A tuberculosis vaccine antigen and vaccine

Abstract

The application relates to the technical field of biological medicine, in particular to a tuberculosis vaccine antigen and a vaccine. Specifically, the application provides an application of a mycobacterium tuberculosis Rv1787 gene in preparation of a vaccine for preventing and / or treating tuberculosis, and the application is characterized in that the sequence of the Rv1787 gene or a coded product thereof is determined, wherein the Rv1787 gene and the coded product thereof are used as antigens, have mycobacterium tuberculosis specificity, can precisely stimulate the immune system of the body to recognize and respond to the mycobacterium tuberculosis, avoid non-specific immune reactions to other normal tissues, provide a new specific molecular target for preventing and treating tuberculosis, and break through the limitation of traditional vaccines depending on single antigens or attenuated strains. The specific expression product of the gene can stimulate the immune system of the body to directionally recognize and attack the mycobacterium tuberculosis, significantly improves the pertinence and effectiveness of the vaccine, and provides a new path for solving the tuberculosis prevention and treatment problem.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to a tuberculosis vaccine antigen and a vaccine. Background Technology

[0002] Tuberculosis is caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis) Mycobacterium tuberculosis , Mtb Tuberculosis is a chronic infectious disease caused by tuberculosis. Although BCG has been used for nearly a century, its protective effect varies significantly among different populations. In particular, its protective efficacy against adult tuberculosis is limited, and it cannot effectively prevent the recurrence of latent infection, making it difficult to meet the needs of prevention and control. Therefore, the development of new and highly effective tuberculosis vaccines has become a major issue that urgently needs to be addressed.

[0003] In the field of tuberculosis vaccine development, screening for highly effective and specific protective antigens from Mycobacterium tuberculosis antigens is a core step. Mycobacterium tuberculosis, an intracellular parasitic Gram-positive bacterium, has a genome containing approximately 4,000 genes. These genes are co-expressed through a complex transcriptional regulatory network, and about 15%-20% of these genes encode proteins with immunogenicity, capable of eliciting a specific immune response in the body, thus becoming potential candidate antigens for vaccine development.

[0004] Existing research indicates that various Mycobacterium tuberculosis proteins, such as the Ag85 complex, ESAT-6, and CFP-10, have been explored for use in subunit vaccine development. The Ag85 complex, composed of Ag85A, Ag85B, and Ag85C, acts as a cell wall arabinotransferase, inducing a strong cellular immune response. ESAT-6, a low-molecular-weight secreted protein, plays a crucial role in the virulence and immune evasion of Mycobacterium tuberculosis; targeting this antigen can overcome host immune tolerance. CFP-10 forms a heterodimer with ESAT-6, and their synergistic effect enhances antigen presentation efficiency. However, current tuberculosis vaccines (such as BCG) offer limited protection, particularly against adult pulmonary tuberculosis. Therefore, there is an urgent need to develop a novel target antigen for tuberculosis vaccines to improve vaccine efficacy. Summary of the Invention

[0005] The purpose of this application is to provide a tuberculosis vaccine antigen and a vaccine, aiming to solve the problem that the protective effect of tuberculosis vaccines in the prior art is limited and the protection rate against adult pulmonary tuberculosis is low.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0007] In a first aspect, this application provides the use of the Mycobacterium tuberculosis Rv1787 gene in the preparation of a vaccine for the prevention and / or treatment of tuberculosis, wherein the Rv1787 gene or its encoded product is selected from any of the following:

[0008] (a) a nucleotide sequence as set forth in SEQ ID NO. 1 ;

[0009] (b) a protein consisting of an amino acid sequence as set forth in SEQ ID NO. 2 encoded by SEQ ID NO. 1 ;

[0010] (c) a derivative protein having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 2, and having the same immunogenic function;

[0011] (d) an immunologically active fragment of the amino acid sequence as set forth in SEQ ID NO. 2, the immunologically active fragment comprising at least 15 contiguous amino acids;

[0012] (e) an mRNA molecule encoding the protein of any one of (b), (c) or (d), the mRNA comprising a 5' cap structure, a 5' untranslated region, a coding region, a 3' untranslated region, and a 3' poly(A) tail structure.

