Compared with the normal saline group: *P<0.05, **P<0.01; compared with the pVAX1 vector group: #P<0.05, ##P<0.01; compared with the microcard vaccine group: &P<0.05.

[0081] Fig.13 It is the IL-2 expression level in the culture supernatant of spleen cells of each group of mice in Example 3 of the present invention. Note: Compared with the microcard vaccine group: &P<0.05, &&P<0.01.

[0082] Fig.14 The expression levels of IL-4 in the culture supernatant of spleen cells of each group of mice in Example 3 of the present invention. Note: Compared with the pVAX1 vector group: #P<0.05.

[0083] Fig.15 The expression levels of IL-6 in the culture supernatant of spleen cells of each group of mice in Example 3 of the present invention. Note: Compared with the pVAX1 vector group: #P<0.05.

[0084] Fig.16 These are the results of colony counts in the lungs of mice at different stages in Example 3 of the present invention.

[0085] Fig.17 The colony count results of mice liver at different stages in Example 3 of the present invention are shown.

[0086] Fig.18 This is the lung tissue pathology of each group of mice in Example 3 of the present invention (H&E, 40×). DETAILED DESCRIPTION

[0087] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0088] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0089] The ag85ab plasmid DNA and construction method in the following embodiments are recorded in the invention patent entitled Mycobacterium tuberculosis Ag85ab chimeric gene vaccine, its preparation method and application, with patent number ZL201010191243.X. The public can obtain it from the Eighth Medical Center of the General Hospital of the Chinese People's Liberation Army. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0090] Example 1: Preparation method of a Mycobacterium tuberculosis-specific tandem DNA vaccine W543

[0091] The Mycobacterium tuberculosis tandem DNA vaccine designed by the present invention is a novel Mycobacterium tuberculosis DNA vaccine with multiple antigen immunodominant epitopes constructed by tandemly linking the immunodominant epitope genes of Mycobacterium tuberculosis proliferation antigens and latency-related antigens through genetic engineering and connecting them to the eukaryotic expression vector pVAX1. The specific construction method is as follows:

[0092] 1. Construction of recombinant plasmid pVAX1-W543

[0093] 1. Design and synthesis of W543 gene sequence

[0094] The DNA sequence suitable for eukaryotic expression of DNA vaccine W543 designed and optimized by the present invention is shown in sequence 2 in the sequence table, and is recorded as W543 gene. The W543 gene sequence is artificially synthesized, and the gene sequence comprises a NheI restriction endonuclease recognition site (positions 1-6 of sequence 2), a start codon (positions 16-18 of sequence 2), a coding gene sequence of 16 antigenic peptides (antigenic epitope A-antigenic epitope P), a stop codon (positions 2242-2244 of sequence 2) and an EcoRI restriction endonuclease recognition site (positions 2245-2250 of sequence 2).

[0095] Among them, the amino acid sequence of antigenic epitope A is shown in the 3rd to 291st positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 22nd to 888th positions of sequence 2 in the sequence listing; the amino acid sequence of antigenic epitope B is shown in the 299th to 313th positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 910th to 954th positions of sequence 2 in the sequence listing; the amino acid sequence of antigenic epitope C is shown in the 319th to 327th positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 970th to 996th positions of sequence 2 in the sequence listing; the amino acid sequence of antigenic epitope D is shown in the 333rd to 368th positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 1012th to 1119th positions of sequence 2 in the sequence listing; The amino acid sequence of E is shown in the 374th to 393rd positions of Sequence 1 in the sequence listing, and the coding gene sequence thereof is shown in the 1135th to 1194th positions of Sequence 2 in the sequence listing; the amino acid sequence of antigen epitope F is shown in the 399th to 407th positions of Sequence 1 in the sequence listing, and the coding gene sequence thereof is shown in the 1210th to 1236th positions of Sequence 2 in the sequence listing; the amino acid sequence of antigen epitope G is shown in the 413th to 426th positions of Sequence 1 in the sequence listing, and the coding gene sequence thereof is shown in the 1252th to 1293rd positions of Sequence 2 in the sequence listing; the amino acid sequence of antigen epitope H is shown in the 432nd to 449th positions of Sequence 1 in the sequence listing, and the coding gene sequence thereof is shown in the 1309th to 1362th positions of Sequence 2 in the sequence listing; The amino acid sequence of epitope I is shown in the 455th to 469th positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 1378th to 1422th positions of sequence 2 in the sequence listing; the amino acid sequence of epitope J is shown in the 475th to 510th positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 1438th to 1545th positions of sequence 2 in the sequence listing; the amino acid sequence of epitope K is shown in the 518th to 641th positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 1567th to 1938th positions of sequence 2 in the sequence listing; the amino acid sequence of epitope L is shown in the 649th to 662th positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 1960th to 2001th positions of sequence 2 in the sequence listing; The amino acid sequence of the original epitope M is shown in the 668th to 680th positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 2017th to 2055th positions of sequence 2 in the sequence listing; the amino acid sequence of the antigenic epitope N is shown in the 686th to 700th positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 2071st to 2115th positions of sequence 2 in the sequence listing; the amino acid sequence of the antigenic epitope O is shown in the 706th to 717th positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 2131st to 2166th positions of sequence 2 in the sequence listing; the amino acid sequence of the antigenic epitope P is shown in the 723rd to 742th positions of sequence 1 in the sequence listing, and its encoding gene sequence is shown in the 2182nd to 2241st positions of sequence 2 in the sequence listing.

