Mycobacterium tuberculosis fusion antigen, nucleic acid vaccine and application thereof

Abstract

The invention provides a mycobacterium tuberculosis fusion antigen which can be used as a subunit vaccine for preventing acute tuberculosis infection and treating latent tuberculosis infection. On the basis, the invention also constructs a nucleic acid vaccine to provide effective anti-tuberculosis protection capability. The mycobacterium tuberculosis fusion antigen and the nucleic acid vaccine thereof are expected to become a novel anti-tuberculosis vaccine.

Description

Technical Field

[0001] The present invention belongs to the field of vaccines and relates to Mycobacterium tuberculosis fusion antigens, their subunit vaccines and nucleic acid vaccines and their medical uses. Background Art

[0002] Tuberculosis (TB) is a respiratory infectious disease caused by infection with Mycobacterium tuberculosis (Mtb).

[0003] According to the 2023 TB report of the World Health Organization (WHO), an estimated 10.6 million people were infected with pulmonary tuberculosis globally in 2022, of which 7.5 million were newly diagnosed with tuberculosis, 16% higher than the number of newly diagnosed cases in 2021, and also the highest single-year figure since the WHO began monitoring the global tuberculosis situation in the 1990s. As a global public health problem, there is still no vaccine that can effectively prevent and treat tuberculosis.

[0004] Mtb is transmitted through droplets generated when tuberculosis patients cough. When the human body inhales droplets containing Mtb, Mtb enters the lungs through the respiratory tract and reaches the lower lobes of the lungs. Subsequently, Mtb that enters the lungs is phagocytosed by alveolar macrophages. Mtb within these macrophages can inhibit the fusion of phagosomes and lysosomes, interfering with the antigen presentation process of macrophages, enabling Mtb to evade the host immune system and further proliferate within macrophages. Among them, the early secreted antigenic target of Mtb (6 kDa early secretory antigenic target, ESAT-6) plays an important role in this process. ESAT-6 can enter the endoplasmic reticulum of macrophages and bind to macroglobulin β2M, inhibiting the presentation of the MHC-I-β2M complex on the cell surface and hindering the occurrence and development of the host's adaptive immunity under pathogen infection.

[0005] After phagocytosing Mtb, macrophages release various cytokines to recruit corresponding immune cells, such as dendritic cells, CD4 + T cells, CD8 +T cells, B cells, neutrophils, etc. These immune cells can gather together to form granuloma structures in the area of lung infection. The internal environment of the granuloma structure is hypoxic and nutrient-deficient, and these conditions will inhibit the proliferation and growth of Mtb within it, and the Mtb within the granuloma structure enters the dormant phase. At this time, Mtb still exists in the host body, but the immune response in the body inhibits the further spread of Mtb, and patients do not show strong clinical symptoms such as weight loss, dyspnea, night sweats, etc. at this time. This state is called the latent tuberculosis infection stage (Latent tuberculosis infection, LTBI).

[0006] According to statistics, there are approximately 360 million LTBI people in China, which is a high-burden country for latent tuberculosis infection in the world. When the immune system function of LTBI people is disordered or damaged, such as aging, infection with HIV, etc., the balance maintained between Mtb and the immune system in the body will be broken. The most direct impact is the death of immune cells inside the pulmonary granuloma, followed by the rupture of the granuloma, the appearance of cavities in the lungs, and the release of Mtb wrapped by the granuloma and contact with oxygen to start resuscitation. Patients transform from latent infection to active pulmonary tuberculosis and show clinical symptoms, and can spread to a new round through droplets. Some patients will also show symptoms of extrapulmonary tuberculosis after the spread of Mtb, and Mtb enters the lymphatic system or other organs for infection, causing further damage to the body

[0007] Bacille Calmette Guerin (BCG) is the only currently licensed anti-tuberculosis vaccine. However, the protective effect of BCG on adults is limited, and the protection period only lasts until about 18 years old in humans, and re-vaccination with BCG after adulthood does not produce a good anti-tuberculosis protective effect. Therefore, in order to prevent and treat tuberculosis, it is urgent to research and develop new anti-tuberculosis vaccines with higher protective effects and protection durations. Summary of the Invention

[0008] In order to solve the problems existing in the prior art, the present invention provides a Mycobacterium tuberculosis fusion antigen, which can be used as a subunit vaccine to prevent acute tuberculosis infection and treat latent tuberculosis infection. On this basis, the present invention also constructs a nucleic acid vaccine to provide effective anti-tuberculosis protection ability. The Mycobacterium tuberculosis fusion antigen and its nucleic acid vaccine involved in the present invention are expected to become new anti-tuberculosis vaccines.

[0009] The first object of the present invention is to provide a Mycobacterium tuberculosis fusion antigen, which can be used as a subunit vaccine to prevent acute tuberculosis infection and treat latent tuberculosis infection.

[0010] The present invention provides a Mycobacterium tuberculosis fusion antigen, which has a structural form of A-linker-B-linker-C, wherein A and B are each independently selected from the sequences shown in SEQ ID NO:1 or SEQ ID NO:2; linker is a linking peptide; C is selected from the sequences shown in any one of SEQ ID NO:6 - SEQ ID NO:15.

[0011] In another preferred embodiment, the present invention provides a Mycobacterium tuberculosis fusion antigen, which has a structural form of A-linker-B-linker-C, wherein A has the sequence shown in SEQ ID NO:1; B has the sequence shown in SEQ ID NO:2; linker is a linking peptide; C is selected from the sequences shown in any one of SEQ ID NO:6 - SEQ ID NO:15.