[0013] In some embodiments, the derivative protein is obtained by modification of the amino acid sequence as set forth in SEQ ID NO. 2 by substitution, deletion or addition of one or more amino acid residues.

[0014] In some embodiments, the immunologically active fragment comprises an antigenic epitope region of the protein encoded by the Rv1787 gene.

[0015] In some embodiments, the vaccine induces a Th1 type immune response.

[0016] In some embodiments, the Th1 type immune response comprises production of IFN-γ and TNF-α.

[0017] In some embodiments, the tuberculosis is pulmonary tuberculosis.

[0018] In some embodiments, the vaccine comprises any one of a DNA vaccine, an mRNA vaccine, a protein subunit vaccine, a recombinant viral vector vaccine.

[0019] In a second aspect, the present application provides a tuberculosis mRNA vaccine comprising a Mycobacterium tuberculosis Rv1787 gene or a coding product thereof, the Rv1787 gene or the coding product thereof being selected from any one of:

[0020] (a) a nucleotide sequence as set forth in SEQ ID NO. 1 ;

[0021] (b) a protein consisting of an amino acid sequence as set forth in SEQ ID NO. 2 encoded by SEQ ID NO. 1 ;

[0022] (c) a derivative protein having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO. 2 and having the same immunogenic function;

[0023] (d) an immunologically active fragment of the amino acid sequence shown in SEQ ID NO. 2, the immunologically active fragment comprising at least 15 contiguous amino acids;

[0024] (e) an mRNA molecule encoding any one of the proteins in (b), (c) or (d), the mRNA comprising a 5' cap structure, a 5' untranslated region, a coding region, a 3' untranslated region and a 3' poly(A) tail structure.

[0025] In some embodiments, the derivative protein is obtained by modifying the amino acid sequence shown in SEQ ID NO. 2 by substitution, deletion or addition of one or more amino acid residues.

[0026] In some embodiments, the immunologically active fragment comprises an antigenic epitope region of the protein encoded by the Rv1787 gene.

[0027] In a third aspect, the present application provides use of the above-mentioned mRNA vaccine for tuberculosis in the preparation of a drug for preventing or treating infectious diseases caused by Mycobacterium tuberculosis.

[0028] The Mycobacterium tuberculosis Rv1787 gene provided in the first aspect of the present application is used in the preparation of a vaccine for preventing and / or treating tuberculosis. In this application, the sequence of the Rv1787 gene or its encoded product is determined. The Rv1787 gene and its encoded product serve as antigens and have specificity for Mycobacterium tuberculosis, enabling precise stimulation of the immune system of the body to recognize and respond to Mycobacterium tuberculosis, thereby avoiding non-specific immune reactions to other normal tissues. This provides a new and specific molecular target for preventing and treating tuberculosis, and breaks through the limitations of traditional vaccines that rely on a single antigen or attenuated strains. The specific expression product of this gene can stimulate the immune system of the body to recognize and attack Mycobacterium tuberculosis in a targeted manner, significantly improving the specificity and effectiveness of the vaccine and providing a new path to solve the problem of tuberculosis prevention and treatment.

[0029] The mRNA vaccine for tuberculosis provided in the second aspect of the present application is constructed as a vaccine system with Rv1787 gene as the core. The antigen can be produced in large quantities through genetic engineering technology, thus eliminating the complex process of traditional vaccines that rely on pathogen culture, reducing production costs and biological safety risks. In addition, the genetic vaccine has strong stability and is convenient for storage and transportation, and is particularly suitable for remote areas where tuberculosis is prevalent.

[0030] The third aspect of the present application provides the use of the tuberculosis mRNA vaccine in preventing or treating Mycobacterium tuberculosis infectious diseases. The tuberculosis mRNA vaccine can be used not only for tuberculosis prevention of healthy people, but also for the treatment of Mycobacterium tuberculosis infected patients, thereby providing a new treatment option for tuberculosis patients. By stimulating the body's own immune system to fight infection, the problems of drug resistance and side effects caused by traditional antibiotic treatment are avoided, thereby providing a new idea and method for solving the problem of tuberculosis treatment, and having important clinical application value and broad market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0032] Figure 1 Figure 1 is a Western blot detection of protein expression of Rv1787 mRNA transfected 293T cells provided by the embodiments of the present application, after 24 hours.