[0096] Artificially synthesized W543 gene sequence.

[0097] 2. Amplification of W543 gene sequence

[0098] Using the W543 gene sequence in step 1 as a template, PCR amplification was performed using W543-F primer and W543-R primer to obtain a PCR amplification product, namely the W543 gene sequence. The primer sequences are as follows:

[0099] W543-F:5'-CTA GCTAGC GCCGCCACCATGGTCTCC-3' (the underlined sequence is the Nhe I restriction site);

[0100] W543-R:5'-CG GAATTC TCAGGCATTGCCCAGTCT-3' (the underlined sequence is the EcoR I restriction site).

[0101] The PCR program was as follows: denaturation at 98°C for 2 min; then denaturation at 98°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 45 s, for a total of 37 cycles; finally extension at 72°C for 5 min; and incubation at 4°C for 5 min.

[0102] Refer to the instructions of the Omega Gel Recovery Kit to recover the PCR amplification product.

[0103] 3. Double enzyme digestion

[0104] The vector plasmid pVAX1 (Invitrogen product of Thermo, Lot: 1724963, purchased from Beijing Yongze Haojia Biotechnology Development Center) and the PCR amplification product obtained in step 2 were double-digested with restriction endonucleases Nhe I and EcoR I (NEB) to obtain pVAX1 double-digested products and W543 gene double-digested products, respectively.

[0105] The enzyme digestion reaction system was as follows: 10xCutSmart 5 μl, Nhe I 2 μl, EcoR I 2 μl, pVAX1 (4 μg) or W543 product (20 μl), water was added to make up to 50 μl, enzyme digestion was carried out at 37°C overnight, and the reaction was terminated at 65°C.

[0106] Refer to the instructions of the Omega Gel Recovery Kit to recover the enzyme digestion product.

[0107] 4. Connect

[0108] The pVAX1 double-enzyme digestion product obtained in step 3 and the W543 gene double-enzyme digestion product were ligated with T4 DNA Ligase (NEB) to obtain a ligation product.

[0109] The ligation reaction system was as follows: 2 μl of 10xT4 Ligase buffer, 1 μl of T4 Ligase, 1 μl of pVAX1 double-enzyme digestion product; 3 μl of W543 gene double-enzyme digestion product, add water to make up to 20 μl, and ligate at 16°C overnight.

[0110] 5. Transformation of ligation product into E. coli DH5a

[0111] Add 1 μl of the ligation product to the E. coli DH5a competent cells, place on ice for 30 min, heat shock at 42°C for 90 s, and quickly place on ice for 5 min; add 500 μL of antibiotic-free LB culture medium, shake at 37°C and 180 rpm for 1 h, centrifuge, spread all on an LB plate containing kanamycin, and culture inverted at 37°C overnight.

[0112] II. Enzyme digestion identification of recombinant plasmid pVAX1-W543

[0113] The single clones on the plate were picked and inoculated into 2 mL of LB culture medium containing kanamycin (Kan+), and cultured overnight at 37°C and 220 rpm with shaking; the next day, the overnight culture bacteria were subjected to plasmid extraction using the Omega Endotoxin-Free Plasmid Extraction Kit. The recombinant plasmid pVAX1-W543 was then double-digested with restriction endonucleases Nhe I and EcoR I (NEB) to obtain the digestion product.

[0114] The enzyme digestion system was as follows: 10xCutSmart 5 μl, Nhe I 2 μl, EcoR I 2 μl, recombinant plasmid pVAX1-W543 (4 μg), made up to 50 μl with water, incubated at 37°C for overnight, and terminated at 65°C.

[0115] The digested products were electrophoresed in 1% agarose gel. Figure 1 .from Figure 1 It can be seen that the sizes of the fragments cut out by enzyme digestion are basically consistent with the theoretical lengths. The results of enzyme digestion identification prove that the W543 gene fragment has been successfully inserted into the pVAX1 plasmid.

[0116] 3. Gene sequencing and identification of recombinant plasmid pVAX1-W543

[0117] The purified recombinant plasmid pVAX1-W543 was sent to the company for gene sequencing. The sequencing results and the designed original sequence were compared and analyzed to show that the two sequences were completely consistent. The recombinant plasmid pVAX1-W543 with the correct sequencing results is the Mycobacterium tuberculosis tandem DNA vaccine W543 of the present invention. The amino acid sequence of the W543 protein expressed by the plasmid is shown in Sequence 1 in the sequence table.

[0118] Example 2: Evaluation of the immune effect of Mycobacterium tuberculosis tandem DNA vaccine W543 as an immunomodulatory agent

[0119] The Mycobacterium tuberculosis tandem DNA vaccine W543 constructed and purified in Example 1 of the present invention was used to immunize mice as an immunomodulatory preparation, and a good immunomodulatory effect was obtained. The immunomodulatory process of mice and the evaluation results of the immune effect are shown below:

[0120] 1. Experimental methods and materials

[0121] 1. Animal selection

[0122] Forty female BALB / c mice aged 56-62 days and weighing 18-20 g with a certificate of conformity were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The weight difference in the same batch of experiments did not exceed 2 g.

