Mycobacterium tuberculosis tandem DNA vaccine W545, and preparation method and application thereof

Abstract

The application discloses a mycobacterium tuberculosis tandem DNA vaccine W545 and a preparation method and application thereof. The application constructs a novel mycobacterium tuberculosis DNA vaccine W545 with a multi-antigen immune dominant epitope by connecting epitope genes of Ag85A protein and Ag85B protein antigen and Rv1419, Rv3407 and Rv2628c together in series through genetic engineering technology and connecting the epitope genes to a eukaryotic expression vector pVAX1. The prepared mycobacterium tuberculosis tandem DNA vaccine W545 can significantly enhance specific cellular immune function of mice, mainly stimulates Th1 type immune response, and can make the lesion range of organs and tissues of a mouse tuberculosis model significantly reduced and the lesion alleviated in treatment, and can be used in preparation of drugs or vaccines for preventing or treating tuberculosis.

Description

TECHNICAL FIELD

[0001] The present application relates to a Mycobacterium tuberculosis specific tandem DNA vaccine W545, a preparation method and application thereof, in particular to a novel Mycobacterium tuberculosis specific tandem DNA vaccine W545 prepared by genetic engineering technology and application thereof, and belongs to the technical field of tuberculosis medical immunology prevention and treatment. BACKGROUND

[0002] Tuberculosis (TB) has been a global health problem that people have been concerned about. The World Health Organization (WHO) Global Tuberculosis Report 2020 pointed out that in 2019, there were 10 million new cases of tuberculosis worldwide, and 1.2 million deaths. About one-third of the world's population has Mycobacterium tuberculosis (M.tb) latent infection, of which 5% to 10% of the infected people will eventually develop into TB patients. The number of TB patients in China ranks third in the world. About one-third of the population in China is infected with tuberculosis, of which 5% may develop early, 5% may develop at any time in their life, and there are many drug-resistant TB patients. Therefore, the prevention and treatment of tuberculosis-infected population can effectively reduce the incidence of pulmonary tuberculosis, and the early diagnosis and effective chemotherapy of tuberculosis patients will control the spread of tuberculosis and tuberculosis bacteria. Effective prevention and treatment of M.tb infection at different stages is crucial.

[0003] Tuberculosis is a chronic respiratory infectious disease with contradictions and challenges in infection, immunity, prevention and treatment, etc. Reasonable and regular chemotherapy can kill most of the tuberculosis bacteria in the lesion within 1-2 months, but a small amount of bacteria remains, especially the tuberculosis bacteria parasitized in macrophages, which are difficult to be killed and need to be treated for 3-4 months or even longer. Due to the spread of drug-resistant strains, the treatment of drug-resistant tuberculosis (especially multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis) is a problem that China will focus on in the future tuberculosis control. The currently used ten or more anti-tuberculosis drugs have been used for decades, and the initial treatment of tuberculosis usually requires 3-4 drugs for more than 6 months, while the retreatment or drug-resistant tuberculosis usually requires 5-6 drugs for more than 1 year, plus the side effects of drugs, the high price of second-line drugs, and the difficulty for some patients to adhere to the treatment. Due to the development of new anti-tuberculosis drugs, not only the investment is large, the period is long, and it is more difficult than the development of new therapeutic vaccines, but also the new drugs may soon develop drug resistance. Vaccine treatment only needs to be injected several times in a few months, which is much more convenient than taking medicine every day, has fewer side effects, and is low in cost. Anti-tuberculosis immunity is mainly cell-mediated immune response, and vaccine treatment can induce the potential of the immune system of tuberculosis patients to achieve the purpose of treating the disease. Therefore, in recent years, the treatment of tuberculosis through immune regulation has become one of the research hotspots, and the research and development of therapeutic vaccines have become an important research direction.

[0004] Vaccination is the most effective way to prevent tuberculosis. The current domestic and foreign tuberculosis vaccine research mainly has the following three kinds: live vaccine, subunit vaccine and inactivated vaccine. Subunit vaccine only uses part of the components of Mycobacterium tuberculosis to cause the body to produce an immune protective response, mainly including DNA vaccine, recombinant protein vaccine or polypeptide vaccine (with adjuvant), other purified main components (such as mycolic acid, glycolipid, etc.) other than polypeptide, which can be used as a booster vaccine for BCG and also as a therapeutic vaccine. Compared with other types of vaccines, nucleic acid vaccines have the following advantages: (1) DNA vaccine can induce a comprehensive immune response: it can activate both humoral immunity and cellular immunity, especially it can induce CTL response, which can recognize, kill and destroy infected cells and eliminate pathogens in cells, which is an effective clearance pathway for killing Mycobacterium tuberculosis parasitized in macrophages, which can make up for the deficiency of BCG in inducing weak CTL response, which is incomparable with recombinant protein subunit vaccine and inactivated vaccine; (2) It can overcome the problem of incomplete glycosylation and incorrect folding of protein subunit vaccine; (3) Multivalent vaccine can be prepared by mixing plasmids expressing multiple antigens or constructing complex plasmids capable of expressing multiple antigens, which meets the requirements of the long-term goal of WHO's child vaccine program (using one vaccine to prevent multiple diseases); (4) It can produce a long-lasting immune response, and once vaccination can obtain long-term immunity without the need for repeated multiple booster immunization; (5) After vaccination with nucleic acid vaccine, protein antigens are expressed in host cells, and there is no risk of disease caused by toxic or residual toxic virus particles, and it is safer to use; (6) Preparation is simple, and production cost is low; (7) The plasmid DNA of nucleic acid vaccine has good stability, and is easy to store and transport. In addition, DNA vaccine preparation and application are simple, and it is safer for immunocompromised individuals than BCG. The key to the development of the vaccine lies in the selection of protective antigens and the determination of the dose, and the selection of antigens as DNA vaccines is mainly whether they can induce protective T cell response to protect people against tuberculosis. The selection and construction of antigens are crucial. The current research proves that the vaccine mixed, fused or chimeric of multiple protective antigens of Mycobacterium tuberculosis stimulates CD4 + and CD8 + T cell response and the protection, treatment effect and long-term survival ability are stronger than single vaccine component. SUMMARY

[0005] The purpose of the present application is to provide a Mycobacterium tuberculosis specific tandem DNA vaccine for making a rapid and effective cellular immune response against Mycobacterium tuberculosis infection, inhibiting the growth of Mycobacterium tuberculosis in vivo, inhibiting the endogenous relapse of M.tb, and for the prevention and treatment of tuberculosis infection, and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned purpose, the present application firstly provides a DNA vaccine of Mycobacterium tuberculosis, and is named as W545.

[0007] The active ingredient of the DNA vaccine of Mycobacterium tuberculosis W545 provided by the present application is a recombinant eukaryotic cell expression vector expressing a fusion protein; the fusion protein comprises antigen epitope A, antigen epitope B, antigen epitope C, antigen epitope D, antigen epitope E, antigen epitope F, antigen epitope G, antigen epitope H, antigen epitope I, antigen epitope J, antigen epitope K and antigen epitope L:

[0008] The amino acid sequence of the antigen epitope A is shown in sequence 1 of the sequence listing as 3-291;

[0009] The amino acid sequence of the antigen epitope B is shown in sequence 1 of the sequence listing as 299-313;

[0010] The amino acid sequence of the antigen epitope C is shown in sequence 1 of the sequence listing as 319-327;

[0011] The amino acid sequence of the antigen epitope D is shown in sequence 1 of the sequence listing as 333-368;

[0012] The amino acid sequence of the antigen epitope E is shown in sequence 1 of the sequence listing as 374-393;

[0013] The amino acid sequence of the antigen epitope F is shown in sequence 1 of the sequence listing as 399-407;

[0014] The amino acid sequence of the antigen epitope G is shown in sequence 1 of the sequence listing as 413-426;

[0015] The amino acid sequence of the antigen epitope H is shown in sequence 1 of the sequence listing as 432-449;

[0016] The amino acid sequence of the antigen epitope I is shown in sequence 1 of the sequence listing as 457-580;

[0017] The amino acid sequence of the antigen epitope J is shown in sequence 1 of the sequence listing as 588-602;

[0018] The amino acid sequence of the antigen epitope K is shown in sequence 1 of the sequence listing as 608-643;

[0019] The amino acid sequence of the antigen epitope L is shown in sequence 1 of the sequence listing as 649-668.

[0020] In the above-mentioned DNA vaccine, the antigen epitope A is an Ag85A protein antigen epitope. The Ag85A protein can not only stimulate the body to produce humoral immunity, but also can stimulate strong Th1 type cellular immunity, cause CD8+ T cell proliferation and an increase in the levels of cytokines such as IL-2 and IFN-γ.

