Mycobacterium tuberculosis tandem DNA vaccine W665, and preparation method and application thereof

Abstract

The application discloses a mycobacterium tuberculosis tandem DNA vaccine W665, a preparation method and application thereof. The mycobacterium tuberculosis tandem DNA vaccine W665 is a novel mycobacterium tuberculosis multi-antigen DNA vaccine which is obtained by the following steps: fusing together immunodominant epitope genes of multiple mycobacterium tuberculosis antigens through genetic engineering means, and cloning the fused genes to a eukaryotic expression vector pVAX1. Experimental results show that after the DNA vaccine W665 is used as an immunomodulator to immunize mice, the innate immunity and cellular immune response of the mice can be significantly enhanced; as a prophylactic preparation, the DNA vaccine W665 can be used for preventing acute tuberculosis infection of mice, can significantly reduce the live bacteria count in the liver, can obviously reduce lung tissue lesions, and can enhance the innate immune response level. The mycobacterium tuberculosis tandem DNA vaccine W665 has a good application prospect in the prevention or / and immunotherapy of mycobacterium tuberculosis infection.

Description

Technical Field

[0001] This invention relates to a Mycobacterium tuberculosis tandem DNA vaccine W665, its preparation method and application, specifically to a novel Mycobacterium tuberculosis-specific tandem DNA vaccine W665 prepared using genetic engineering technology and its application, belonging to the field of tuberculosis medical immunology prevention and treatment technology. Background Technology

[0002] Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacterium tuberculosis Tuberculosis (TB) is a chronic infectious disease caused by tuberculosis (M.tb.) infection and is one of the most serious infectious diseases threatening human health worldwide. According to the World Health Organization's "Global Tuberculosis Report 2025," approximately 10.7 million new cases of TB and 1.23 million deaths occurred globally in 2024. my country remains one of the countries with a high burden of TB globally. Numerous epidemiological studies have shown a large number of people with latent tuberculosis infection (LTBI), some of whom can further develop active TB, becoming a significant source of continued TB transmission. Furthermore, the continued increase in multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) poses greater challenges to TB prevention and treatment. Therefore, developing safe and effective new TB prophylactic agents is of great significance for reducing TB incidence and transmission risk.

[0003] Vaccination is one of the most effective public health measures for preventing tuberculosis (TB). Currently, TB vaccines mainly include live attenuated vaccines, inactivated vaccines, subunit vaccines, vector live vaccines, and nucleic acid vaccines. BCG is currently the only approved and widely used TB vaccine, offering good protection against severe TB in children, but its protective efficacy against adult pulmonary TB is unstable, and its preventive effect against LTBI and drug-resistant TB is also insufficient. Furthermore, BCG vaccination can lead to a positive tuberculin skin test, interfering with the diagnosis of TB infection; its use is also limited in immunocompromised populations.

[0004] Among novel tuberculosis vaccines, subunit vaccines have been extensively studied. They primarily utilize antigens such as recombinant proteins, peptides, or glycolipids to induce a specific immune response in the body, offering advantages such as high safety. However, these vaccines typically have weak immunogenicity and often require the use of adjuvants. Furthermore, due to the relatively limited range of antigens, their induced immune protection spectrum is narrow, and their cross-protective ability against different strains, especially drug-resistant strains, still needs improvement.

[0005] In recent years, DNA vaccines have become an important direction in tuberculosis vaccine research due to their ability to induce humoral and cellular immunity, facilitate the co-expression of multiple antigens, have relatively simple preparation processes, and possess high safety. DNA vaccines can utilize host cells to express target antigens, maintain the native conformation of antigens to a certain extent, and achieve the co-expression of multiple protective antigens by constructing multi-antigen expression vectors, which is beneficial for improving the immune protection effect.

[0006] With the development of tuberculosis vaccine research, multi-stage, multi-antigen DNA vaccines have gradually become a research hotspot. Current technologies typically combine active and latent antigens to cover different stages of Mycobacterium tuberculosis infection. However, most publicly reported multi-stage DNA vaccines still primarily employ simple antigen combinations, lacking targeted design for innate immune signaling pathways, particularly optimizing strategies for Toll-like receptor 2 (TLR2)-mediated immune responses. Furthermore, existing vaccines still have limited activation capacity for innate immune cells such as macrophages, and their clearance efficiency against intracellular Mycobacterium tuberculosis, especially latent bacteria, needs further improvement. Therefore, it is difficult to simultaneously address the protective needs against latent infection, active infection, and drug-resistant strain infection.

[0007] Mycobacterium tuberculosis primarily resides within host macrophages, and its long-term survival and continuous replication are crucial factors contributing to latent infection and disease progression. Toll-like receptors (TLRs) are important pattern recognition receptors in the host's innate immunity. TLR2, in particular, recognizes various pathogen-associated molecular patterns (PAMPs) derived from Mycobacterium tuberculosis, mediating macrophage and dendritic cell activation and promoting antigen presentation and subsequent adaptive immune responses. Therefore, how to enhance TLR2-mediated innate immune activation through the rational design of antigen combinations, thereby further strengthening the host's ability to clear intracellular Mycobacterium tuberculosis, especially latent bacteria, remains a challenge in current tuberculosis DNA vaccine technologies. Summary of the Invention

[0008] The purpose of this invention is to provide a novel Mycobacterium tuberculosis-specific tandem DNA vaccine, W665, which can enhance the host's innate immunity, especially macrophage immune function and Th1-type-dominant specific cellular immune function, and maintain the immune memory capacity of long-acting cells, especially effector memory T cells.