[0123] 2. Immunogenicity of Mycobacterium tuberculosis tandem DNA vaccine W543

[0124] (1) Animal immunization

[0125] The mice were divided into 5 groups, 8 in each group, and the treatment methods were as follows:

[0126] ①Normal saline group: Each mouse was injected intramuscularly with 100 μl of normal saline.

[0127] ②pVAX1 vector group (pVAX-1 vector group): each mouse was intramuscularly injected with 100 μl of pVAX1 solution (pVAX1 solution is a solution obtained by dissolving 100 μg of pVAX1 vector plasmid DNA in 100 μl of physiological saline).

[0128] ③ Microcard vaccine group: each mouse was injected intramuscularly with 100 μl of microcard vaccine solution (the microcard vaccine solution was a solution obtained by dissolving 22.5 μg of microcard vaccine (Anhui Longcoma Bio-Pharmaceutical Co., Ltd., batch number: 20191001) in 100 μl of normal saline).

[0129] ④ag85ab plasmid DNA group: each mouse was intramuscularly injected with 100 μl ag85ab plasmid DNA solution (ag85ab plasmid DNA solution is a solution obtained by dissolving 100 μg ag85ab plasmid DNA in 100 μl physiological saline).

[0130] ⑤W543 plasmid DNA group: Each mouse was intramuscularly injected with 100 μl of W543 plasmid DNA solution (W543 plasmid DNA solution is a solution obtained by dissolving 100 μg of W543 plasmid DNA in 100 μl of physiological saline).

[0131] The mice in each group were injected intramuscularly once every 2 weeks, for a total of 3 times.

[0132] (2) Detection of antibody levels in mouse plasma by ELISA

[0133] Three weeks after the third immunization, the mice were killed and blood was collected and placed in lithium heparin anticoagulant tubes, and the plasma was separated and frozen at -20°C. The level of anti-W543 protein specific antibodies in mouse plasma was simultaneously detected by ELISA.

[0134] (3) Detection of interferon-γ secreting T lymphocyte spots in mice by ELISPOT assay

[0135] The Mouse IFN-γ ELISPOT PLUS kit (Mabtech Technology Co., Ltd.) was used for enzyme-linked immunosorbent assay (ELISPOT) according to the instructions of the kit. The specific steps were as follows: 5 weeks after the third immunization, the mice were killed and spleens were obtained, spleen cells were isolated, and the concentration of spleen lymphocytes was adjusted to 3×10 6 In the test plate, 1640 complete cell culture medium, W543 protein (amino acid sequence as shown in sequence 1 in the sequence table, final concentration of 30 μg / ml), phytohemagglutinin (PHA) (final concentration of 30 μg / ml), and spleen lymphocytes were added at 3×10 5 / well, incubate in a CO2 incubator at 37℃ for 20h, detect the number of T lymphocyte spots secreting IFN-γ in each group by ELISPOT method, and calculate the number of cell spots in the detection wells of each group of mice with the blank control wells of each group as the control.

[0136] (4) Detection of Th1 and Th2 cytokine expression levels in mouse spleen lymphocyte culture supernatant using CBA method

[0137] Five weeks after the third immunotherapy, mice were killed and spleens were obtained to separate spleen cells. Spleen lymphocytes separated from mice in each group were mixed, cultured with protein stimulation, and the culture supernatant was aspirated and frozen. The expression levels of Th1 and Th2 cytokines in the supernatant of spleen cell culture were detected by CBA method.

[0138] 3. Statistical analysis

[0139] SAS9.1 software was used to process the data. One-way analysis of variance was used for the normally distributed quantitative data, and Dunnett's t test was used for pairwise comparison. Kruskal-Wallis test was used for the skewed distributed quantitative data, and q (Student-Newman-Keuls) test was used for pairwise comparison.

[0140] 2. Experimental Results

[0141] 1. Anti-W543 antibody levels in plasma of mice in each group

[0142] Results Figure 2-Figure 4 The results showed that 3 weeks after the third immunization, the levels of anti-W543 IgG antibodies and their subtypes IgG1 and IgG2a in the W543 plasmid DNA group and the ag85ab plasmid DNA group were higher than those in the saline group and the microcardia vaccine group. The levels of anti-W543 IgG antibodies and their subtypes IgG1 and IgG2a in the ag85ab plasmid DNA group were significantly increased (P<0.0001 or P<0.01 or P<0.05), and the level of anti-W543 IgG2a in the ag85ab plasmid DNA group was also significantly higher than that in the W543 plasmid DNA group (P<0.01). However, there was no significant difference in the levels of anti-W543 IgG antibodies and their subtypes IgG1 and IgG2a in the W543 plasmid DNA group compared with the saline group and the pVAX-1 vector group (P>0.05).

[0143] 2. Number of T lymphocyte spots secreting interferon-γ in spleen lymphocytes of mice in each group

[0144] Results Figure 5 The results showed that a small amount of W543 protein-specific IFN-γ-secreting T lymphocytes were observed in the saline group, pVAX-1 vector group, and microcard vaccine group; a moderate amount of W543 protein-specific IFN-γ-secreting T lymphocytes were observed in the ag85ab plasmid DNA group and W543 plasmid DNA group. Compared with the saline group, pVAX-1 vector group, and microcard vaccine group, the ag85ab plasmid DNA group and the W543 plasmid DNA group could induce a moderate amount of IFN-γ-secreting T lymphocytes (P<0.0001 or P<0.01), and the IFN-γ-secreting T lymphocytes in the ag85ab plasmid DNA group were significantly higher than those in the W543 plasmid DNA group (P<0.05).