[0021] Antigen epitope B, antigen epitope C, antigen epitope D, antigen epitope E, antigen epitope F, antigen epitope G and antigen epitope H are all antigen epitopes of Ag85B protein. Ag85B protein can induce Th1 type cellular immune response in experimental animals, produce high concentrations of IFN-γ and TNF-α, and its ability to resist Mycobacterium tuberculosis reinfection is superior to BCG. Ag85A and Ag85B belong to the Ag85 complex, which is the main component in the culture filtrate protein. Members of the complex are involved in cellular activities during the pathogenic process of M.tb, and inhibit phagosome maturation. Studies have shown that the Ag85 complex has strong immunogenicity and can resist M.tb infection by inducing strong T cell immune response.

[0022] Antigen epitope I is an antigen epitope of Rv1419 protein. Rv1419 protein is a secreted ricin-like agglutinin, which exists in the culture filtrate of M.tb H37Rv. This agglutinin can be involved in the regulation of cell adhesion, cell mitosis and innate immunity and other biological processes, and plays an important role in host-pathogen interaction. In active pulmonary tuberculosis patients, Rv1419 protein can induce a large amount of IFN-γ.

[0023] Antigen epitope J and antigen epitope K are both antigen epitopes of Rv3407 protein. Rv3407 protein is a dormancy-related (DosR) antigen, which is a specific protein produced when M.tb is reactivated from latent state. Studies have shown that it can enhance the killing ability of macrophages to M.tb, and can induce Th1 type immune response in Rv3407 DNA vaccinated mice, and reduce lung bacterial load.

[0024] Antigen epitope L is an antigen epitope of Rv2628c protein. Rv2628c protein is a latency-related antigen, and studies have shown that pVAX1-Rv2628c DNA can cause strong humoral immune response and strong T cell immune response in animal models infected with M.tb, and has certain therapeutic effect on endogenous relapse mouse TB model.

[0025] Further, the nucleic acid molecule encoding the antigen epitope A is shown as SEQ ID No. 2, 7-873 in the sequence listing;

[0026] The nucleic acid molecule encoding the antigen epitope B is shown as SEQ ID No. 2, 895-939 in the sequence listing;

[0027] The nucleic acid molecule encoding the antigen epitope C is shown as SEQ ID No. 2, 955-981 in the sequence listing;

[0028] The nucleic acid molecule encoding the antigenic epitope D is shown as SEQ ID NO: 2, 997-1104 in the sequence listing;

[0029] The nucleic acid molecule encoding the antigenic epitope E is shown as SEQ ID NO: 2, 1120-1179 in the sequence listing;

[0030] The nucleic acid molecule encoding the antigenic epitope F is shown as SEQ ID NO: 2, 1195-1221 in the sequence listing;

[0031] The nucleic acid molecule encoding the antigenic epitope G is shown as SEQ ID NO: 2, 1237-1278 in the sequence listing;

[0032] The nucleic acid molecule encoding the antigenic epitope H is shown as SEQ ID NO: 2, 1294-1347 in the sequence listing;

[0033] The nucleic acid molecule encoding the antigenic epitope I is shown as SEQ ID NO: 2, 1369-1740 in the sequence listing;

[0034] The nucleic acid molecule encoding the antigenic epitope J is shown as SEQ ID NO: 2, 1762-1806 in the sequence listing;

[0035] The nucleic acid molecule encoding the antigenic epitope K is shown as SEQ ID NO: 2, 1822-1929 in the sequence listing;

[0036] The nucleic acid molecule encoding the antigenic epitope L is shown as SEQ ID NO: 2, 1945-2004 in the sequence listing.

[0037] In the above DNA vaccine, the fusion protein is specifically the protein shown in R1) to R4) as follows:

[0038] R1) the protein with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;

[0039] R2) the fusion protein with the same function obtained by fusing a tag protein to the carboxyl terminal or / and amino terminal of the protein shown in R1);

[0040] R3) the protein with the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence shown in R1) or R2);

[0041] R4) the protein with the same function having 75% or more homology with the amino acid sequence shown in R1) or R2).

[0042] In the protein of R2), the tag refers to a polypeptide or protein fused with the target protein by DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0043] In the protein of R3), the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0044] In the protein of R4), the "identity" includes an amino acid sequence having 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown in Sequence 1 of the present application.

[0045] The protein of R1), R2), R3) or R4) can be artificially synthesized, or the encoding gene thereof can be synthesized first and then expressed biologically.

[0046] In the DNA vaccine, the recombinant eukaryotic cell expression vector is a vector obtained by inserting a nucleic acid molecule encoding the fusion protein into a restriction site of a eukaryotic expression vector.

[0047] Further, the nucleic acid molecule encoding the fusion protein is a DNA molecule shown in Sequence 2, 1-2004 in the sequence listing.

[0048] Still further, the eukaryotic expression vector includes all vectors that can express the target protein in eukaryotic cells, such as a retrovirus vector, an adenovirus vector, a plasmid vector, etc. The plasmid vector is specifically pVAX1.

[0049] In a specific embodiment of the present application, the recombinant eukaryotic cell expression vector is pVAX1-W545, which is a vector obtained by replacing the DNA molecule between the NheI and EcoRI restriction sites in the plasmid vector pVAX1 with the DNA molecule shown in Sequence 2, 1-2004 in the sequence listing.

[0050] The recombinant eukaryotic cell expression vector, the fusion protein, and the nucleic acid molecule encoding the fusion protein or the expression cassette or recombinant vector containing the nucleic acid molecule all belong to the protection scope of the present application.

[0051] In order to achieve the above-mentioned purpose, the present application further provides a new use of the above-mentioned DNA vaccine or recombinant eukaryotic cell expression vector or fusion protein or nucleic acid molecule or expression cassette containing the nucleic acid molecule or recombinant vector.

[0052] The present application provides the use of the above-mentioned DNA vaccine or recombinant eukaryotic cell expression vector or fusion protein or nucleic acid molecule or expression cassette containing the nucleic acid molecule or recombinant vector in any one of S1) to S7) as follows:

[0053] S1) preparing a product for preventing and / or treating tuberculosis;

[0054] S2) preventing and / or treating tuberculosis;

[0055] S3) preparing a product for inhibiting the growth and / or proliferation and / or endogenous relapse of Mycobacterium tuberculosis;

[0056] S4) inhibiting the growth and / or proliferation and / or endogenous relapse of Mycobacterium tuberculosis;

[0057] S5) preparing a product for improving or enhancing the level of cellular immune function or cellular immune response;

[0058] S6) improving or enhancing the level of cellular immune function or cellular immune response;

[0059] S7) serving as an anti-Mycobacterium tuberculosis preparation or Mycobacterium tuberculosis treatment preparation.

[0060] In any one of the above-mentioned uses, the improvement or enhancement of the level of cellular immune function or cellular immune response is the improvement or enhancement of Th1 type immune response.

[0061] In any one of the above-mentioned uses, the product can be a drug or a vaccine.

[0062] In order to achieve the above-mentioned purpose, the present application finally provides an anti-Mycobacterium tuberculosis preparation or Mycobacterium tuberculosis treatment preparation.

[0063] The anti-Mycobacterium tuberculosis preparation or Mycobacterium tuberculosis treatment preparation provided by the present application contains the above-mentioned DNA vaccine or recombinant eukaryotic cell expression vector or fusion protein or nucleic acid molecule or expression cassette containing the nucleic acid molecule or recombinant vector.

[0064] The present application has the following beneficial effects: the present application clones, expresses and purifies a novel Mycobacterium tuberculosis tandem DNA vaccine W545 through genetic engineering technology, can stimulate the humoral immune and cellular immune response of the body, inhibit the growth of Mycobacterium tuberculosis in the body, inhibit the endogenous relapse of M.tb, and can be used for the prevention and treatment of tuberculosis infection.

[0065] The present application provides a mycobacterium tuberculosis tandem DNA vaccine W545, which is a novel mycobacterium tuberculosis DNA vaccine of multi-antigen immunodominant epitopes obtained by genetically fusing Ag85A protein and Ag85B protein antigen epitopes, epitope genes of Rv1419, Rv3407 and Rv2628c, and connecting to a eukaryotic expression vector pVAX1. The mycobacterium tuberculosis specific tandem DNA vaccine W545 of the present application can significantly increase the number of T lymphocyte spots secreting interferon gamma when used as an immunomodulatory agent to immunize mice, slightly increase the level of anti-W545 protein antibody and subtype in plasma, present a Th1 type immune response (mainly stimulate Th1 type immune response), obtain better immune regulation effect, and significantly enhance the specific cellular immune function of mice; when used as a therapeutic agent to treat a mouse tuberculosis model, it can reduce the organ tissue lesions, and the treatment effect is equivalent to that of the existing commercial tuberculosis immunotherapy agent of the M. bovis BCG vaccine, and can be used to prepare a drug or vaccine for preventing or treating tuberculosis patients, and has a wide application prospect in the prevention and treatment of tuberculosis.