[0009] In a first aspect, the present invention claims protection for a Mycobacterium tuberculosis DNA vaccine.

[0010] The active ingredient of the Mycobacterium tuberculosis DNA vaccine claimed in this invention includes a recombinant expression vector containing a nucleic acid molecule encoding antigenic polypeptide A, a nucleic acid molecule encoding antigenic polypeptide B, a nucleic acid molecule encoding antigenic polypeptide C, and a nucleic acid molecule encoding antigenic polypeptide D. The amino acid sequence of the antigenic polypeptide A is shown in positions 1-134 of SEQ ID NO:1; The amino acid sequence of the antigenic polypeptide B is shown in positions 143-287 of SEQ ID NO:1; The amino acid sequence of the antigenic polypeptide C is shown in positions 288-444 of SEQ ID NO:1; The amino acid sequence of the antigenic polypeptide D is shown in positions 445-594 of SEQ ID NO:1.

[0011] Furthermore, the nucleic acid molecule encoding the antigenic polypeptide A is shown in positions 22-423 of SEQ ID NO:2; The nucleic acid molecule encoding the antigenic polypeptide B is shown in positions 448-882 of SEQ ID NO:2; The nucleic acid molecule encoding the antigenic polypeptide C is shown in positions 883-1353 of SEQ ID NO:2; The nucleic acid molecule encoding the antigenic polypeptide D is shown in positions 1354-1803 of SEQ ID NO:2.

[0012] Furthermore, the recombinant expression vector expresses a fusion protein composed of antigenic polypeptide A, antigenic polypeptide B, antigenic polypeptide C, and antigenic polypeptide D in tandem.

[0013] Furthermore, antigenic polypeptide A and antigenic polypeptide B are linked by a linker peptide, and antigenic polypeptide B and antigenic polypeptide C, as well as antigenic polypeptide C and antigenic polypeptide D, are directly linked.

[0014] In some embodiments, the amino acid sequence of the linker peptide is shown in SEQ ID NO:3.

[0015] In some embodiments, the fusion protein is any of the following: X1) The amino acid sequence is that of the protein in SEQ ID NO:1; X2) A protein that has more than 80% identity with the amino acid sequence shown in SEQ ID NO:1 and has the same function; X3) A fusion protein obtained by attaching a tag to the C-terminus and / or N-terminus of the protein described in X1) or X2).

[0016] In this invention, the identity refers to the identity of the amino acid sequence, which includes amino acid sequences having an identity of 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher.

[0017] In this invention, to facilitate the expression, detection, tracing, and / or purification of the target fusion protein, a tag protein, such as GST tag protein, HI6 tag protein, MBP tag protein, FLAG tag protein, SUMO tag protein, HA tag protein, MYC tag protein, GFP tag protein, etc., can be attached to the C-terminus and / or N-terminus of the target fusion protein when it is prepared using DNA in vitro recombination technology.

[0018] In some implementations, the recombinant expression vector is a recombinant eukaryotic cell expression vector.

[0019] In some specific embodiments, the recombinant eukaryotic cell expression vector is a vector obtained by inserting the DNA molecule shown in positions 7-1806 of SEQ ID NO:2 into a eukaryotic expression vector.

[0020] In some preferred embodiments, the recombinant eukaryotic cell expression vector is a vector obtained by inserting the DNA molecule shown in positions 7-1806 of SEQ ID NO:2 into the Nhe I and EcoR I restriction sites of the eukaryotic expression vector pVAX1.

[0021] Secondly, the present invention claims protection for the above-mentioned fusion protein and the nucleic acid molecule encoding the above-mentioned fusion protein or the expression cassette or recombinant vector containing the nucleic acid molecule.

[0022] The nucleic acid molecule encoding the aforementioned fusion protein can be either a DNA molecule or an RNA molecule.

[0023] In some embodiments, the nucleic acid molecule encoding the fusion protein is the DNA molecule shown in positions 22-1803 of SEQ ID NO:2.

[0024] The expression cassette may include a promoter, the aforementioned nucleic acid molecule, and a terminator. Furthermore, the expression cassette may also include an enhancer sequence.

[0025] The aforementioned vector refers to a eukaryotic cell expression vector capable of delivering the aforementioned nucleic acid molecules into host cells for amplification and expression. The recombinant vector refers to a recombinant DNA molecule constructed by in vitro ligation of the aforementioned nucleic acid molecules with the eukaryotic cell expression vector. Those skilled in the art can construct recombinant vectors containing the aforementioned nucleic acid molecules using any existing eukaryotic cell expression vector.

[0026] In some implementations, the eukaryotic cell expression vector is a pVAX1 vector.