[0145] 3. Expression levels of Th1 and Th2 cytokines in the supernatant of spleen lymphocytes of mice in each group

[0146] Five weeks after the end of immunization, the expression levels of Th1 cytokines (IFN-γ, TNF) and Th2 cytokines (IL-4, IL-6) in the culture supernatant of mouse spleen lymphocytes were detected.

[0147] The results of IFN-γ level detection are shown in Figure 6The results showed that the IFN-γ levels in the W543 plasmid DNA group (7.28pg / ml±1.38pg / ml) and the ag85ab plasmid DNA group (38.52pg / ml±17.16pg / ml) were higher than those in the normal saline group (0.16pg / ml±0.28pg / ml), the pVAX-1 vector group (0.15pg / ml±0.15pg / ml) and the microcard vaccine group (0.05pg / ml±0.09pg / ml), and the ag85ab plasmid DNA group was significantly higher than all the groups (P<0.0001 or P<0.01).

[0148] TNF level test results are shown in Figure 7 The results showed that the TNF level in the W543 plasmid DNA group (587.13pg / ml±12.93pg / ml) was higher than that in the normal saline group (527.87pg / ml±61.63pg / ml), the pVAX-1 vector group (412.27pg / ml±24.80pg / ml) and the ag85ab plasmid DNA group (510.24pg / ml±51.50pg / ml), and the W543 plasmid DNA group and the microcard vaccine group were significantly higher than those in the pVAX-1 vector group (P<0.01).

[0149] The results of IL-4 level test are shown in Figure 8 The results showed that the IL-4 levels in the microcardia vaccine group (0.42pg / ml±0.38pg / ml) and W543 plasmid DNA group (0.64pg / ml±0.91pg / ml) were lower than those in the normal saline group (2.71pg / ml±1.90pg / ml), pVAX-1 vector group (0.85pg / ml±1.06pg / ml) and ag85ab plasmid DNA group (12.07pg / ml±2.55pg / ml), but the differences were not statistically significant (P>0.05).

[0150] The results of IL-6 level test are shown in Fig. 9 The results showed that compared with the IL-6 level in the normal saline group (248.54pg / ml±47.51pg / ml), the IL-6 levels in the pVAX-1 vector group (184.62pg / ml±15.85pg / ml), the microcardia vaccine group (186.24pg / ml±31.17pg / ml), the W543 plasmid DNA group (196.79pg / ml±10.60pg / ml) and the ag85ab plasmid DNA group (246.59pg / ml±23.03pg / ml) were all reduced to varying degrees, but the differences were not statistically significant (P>0.05).

[0151] Example 3: Application of Mycobacterium tuberculosis tandem DNA vaccine W543 in the preventive treatment of latent tuberculosis infection

[0152] The Mycobacterium tuberculosis tandem DNA vaccine W543 constructed and purified in Example 1 of the present invention was applied to the preventive treatment of the mouse latent tuberculosis infection model. As a therapeutic preparation, it achieved a good therapeutic effect, which was comparable to the therapeutic effect of the existing commercialized tuberculosis latent infection preventive treatment preparation, Microcarb vaccine. The immunotherapy process and preventive treatment effect of the mouse latent tuberculosis infection model are as follows:

[0153] 1. Experimental methods and materials

[0154] 1. Animal selection

[0155] A total of 101 certified female BALB / c mice aged 42-62 days were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0156] 2. Preventive and therapeutic effects of the vaccine on the latent tuberculosis infection model in mice

[0157] (1) Preparation of latent tuberculosis infection model in mice

[0158] ① Each of the 91 mice was injected via the tail vein with 0.4 ml of 3.6×10 5 The tuberculosis infection model in mice was prepared by using a suspension of the standard strain of Mycobacterium tuberculosis H37Rv with CFUs.

[0159] ② Chemotherapy: 4 weeks after infection, mice were treated with pyrazinamide (pyrazinamide tablets: specification: 0.25 g / tablet, batch number: 2004051, Shenyang Hongqi Pharmaceutical Co., Ltd.) and isoniazid (isoniazid tablets: specification: 100 mg / tablet, batch number: 200811, Taiji Southwest Pharmaceutical Co., Ltd.). Every day, 2 g of pyrazinamide and 0.03 g of isoniazid were dissolved in 250 ml of sterile distilled water to make the final concentration of pyrazinamide 8 g / L and the final concentration of isoniazid 0.12 g / L. The treatment lasted for 12 weeks to establish a latent tuberculosis infection model in mice.

[0160] ③ Hormone treatment: In order to verify whether the latent tuberculosis infection model was successfully established, each group of mice was injected intramuscularly with hydrocortisone sodium succinate (National Medicine Standard H20093293, Batch Number: 20200311D1, Changzhou Siyao Pharmaceutical Co., Ltd.) 3 weeks after the third DNA vaccine preventive treatment, with each mouse injected with 0.5 mg / 0.1 ml, 3 times a week for 3 consecutive weeks.