[0066] The present application is further described below in combination with the accompanying drawings and specific embodiments, but does not mean to limit the protection scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 The agarose electrophoresis map results of the vector plasmid pVAX1 and the recombinant plasmid pVAX1-W545 after digestion by restriction endonuclease Nhe I and Eco R I. M: DM10000 DNA molecular weight marker; pVAX1: pVAX1 vector plasmid; W545: pVAX1-W545 recombinant plasmid. Lane 1 indicates that the vector plasmid pVAX1 or the recombinant plasmid pVAX1-W545 is digested by restriction endonuclease Eco R I; lane 2 indicates that the vector plasmid pVAX1 or the recombinant plasmid pVAX1-W545 is digested by restriction endonuclease Nhe I; lane 3 indicates that the vector plasmid pVAX1 or the recombinant plasmid pVAX1-W545 is digested by restriction endonucleases Nhe I and Eco R I.

[0068] Figure 2 The number of T lymphocyte spots secreting IFN-γ in the spleen lymphocytes of each group of mice in Example 2 after the third immunization. From Figure 2 It can be seen that, after the third immunization, compared with the normal saline group, the pVAX-1 vector group and the M. bovis BCG vaccine group, the ag85ab plasmid DNA group and the W545 plasmid DNA group can induce the production of moderate amount of T lymphocytes secreting IFN-γ (P<0.0001).

[0069] Figure 3The anti-W545 protein IgG antibody levels in the plasma of each group of mice in Example 2 before and after immunization were determined. Figure 3 It can be seen that after the third immunization, compared with the physiological saline group, the pVAX-1 vector group and the micro-calc vaccine group, the anti-W545 protein IgG antibody level of the W545 plasmid DNA group was slightly increased, and the anti-W545 protein IgG antibody level of the ag85ab plasmid DNA group was significantly increased (P<0.001 or P<0.0001).

[0070] Figure 4 The anti-W545 protein IgG1 antibody level in the plasma of each group of mice in Example 2 after the third immunization was determined. Figure 4 It can be seen that after the third immunization, compared with the physiological saline group, the pVAX-1 vector group and the micro-calc vaccine group, the anti-W545 protein IgG1 level of the ag85ab plasmid DNA group and the W545 plasmid DNA group was slightly increased, but the difference was not statistically significant.

[0071] Figure 5 The anti-W545 protein IgG2a antibody level in the plasma of each group of mice in Example 2 after the third immunization was determined. Figure 5 It can be seen that after the third immunization, compared with the physiological saline group, the pVAX-1 vector group and the micro-calc vaccine group, the anti-W545 protein IgG2a level of the W545 plasmid DNA group was slightly increased, and the anti-W545 protein IgG2a level of the ag85ab plasmid DNA group was significantly increased (P<0.001 or P<0.0001).

[0072] Figure 6 The IFN-γ expression level in the culture supernatant of the spleen cells of each group of mice in Example 2 was determined. Figure 6 It can be seen that 3 weeks after the end of immunization, compared with the physiological saline group, the pVAX-1 vector group and the micro-calc vaccine group, the IFN-γ expression level in the culture supernatant of the spleen cells of the mice of the W545 plasmid DNA group and the ag85ab plasmid DNA group was increased to different degrees, but only the ag85ab plasmid DNA group was significantly increased (P<0.0001).

[0073] Figure 7 The IL-2 expression level in the culture supernatant of the spleen cells of each group of mice in Example 2 was determined. Figure 7 It can be seen that 3 weeks after the end of immunization, compared with the physiological saline group, the pVAX-1 vector group and the micro-calc vaccine group, the IL-2 level in the culture supernatant of the spleen cells of the mice of the ag85ab plasmid DNA group was significantly increased (P<0.01), and there was no statistically significant difference between the W545 plasmid DNA group and the control group (P>0.05).

[0074] Figure 8The IL-4 expression levels in the culture supernatant of the spleen cells of the mice in Example 2 of the present application were determined. Figure 8 It can be seen that 3 weeks after the end of immunization, the spleen lymphocytes of only a small part of the mice in each group were detectable for IL-4 expression level, but there was no statistical difference between the groups (P>0.05).

[0075] Figure 9 The IL-6 expression levels in the culture supernatant of the spleen cells of the mice in Example 2 of the present application were determined. Figure 9 It can be seen that 3 weeks after the end of immunization, the IL-6 levels in the culture supernatant of the spleen cells of the mice in the W545 plasmid DNA group, the pVAX-1 vector group and the ag85ab plasmid DNA group were lower than that in the physiological saline group, but only the W545 plasmid DNA group was significantly lower than that in the physiological saline group (P<0.05).

[0076] Figure 10 The body weight changes of the mice in Example 3 of the present application were determined. Figure 10 It can be seen that 3 weeks after the end of immunotherapy, the body weight of the mice in each group was significantly increased compared with that before immunization (P<0.05), and the body weight of the mice in the W545 plasmid DNA group was significantly higher than that in the physiological saline group (P<0.05); the body weight of the mice in the ag85ab plasmid DNA group, the pVAX-1 vector group and the M. bovis BCG group was higher than that in the physiological saline group, but the difference was not statistically significant (P>0.05).

[0077] Figure 11 The lung weight indices of the mice in Example 3 of the present application were determined. Figure 11 It can be seen that 3 weeks after the end of immunotherapy, compared with the physiological saline group, the lung weight indices of the mice in the pVAX-1 vector group, the M. bovis BCG group, the ag85ab plasmid DNA group and the W545 plasmid DNA group showed a decreasing trend, but only the W545 plasmid DNA group had statistical significance (P<0.05).

[0078] Figure 12 The liver weight indices of the mice in Example 3 of the present application were determined. Figure 12 It can be seen that 3 weeks after the end of immunotherapy, compared with the physiological saline group and the pVAX-1 vector group, the liver weight indices of the mice in the M. bovis BCG group, the ag85ab plasmid DNA group and the W545 plasmid DNA group were decreased, but there was no statistical difference (P>0.05).

[0079] Figure 13 The spleen weight indices of the mice in Example 3 of the present application were determined. Figure 13 It can be seen that 3 weeks after the end of immunotherapy, compared with the physiological saline group and the pVAX-1 vector group, the spleen weight indices of the mice in the M. bovis BCG group, the ag85ab plasmid DNA group and the W545 plasmid DNA group were decreased, but there was no statistical difference (P>0.05).

[0080] Figure 14 The lung tissue pathology of each group of mice in Example 3 of the present application is shown in Table 3. Figure 14 It can be seen that 3 weeks after the end of immunotherapy, the saline group and the pVAX-1 vector group had more severe lesions, with severe alveolar structure damage, thickening of the alveolar septum, congestion, and a large number of lymphocyte infiltration. The BCG vaccine group, the ag85ab plasmid DNA group, and the W545 plasmid DNA group had more localized lung tissue lesions, with varying degrees of reduction in lesions, and a small amount of lymphocyte infiltration.

[0081] Figure 15 The lung tissue lesion range of each group of mice in Example 3 of the present application is shown in Table 4. Figure 15 It can be seen that the lung lesion range of the W545 plasmid DNA group, the BCG vaccine group, and the ag85ab plasmid DNA group was smaller than that of the saline group and the pVAX-1 vector group (P<0.05).

[0082] Figure 16 The IFN-γ expression level in the spleen cell culture supernatant of each group of mice in Example 3 of the present application is shown in Table 5. Figure 16 It can be seen that 3 weeks after the end of immunotherapy, compared with the saline group, the IFN-γ expression level in the spleen cell culture supernatant of the pVAX-1 vector group and the ag85ab plasmid DNA group was increased, but only statistically different from the pVAX-1 vector group (P<0.01); the W545 plasmid DNA group and the BCG vaccine group showed a downward trend, but no statistical difference (P>0.05), and the pVAX-1 vector group was significantly higher than the W545 plasmid DNA group and the BCG vaccine group (P<0.01).

[0083] Figure 17 The IL-2 expression level in the spleen cell culture supernatant of each group of mice in Example 3 of the present application is shown in Table 6. Figure 17 It can be seen that 3 weeks after the end of immunotherapy, IL-2 expression was detectable in the spleen lymphocyte culture supernatant of only some mice in each group. The IL-2 level of the W545 plasmid DNA group was significantly higher than that of the saline group, the pVAX-1 vector group, and the BCG vaccine group (P<0.05), but not statistically different from the ag85ab plasmid DNA group.