[0027] In some embodiments, the recombinant vector is a vector obtained by inserting the DNA molecule shown in positions 7-1806 of SEQ ID NO:2 into the Nhe I and EcoR I restriction sites of the eukaryotic expression vector pVAX1.

[0028] Thirdly, the present invention claims protection for any of the following applications: Y1) The application of the above-mentioned Mycobacterium tuberculosis DNA vaccine or the above-mentioned fusion protein or the above-mentioned nucleic acid molecule, expression cassette, recombinant vector in the prevention and / or treatment of Mycobacterium tuberculosis infection; Y2) The use of the above-mentioned Mycobacterium tuberculosis DNA vaccine, or the above-mentioned fusion protein, or the above-mentioned nucleic acid molecule, expression cassette, or recombinant vector in the preparation of products for the prevention and / or treatment of Mycobacterium tuberculosis infection.

[0029] Fourthly, the present invention claims protection for an agent for the prevention and / or treatment of Mycobacterium tuberculosis infection.

[0030] The preparations provided by this invention for the prevention and / or treatment of Mycobacterium tuberculosis infection contain the above-mentioned Mycobacterium tuberculosis DNA vaccine or the above-mentioned fusion protein or the above-mentioned nucleic acid molecule, expression cassette, or recombinant vector.

[0031] The preparation may be an immunomodulatory agent for Mycobacterium tuberculosis, a prophylactic preparation for Mycobacterium tuberculosis, or a therapeutic preparation for Mycobacterium tuberculosis.

[0032] When preparing the formulation, excipients such as diluents, solubilizers, buffers, and pH adjusters, such as physiological saline, may also be added.

[0033] The formulation can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, or by respiratory administration, including nasal spray, oral inhalation and other methods.

[0034] When using the aforementioned formulation to prevent or / and treat Mycobacterium tuberculosis infection, an effective amount of the aforementioned DNA vaccine, fusion protein, nucleic acid molecule, expression cassette, or recombinant vector is administered to the subject organism.

[0035] Any of the products mentioned above may be drugs or vaccines.

[0036] The above-mentioned Mycobacterium tuberculosis infection can be an acute Mycobacterium tuberculosis infection.

[0037] This invention utilizes genetic engineering technology to tandem the immunodominant epitopes of multiple Mycobacterium tuberculosis antigens and clone them into the eukaryotic expression vector pVAX1, constructing the Mycobacterium tuberculosis tandem DNA vaccine W665. Animal experiments show that, when used as an immunomodulator in mice, the Mycobacterium tuberculosis tandem DNA vaccine W665 enhances the expression of TLR2 protein on the surface of spleen and lung macrophages; simultaneously, it significantly increases the number of speckled T lymphocytes secreting interferon-gamma, induces a Th1-dominant specific cellular immune response, and maintains a high level of T lymphocyte immune memory, thus exhibiting good immunomodulatory effects and significantly enhancing the innate and cellular immune responses in mice. Further challenge protection experiments show that, when used as a prophylactic vaccine to prevent acute tuberculosis infection in mice, the Mycobacterium tuberculosis tandem DNA vaccine W665 significantly reduces the live Mycobacterium tuberculosis count in mouse liver tissue and significantly alleviates pathological damage in lung tissue, demonstrating good immunoprotective effects. Therefore, the Mycobacterium tuberculosis tandem DNA vaccine W665 provided by the present invention can simultaneously enhance the host's innate immunity and specific cellular immune response, improve the body's immune protection against Mycobacterium tuberculosis infection, and can be used to prepare biological agents for the prevention and / or treatment of Mycobacterium tuberculosis infection. Attached Figure Description

[0038] Figure 1 The PCR amplification product of recombinant plasmid pVAX1-W665 was identified by 1% agarose gel electrophoresis.

[0039] Figure 2 The proportion of TLR2 expression on the surface of splenic macrophages in each group of mice 4 weeks after the third immunization with the vaccine. A: Representative flow cytometry results of each group. B: Analysis of TLR2 expression proportions in macrophages among groups. Data are expressed as mean ± standard deviation (n=3). * indicates... P <0.05, ** is P <0.01, *** is P <0.001, **** is P <0.0001. Figure 3 To detect the number of IFN-γ-secreting splenic lymphocyte spots in mice 4 weeks after the third immunization with the vaccine using ELISPOT. I: Representative splenic lymphocyte spot scans from each group, where A: unstimulated blank control wells; B: non-specific stimulation wells with PHA; C, D, and E: specific stimulation wells with W665 protein. II: Analysis results of splenic cell spot counts in each group after W665 protein stimulation. Data are expressed as mean ± standard deviation (n=3). **For P<0.01.

[0040] Figure 4 The results show the viable bacterial counts in the lungs and liver of mice in each group 9 weeks after infection with Mycobacterium tuberculosis. A: Lung colony count. B: Liver colony count. * indicates... P <0.05, ** is P <0.01.

[0041] Figure 5 The images show representative lung tissue histopathological (H&E) examinations of mice in each group 9 weeks after infection with Mycobacterium tuberculosis. A, B, and C: Representative lung tissue histopathological examinations of each group.