[0161] (2) Experimental groups

[0162] ① Mouse infection group

[0163] A, 4-week infection group: 5 mice, 4 weeks after infection with Mycobacterium tuberculosis;

[0164] B, 16-week infection group: 8 mice, 16 weeks after infection with Mycobacterium tuberculosis;

[0165] C, 29-week infection group: 10 mice, 29 weeks after infection with Mycobacterium tuberculosis.

[0166] ② Chemotherapy group

[0167] A. 12-week chemotherapy group: 8 mice, 4 weeks after infection with tuberculosis, were given chemotherapy for 12 weeks;

[0168] B. Chemotherapy-withdrawal 13-week group: 10 mice, 4 weeks after infection with tuberculosis bacteria, given chemotherapy for 12 weeks, and stopped chemotherapy for 13 weeks;

[0169] C. Hormone treatment and 3-week discontinuation group (i.e., normal saline group): 10 mice were infected with tuberculosis bacteria 4 weeks ago, given chemotherapy for 12 weeks, and then treated with hormones for 3 weeks, and then discontinued hormones for 3 weeks.

[0170] ③DNA vaccine immunotherapy + hormone therapy for 3 weeks and drug withdrawal for 3 weeks group

[0171] 50 mice were infected with tuberculosis for 4 weeks and given chemotherapy for 12 weeks. The mice were randomly divided into 5 groups, with 10 mice in each group, and different immunoprophylactic treatments were performed. The specific treatment methods were as follows:

[0172] A, Normal saline group: Each mouse was intramuscularly injected with 100 μl normal saline each time, once every 2 weeks, for a total of 3 times;

[0173] B. pVAX1 vector group: Each mouse was intramuscularly injected with 100 μl of pVAX1 solution (pVAX1 solution is a solution obtained by dissolving 100 μg of pVAX1 vector plasmid DNA in 100 μl of normal saline), once every 2 weeks, for a total of 3 times;

[0174] C. Microcard vaccine group: Each mouse was intramuscularly injected with 100 μl of microcard vaccine solution each time (the microcard vaccine solution is a solution obtained by dissolving 22.5 μg of microcard vaccine (Anhui Longcoma Biopharmaceutical Co., Ltd., batch number: 20191001) in 100 μl of normal saline), immunized once every 2 weeks, for a total of 3 times;

[0175] D. ag85ab plasmid DNA group: Each mouse was intramuscularly injected with 100 μl ag85ab plasmid DNA solution (ag85ab plasmid DNA solution is a solution obtained by dissolving 100 μg ag85ab plasmid DNA in 100 μl physiological saline), once every 2 weeks, for a total of 3 times;

[0176] E. W543 plasmid DNA group: Each mouse was intramuscularly injected with 100 μl of W543 plasmid DNA solution (W543 plasmid DNA solution is a solution obtained by dissolving 100 μg of W543 plasmid DNA in 100 μl of physiological saline) each time, and immunization was performed once every 2 weeks, for a total of 3 times.

[0177] Each group of mice received hormone treatment 3 weeks after the third preventive treatment.

[0178] (3) Detection of the number of lymphocyte spots secreting IFN-γ in spleen lymphocytes by ELISPOT method

[0179] The mice in the DNA vaccine immunotherapy + hormone therapy group for 3 weeks and the drug withdrawal group for 3 weeks and the normal control group were killed and spleens were obtained. Splenocytes were isolated and the concentration of spleen lymphocytes was adjusted to 3×10 6 In the test plate, 1640 complete cell culture medium, W543 protein (amino acid sequence as shown in sequence 1 in the sequence table, final concentration of 30 μg / ml), phytohemagglutinin (PHA) (final concentration of 30 μg / ml), and spleen lymphocytes were added at 3×10 5 / well, incubate in a CO2 incubator at 37℃ for 20h, detect the number of T lymphocyte spots secreting IFN-γ in each group by ELISPOT method, and calculate the number of cell spots in the detection wells of each group of mice with the blank control wells of each group as the control.

[0180] (4) Detection of the proportion of FoxP3 regulatory T cells in mouse spleen cells by flow cytometry

[0181] After killing mice in the DNA vaccine immunotherapy + hormone treatment 3 weeks and 3 weeks of drug withdrawal group and the normal control group, flow cytometry was used to detect the CD4 + CD25 + FOXP3 + Cell percentage.

[0182] (5) CBA method was used to detect the expression levels of Th1 and Th2 cytokines in the culture supernatant of mouse spleen lymphocytes. After killing mice in the DNA vaccine immunotherapy + hormone treatment 3 weeks and 3 weeks of drug withdrawal group and the normal control group, the spleen lymphocytes of each group were mixed, cultured with protein stimulation, and the culture supernatant was aspirated and frozen. The expression levels of Th1 and Th2 cytokines in the culture supernatant of spleen cells were detected by CBA method.