[0084] Figure 18 The IL-4 expression level in the spleen cell culture supernatant of each group of mice in Example 3 of the present application is shown in Table 7. Figure 18It can be seen that 3 weeks after the end of immunotherapy, only a small part of the spleen lymphocytes of mice in each group can detect the expression level of IL-4, and the expression level of the micro-carcinoma vaccine group and the W545 plasmid DNA group is higher than that of the physiological saline group, the pVAX-1 carrier group and the ag85ab plasmid DNA group, and only the micro-carcinoma vaccine group is significantly higher than the ag85ab plasmid DNA group (P<0.05), and the difference between the other groups is not statistically significant (P>0.05).

[0085] Figure 19 The IL-6 expression level in the culture supernatant of the spleen cells of each group of mice in Example 3 is shown in Table 3. Figure 19 It can be seen that 3 weeks after the end of immunotherapy, only a small part of the spleen lymphocytes of mice in each group can detect the expression level of IL-6, and the expression level of the pVAX-1 carrier group and the ag85ab plasmid DNA group is higher than that of the W545 plasmid DNA group, the physiological saline group and the micro-carcinoma vaccine group, but the difference is not statistically significant (P>0.05). DETAILED DESCRIPTION

[0086] The application will be further described in detail below in combination with specific embodiments, and the examples given are only for illustrating the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application in any way.

[0087] In the following examples, the experimental methods are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0088] The ag85ab plasmid DNA and the construction method in the following examples are described in the invention patent with the title of Mycobacterium tuberculosis Ag85ab Chimeric Gene Vaccine, Its Preparation Method and Application, and the patent number is ZL201010191243.X, which can be obtained from the Eighth Medical Center of General Hospital of People's Liberation Army, and the biological material is only used for repeating the related experiments of the application, and cannot be used for other purposes.

[0089] Example 1, a preparation method of a Mycobacterium tuberculosis specific tandem DNA vaccine W545

[0090] The Mycobacterium tuberculosis tandem DNA vaccine designed in the application is a novel Mycobacterium tuberculosis DNA vaccine with multi-antigen immunodominant epitopes, which is obtained by connecting the immunodominant epitope genes of Mycobacterium tuberculosis proliferation phase antigens Ag85A, Ag85B, Rv1419, activation-related antigen Rv3407 and latent-related antigen Rv2628 together in series through genetic engineering, and connecting to the eukaryotic expression vector pVAX1. The specific construction method is as follows:

[0091] I. Construction of recombinant plasmid pVAX1-W545

[0092] 1. Gene design and synthesis

[0093] The DNA sequence of the eukaryotic expression DNA vaccine W545 designed and optimized in the application is shown in sequence 2, 1-2004 in the sequence list, and is recorded as W545 gene. The sequence 2, 7-873 is the coding gene of Ag85A antigen epitope, the sequence 2, 895-939, 955-981, 997-1104, 1120-1179, 1195-1221, 1237-1278, 1294-1347 are the coding genes of Ag85B antigen epitope, the sequence 2, 1369-1740 is the coding gene of Rv1419 antigen epitope, the sequence 2, 1762-1806, 1822-1929 are the coding genes of Rv3407 antigen epitope, and the sequence 2, 1945-2004 is the coding gene of Rv2628 antigen epitope. The W545 gene sequence is artificially synthesized.

[0094] 2. PCR amplification of W545 gene

[0095] The W545 gene sequence in step 1 is used as a template, and W545-F and W545-R primers are used for PCR amplification, and the primer sequences are as follows:

[0096] W545-F: 5'-CTA GCTAGC GCCGCCACCATGGTCTCC-3' (the sequence represented by underlining is the Nhe I enzyme digestion site);

[0097] W545-R: 5'-CG GAATTC TCAGCCCCACCTGCCGAT-3' (the sequence represented by underlining is the EcoR I enzyme digestion site).

[0098] The PCR program is as follows: denaturation at 98℃ for 2min; then denaturation at 98℃ for 10s, annealing at 60℃ for 20s, extension at 72℃ for 45s, a total of 37 cycles; finally, extension at 72℃ for 5min; and placed at 4℃ for 5min.

[0099] According to the Omega gel recovery kit instructions, the PCR amplification product of W545 was recovered.

[0100] 3. Double enzyme digestion of the vector plasmid pVAX1 and the W545 gene

[0101] The vector plasmid pVAX1 (Thermo's invitrogen product, Lot: 1724963, purchased from Beijing Yongzehaojia Biotechnology Development Center) and the PCR amplification product of W545 obtained in step 2 were double enzyme digested with NEB's restriction endonuclease NheI and EcoRI. The enzyme digestion system was as follows: 10x CutSmart 5 μl, NheI 2 μl, EcoRI 2 μl, pVAX1 (4 μg) or W545 product (20 μl), add water to 50 μl, 37°C enzyme digestion overnight, 65°C termination reaction. According to the Omega gel recovery kit instructions, the enzyme digestion product was recovered.

[0102] 4. Ligation reaction

[0103] The pVAX1 double enzyme digestion product and the W545 gene double enzyme digestion product were ligated with NEB's T4 DNA Ligase to obtain the ligation product.

[0104] The ligation reaction system was as follows: 10x T4 Ligase buffer 2 μl, T4 Ligase 1 μl, pVAX1 double enzyme digestion product 1 μl; W545 gene double enzyme digestion product 3 μl, add water to 20 μl, 16°C ligation overnight.

[0105] 5. Transformation of E. coli DH5a with ligation product

[0106] 1 μl of ligation product was added to E. coli DH5a competent cells, ice bath for 30 min, 42°C heat shock for 90 s, quickly placed on ice for 5 min; add 500 μL of antibiotic-free LB culture solution, 37°C, 180 rpm shaking for 1 h, centrifuged and all plated on LB plates containing kanamycin, 37°C inverted culture overnight.

[0107] II. Enzyme digestion identification of recombinant plasmid pVAX1-W545

[0108] The single clone on the plate was inoculated into a test tube containing 2 mL of LB culture solution containing Kan+, and was cultured at 37°C and 220 rpm overnight. The next day, the overnight bacteria were subjected to plasmid extraction using an omega endotoxin-free plasmid extraction kit. Then, the recombinant plasmid pVAX1-W545 was subjected to double enzyme digestion using NEB restriction enzymes NheI and EcoRI, and the enzyme digestion system was as follows: 10 x CutSmart 5 μl, NheI 2 μl, EcoRI 2 μl, recombinant plasmid pVAX1-W545 (4 μg) or pVAX1 (4 μg), and water was added to 50 μl, and the enzyme digestion was performed at 37°C overnight, and the reaction was terminated at 65°C. The enzyme digestion product was electrophoresed in a 1% agarose gel, and the results are shown in Figure 1 Figure 1 As can be seen from the above table, the sizes of the enzyme-digested fragments are basically consistent with the theoretical lengths, and the enzyme digestion identification results are correct.

[0109] III. Sequencing identification of the recombinant plasmid pVAX1-W545

[0110] The purified recombinant plasmid pVAX1-W545 was sent to a company for gene sequencing, and the sequencing results were compared and analyzed with the original designed sequence, and it was found that the two sequences were completely identical. The correct recombinant plasmid pVAX1-W545 obtained by the sequencing results is the Mycobacterium tuberculosis tandem DNA vaccine W545 of the present application.

[0111] The recombinant plasmid pVAX1-W545 is obtained by replacing the DNA molecule between the NheI and EcoRI enzyme digestion sites in the vector plasmid pVAX1 with the DNA molecule shown in sequence 2 in the sequence table. The amino acid sequence of the W545 protein expressed by the plasmid is shown in sequence 1 in the sequence table, wherein sequence 1 is the Ag85A antigen epitope amino acid sequence, sequence 1 is the Ag85B antigen epitope amino acid sequence, sequence 1 is the Rv1419 antigen epitope amino acid sequence, sequence 1 is the Rv3407 antigen epitope amino acid sequence, and sequence 1 is the Rv2628 antigen epitope amino acid sequence.

[0112] Example 2, evaluation of the immune protection effect of the Mycobacterium tuberculosis tandem DNA vaccine W545 as an immunomodulatory agent

[0113] The Mycobacterium tuberculosis tandem DNA vaccine W545 constructed and purified in Example 1 of the present application was used to immunize mice, and it was used as an immunomodulatory agent, and good immunomodulatory effect was obtained. The mouse immunization immunomodulation process is as follows: ​

[0114] I. Experimental methods and materials

[0115] 1. Animal selection

[0116] Forty 56-62-day-old female BALB / c mice with a weight of 18-20 g and a certificate of qualification were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The weight difference of the mice in the same batch was not more than 2 g.