[0042] Figure 6 Western blot analysis of TLR2 protein expression in lung tissues of mice in each group 9 weeks after Mycobacterium tuberculosis infection. A: TLR2 protein bands in each group. B: β-actin internal control bands in each group. The leftmost value represents the protein molecular weight standard. The actual molecular weight of TLR2 protein is 89.48 kD; the measured molecular weight of β-actin protein is 41.74 kD. C: TLR2 / β-actin grayscale ratio analysis. Data are expressed as mean ± standard deviation (n=3), and one-way ANOVA was used for inter-group comparisons.

[0043] Figure 7 This study analyzed the proportions of spleen memory T cell subsets in mice at 9 weeks post-infection with Mycobacterium tuberculosis. A–C: CD3 + T cells, CD3 + Central memory T cells and CD3 + The proportion of effector memory T cells. D–F: CD4 + T cells, CD4 + Central memory T cells and CD4 + The proportion of effector memory T cells. G–I: CD8 + T cells, CD8 + Central memory T cells and CD8 + The proportion of effector memory T cells. Data are expressed as mean ± standard deviation (n=3), and one-way ANOVA was used for inter-group comparisons. * indicates... P <0.05, ** is P <0.01, *** is P <0.001, **** is P <0.0001. Detailed Implementation

[0044] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0045] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the experimental methods in the following embodiments are performed at least three times.

[0046] Example 1: A method for preparing a Mycobacterium tuberculosis-specific tandem DNA vaccine, W665. This invention utilizes genetic engineering technology to tandem the immunodominant epitope genes of multiple Mycobacterium tuberculosis antigens and clone them into the eukaryotic expression vector pVAX1, constructing a novel Mycobacterium tuberculosis DNA vaccine, W665, which possesses multiple immunodominant epitopes. The specific construction method is as follows: I. Design and Synthesis of the W665 Gene Sequence The DNA sequence of the W665 eukaryotic expression DNA vaccine designed and optimized by this invention is shown in SEQ ID NO:2. It contains, in sequence, an NheI restriction endonuclease recognition site (SEQ ID NO:2, positions 1-6), a start codon (SEQ ID NO:2, positions 16-18), the coding gene sequences of four antigenic polypeptides (antigenic polypeptide A, antigenic polypeptide B, antigenic polypeptide C, and antigenic polypeptide D) (SEQ ID NO:2, positions 22-1803), a stop codon (SEQ ID NO:2, positions 1804-1806), and an EcoRI restriction endonuclease recognition site (SEQ ID NO:2, positions 1807-1812). The gene shown in SEQ ID NO:2 is named the W665 gene.

[0047] The amino acid sequence of antigenic polypeptide A is shown in positions 1-134 of SEQ ID NO:1, and its coding gene sequence is shown in positions 22-423 of SEQ ID NO:2; the amino acid sequence of antigenic polypeptide B is shown in positions 143-287 of SEQ ID NO:1, and its coding gene sequence is shown in positions 448-882 of SEQ ID NO:2; the amino acid sequence of antigenic polypeptide C is shown in positions 288-444 of SEQ ID NO:1, and its coding gene sequence is shown in positions 883-1353 of SEQ ID NO:2; the amino acid sequence of antigenic polypeptide D is shown in positions 445-594 of SEQ ID NO:1, and its coding gene sequence is shown in positions 1354-1803 of SEQ ID NO:2.

[0048] II. Construction of recombinant plasmid pVAX1-W665 1. PCR amplification of the W665 gene The W665 gene sequence shown in SEQ ID NO:2 was artificially synthesized and used as a template for PCR amplification using primers W665-F and W665-R. The PCR amplification product (i.e., the W665 gene sequence) was obtained, and the PCR amplification product was recovered according to the Omega gel extraction kit instructions. The primer sequences are as follows: W665-F:5'-CG GCTAGC GCCGCCACCATGGCCATGCC-3' (The underlined sequence is the Nhe I restriction site); W665-R: 5'-CG GAATTC TCACTGAGGAGCTGGGCCTGT-3' (The underlined sequence is the EcoR I restriction site).

[0049] The PCR procedure was as follows: denature at 98℃ for 2 min; then denature at 98℃ for 10 s, anneal at 60℃ for 20 s, extend at 72℃ for 30 s, for a total of 37 cycles; finally extend at 72℃ for 5 min; incubate at 4℃ for 5 min.

[0050] 2. Double enzyme digestion of vector plasmid and W665 gene The vector plasmid pVAX1 (an invitrogen product from Thermo, Lot: 1724963, purchased from Beijing Yongze Haojia Biotechnology Development Center) was double-digested with restriction endonucleases Nhe I and EcoRI to obtain the double-digested pVAX1 product.

[0051] The PCR amplification product of the W665 gene obtained in step 1 was double-digested with restriction endonucleases Nhe I and EcoRI to obtain the double-digested product of the W665 gene.

[0052] The reaction system for double enzyme digestion is as follows: 5 μL of 10×CutSmart, 2 μL of Nhe I, 2 μL of EcoRI, 20 μL of PCR amplification product of pVAX1 (4 μg) or W665, and water to make up to a total volume of 50 μL.

[0053] The reaction conditions for double enzyme digestion are as follows: digestion overnight at 37℃, and termination of the reaction at 65℃.