[0033] Figure 2 Figure 2 is a flow cytometry detection of CD4+ T cell IFNγ, TNFα and IL2 expression levels of mice after the last immunization for 14 days, and the spleen was taken to prepare a cell suspension, which was stimulated in vitro by the protein encoded by Rv1787 gene.

[0034] Figure 3 Figure 3 is the lung tissue Mycobacterium tuberculosis count result of the Rv1787 mRNA candidate vaccine immunized mice after 4 weeks of challenge provided by the embodiments of the present application.

[0035] Figure 4 Figure 4 is the lung tissue HE staining pathological result of the Rv1787 mRNA candidate vaccine immunized mice after 4 weeks of challenge provided by the embodiments of the present application. DETAILED DESCRIPTION

[0036] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application will be further described in detail in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0037] In the present application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B can represent the following three cases: A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after it.

[0038] In the present application, "at least one" means one or more, and "multiple" means two or more. "At least one" or the like means any combination of the items, including any combination of single item or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can represent a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, wherein a, b, and c can be single or multiple.

[0039] It should be understood that the size of the sequence number of the above-mentioned processes in various embodiments of the present application does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence. The execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0040] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a" and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0041] The weight of the related components mentioned in the specification of the embodiments of the present application can not only refer to the specific content of each component, but also represent the weight ratio relationship between each component. Therefore, as long as the content of the related components in the specification of the embodiments of the present application is enlarged or reduced in proportion, it is within the scope disclosed in the specification of the embodiments of the present application. Specifically, the mass in the specification of the embodiments of the present application can be µg, mg, g, kg, etc. mass units commonly known in the chemical field.

[0042] The terms "first", "second" are only used for descriptive purposes, to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX can also be referred to as the second XX, and similarly, the second XX can also be referred to as the first XX. Therefore, the features limited by "first" and "second" can explicitly or implicitly include one or more of the features.

[0043] The application provides an application of a Mycobacterium tuberculosis Rv1787 gene in preparation of a vaccine for preventing and / or treating tuberculosis, and the Rv1787 gene or a coded product thereof is selected from any one of the following:

[0044] (a) a nucleotide sequence as shown in SEQ ID NO. 1;

[0045] (b) a protein consisting of an amino acid sequence as shown in SEQ ID NO. 2 encoded by SEQ ID NO. 1;

[0046] (c) a derivative protein having at least 80% sequence identity with the amino acid sequence as shown in SEQ ID NO. 2 and having the same immunogenic function;

[0047] (d) an immunologically active fragment of the amino acid sequence as shown in SEQ ID NO. 2, wherein the immunologically active fragment comprises at least 15 continuous amino acids;

[0048] (e) an mRNA molecule encoding any one of the proteins in (b), (c) or (d), wherein the mRNA comprises a 5' cap structure, a 5' untranslated region, a coding region, a 3' untranslated region and a 3' poly-A tail structure.

[0049] The application provides the application of the Mycobacterium tuberculosis Rv1787 gene in preparation of the vaccine for preventing and / or treating tuberculosis, and the sequence of the Rv1787 gene or the coded product thereof is determined, wherein the Rv1787 gene and the coded product thereof are used as antigens, have Mycobacterium tuberculosis specificity, can precisely stimulate the immune system of the body to recognize and respond to Mycobacterium tuberculosis, avoid non-specific immune reactions to other normal tissues, provide a new specific molecular target for preventing and treating tuberculosis, and break the limitation of traditional vaccines depending on single antigens or attenuated strains. The specific expression product of the gene can stimulate the immune system of the body to recognize and attack Mycobacterium tuberculosis in a directional manner, significantly improve the specificity and effectiveness of the vaccine, and provide a new path for solving the tuberculosis prevention and treatment problem.