[0117] 2. Immunogenicity of the Mycobacterium tuberculosis tandem DNA vaccine W545

[0118] (1) Animal immunization

[0119] The mice were divided into five groups, with eight mice in each group, and the treatment methods were as follows:

[0120] ① Saline group: each mouse was injected intramuscularly with 100 μl of saline;

[0121] ② pVAX1 vector group (pVAX-1 vector group): each mouse was injected intramuscularly with 100 μg / 100 μl of pVAX1 solution (pVAX1 vector was dissolved in saline to prepare 100 μg / 100 μl of pVAX1 solution);

[0122] ③ Microcalmette-Guerin vaccine group: each mouse was injected intramuscularly with 22.5 μg / 100 μl of microcalmette-Guerin vaccine (Anhui Longkemabio Pharmaceutical Co., Ltd., batch number: 20191001);

[0123] ④ ag85ab plasmid DNA group: each mouse was injected intramuscularly with 100 μg / 100 μl of ag85ab plasmid DNA;

[0124] ⑤ W545 plasmid DNA group: each mouse was injected intramuscularly with 100 μg / 100 μl of W545 plasmid DNA (constructed in Example 1).

[0125] The mice in each group were injected intramuscularly once every two weeks, for a total of three times.

[0126] (2) Detection of the number of T lymphocyte spots secreting interferon gamma in mice by enzyme-linked immunospot assay (ELISPOT)

[0127] The Mouse IFN-γ ELISPOT PLUS kit (purchased from Mabtech Technology Co., Ltd.) was used to perform the enzyme-linked immunospot assay (ELISPOT), and the kit instructions were followed. The specific steps were as follows: five weeks after the third immunization, the mice were killed to obtain the spleen, the spleen cells were isolated, and the concentration of the spleen lymphocytes was adjusted to 3×10 6 / ml. In the test plate, add 1640 complete cell culture medium, W545 protein (final concentration 30 μg / ml), phytohemagglutinin (PHA) (final concentration 30 μg / ml), and 3 × 102 spleen lymphocytes. 5 / well, incubated in a CO2 incubator at 37°C for 20h, and the number of T lymphocyte spots secreting IFN-γ in each group was detected by the ELISPOT method. The blank control wells of each group of mice were used as controls, and the number of cell spots in the detection wells of each group of mice was calculated.

[0128] (3) Antibody levels in mouse plasma were detected by ELISA.

[0129] Five weeks after the third immunization, mice were euthanized, and blood was collected in lithium heparin anticoagulant tubes. Plasma was separated and stored at -20°C. The level of anti-W545 protein-specific antibodies in mouse plasma was simultaneously detected using ELISA.

[0130] (4) The expression levels of Th1 and Th2 cytokines in the supernatant of mouse spleen lymphocyte culture were detected by the CBA method.

[0131] Three weeks after the third immunotherapy, the mice were sacrificed, and spleens were harvested. Splenic cells were isolated, and each group of splenic lymphocytes was mixed, cultured with protein stimulation, and the culture supernatant was aspirated and frozen. The expression levels of Th1 and Th2 cytokines in the spleen cell culture supernatant were detected using the CBA method.

[0132] 3. Statistical Analysis

[0133] Data were processed using SAS 6.12 software. Normally distributed quantitative data were analyzed using one-way ANOVA, and pairwise comparisons were performed using Dunnett's t-test. Skewed quantitative data were analyzed using the Kruskal-Wallis rank-sum test, and pairwise comparisons were performed using the Student-Newman-Keuls test.

[0134] II. Results

[0135] 1. Number of T lymphocyte spots secreting interferon-gamma in the spleen lymphocytes of mice in each group

[0136] See results Figure 2 The results showed that a small number of T lymphocytes specifically secreting IFN-γ, derived from W545 protein, were observed in the saline group, pVAX-1 vector group, and BCG vaccine group; a moderate number of T lymphocytes specifically secreting IFN-γ, derived from W545 protein, were observed in the ag85ab plasmid DNA group and W545 plasmid DNA group. Compared with the saline group, pVAX-1 vector group, and BCG vaccine group, both the ag85ab plasmid DNA group and W545 plasmid DNA group could induce a moderate number of T lymphocytes secreting IFN-γ (P<0.0001).

[0137] 2. Anti-W545 antibody levels in the plasma of mice in each group

[0138] Results are shown in Figures 3-5 The results showed that, after the third immunization, the levels of anti-W545 protein IgG antibody and its subtypes IgG2a and IgG1 were slightly higher in the W545 plasmid DNA group than in the normal saline group, the pVAX-1 vector group and the M. tuberculosis vaccine group, but the difference was not statistically significant (P>0.05), and the levels of anti-W545 protein IgG antibody and its subtype IgG2a were significantly higher in the ag85ab plasmid DNA group (P<0.01 or P<0.0001).

[0139] 3. Expression levels of Th1 and Th2 cytokines in the culture supernatant of spleen lymphocytes of mice in each group

[0140] Three weeks after the end of immunization, the expression levels of Th1 type cytokines (IL-2, IFN-γ) and Th2 type cytokines (IL-4, IL-6) in the culture supernatant of spleen lymphocytes of mice were detected.

[0141] The results of detection of the level of IFN-γ are shown in Figure 6 The results showed that, compared with the normal saline group (0.24 pg / ml ± 0.20 pg / ml), the pVAX-1 vector group (0.05 pg / ml ± 0.09 pg / ml) and the M. tuberculosis vaccine group (0.32 pg / ml ± 0.56 pg / ml), the levels of IFN-γ were increased to different degrees in the W545 plasmid DNA group (5.38 pg / ml ± 0.89 pg / ml) and the ag85ab plasmid DNA group (24.50 pg / ml ± 6.60 pg / ml), but only the ag85ab plasmid DNA group was significantly increased (P<0.0001).

[0142] The results of detection of the level of IL-2 are shown in Figure 7 The results showed that, compared with the normal saline group (2.46 pg / ml ± 1.48 pg / ml), the pVAX-1 vector group (1.19 pg / ml ± 1.28 pg / ml) and the M. tuberculosis vaccine group (1.25 pg / ml ± 0.99 pg / ml), the level of IL-2 was significantly increased in the ag85ab plasmid DNA group (9.28 pg / ml ± 3.36 pg / ml) (P<0.01), but there was no statistically significant difference between the W545 plasmid DNA group (0.76 pg / ml ± 0.25 pg / ml) and the control groups (P>0.05).

[0143] The results of detection of the level of IL-4 are shown in Figure 8 The results showed that only a small part of spleen lymphocytes of mice in each group could detect the expression level of IL-4, but there was no statistically significant difference between the groups (P>0.05).

[0144] IL-6 level detection results are shown in Table 4. Figure 9 The results show that the IL-6 levels of the W545 plasmid DNA group (110.23 pg / ml ± 10.18 pg / ml), the pVAX-1 vector group (179.19 pg / ml ± 16.46 pg / ml), the microcalmette vaccine group (171.63 pg / ml ± 21.65 pg / ml), and the ag85ab plasmid DNA group (197.66 pg / ml ± 39.30 pg / ml) are lower than that of the physiological saline group (207.09 pg / ml ± 61.58 pg / ml), but only the W545 plasmid DNA group is significantly lower than that of the physiological saline group (P < 0.05).

[0145] Example 3, Application of Mycobacterium tuberculosis tandem DNA vaccine W545 as a therapeutic preparation in the treatment of tuberculosis

[0146] The Mycobacterium tuberculosis tandem DNA vaccine W545 constructed and purified in Example 1 is applied to the treatment of a mouse tuberculosis model, and as a therapeutic preparation, a good therapeutic effect is obtained, which is comparable to the therapeutic effect of the existing commercial tuberculosis immunotherapy preparation microcalmette vaccine. The immunotherapy process of the mouse tuberculosis model is as follows:

[0147] I. Experimental methods and materials

[0148] 1. Animal selection

[0149] Fifty-three 42-62-day-old female BALB / c mice with a certificate were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0150] 2. Therapeutic effect of vaccine

[0151] (1) Preparation of mouse tuberculosis infection model

[0152] Each mouse was injected with 0.4 ml of a Mycobacterium tuberculosis standard strain H37Rv suspension containing 1.4 × 10 5 CFUs, to prepare a mouse tuberculosis model.