[0054] The pVAX1 double digestion product and the W665 gene double digestion product were recovered according to the instructions of the Omega gel recovery kit.

[0055] 3. Connection reaction The pVAX1 double digestion product and the W665 gene double digestion product obtained in step 2 were ligated using T4 DNA Ligase (NEB) to obtain the ligation product.

[0056] The ligation reaction system is as follows: 2 μL of 10×T4 Ligase buffer, 1 μL of T4 Ligase, 1 μL of pVAX1 double digestion product, 3 μL of W665 gene double digestion product, and water to make up to a total volume of 20 μL.

[0057] The reaction conditions for connection are as follows: overnight at 16°C.

[0058] 4. PCR identification of recombinant plasmid pVAX1-W665 Add 1 μL of the ligation product obtained in step 3 to E. coli DH5α competent cells and incubate on ice for 30 min, heat shock at 42°C for 90 s, and then immediately place on ice for 5 min. Then add 500 μL of antibiotic-free LB medium, shake at 37°C and 180 rpm for 1 h, centrifuge, and spread the entire mixture onto LB agar plates containing kanamycin and incubate upside down at 37°C overnight.

[0059] Single clones were picked from the plate and inoculated into test tubes containing 2 mL of LB medium containing Kan+. The culture was incubated overnight at 37°C and 220 rpm with shaking. Using the bacterial culture as a template, the target gene was amplified using PCR primers for the W665 gene, and the PCR products were subjected to 1% agarose gel electrophoresis.

[0060] The results are as follows Figure 1 As shown. From Figure 1 It can be seen that the size of the PCR amplification product is 1812 bp, which is basically consistent with the theoretical length, and the PCR identification result is correct.

[0061] 5. Gene sequencing identification of recombinant plasmid pVAX1-W665 The purified recombinant plasmid pVAX1-W665 was sent to the company for gene sequencing. Comparison analysis of the sequencing results with the designed original sequence showed that the two sequences were completely identical, proving that the W665 gene fragment had been successfully inserted into the pVAX1 plasmid. This correctly sequenced recombinant plasmid pVAX1-W665 is the Mycobacterium tuberculosis tandem DNA vaccine W665 of this invention. The pVAX1-W665 plasmid expresses the W665 protein, the amino acid sequence of which is shown in SEQ ID NO:1.

[0062] Example 2: Evaluation of the immunogenicity of the Mycobacterium tuberculosis tandem DNA vaccine W665 The Mycobacterium tuberculosis tandem DNA vaccine W665 prepared in Example 1 was used as an immunomodulatory agent to immunize mice, and its immunomodulatory effect was evaluated. The specific steps are as follows: I. Experimental Methods and Materials 1. Animal selection Eighteen female BALB / c mice aged 56-62 days, weighing 18-20 g, with certificates of conformity were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The weight difference within the same batch of mice should not exceed 2 g.

[0063] 2. Immunogenicity evaluation of the Mycobacterium tuberculosis tandem DNA vaccine W665 (1) Animal immunization The mice from step 1 were divided into 3 groups of 6 mice each, and the treatment methods were as follows: ① Saline group: Each mouse was injected intramuscularly with 100 μL of saline.

[0064] ② pVAX1 vector group: Each mouse was injected intramuscularly with 100 μg / 100 μL pVAX1 solution (pVAX1 vector was dissolved in physiological saline to prepare 100 μg / 100 μL pVAX1 solution).

[0065] ③W665 DNA vaccine group: Each mouse was injected intramuscularly with 100μg / 100μL W665 DNA vaccine (recombinant plasmid pVAX1-W665 was dissolved in physiological saline to prepare a 100μg / 100μL W665 DNA solution).

[0066] Each group of mice received an intramuscular injection every 2 weeks for a total of 3 injections. Four weeks after the third immunization, mice in each group were sacrificed by cervical dislocation.

[0067] (2) The expression ratio of TLR2 on the surface of mouse spleen macrophages was detected by flow cytometry. Four weeks after the third immunization, mice in each group were sacrificed, and spleens were harvested and spleen cells were isolated. 600 μL of mouse spleen cells were then inoculated at 1.5 × 10⁻⁶ cells per cell line. 6Spinal macrophages were seeded at a density of 10 cells / well in 12-well cell culture plates. One well was added with 300 μL of RPMI-1640 complete culture medium, two wells with 300 ng / mL lipopolysaccharide (LPS), and two wells with 90 μg / mL (working concentration) W665 protein (final concentration 30 μg / mL). The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. Splenic macrophages were then collected and cultured at 1 × 10⁻⁶ cells / well. 6 Each cell / tube was added to a flow cytometry tube, and F4 / 80, CD11b, and TLR2 (CD282) fluorescent monoclonal antibodies were added sequentially for staining to analyze the expression level of TLR2 (CD282) on the surface of splenic macrophages.