[0183] (6) Lung and liver colony counts

[0184] The mice in the infection group, chemotherapy group, DNA vaccine immunotherapy + hormone treatment for 3 weeks and drug withdrawal for 3 weeks, and normal control group were killed and dissected to obtain the left lobe of the lung and the upper part of the liver, weighed, and counted for colonies. The left lobe of the lung and the upper part of the liver were placed in a sterile grinder, homogenized with physiological saline, and 100 μl was inoculated in Roche plate culture medium after multiple dilution. The colony growth was observed and counted in a 37°C incubator for 4 weeks. The number of colonies in the left lobe of the lung was converted to the number of colonies in the whole lung according to the weight of the left lobe of the lung and the whole lung, and the number of colonies in the upper part of the liver was converted to the number of colonies in the whole liver according to the weight of the upper part of the liver and the whole liver.

[0185] (7) Pathological examination of lung tissue

[0186] The mice in the infection group, chemotherapy group, DNA vaccine immunotherapy + hormone treatment for 3 weeks followed by 3 weeks of drug withdrawal, and normal control group were killed, and the right lobe of the mouse lung was fixed in a pathology centrifuge tube containing 10% neutral formaldehyde, embedded in paraffin, and sliced. After hematoxylin / eosin (HE) staining, 5 μm continuous sections were made, and the histopathological lesions of the lung tissue were observed under a microscope, and the lesion area of ​​the lung was calculated using Image-Pro Plus software (Media Cybernetics, Inc., Rockville, Maryland, USA).

[0187] 3. Statistical analysis

[0188] SAS9.1 software was used to process the data. One-way analysis of variance was used for the normally distributed quantitative data, and Dunnett's t test was used for pairwise comparison. Kruskal-Wallis test was used for the skewed distributed quantitative data, and q (Student-Newman-Keuls) test was used for pairwise comparison.

[0189] 2. Experimental Results

[0190] 1. Death of mice in each group

[0191] The statistical results of the mortality rate of mice in each group are as follows: 5 mice died in the 16-week infection group, with a mortality rate of 62.5%; 1 mouse died in the 12-week chemotherapy group, with a mortality rate of 12.5%; the mortality rate of mice in the 29-week infection group was 80%; the mortality rate of mice in other groups was 0%.

[0192] 2. Number of lymphocyte spots secreting IFN-γ in spleen lymphocytes of mice in each group

[0193] Results Fig.10The results showed that 3 weeks after the cessation of hormone treatment in mice, a small number of W543 protein-specific T lymphocytes secreting IFN-γ were observed in the saline group, pVAX-1 vector group, and microcard vaccine group; a moderate number of W543 protein-specific T lymphocytes secreting IFN-γ were observed in the ag85ab plasmid DNA group and the W543 plasmid DNA group. Compared with the normal saline group, pVAX-1 vector group and microcard vaccine group, the number of T lymphocytes secreting IFN-γ in the ag85ab plasmid DNA group was significantly increased (P<0.0001); compared with the pVAX-1 vector group and microcard vaccine group, the number of T lymphocytes secreting IFN-γ in the W543 plasmid DNA group was significantly increased (P<0.0001 or P<0.01); the number of T lymphocytes secreting IFN-γ in the ag85ab plasmid DNA group was also significantly higher than that in the W543 plasmid DNA group (P<0.05), and the number of T lymphocytes secreting IFN-γ in the normal saline group was significantly higher than that in the pVAX-1 vector group (P<0.01).

[0194] 3. Percentage of FoxP3 regulatory T cells in spleen cells of mice in each group

[0195] Results Fig.11 The results showed that 3 weeks after the hormone treatment was stopped, the CD4 + CD25 + FOXP3 + The percentage was significantly lower than that of the microcard vaccine group (P<0.05), and there was no significant difference among the other groups (P>0.05).

[0196] 4. Expression levels of Th1 and Th2 cytokines in the supernatant of spleen lymphocytes from mice in each group

[0197] The results of IFN-γ level detection are shown in Fig.12 The results showed that 3 weeks after the hormone treatment of mice was stopped, the IFN-γ level in the supernatant of spleen lymphocytes in the ag85ab plasmid DNA group was significantly higher than that in the pVAX-1 vector group, the microcard vaccine group and the W543 plasmid DNA group (P<0.05). The IFN-γ level in the supernatant of spleen lymphocytes in the W543 plasmid DNA group was higher than that in the normal saline group, the pVAX-1 vector group and the microcard vaccine group (P<0.05 or P<0.01).

[0198] The results of IL-2 level test are shown in Fig.13 The results showed that the IL-2 level in the supernatant of spleen lymphocytes in the ag85ab plasmid DNA group and the W543 plasmid DNA group was significantly higher than that in the microcardia vaccine group (P<0.05 or P<0.01).

[0199] The results of IL-4 level test are shown in Fig.14The results showed that compared with the pVAX-1 vector group, the IL-4 level in the supernatant of spleen lymphocytes in the microcardia vaccine group was significantly reduced (P<0.05), and the IL-4 level in the supernatant of spleen lymphocytes in the ag85ab plasmid DNA group and the W543 plasmid DNA group was slightly reduced, but the difference was not statistically significant (P>0.05).

[0200] The results of IL-6 level test are shown in Fig.15 The results showed that the IL-6 level in the culture supernatant of spleen lymphocytes in the W543 plasmid DNA group was significantly increased compared with that in the pVAX-1 vector group (P<0.05).