[0153] (2) Experimental grouping

[0154] After the mice were infected, the mice were randomly and evenly divided into 5 groups, 10 mice in each group, and treatment began on the 5th day after infection. The treatment method of each group is as follows:

[0155] ① Saline group: each mouse was injected with 100 μl of saline;

[0156] ② pVAX1 vector group (pVAX-1 vector group): each mouse was injected with 100 μg / 100 μl of pVAX1 vector solution;

[0157] ③Micrococcinum group: each mouse was injected intramuscularly with 22.5 μg / 100 μl of micrococcinum;

[0158] ④ag85ab plasmid DNA group: each mouse was injected intramuscularly with 100 μg / 100 μl of ag85ab plasmid DNA;

[0159] ⑤W545 plasmid DNA group: each mouse was injected intramuscularly with 100 μg / 100 μl of W545 plasmid DNA (constructed in Example 1).

[0160] Each treatment group was injected intramuscularly once every 3 weeks, for a total of 3 injections.

[0161] (3) Weight index of mice in each group

[0162] Three weeks after the end of treatment, the mice were accurately weighed before dissection, and the weights of the lungs, liver, and spleen were accurately measured, and the organ (lungs, liver, and spleen) weight index (WI) was calculated, according to the following formula: organ weight index (WI) = (organ weight / mouse weight) x 100%.

[0163] (4) Lung tissue pathological examination

[0164] Three mice were killed using the cervical dislocation method 5 days after infection. Three weeks after the end of immunotherapy, 50 mice were killed, and the right lobe of the lung was fixed in a pathological centrifuge tube containing 10% neutral formaldehyde and embedded in paraffin for sectioning. After hematoxylin / eosin (HE) staining, 5 μm serial sections were observed under a microscope for histopathological lesions of the lung tissue, and the lesion area of the lung was calculated by Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA).

[0165] (5) Detection of Th1 and Th2 cytokine expression levels in mouse spleen lymphocyte culture supernatant by CBA method

[0166] Three weeks after the third immunotherapy, the mice were killed, and 10 mice from each group were mixed, and after stimulation with protein, the culture supernatant was aspirated and frozen. The Th1 and Th2 cytokine expression levels in the spleen cell culture supernatant were detected by the CBA method.

[0167] 3. Statistical analysis

[0168] SAS6.12 software was used to process the data, and the quantitative data with normal distribution were analyzed by one-way ANOVA, and Dunnett's t test was used for pairwise comparison; the quantitative data with skew distribution were analyzed by rank sum (Kruskal-Wallis) test, and q (Student-Newman-Keuls) test was used for pairwise comparison.

[0169] II. Results

[0170] 1. Changes in body weight of mice in each group

[0171] Results are shown in Table 2. Figure 10 Results showed that the body weight of mice in each group slowly increased after challenge with Mycobacterium tuberculosis. Three weeks after the third immunization, the body weight of mice in each group was significantly increased (P < 0.05) compared with that before immunization. The body weight of mice in the W545 plasmid DNA group, the ag85ab plasmid DNA group, the pVAX-1 vector group, and the BCG vaccine group was higher than that in the normal saline group, but the difference was not statistically significant (P > 0.05).

[0172] 2. Organ weight index of mice in each group

[0173] Three weeks after the third immunization, the lungs, livers, and spleens of mice in each group were taken to calculate the organ weight index. Results are shown in Table 3. Figures 11-13 Results showed that compared with the normal saline group, the lung weight index of the pVAX-1 vector group, the BCG vaccine group, the ag85ab plasmid DNA group, and the W545 plasmid DNA group showed a decreasing trend, but only the W545 plasmid DNA group had statistical significance (P < 0.05). Compared with the normal saline group and the pVAX-1 vector group, the liver and spleen weight index of the BCG vaccine group, the ag85ab plasmid DNA group, and the W545 plasmid DNA group was decreased, but there was no statistical difference (P > 0.05).

[0174] 3. Lung tissue pathology of mice in each group

[0175] Representative pictures of lung tissue pathology of mice in each group are shown in Figure 2. Figures 14-15 Results showed that the lesions in the normal saline group and the pVAX-1 vector group were more serious, with more serious alveolar structure destruction, thickening of alveolar septum, congestion, and a large number of lymphocyte infiltration. The lung tissue lesions in the BCG vaccine group, the ag85ab plasmid DNA group, and the W545 plasmid DNA group were limited, with varying degrees of lesion alleviation, and a small amount of lymphocyte infiltration. The lesion range in the normal saline group was 69.37 ± 23.29, the lesion range in the pVAX-1 vector group was 65.88 ± 24.41, the lesion range in the BCG vaccine group was 53.16 ± 28.07, the lesion range in the ag85ab plasmid DNA group was 54.6 ± 28.69, and the lesion range in the W545 plasmid DNA group was 57.81 ± 26.64. Results suggested that the lung lesion range of mice in the W545 plasmid DNA group, the BCG vaccine group, and the ag85ab plasmid DNA group was smaller than that in the normal saline group and the pVAX-1 vector group (P < 0.05).

[0176] 4. Expression level of Th1 and Th2 cytokines in the culture supernatant of spleen lymphocytes of mice in each group

[0177] The results of detecting the level of IFN-γ in the culture supernatant of spleen lymphocytes of mice 3 weeks after the end of treatment are shown in Table 1. Figure 16 The results show that, compared with the physiological saline group (21.88 pg / ml ± 10.48 pg / ml), the pVAX-1 vector group (56.13 pg / ml ± 14.35 pg / ml) and the ag85ab plasmid DNA group (34.01 pg / ml ± 29.10 pg / ml) are increased, but only compared with the pVAX-1 vector group, there is a statistical difference (P < 0.01); the W545 plasmid DNA group (15.12 pg / ml ± 7.89 pg / ml) and the micro-carcinoma vaccine group (18.96 pg / ml ± 6.16 pg / ml) are decreased, but there is no statistical difference (P > 0.05), and the pVAX-1 vector group is significantly higher than the W545 plasmid DNA group and the micro-carcinoma vaccine group (P < 0.01).

[0178] The results of detecting the level of IL-2 in the culture supernatant of spleen lymphocytes of mice 3 weeks after the end of treatment are shown in Table 2. Figure 17 The results show that only part of the mice in each group can detect the expression of IL-2 in the culture supernatant of spleen lymphocytes, and the IL-2 level of the W545 plasmid DNA group (3.97 pg / ml ± 5.06 pg / ml) is significantly higher than that of the physiological saline group (0.38 pg / ml ± 0.85 pg / ml), the pVAX-1 vector group (0.72 pg / ml ± 1.08 pg / ml), and the micro-carcinoma vaccine group (0.48 pg / ml ± 0.79 pg / ml) (P < 0.05), but there is no statistical difference with the ag85ab plasmid DNA group (0.93 pg / ml ± 1.77 pg / ml).

[0179] The results of detecting the level of IL-4 in the culture supernatant of spleen lymphocytes of mice 3 weeks after the end of treatment are shown in Table 3. Figure 18 The results show that only a small part of the mice in each group can detect the expression level of IL-4 in the culture supernatant of spleen lymphocytes, and the micro-carcinoma vaccine group (2.70 pg / ml ± 2.75 pg / ml) and the W545 plasmid DNA group (1.18 pg / ml ± 1.73 pg / ml) are higher than the physiological saline group (0.60 pg / ml ± 1.02 pg / ml), the pVAX-1 vector group (0.59 pg / ml ± 0.70 pg / ml), and the ag85ab plasmid DNA group (0 ± 0), and only the micro-carcinoma vaccine group is significantly higher than the ag85ab plasmid DNA group (P < 0.05), and there is no statistical difference between the other groups (P > 0.05).

[0180] The results of detecting the level of IL-6 in the culture supernatant of spleen lymphocytes of mice 3 weeks after the end of treatment are shown in Table 4. Figure 19The results show that: only a small part of mice in each group can detect the expression level of IL-6 in spleen lymphocytes, the IL-6 level of pVAX-1 vector group (12.80 pg / ml ± 7.35 pg / ml) and ag85ab plasmid DNA group (13.32 pg / ml ± 13.52 pg / ml) is higher than that of W545 plasmid DNA group (8.22 pg / ml ± 8.56 pg / ml), the physiological saline group (4.42 pg / ml ± 5.17 pg / ml) and the micro-carcinoma vaccine group (1.89 pg / ml ± 2.73 pg / ml), but the difference is not statistically significant (P>0.05).