[0068] (3) The number of T lymphocyte spots secreting interferon-gamma in mice was detected by enzyme-linked immunospot assay (ELISPOT). Four weeks after the third immunization, the mice were sacrificed, their spleens were harvested, and spleen cells were isolated. The concentration of spleen lymphocytes was adjusted to 3 × 10⁻⁶. 6 / mL. In an ELISPOT plate, add 1640 complete cell culture medium, W665 protein (final concentration 30 μg / mL), and phytohemagglutinin (PHA) (final concentration 30 μg / mL), with 3 × 102 spleen lymphocytes. 5 / well, incubated in a CO2 incubator at 37℃ for 24h, and performed enzyme-linked immunospot assay using the Mouse IFN-γ ELISPOT PLUS kit (product of Mabtech AG, Sweden, purchased from Beijing Zuorun Chengye Biotechnology Co., Ltd.). The operation was performed according to the kit instructions. The number of T lymphocyte spots secreting IFN-γ in each group was detected. The blank control wells of each group of mice were used as controls, and the number of cell spots in the test wells of each group of mice was calculated.

[0069] 3. Statistical Analysis Data were processed using Graphpad Prism software. One-way ANOVA was used for normally distributed quantitative data, and t-tests were used for pairwise comparisons. II. Experimental Results 1. Proportion of TLR2 expression on the surface of splenic macrophages The expression ratio of TLR2 (CD282) on the surface of splenic macrophages in each group of mice was detected by flow cytometry, and the results are as follows: Figure 2 As shown. The results showed that the proportion of cells expressing TLR2 in the W665 DNA vaccine group was significantly higher than that in the saline group and the pVAX1 vector group ( P <0.0001, P <0.005). This indicates that the W665 DNA vaccine can significantly induce TLR2 expression on the surface of macrophages.

[0070] 2. Number of splenic T lymphocytes secreting gamma interferon The number of splenic T lymphocytes secreting interferon-gamma in the spleen cells of mice in each group was detected by the ELISPOT assay. The results are as follows: Figure 3 As shown. The results showed that after stimulation with W665 protein, the number of spots in the W665 DNA vaccine group was significantly higher than that in the saline group and the pVAX1 vector group ( Figure 3 (Middle II) P <0.01, P <0.01). This indicates that the W665 DNA vaccine can express the antigen in mice and significantly stimulate splenic lymphocytes to secrete gamma interferon.

[0071] Example 3: Application of Mycobacterium tuberculosis tandem DNA vaccine W665 in the prevention of acute Mycobacterium tuberculosis infection The Mycobacterium tuberculosis tandem DNA vaccine W665 prepared in Example 1 was used as a prophylactic agent to immunize a mouse model of acute tuberculosis infection, and its preventive effect in acute Mycobacterium tuberculosis infection was evaluated. The specific steps are as follows: I. Experimental Methods and Materials 1. Animal selection Forty female BALB / c mice aged 42-62 days with valid certificates were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0072] 2. The preventive effect of W665 DNA vaccine against acute infection of Mycobacterium tuberculosis. (1) Experimental grouping After weighing the 42-62 day old female BALB / c mice from step 1, they were randomly and evenly divided into 4 groups of 10 mice each. The treatment method for each group was as follows: ① Saline group: Each mouse was injected intramuscularly with 100 μL of saline each time, and immunized once every 3 weeks for a total of 3 times.

[0073] ② pVAX1 vector group: Each mouse was injected intramuscularly with 100 μg / 100 μL pVAX1 solution (pVAX1 vector was dissolved in physiological saline to prepare 100 μg / 100 μL pVAX1 solution) once every 3 weeks, for a total of 3 times.

[0074] ③W665 DNA vaccine group: Each mouse was injected intramuscularly with 100μg / 100μL W665 DNA vaccine (recombinant plasmid pVAX1-W665 dissolved in physiological saline to prepare a 100μg / 100μL W665 DNA solution) once every 3 weeks for a total of 3 times.

[0075] ④ Normal control group: 10 mice were raised normally in the animal laboratory, without being vaccinated or infected with Mycobacterium tuberculosis.

[0076] (2) Attacking the poison Mice in groups ①-③ were challenged 32 days after their third immunization. The challenge method was as follows: 30 mice were injected via the tail vein with 0.4 mL of a solution containing 1... 10 6 A mouse model of acute tuberculosis infection was prepared using a suspension of the attenuated Mycobacterium tuberculosis strain H37Ra containing CFUs. The mice were then challenged with the virus and housed in a biosafety level II negative pressure laboratory.

[0077] (3) Viable bacterial count in the lungs and liver Nine weeks after challenge, mice from all groups were sacrificed, and the left lung and left half of the liver were dissected for colony counting. The specific steps were as follows: The left lung lobe and left half of the liver of each mouse were placed in sterile grinding tubes, and 3 mL of sterile physiological saline was added to each organ. The tissue homogenate was then homogenized using a tissue homogenizer. One mL of the tissue homogenate was aspirated and an equal volume of 4% NaOH was added. After digestion for 30 min, a 10-fold serial dilution was performed, with two concentrations for each organ: the original concentration and the 10-fold serial dilution. -1 10 -2 Three concentrations were tested. 0.1 mL of each concentration was spread onto two Löwenkel agar plates and incubated at 37°C for 4 weeks. Colony growth was observed, and colonies were counted. The colony counts in the left lung lobe were converted to the whole lung colony count based on the weight of the left lung lobe and the whole lung. Similarly, the colony counts in the left hepatic half were converted to the whole liver colony count based on the weight of the left hepatic half and the whole liver.