[0050] The method for screening antigens with potential protective effect by bioinformatics analysis is as follows: 1. The number of peptide fragments that can be combined with HLA I and HLA II with high affinity (IC50 value <10 nM) in 4000 genes (reference genome: GCF_000195955.2) of Mycobacterium tuberculosis is calculated by using software NetMHCpan-4.1 and NetMHCIIpan-4.0 respectively. The reference data set of selected HLA I and II alleles can cover 97% and 99% of the population. 2. The ratio of the number of effective epitopes in each gene to the length of the gene sequence is calculated to determine the effective epitope density of each gene, and the genes are sorted in descending order according to this value. 3. The mouse MHC molecule is used as a reference for prediction and gene sorting as a supplement. 4. The genes that present high effective epitope density for both mice and humans are selected and sorted.

[0051] Therefore, according to the above steps, the Rv1787 gene is selected.

[0052] In some embodiments, the Rv1787 gene is selected from the nucleotide sequence as shown in SEQ ID NO. 1. Wherein, SEQ ID NO. 1 is specifically as follows:

[0053]

[0054] In some embodiments, the Rv1787 gene is selected from a protein consisting of an amino acid sequence as shown in SEQ ID NO. 2 encoded by SEQ ID NO. 1. Wherein, the SEQ ID NO. 2 is specifically as follows: LDFGALPPEINSGRMYCGPGSGPMLAAAAAWDGVAVELGLAATGYASVIAELTGAPWVGAASLSMVAAATPYVAWLSQAAARAEQAGMQAAAAAAAYEAAFVMTVPPPVITANRVLVMTLIATNFFGQNSAAIAVAEAQYAEMWAQDAVAMYGYAAASASASRLIPFAAPPKTTNSAGVVAQVAAVAAMPGLLQRLSSAASVSWSNPNDWWLVRLLGSITPTERTTIVRLLGQSYFATGMAQFFASIAQQLTFGPGGTTAGSGGAWYPTPQFAGLGASRAVSASLARANKIGALSVPPSWVKTTALTESPVAHAVSANPTVGSSHGPHGLLRGLPLGSRITRRSGAFAHRYGFRHSVVARPPSAG.

[0055] In some embodiments, the Rv1787 gene is selected from a derivative protein having at least 80% sequence identity to the amino acid sequence as shown in SEQ ID NO. 2, and having the same immunogenic function.

[0056] In some embodiments, the derivative protein (c) is obtained by modifying the amino acid sequence as shown in SEQ ID NO. 2 through substitution, deletion or addition of one or more amino acid residues.

[0057] In some embodiments, the Rv1787 gene is selected from an immunologically active fragment of the amino acid sequence as shown in SEQ ID NO. 2, and the immunologically active fragment comprises at least 15 consecutive amino acids.

[0058] In some embodiments, the immunologically active fragment (d) comprises an antigenic epitope region of the protein encoded by the Rv1787 gene.

[0059] In some embodiments, the immunologically active fragment sequence encoded by the Rv1787 gene is as shown below, specifically including:

[0060] (1) as shown in SEQ ID NO. 3, and the SEQ ID NO. 3 is specifically as follows: MVAAATPYVAWLSQAAARAE;

[0061] (2) as shown in SEQ ID NO. 4, and SEQ ID NO. 4 is specifically FVMTVPPPVITANRVLVMTL;

[0062] (3) as shown in SEQ ID NO. 5, and SEQ ID NO. 5 is specifically GLLQRLSSAASVSWSNPNDW;

[0063] (4) as shown in SEQ ID NO. 6, and SEQ ID NO. 6 is specifically FATGMAQFFASIAQQLTFGP;

[0064] (5) as shown in SEQ ID NO. 7, and SEQ ID NO. 7 is specifically WYPTPQFAGLGASRAVSASL;

[0065] (6) as shown in SEQ ID NO. 8, and SEQ ID NO. 8 is specifically FFGQNSAAIAVAEAQYAEMW;

[0066] (7) as shown in SEQ ID NO. 9, and SEQ ID NO. 9 is specifically EQAGMQAAAAAAAYEAAFVM;

[0067] (8) as shown in SEQ ID NO. 10, and SEQ ID NO. 10 is specifically NSAGVVAQVAAVAAMPGLLQ;

[0068] (9) as shown in SEQ ID NO. 11, and SEQ ID NO. 11 is specifically IGALSVPPSWVKTTALTESP;

[0069] (10) as shown in SEQ ID NO. 12, and SEQ ID NO. 12 is specifically WAQDAVAMYGYAAASASASR;

[0070] (11) as shown in SEQ ID NO. 13, and SEQ ID NO. 13 is specifically LGSRITRRSGAFAHRYGFRH;

[0071] (12) as shown in SEQ ID NO. 14, and SEQ ID NO. 14 is specifically DWWLVRLLGSITPTERTTIV.