[0201] 5. Results of colony counts in the lungs and livers of mice in each group

[0202] The statistical results of the number of colonies in the lungs and livers of mice in each group are shown in Figure 16-17 And Table 1. From the results of lung colony counts, the number of lung colonies in the mice infected for 4 weeks was 7.78Log 10 CFU, indicating that the mouse tuberculosis infection model was successfully established. The number of lung colonies in the chemotherapy group after 12 weeks decreased to 0 CFU, indicating that chemotherapy was effective. As the infection time prolonged, the number of lung colonies in the infection group after 16 weeks and the infection group after 29 weeks reached 6.90Log 10 CFU and 7.33Log 10 CFU decreased slightly, which may be due to the natural resistance of BALB / c mice to Mycobacterium tuberculosis. After 3 weeks of hormone treatment, the lung CFU value of the saline group and the pVAX-1 vector group increased to 4.07Log 10 CFU and 5.20Log 10 CFU, indicating that the endogenous reactivation model of latent tuberculosis infection in mice was successfully established, proving that the latent tuberculosis infection model in mice was successfully established. The lung colony count in the chemotherapy withdrawal group for 13 weeks was 1.95Log 10 CFU, indicating that the latent Mycobacterium tuberculosis in mice has reactivated. After the mice were treated with DNA vaccine, hormones were used to induce endogenous activation of latent MTB in mice to evaluate the therapeutic effect of the vaccine in preventing the reactivation of latent MTB. Compared with the saline group and the pVAX-1 vector group, the lung colony counts and liver colony counts of mice in the microcard vaccine group, ag85ab plasmid DNA group, and W543 plasmid DNA group were reduced to varying degrees, and the liver colony counts of mice in the ag85ab plasmid DNA group and W543 plasmid DNA group were 0 CFU, indicating that the microcard vaccine group, ag85ab plasmid DNA group, and W543 plasmid DNA group have a certain immunosuppressive and clearing effect on latent MTB infection, can reduce the bacterial load in the organs of mice, inhibit the reactivation of latent MTB, and prevent the recurrence of TB.

[0203] Table 1. Results of colony counts in lungs and livers of mice in each group

[0204] Group <![CDATA[Lung colony count (Log 10 CFU)]]> <![CDATA[Liver colony-forming unit count (Log 10 CFU)]]> Normal saline group 4.07±1.55 0.63±1.32 pVAX-1 vector group 5.20±0.73 0.71±1.50 Microcard vaccine group 2.43±2.66 0.31±0.99 ag85ab plasmid DNA group 0.83±1.75 0±0 W543 plasmid DNA group 2.39±2.65 0±0

[0205] 6. Lung tissue pathology of mice in each group

[0206] Representative images of lung tissue pathology of mice in each group are shown in Fig.18 The results showed that the lesions in the saline group were more severe, with a range of 55.91% ± 13.96%; the lesions in the pVAX-1 vector group were more severe, with a range of 51.05% ± 9.75%; the lesions in the microcard vaccine group were limited, with a milder range of 45.65% ± 17.17%; the lesions in the ag85ab plasmid DNA group were limited, with a milder range of 45.06% ± 8.33%; the lesions in the W543 plasmid DNA group were limited, with a milder range of 45.33% ± 17.27%. The results showed that the range of lung lesions in the W543 plasmid DNA group, the microcard vaccine group, and the ag85ab plasmid DNA group were smaller than those in the saline group and the pVAX-1 vector group.

[0207] The above experimental results show that the Mycobacterium tuberculosis tandem DNA vaccine W543 of the present invention can significantly enhance the specific cellular immune function of mice, mainly stimulate the Th1 type immune response, and preventively treat the mouse latent tuberculosis infection model, which can reduce the number of bacteria in the lung and liver tissues, significantly reduce the scope of lung tissue lesions, and alleviate the lesions. Its therapeutic effect is slightly better than the therapeutic effect of the existing commercialized tuberculosis latent infection prevention and treatment preparation Microcar vaccine. Therefore, the Mycobacterium tuberculosis tandem DNA vaccine W543 of the present invention will have broad application prospects in the preventive treatment of latent tuberculosis infection.