[0078] (4) Lung tissue pathological examination Nine weeks after the viral challenge, mice in each group were sacrificed. The right lobe of the lungs of the mice was fixed in a pathological centrifuge tube containing 10% neutral formaldehyde, embedded in paraffin, sectioned, stained with hematoxylin / eosin (HE), and serially sectioned at 5 μm. Histopathological lesions of the lung tissue were observed under a microscope.

[0079] (5) Expression of TLR2 protein in lung tissue 1 mL of mouse lung tissue homogenate was placed into an ice-cold EP tube, and 1 mL of RIPA lysis buffer (containing PMSF and protease inhibitor) was added to the tube. After lysis on ice for 30 min, the lysis buffer was transferred to a 2 mL EP tube and centrifuged at 4 °C and 12,000 rpm for 5 min. The supernatant was aliquoted into 2 mL EP tubes, and the expression level of TLR2 in each group was quantitatively detected by digital Western blot.

[0080] (6) Analysis of mouse spleen cell memory T cell subsets using flow cytometry Nine weeks after challenge, mice in each group were sacrificed, spleen cells were isolated, and spleen cell suspensions were prepared, with the cell concentration adjusted to 1×10⁻⁶. 6 Each spleen cell suspension was stained and incubated with 2.5 μL of CD3 fluorescent monoclonal antibody, 1.25 μL of CD4 fluorescent monoclonal antibody, 1.25 μL of CD8 fluorescent monoclonal antibody, 1.25 μL of CD44 fluorescent monoclonal antibody, 1.25 μL of CD45 fluorescent monoclonal antibody, and 1.25 μL of CD62L fluorescent monoclonal antibody (all six fluorescent monoclonal antibodies were products of Biolegend and purchased from Beijing Dakewei Biotechnology Co., Ltd.). Each spleen cell suspension sample was repeated three times for each group. After thorough mixing, the proportions of each subset of spleen memory T lymphocytes were analyzed by flow cytometry.

[0081] 3. Statistical Analysis Data were processed using Graphpad Prism software. One-way ANOVA was used for normally distributed quantitative data, and t-tests were used for pairwise comparisons. II. Experimental Results 1. Live bacterial count in lung and liver tissues Mice in each group were sacrificed 9 weeks after infection with Mycobacterium tuberculosis. The results of viable bacterial counts in the lungs and livers of mice in each group are as follows: Figure 4 As shown. Lung tissue viable bacterial count results showed that there was no statistically significant difference in viable bacterial count between the W665 DNA vaccine group and the saline group and the pVAX1 group. P >0.05). Liver tissue viable count results showed that the viable counts in the W665 DNA vaccine group and the pVAX1 group were significantly lower than those in the saline group ( P <0.01, P <0.05); and the viable bacterial count in the W665 DNA vaccine group was also lower than that in the pVAX1 group, but the difference between the two groups was not statistically significant. P >0.05). The above results indicate that W665 DNA vaccine immunization can significantly reduce the Mycobacterium tuberculosis load in mouse liver tissue, but has no significant effect on clearing live bacteria in the lungs.

[0082] 2. Lung tissue pathological examination Mice from each group were sacrificed 9 weeks after infection with Mycobacterium tuberculosis. Pathological examination results of representative lung tissue from each group are as follows: Figure 5 As shown in the figure. The results indicated that the saline group and the pVAX1 vector group showed more significant inflammatory infiltration and tissue structure destruction in the lung tissue, while the W665 DNA group showed relatively milder lung tissue lesions. These results suggest that W665 DNA vaccine immunization can significantly reduce pathological damage to lung tissue.

[0083] 3. Lung tissue TLR2 protein expression Nine weeks after infection with Mycobacterium tuberculosis, mice in each group were sacrificed, and total protein was extracted from lung tissue. Western blot analysis was used to detect the expression level of TLR2 protein in mouse lung tissue. The results are as follows: Figure 6 As shown in the figure. The results showed that TLR2 protein expression could be detected in the lung tissue of each group. Compared with the saline group and the pVAX1 vector group, the TLR2 protein expression level in the W665 DNA vaccine group showed an increasing trend, and the gray ratio of TLR2 to β-actin was higher than that in the two control groups, but the differences were not statistically significant. P >0.05). The above results indicate that W665 DNA vaccine immunization can promote TLR2-related immune responses in lung tissue, thereby enhancing the innate immune response of lung tissue after infection.