[0072] In some embodiments, the encoded product of Rv1787 is selected from an mRNA molecule encoding a protein of any one of (b), (c), or (d), the mRNA comprising a 5' cap structure, a 5' untranslated region, a coding region, a 3' untranslated region, and a 3' poly(A) tail structure.

[0073] wherein the mRNA comprises a 5' cap structure, a 5' untranslated region, a coding region, a 3' untranslated region, and a 3' poly-A tail structure, all of which are routinely used by those skilled in the art and are not described here in detail for the sake of brevity.

[0074] In some embodiments, the tuberculosis vaccine can induce a Th1 type immune response. The Th1 type immune response is a key defense line of the body to eliminate intracellular parasitic bacteria (such as Mycobacterium tuberculosis). The vaccine activates Th1 cells, promotes macrophage activation, enhances the killing effect of cytotoxic T lymphocytes (CTL), effectively inhibits the survival and reproduction of Mycobacterium tuberculosis in macrophages, and guarantees the prevention and treatment effect of the vaccine on tuberculosis from the immune mechanism level.

[0075] In some embodiments, the Th1 type immune response includes the production of IFN-γ and TNF-α. IFN-γ and TNF-α are core cytokines in the Th1 type immune response. The former can activate the bactericidal activity of macrophages and regulate T cell differentiation, and the latter can promote inflammatory response and enhance immune cell recruitment. The production of these two cytokines quantifies the immune activation effect of the vaccine, provides objective indicators for vaccine efficacy evaluation, and provides scientific basis for clinical monitoring and efficacy prediction.

[0076] In some embodiments, the tuberculosis is pulmonary tuberculosis. The tuberculosis provided by the embodiments of the present application is pulmonary tuberculosis, which can determine the application of the vaccine to pulmonary tuberculosis disease.

[0077] In some embodiments, the vaccine includes any one of a DNA vaccine, an mRNA vaccine, a protein subunit vaccine, and a recombinant virus vector vaccine.

[0078] The second aspect of the embodiments of the present application provides a tuberculosis mRNA vaccine, comprising a Mycobacterium tuberculosis Rv1787 gene or a coding product thereof, and the Rv1787 gene or the coding product thereof is selected from any one of the following:

[0079] (a) a nucleotide sequence as shown in SEQ ID NO. 1;

[0080] (b) a protein consisting of an amino acid sequence as shown in SEQ ID NO. 2 encoded by SEQ ID NO. 1;

[0081] (c) a derivative protein having at least 80% sequence identity with the amino acid sequence as shown in SEQ ID NO. 2 and having the same immunogenic function;

[0082] (d) an immunologically active fragment of the amino acid sequence as shown in SEQ ID NO. 2, the immunologically active fragment comprising at least 15 consecutive amino acids;

[0083] (e) an mRNA molecule encoding the protein of any one of (b), (c), or (d), the mRNA comprising a 5' cap structure, a 5' untranslated region, a coding region, a 3' untranslated region, and a 3' poly(A) tail structure.

[0084] The tuberculosis mRNA vaccine provided in the second aspect of the embodiments of the present application is constructed with the Rv1787 gene as the core of the vaccine system, and the antigens can be produced in batches through genetic engineering technology, thus avoiding the complex process of traditional vaccines depending on pathogen culture, reducing the production cost and biological safety risk. In addition, the genetic vaccine has strong stability and is convenient for storage and transportation, and is particularly suitable for remote areas with high incidence of tuberculosis.

[0085] In some embodiments, the derivative protein (c) is obtained by modifying the amino acid sequence shown in SEQ ID NO. 2 through substitution, deletion, or addition of one or more amino acid residues.

[0086] In some embodiments, the immunologically active fragment (d) comprises an antigenic epitope region of the protein encoded by the Rv1787 gene.

[0087] The related selected sequences of the Rv1787 gene or its encoding product are consistent with the above, and are not described again here in order to save space.