[0181] The above experimental results show that the Mycobacterium tuberculosis tandem DNA vaccine W545 can significantly enhance the specific cellular immune function of mice, mainly stimulate Th1 type immune response, and the treatment of the mouse tuberculosis model can significantly reduce the lesion range of the organ tissue, and the lesion is reduced, and the treatment effect is equivalent to that of the existing commercial tuberculosis immunotherapy preparation micro-carcinoma vaccine, and can be used for preparing a drug or vaccine for preventing or treating tuberculosis patients. Therefore, the application will have a wide application prospect in the adjuvant therapy of tuberculosis.

[0182] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the present application. Some basic features can be applied according to the scope of the following attached claims. SEQUENCE LISTING <110> The Eighth Medical Center of General Hospital of Chinese People's Liberation Army <120> A Mycobacterium tuberculosis tandem DNA vaccine W545 and a preparation method and application thereof <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 668 <212> PRT <213> Artificial Sequence <400> 1 Met Val Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser 1 5 10 15 Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Ala 20 25 30 Asn Ser Pro Ala Leu Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp 35 40 45 Phe Ser Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gln 50 55 60 Ser Gly Leu Ser Val Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr 65 70 75 80 Ser Asp Trp Tyr Gln Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr 85 90 95 Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gly Trp Leu Gln Ala 100 105 110 Asn Arg His Val Lys Pro Thr Gly Ser Ala Val Val Gly Leu Ser Met 115 120 125 Ala Ala Ser Ser Ala Leu Thr Leu Ala Ile Tyr His Pro Gln Gln Phe 130 135 140 Val Tyr Ala Gly Ala Met Ser Gly Leu Leu Asp Pro Ser Gln Ala Met 145 150 155 160 Gly Pro Thr Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys 165 170 175 Ala Ser Asp Met Trp Gly Pro Lys Glu Asp Pro Ala Pro Leu Leu Asn 180 185 190 Val Gly Lys Leu Ile Ala Asn Asn Thr Arg Val Trp Val Tyr Cys Gly 195 200 205 Asn Gly Lys Pro Ser Asp Leu Gly Gly Asn Asn Leu Pro Ala Lys Phe 210 215 220 Leu Glu Gly Phe Val Arg Thr Ser Asn Ile Lys Phe Gln Asp Ala Tyr 225 230 235 240 Asn Ala Gly Gly Gly His Asn Gly Val Phe Asp Phe Pro Asp Ser Gly 245 250 255 Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Pro 260 265 270 Asp Leu Gln Arg Ala Leu Gly Ala Thr Pro Asn Thr Gly Pro Ala Pro 275 280 285 Gln Gly Ala Gly Ser Gly Gly Gly Ser Gly Gly Arg Arg Leu Met Ile 290 295 300 Gly Thr Ala Ala Ala Val Val Leu Pro Gly Pro Gly Pro Gly Pro Ser 305 310 315 320 Met Gly Arg Asp Ile Lys Val Gly Pro Gly Pro Gly Trp Tyr Ser Pro 325 330 335 Ala Cys Gly Lys Ala Gly Cys Gin Thr Tyr Lys Trp Glu Thr Phe Leu 340 345 350 Thr Ser Glu Leu Pro Gin Trp Leu Ser Ala Asn Arg Ala Val Lys Pro 355 360 365 Gly Pro Gly Pro Gly Gin Gin Phe He Tyr Ala Gly Ser Leu Ser Ala 370 375 380 Leu Leu Asp Pro Ser Gin Gly Met Gly Gly Pro Gly Pro Gly Ala Asn 385 390 395 400 Asn Thr Arg Leu Trp Val Tyr Gly Pro Gly Pro Gly Gin Asp Ala Tyr 405 410 415 Asn Ala Ala Gly Gly His Asn Ala Val Phe Gly Pro Gly Pro Gly His 420 425 430 Ser Trp Glu Tyr Trp Gly Ala Gin Leu Asn Ala Met Lys Gly Asp Leu 435 440 445 Gln Gly Ser Gly Gly Gly Ser Gly Asp Gly Pro Val Gin Leu Lys Ser 450 455 460 Arg Leu Gly Asp Val Cys Leu Asp Ala Pro Ser Gly Ser Trp Phe Ser 465 470 475 480 Pro Leu Val lie Asn Pro Cys Asn Gly Thr Asp Phe Gin Arg Trp Asn 485 490 495 Leu Thr Asp Asp Arg Gin Val Glu Ser Val Ala Phe Pro Gly Glu Cys 500 505 510 Val Asn lie Gly Asn Ala Leu Trp Ala Arg Leu Gin Pro Cys Val Asn 515 520 525 Trp lie Ser Gin His Trp Thr Val Gin Pro Asp Gly Leu Val Lys Ser 530 535 540 Asp Leu Asp Ala Cys Leu Thr Val Leu Gly Gly Pro Asp Pro Gly Thr 545 550 555 560 Trp Val Ser Thr Arg Trp Cys Asp Pro Asn Ala Pro Asp Gin Gin Trp 565 570 575 Asp Ser Val Pro Gly Ser Gly Gly Gly Ser Gly Leu Arg Gin His Ala 580 585 590 Ser Arg Tyr Leu Ala Arg Val Glu Ala Gly Gly Pro Gly Pro Gly Ser 595 600 605 Gly Val Leu lie Pro Ala Arg Arg Pro Gin Asn Leu Leu Asp Val Thr 610 615 620 Ala Glu Pro Ala Arg Gly Arg Lys Arg Thr Leu Ser Asp Val Leu Asn 625 630 635 640 Glu Met Arg Gly Pro Gly Pro Gly Ile Arg Ala Val Gly Pro Tyr Ala 645,650,655 Trp Ala Gly Arg Cys Gly Arg Ile Gly Arg Trp Gly 660 665 <210> 2 <211> 2007 <212> DNA <213> Artificial Sequence <400> 2 atggtctcca ggcccggcct gcctgtggag tacctgcagg tgcctagccc tagcatgggc 60 agggacatca aggtgcagtt tcagagcggc ggcgccaaca gccccgccct gtatctgctg 120 gatggcctga gggcccagga cgacttcagc ggctgggata tcaatacccc cgcctttgag 180 tggtacgatc agagcggcct gagcgtggtg atgcctgtgg gcggccagag cagcttctac 240 agcgattggt accagcccgc ctgtggcaag gccggctgcc aaacctacaa gtgggagacc 300 ttcctgacaa gcgagctgcc cggctggctg caggccaaca ggcacgtgaa gcctaccggc 360 tccgccgtgg tgggcctgtc tatggccgcc agctccgccc tgaccctggc tatctaccac 420 cctcagcagt ttgtgtacgc cggcgccatg tccggcctgc tggaccctag ccaggccatg 480 ggccctaccc tgatcggcct ggccatgggc gatgccggcg gatacaaggc ctccgatatg 540 tggggcccca aggaggaccc cgcccctctg ctgaacgtgg gcaagctgat cgccaacaat 600 accagggtgt gggtgtactg tggcaatggc aagcctagcg atctgggcgg caacaacctg 660 cccgccaagt ttctggaggg cttcgtgagg acaagcaaca tcaagttcca ggatgcctac 720 aacgccggcg gcggccacaa tggcgtgttc gactttcctg atagcggcac acactcctgg 780 gagtactggg gcgcccagct gaacgccatg aagcccgatc tgcagagagc cctgggcgcc 840 acacccaaca caggccctgc ccctcagggc gctggctctg gaggaggaag cggcggaagg 900 aggctgatga tcggcaccgc cgccgccgtg gtgctgcctg gacctggacc aggcccttcc 960 atgggcaggg atatcaaggt gggccccggc cctggctggt actcccctgc ttgtggcaag 1020 gctggctgcc agacctacaa gtgggaaaca tttctgacca gcgagctgcc acagtggctg 1080 agcgccaaca gagccgtgaa gcctggcccc ggcccaggac aacagttcat ctacgccggc 1140 agcctgagcg ccctgctgga cccaagccag ggcatgggcg gcccaggacc aggagctaac 1200 aacacaaggc tgtgggtgta cggccctggc cccggacagg atgcctataa cgccgccggc 1260 ggccacaacg ccgtgtttgg acccggcccc ggccactctt gggagtactg gggagcccag 1320 ctgaatgcca tgaagggcga tctgcagggc agcggcggcg gatctggcga tggacctgtg 1380 cagctgaagt ccaggctggg cgacgtgtgt ctggacgccc cctccggaag ctggttctcc 1440 cctctggtga tcaatccctg caatggcaca gactttcaga ggtggaatct gacagacgat 1500 aggcaggtgg agagcgtggc cttccctggc gagtgcgtga acatcggcaa cgccctgtgg 1560 gccagactgc agccctgcgt gaattggatc agccagcact ggacagtgca gcccgacggc 1620 ctggtgaaga gcgacctgga tgcctgtctg accgtgctgg gcggccccga tcccggaaca 1680 tgggtgtcca ccagatggtg cgaccctaac gcccccgatc agcagtggga ctccgtgcct 1740 ggctccggcg gaggatccgg actgagacag cacgccagca ggtacctggc cagagtggag 1800 gccggcggcc caggccctgg aagcggagtg ctgatccccg ccaggagacc tcagaacctg 1860 ctggacgtga ccgccgagcc cgccagagga aggaagagaa ccctgagcga cgtgctgaat 1920 GAGATGAGAG GCCCTGGCCC TGGCATCAGA GCCGTGGGAC CTACGCCTGG CC GGAAGA 1980 TGTGGCAGAA TC GGC AGGT GGGGCTGA 2007