[0084] 4. Analysis of spleen memory T cell subsets Nine weeks after infection with Mycobacterium tuberculosis, mice in each group were sacrificed. Flow cytometry results of the proportions of different subsets of spleen memory T lymphocytes are as follows: Figure 7 As shown. The results show that CD4 in each group + T cells and CD8 + There was no significant difference in the proportion of T cells between the groups. P >0.05); W665 DNA vaccine group CD4 + The proportion of central memory T cells was significantly lower in the saline group than in the central memory T cell group. P <0.0001) and pVAX1 vector group ( P <0.05), CD4 + The proportion of effector memory T cells was significantly higher in the saline group than in the saline group. P <0.0001) and pVAX1 vector group ( P <0.01); CD8 + The proportion of central memory T cells was significantly lower in the saline group than in the central memory T cell group. P <0.0001) and pVAX1 vector group ( P <0.001), CD8 + The proportion of effector memory T cells was significantly higher in the saline group and the pVAX1 vector group than in the saline group. P <0.0001). The above results indicate that the W665 DNA vaccine can effectively induce the body to produce long-lasting and stable specific T cell immune memory, mainly effector memory T cells, enabling the immune memory cells to remain in the body for a long time and continuously maintain the body's specific immune defense against Mycobacterium tuberculosis. This fully demonstrates that the vaccine has excellent long-lasting immune activation and immune memory induction functions.

[0085] In summary, the W665 DNA vaccine of this invention can significantly enhance the innate immunity, especially macrophage immunity and specific cellular immunity, in mice, primarily stimulating Th1-type cellular immune responses. Vaccination with the W665 DNA vaccine significantly reduces the number of viable bacteria in the liver tissue and significantly alleviates lung lesions in a mouse model of acute Mycobacterium tuberculosis infection. Therefore, the W665 DNA vaccine of this invention has broad application prospects in the prevention and treatment of tuberculosis.

[0086] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A Mycobacterium tuberculosis DNA vaccine, characterized in that, The active ingredient of the Mycobacterium tuberculosis DNA vaccine includes a recombinant expression vector containing nucleic acid molecules encoding antigenic polypeptide A, antigenic polypeptide B, antigenic polypeptide C, and antigenic polypeptide D. The amino acid sequence of the antigenic polypeptide A is shown in positions 1-134 of SEQ ID NO:1; The amino acid sequence of the antigenic polypeptide B is shown in positions 143-287 of SEQ ID NO:1; The amino acid sequence of the antigenic polypeptide C is shown in positions 288-444 of SEQ ID NO:1; The amino acid sequence of the antigenic polypeptide D is shown in positions 445-594 of SEQ ID NO:

1.

2. The Mycobacterium tuberculosis DNA vaccine as described in claim 1, characterized in that: The nucleic acid molecule encoding the antigenic polypeptide A is shown in positions 22-423 of SEQ ID NO:2; The nucleic acid molecule encoding the antigenic polypeptide B is shown in positions 448-882 of SEQ ID NO:2; The nucleic acid molecule encoding the antigenic polypeptide C is shown in positions 883-1353 of SEQ ID NO:2; The nucleic acid molecule encoding the antigenic polypeptide D is shown in positions 1354-1803 of SEQ ID NO:

2.

3. The Mycobacterium tuberculosis DNA vaccine as described in claim 1 or 2, characterized in that: The recombinant expression vector expresses a fusion protein consisting of antigenic polypeptide A, antigenic polypeptide B, antigenic polypeptide C, and antigenic polypeptide D in tandem.

4. The Mycobacterium tuberculosis DNA vaccine as described in claim 3, characterized in that: In the fusion protein, antigenic polypeptide A and antigenic polypeptide B are linked by a linker peptide, and antigenic polypeptide B and antigenic polypeptide C and antigenic polypeptide C and antigenic polypeptide D are directly linked. Furthermore, the amino acid sequence of the linker peptide is shown in SEQ ID NO:

3.

5. The Mycobacterium tuberculosis DNA vaccine as described in claim 3 or 4, characterized in that: The fusion protein is any one of the following: X1) The amino acid sequence is that of the protein in SEQ ID NO:1; X2) A protein that has more than 80% identity with the amino acid sequence shown in SEQ ID NO:1 and has the same function; X3) A fusion protein obtained by attaching a tag to the C-terminus and / or N-terminus of the protein described in X1) or X2).

6. The Mycobacterium tuberculosis DNA vaccine as described in any one of claims 1-5, characterized in that: The recombinant expression vector is a vector obtained by inserting the DNA molecule shown in positions 7-1806 of SEQ ID NO:2 into a eukaryotic expression vector.

7. The fusion protein according to any one of claims 1-6.

8. A nucleic acid molecule encoding any of the fusion proteins described in claims 1-6, or an expression cassette or recombinant vector containing said nucleic acid molecule.

9. Any of the following applications: Y1) The use of the Mycobacterium tuberculosis DNA vaccine according to any one of claims 1-6, or the fusion protein according to claim 7, or the nucleic acid molecule, expression cassette, or recombinant vector according to claim 8 in the prevention and / or treatment of Mycobacterium tuberculosis infection; Y2) The use of the Mycobacterium tuberculosis DNA vaccine according to any one of claims 1-6, or the fusion protein according to claim 7, or the nucleic acid molecule, expression cassette, or recombinant vector according to claim 8 in the preparation of products for the prevention and / or treatment of Mycobacterium tuberculosis infection.

10. An agent for the prevention and / or treatment of Mycobacterium tuberculosis infection, comprising the Mycobacterium tuberculosis DNA vaccine of any one of claims 1-6, the fusion protein of claim 7, or the nucleic acid molecule, expression cassette, or recombinant vector of claim 8.

Images