[0208] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A Mycobacterium tuberculosis DNA vaccine, wherein the active ingredient is a recombinant eukaryotic cell expression vector, wherein the recombinant eukaryotic cell expression vector comprises a gene sequence encoding an antigenic epitope A, a gene sequence encoding an antigenic epitope B, a gene sequence encoding an antigenic epitope C, a gene sequence encoding an antigenic epitope D, a gene sequence encoding an antigenic epitope E, a gene sequence encoding an antigenic epitope F, a gene sequence encoding an antigenic epitope G, a gene sequence encoding an antigenic epitope H, a gene sequence encoding an antigenic epitope I, a gene sequence encoding an antigenic epitope J, a gene sequence encoding an antigenic epitope K, a gene sequence encoding an antigenic epitope L, a gene sequence encoding an antigenic epitope M, a gene sequence encoding an antigenic epitope N, a gene sequence encoding an antigenic epitope O, and a gene sequence encoding an antigenic epitope P; The amino acid sequence of the antigen epitope A is shown in sequence 1, positions 3-291 in the sequence listing; The amino acid sequence of the antigenic epitope B is shown in sequence 1 at positions 299-313 in the sequence listing; The amino acid sequence of the antigen epitope C is shown in sequence 1 at positions 319-327 in the sequence list; The amino acid sequence of the antigen epitope D is shown in sequence 1 at positions 333-368 in the sequence list; The amino acid sequence of the antigenic epitope E is shown in sequence 1 at positions 374-393 in the sequence list; The amino acid sequence of the antigen epitope F is shown in sequence 1 at positions 399-407 in the sequence list; The amino acid sequence of the antigen epitope G is shown in sequence 1, positions 413-426 in the sequence list; The amino acid sequence of the antigenic epitope H is shown in sequence 1, positions 432-449 in the sequence listing; The amino acid sequence of the antigenic epitope I is shown in sequence 1, positions 455-469 in the sequence listing; The amino acid sequence of the antigen epitope J is shown in sequence 1, positions 475-510 in the sequence listing; The amino acid sequence of the antigen epitope K is shown in sequence 1 at positions 518-641 in the sequence list; The amino acid sequence of the antigen epitope L is shown in sequence 1, positions 649-662 in the sequence list; The amino acid sequence of the antigen epitope M is shown in sequence 1 at positions 668-680 in the sequence list; The amino acid sequence of the antigen epitope N is shown in sequence 1, positions 686-700 in the sequence listing; The amino acid sequence of the antigenic epitope O is shown in positions 706-717 of sequence 1 in the sequence list; The amino acid sequence of the antigen epitope P is shown in the sequence 1 at positions 723-742 in the sequence listing.

2. The DNA vaccine according to claim 1, characterized in that: The coding gene sequence of the antigen epitope A is shown in sequence 2, positions 22-888 in the sequence list; The coding gene sequence of the antigen epitope B is shown in sequence 2, positions 910-954 in the sequence list; The coding gene sequence of the antigen epitope C is shown in sequence 2, positions 970-996 in the sequence list; The coding gene sequence of the antigen epitope D is shown in sequence 2, positions 1012-1119 in the sequence list; The coding gene sequence of the antigenic epitope E is shown in sequence 2, positions 1135-1194 in the sequence list; The coding gene sequence of the antigen epitope F is shown in sequence 2, positions 1210-1236 in the sequence list; The coding gene sequence of the antigen epitope G is shown in sequence 2, positions 1252-1293 in the sequence list; The coding gene sequence of the antigen epitope H is shown in sequence 2, positions 1309-1362 in the sequence list; The coding gene sequence of the antigen epitope I is shown in sequence 2, positions 1378-1422 in the sequence list; The coding gene sequence of the antigen epitope J is shown in sequence 2, positions 1438-1545 in the sequence list; The coding gene sequence of the antigen epitope K is shown in sequence 2, positions 1567-1938 in the sequence list; The coding gene sequence of the antigen epitope L is shown in sequence 2, positions 1960-2001 in the sequence list; The coding gene sequence of the antigen epitope M is shown in sequence 2, positions 2017-2055 in the sequence list; The coding gene sequence of the antigen epitope N is shown in sequence 2, positions 2071-2115 in the sequence list; The coding gene sequence of the antigenic epitope O is shown in sequence 2, positions 2131-2166 in the sequence list; The coding gene sequence of the antigen epitope P is shown in sequence 2, positions 2182-2241 in the sequence list.

3. The DNA vaccine according to claim 1 or 2, characterized in that: The recombinant eukaryotic cell expression vector expresses a fusion protein; the amino acid sequence of the fusion protein is any one of the following P1) or P2): P1) The amino acid sequence is the protein shown in SEQ ID NO:1; P2) A fusion protein having the same function is obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:

1.

4. The DNA vaccine according to any one of claims 1 to 3, characterized in that: The recombinant eukaryotic cell expression vector is a vector obtained by inserting the DNA molecule shown in positions 7 to 2244 of sequence 2 in the sequence table into the restriction site of the eukaryotic expression vector.

5. Any of the following biological materials: Q1) The fusion protein of claim 3; Q2) a nucleic acid molecule encoding the fusion protein of claim 3; Q3) an expression cassette containing a nucleic acid molecule encoding the fusion protein of claim 3; Q4) A recombinant vector containing a nucleic acid molecule encoding the fusion protein of claim 3.

6. The biomaterial according to claim 5, characterized in that: The recombinant vector is the recombinant eukaryotic cell expression vector described in claim 4.

7. A preparation for preventing and treating latent tuberculosis infection, wherein the active ingredient is the DNA vaccine according to any one of claims 1 to 4 or the biological material according to claim 5 or 6.

8. Use of the DNA vaccine according to any one of claims 1 to 4, or the biological material according to claim 5 or 6, or the preparation for preventing and treating latent tuberculosis infection according to claim 7 in the preparation of a product for preventing and / or treating latent tuberculosis infection.

9. Use of the DNA vaccine according to any one of claims 1 to 4, or the biological material according to claim 5 or 6, or the preparation for preventing and treating latent tuberculosis infection according to claim 7 in the preparation of a product for inhibiting the growth and / or proliferation and / or endogenous recurrence of Mycobacterium tuberculosis.

10. Use of the DNA vaccine according to any one of claims 1 to 4, or the biological material according to claim 5 or 6, or the preparation for preventing and treating latent tuberculosis infection according to claim 7 in the preparation of a product for improving or enhancing cellular immune function or cellular immune response level.

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