Claims

1. A Mycobacterium tuberculosis DNA vaccine, the active ingredient of which is a recombinant eukaryotic cell expression vector expressing a fusion protein; said fusion protein includes epitopes A, B, C, D, E, F, G, H, I, J, K, and L: The amino acid sequence of the antigen epitope A is shown as positions 3-291 of sequence 1 in the sequence listing; The amino acid sequence of the antigen epitope B is shown as positions 299-313 of sequence 1 in the sequence listing; The amino acid sequence of the antigen epitope C is shown as positions 319-327 of sequence 1 in the sequence listing; The amino acid sequence of the antigenic epitope D is shown as positions 333-368 of sequence 1 in the sequence listing; The amino acid sequence of the antigenic epitope E is shown as positions 374-393 of sequence 1 in the sequence listing; The amino acid sequence of the antigenic epitope F is shown as positions 399-407 of sequence 1 in the sequence listing; The amino acid sequence of the antigen epitope G is shown as positions 413-426 of sequence 1 in the sequence listing; The amino acid sequence of the antigen epitope H is shown as positions 432-449 of sequence 1 in the sequence listing; The amino acid sequence of antigen epitope I is shown as positions 457-580 of sequence 1 in the sequence listing; The amino acid sequence of the antigen epitope J is shown as positions 588-602 of sequence 1 in the sequence listing; The amino acid sequence of the antigenic epitope K is shown as positions 608-643 of sequence 1 in the sequence listing; The amino acid sequence of the antigen epitope L is shown as positions 649-668 of sequence 1 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope A is shown as positions 7-873 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope B is shown as positions 895-939 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope C is shown as positions 955-981 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope D is shown at positions 997-1104 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope E is shown in positions 1120-1179 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope F is shown in positions 1195-1221 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope G is shown at positions 1237-1278 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope H is shown as positions 1294-1347 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope I is shown as positions 1369-1740 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope J is shown as positions 1762-1806 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope K is shown as positions 1822-1929 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope L is shown as positions 1945-2004 of sequence 2 in the sequence listing; The amino acid sequence of the fusion protein is shown in Sequence 1 of the sequence listing; The recombinant eukaryotic cell expression vector is a vector obtained by inserting a nucleic acid molecule encoding the fusion protein into the restriction enzyme site of a eukaryotic expression vector.

2. The DNA vaccine according to claim 1, characterized in that: The nucleic acid molecule encoding the fusion protein is the DNA molecule shown in positions 1-2004 of sequence 2 in the sequence listing.

3. A recombinant eukaryotic cell expression vector, wherein the recombinant eukaryotic cell expression vector expresses a fusion protein, the fusion protein comprising epitopes A, B, C, D, E, F, G, H, I, J, K, and L: The nucleic acid molecule encoding the antigenic epitope A is shown as positions 7-873 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope B is shown as positions 895-939 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope C is shown as positions 955-981 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope D is shown at positions 997-1104 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope E is shown in positions 1120-1179 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope F is shown in positions 1195-1221 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope G is shown at positions 1237-1278 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope H is shown as positions 1294-1347 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope I is shown as positions 1369-1740 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope J is shown as positions 1762-1806 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope K is shown as positions 1822-1929 of sequence 2 in the sequence listing; The nucleic acid molecule encoding the antigenic epitope L is shown as positions 1945-2004 of sequence 2 in the sequence listing; The amino acid sequence of the fusion protein is shown in Sequence 1 of the sequence listing.

4. A fusion protein, said fusion protein comprising epitope A, epitope B, epitope C, epitope D, epitope E, epitope F, epitope G, epitope H, epitope I, epitope J, epitope K and epitope L. The antigenic epitope A is encoded by the nucleic acid molecule shown in positions 7-873 of sequence 2 in the sequence listing; The antigenic epitope B is encoded by the nucleic acid molecule shown in positions 895-939 of sequence 2 in the sequence listing; The antigenic epitope C is encoded by the nucleic acid molecule shown at positions 955-981 of sequence 2 in the sequence listing; The antigenic epitope D is encoded by the nucleic acid molecule shown at positions 997-1104 of sequence 2 in the sequence listing; The antigenic epitope E is encoded by the nucleic acid molecule shown in positions 1120-1179 of sequence 2 in the sequence listing; The antigenic epitope F is encoded by the nucleic acid molecule shown in positions 1195-1221 of sequence 2 in the sequence listing; The antigenic epitope G is encoded by the nucleic acid molecule shown at positions 1237-1278 of sequence 2 in the sequence listing; The antigenic epitope H is encoded by the nucleic acid molecule shown in positions 1294-1347 of sequence 2 in the sequence listing; The antigenic epitope I is encoded by the nucleic acid molecule shown in positions 1369-1740 of sequence 2 in the sequence listing; The antigenic epitope J is encoded by the nucleic acid molecule shown in positions 1762-1806 of sequence 2 in the sequence listing; The antigenic epitope K is encoded by the nucleic acid molecule shown in positions 1822-1929 of sequence 2 in the sequence listing; The antigenic epitope L is encoded by the nucleic acid molecule shown in positions 1945-2004 of sequence 2 in the sequence listing; The amino acid sequence of the fusion protein is shown in Sequence 1 of the sequence listing.

5. A nucleic acid molecule encoding a fusion protein or an expression cassette or recombinant vector containing said nucleic acid molecule; said fusion protein includes antigenic epitope A, antigenic epitope B, antigenic epitope C, antigenic epitope D, antigenic epitope E, antigenic epitope F, antigenic epitope G, antigenic epitope H, antigenic epitope I, antigenic epitope J, antigenic epitope K and antigenic epitope L; The antigenic epitope A is encoded by the nucleic acid molecule shown in positions 7-873 of sequence 2 in the sequence listing; The antigenic epitope B is encoded by the nucleic acid molecule shown in positions 895-939 of sequence 2 in the sequence listing; The antigenic epitope C is encoded by the nucleic acid molecule shown at positions 955-981 of sequence 2 in the sequence listing; The antigenic epitope D is encoded by the nucleic acid molecule shown at positions 997-1104 of sequence 2 in the sequence listing; The antigenic epitope E is encoded by the nucleic acid molecule shown in positions 1120-1179 of sequence 2 in the sequence listing; The antigenic epitope F is encoded by the nucleic acid molecule shown in positions 1195-1221 of sequence 2 in the sequence listing; The antigenic epitope G is encoded by the nucleic acid molecule shown at positions 1237-1278 of sequence 2 in the sequence listing; The antigenic epitope H is encoded by the nucleic acid molecule shown in positions 1294-1347 of sequence 2 in the sequence listing; The antigenic epitope I is encoded by the nucleic acid molecule shown in positions 1369-1740 of sequence 2 in the sequence listing; The antigenic epitope J is encoded by the nucleic acid molecule shown in positions 1762-1806 of sequence 2 in the sequence listing; The antigenic epitope K is encoded by the nucleic acid molecule shown in positions 1822-1929 of sequence 2 in the sequence listing; The antigenic epitope L is encoded by the nucleic acid molecule shown in positions 1945-2004 of sequence 2 in the sequence listing; The amino acid sequence of the fusion protein is shown in Sequence 1 of the sequence listing.

6. The use of the DNA vaccine of claim 1 or 2, the recombinant eukaryotic cell expression vector of claim 3, the fusion protein of claim 4, or the nucleic acid molecule, expression cassette, or recombinant vector of claim 5 in any of the following S1)-S2): S1) Prepare products for the prevention and / or treatment of tuberculosis; S2) Prepare products that inhibit the growth and / or proliferation and / or endogenous re-ignition of Mycobacterium tuberculosis.

7. An anti-tuberculosis mycobacterium preparation or a mycobacterium tuberculosis treatment preparation, comprising the DNA vaccine of claim 1 or 2 or the recombinant eukaryotic cell expression vector of claim 3.

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