[0088] In some embodiments, the tuberculosis vaccine further comprises a pharmaceutically acceptable adjuvant or carrier.

[0089] In some embodiments, the adjuvant comprises any one of an aluminum salt adjuvant, an oil emulsion adjuvant, a cytokine adjuvant, a hydrocolloid adjuvant, and a nano adjuvant.

[0090] In some embodiments, the carrier comprises any one of a viral vector, a lentivirus vector, a lipid nano-carrier, a cationic polymer carrier, a protein carrier, and a nanoparticle carrier.

[0091] In some embodiments, the dosage form of the vaccine comprises a subcutaneous injection preparation, an intramuscular injection preparation, an oral or nasal inhalation preparation.

[0092] The third aspect of the embodiments of the present application provides a use of the above-mentioned tuberculosis mRNA vaccine in the preparation of a drug for preventing or treating infectious diseases caused by Mycobacterium tuberculosis.

[0093] The tuberculosis mRNA vaccine provided in the third aspect of the embodiments of the present application is used in the prevention or treatment of Mycobacterium tuberculosis infectious diseases. The tuberculosis mRNA vaccine can be used not only for tuberculosis prevention of healthy people, but also for the treatment of Mycobacterium tuberculosis infected patients, thereby providing a new treatment option for tuberculosis patients. The tuberculosis mRNA vaccine stimulates the body's own immune system to fight infection, thereby avoiding the problems of drug resistance and side effects that may be caused by traditional antibiotic treatment, and providing a new idea and method for solving the tuberculosis treatment problem. The tuberculosis mRNA vaccine has important clinical application value and broad market prospects.

[0094] The specific embodiments are described below.

[0095] Embodiment 1

[0096] Through systematic bioinformatics analysis, it can be analyzed that the Mycobacterium tuberculosis Rv1787 gene is used in the preparation of a vaccine for preventing and / or treating tuberculosis. The nucleotide sequence of the Rv1787 gene is shown in SEQ ID NO. 1. SEQ ID NO. 1 is specifically as follows:

[0097]

[0098] The amino acid sequence of the protein encoded by the Rv1787 gene is shown in SEQ ID NO. 2. SEQ ID NO. 2 is specifically

[0099] LDFGALPPEINSGRMYCGPGSGPMLAAAAAWDGVAVELGLAATGYASVIAELTGAPWVGAASLSMVAAATPYVAWLSQAAARAEQAGMQAAAAAAAYEAAFVMTVPPPVITANRVLVMTLIATNFFGQNSAAIAVAEAQYAEMWAQDAVAMYGYAAASASASRLIPFAAPPKTTNSAGVVAQVAAVAAMPGLLQRLSSAASVSWSNPNDWWLVRLLGSITPTERTTIVRLLGQSYFATGMAQFFASIAQQLTFGPGGTTAGSGGAWYPTPQFAGLGASRAVSASLARANKIGALSVPPSWVKTTALTESPVAHAVSANPTVGSSHGPHGLLRGLPLGSRITRRSGAFAHRYGFRHSVVARPPSAG.

[0100] Example 2

[0101] In vitro expression verification of mRNA vaccine

[0102] 1. Cell transfection: HEK293T cells were seeded in a 6-well plate, and when the cell density reached 70-80%, Rv1787 mRNA and control mRNA (encoding GFP) were transfected with Lipo2000 transfection reagent, respectively.

[0103] 2. Expression detection: 24 hours after transfection: collect cell lysate, detect the expression of the protein encoded by the Rv1787 gene by Western blot, use anti-Rv1787 polyclonal antibody (1:1000 dilution) as the primary antibody; Rv1787 mRNA transfection 293T cells, 24h after transfection, discard the cell supernatant, lyse the cells with RIPA lysis buffer for 2min, add 6xloadingbuffer according to the proportion, heat in 95℃ metal bath for 10min, centrifuge at 12000rpm for 5min, then take the supernatant for Western blot detection. The experimental results are shown in Figure 1 As shown in the figure, Western blot detected a specific band at about 35kDa, which was consistent with the predicted molecular weight of the protein encoded by the Rv1787 gene;

[0104] Example 3

[0105] Immunogenicity evaluation of Rv1787 mRNA vaccine

[0106] Property test method:

[0107] 1. Animal immunization: 6-8 weeks old female C57BL / 6 mice were randomly divided into 3 groups (n=6):

[0108] Settings: Experimental group: Rv1787 mRNA vaccine (10 ug / dose);

[0109] Positive control group: BCG vaccine (10 x 6 CFU);

[0110] Negative control group: placebo control group.

[0111] Immunization scheme: intramuscular injection of corresponding injection at day 0 and day 14, a total of 2 doses.

[0112] 2. Detection of cellular immune response: 14 days after the last immunization, the mice were sacrificed, and the spleen was taken to prepare a single cell suspension:

[0113] The spleen cells were stimulated with Rv1787 gene encoded protein (10 ug / mL) for 72 hours, and the expression levels of antigen specific CD4+ T cell IFNγ, TNFα and IL2 were detected by flow cytometry.

[0114] Experimental results: as Figure 2 shown, flow detection showed that the vaccine induced a high proportion of antigen specific polyfunctional T cells (producing IFN-γ, TNF-α and IL-2 at the same time).

[0115] Example 4

[0116] Evaluation of the protective efficacy of mRNA vaccine

[0117] Performance test method:

[0118] Mycobacterium tuberculosis challenge experiment:

[0119] Two weeks after immunization, 10 ug of mRNA candidate vaccine was used to immunize mice (10 ug each) by leg muscle injection, and the second immunization was performed 2 weeks after the initial immunization. Four weeks after the last immunization, all mice were exposed to Mycobacterium tuberculosis H37Rv strain (about 100 CFU) by aerosol infection. Four weeks after infection, the mice were sacrificed:

[0120] The lung and spleen were taken to prepare homogenate, which was spread on 7H10 agar plates to count the colony forming units (CFU);

[0121] Part of the lung tissue was fixed with polyformaldehyde and then HE staining was performed to prepare pathological sections.

[0122] Experimental results: such as Figure 3 As shown, the bacterial load in the lung tissue of the Rv1787 mRNA vaccine group was reduced by 0.7 log10 CFU compared with the Placebo control group (p<0.001), which was comparable to the protective effect of the positive control Ag85b mRNA vaccine; pathological examination showed that the inflammatory infiltration and lesion extent in the lungs of the vaccine group were significantly reduced. Figure 4 As shown, Figure 4 The results of HE staining of lung tissue from mice immunized with the Rv1787 mRNA candidate vaccine provided in this application embodiment are as follows: compared with the control group, the Rv1787 vaccine group showed reduced inflammatory cell infiltration and less pathological changes in the lungs of mice, indicating that it has a good protective effect on the lungs of mice.

[0123] In summary, the application of the Mycobacterium tuberculosis Rv1787 gene provided in this application for the preparation of vaccines for the prevention and / or treatment of tuberculosis demonstrates that this application clarifies the sequence of the Rv1787 gene or its encoded product. The Rv1787 gene and its encoded product, as antigens, possess Mycobacterium tuberculosis specificity, precisely stimulating the body's immune system to recognize and respond to Mycobacterium tuberculosis, avoiding non-specific immune responses to other normal tissues. This provides a novel and specific molecular target for the prevention and treatment of tuberculosis, overcoming the limitations of traditional vaccines that rely on single antigens or attenuated strains. The specific expression product of this gene can stimulate the body's immune system to target and attack Mycobacterium tuberculosis, significantly improving the vaccine's specificity and effectiveness, and providing a new approach to solving the challenges of tuberculosis prevention and control.

[0124] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of the Mycobacterium tuberculosis Rv1787 gene in the preparation of a vaccine for the prevention of tuberculosis, characterized in that, The sequence of the Rv1787 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The vaccine can induce a Th1 immune response.

3. The application according to claim 2, characterized in that, The Th1 immune response includes the production of IFN-γ and TNF-α.

4. The application according to claim 1, characterized in that, The tuberculosis mentioned is pulmonary tuberculosis.

5. The application according to claim 1, characterized in that, The vaccine includes any one of the following: DNA vaccine, mRNA vaccine, protein subunit vaccine, and recombinant viral vector vaccine.

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