[0012] In another preferred embodiment, in the Mycobacterium tuberculosis fusion antigen provided by the present invention, the linker is a linking peptide having 5 - 25 amino acids, and the in vivo function and stability of the fusion protein of the present invention are optimized by adding a small linking peptide to prevent potential unnecessary domain interactions between the A, B, and C sequences. The linking peptide is more preferably a short peptide having 10 - 15 amino acids. Preferably, the linker has a sequence general formula of (GSGGS) n , (G4S) n , (SG4) n or G4(SG4) n , where n is a number between 1 and 4, preferably 2. Most preferably, the linking peptide of the present invention is GSGGSGSGGS (SEQ ID NO:16).

[0013] In another preferred embodiment, the present invention provides a Mycobacterium tuberculosis fusion antigen, which has a structural form of A-linker-B-linker-C, wherein A has the sequence shown in SEQ ID NO:1; B has the sequence shown in SEQ ID NO:2; linker is a linking peptide having the sequence shown in SEQ ID NO:16; C is selected from the sequences shown in any one of SEQ ID NO:6 - SEQ ID NO:15.

[0014] In another preferred embodiment, the present invention provides a Mycobacterium tuberculosis fusion antigen, which is selected from the sequences shown in any one of SEQ ID NO:17 - SEQ ID NO:26.

[0015] The present invention provides a screening process for Mycobacterium tuberculosis antigen epitopes. The epitope screening process includes predicting antigen B cell epitopes using the SEPPA3.0 platform (http: / / bidd2.nus.edu.sg / SEPPA3) and predicting and scoring Mycobacterium tuberculosis antigen T cell epitopes using the IEDB database (www.iedb.org), and screening the best B cell and T cell epitopes in each antigen.

[0016] After screening, in the fusion antigen main body provided by the present invention, the A structure is derived from Rv3875, and its sequence is shown in SEQ ID NO:1; the B structure is derived from Rv3874, and its sequence is shown in SEQ ID NO:2. The present invention screened 72 Mycobacterium tuberculosis antigens as potential candidate antigens. As an example, through the prediction of B cell and T cell epitopes of 72 antigens, the preferred candidate sequences Rv1174c, Rv2290 and Rv1793 (sequences are shown in SEQ ID NO:3 - SEQ ID NO:5) were obtained. Using AI design, the antigen sequences of the candidate sequences Rv1174c, Rv2290 and Rv1793 were truncated, and only the dominant epitope sequences were retained, and the sequences shown in SEQ ID NO:6 - SEQ ID NO:8 were obtained after transformation, thereby obtaining the C structure.

[0017] Similarly, the present invention also optimized other candidate sequences through AI design to obtain other dominant epitope sequences shown in SEQ ID NO:9 - SEQ ID NO:15.

[0018] The present invention combines the optimal B cell and T cell sequences screened from each Mycobacterium tuberculosis antigen with

[0019] Rv3875 and Rv3874 are connected by a linker (GSGGSGSGGS) (SEQ ID NO: 16) and recombined into 72 fusion antigens. After evaluating the immune protection induced by the fusion antigen plasmids using a zebrafish model, we found that BM-13 (Rv3875 + Rv3874 + Rv1174c), BM-31 (Rv3875 + Rv3874 + Rv2290), BM-20 (Rv3875 + Rv3874 + Rv1793), BM-14 (Rv3875 + Rv3874 + Rv1184c), BM-30 (Rv3875 + Rv3874 + Rv2223c), BM-08 (Rv3875 + Rv3874 + Rv0667), BM-07 (Rv3875 + Rv3874 + Rv0309), BM-36 (Rv3875 + Rv3874 + Rv2666), BM-10 (Rv3875 + Rv3874 + Rv0934), and BM-46 (Rv3875 + Rv3874 + Rv3418c) had good anti-tuberculosis protection effects. Among them, the sequences of the fusion antigens are shown in SEQ ID NOs: 17 - SEQ ID NOs: 26, respectively.

[0020] In another preferred embodiment, the present invention selects BM-13, BM-20, and BM-31 as candidate anti-tuberculosis nucleic acid vaccines for subsequent verification. These three fusion antigen plasmids can significantly increase the survival rate of zebrafish and effectively reduce the bacterial load in zebrafish. After expression and purification, BM-13, BM-20, and BM-31 were proven to be fusion antigens with good immunogenicity in mouse experiments. They can form a good immune response in mice and effectively reduce the infection symptoms of Mycobacterium tuberculosis, showing good development prospects.

[0021] Therefore, in the most preferred embodiment of the present invention, the present invention provides a Mycobacterium tuberculosis fusion antigen with a structural form of A-linker-B-linker-C, where A has the sequence shown in SEQ ID NO: 1; B has the sequence shown in SEQ ID NO: 2; the linker has the sequence shown in SEQ ID NO: 16; and C is selected from the sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. More specifically, the Mycobacterium tuberculosis fusion antigen provided by the present invention has the sequences shown in SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0022] Without affecting the structure or activity of the fusion antigen, mutants can be obtained by making various substitutions, additions, and / or deletions of one or several amino acids in the amino acid sequence of the fusion antigen far from the functional domain as shown above. According to the common general knowledge in the art, the biological activity of a protein is closely related to its functional domain. Generally speaking, only site mutations occurring in the functional domain may affect the two-dimensional and three-dimensional structures of the protein, thereby affecting its biological function. For amino acid sites far from the functional domain, since this region does not participate in the functional conformation of the protein, individual point mutations of amino acids will not have a substantial impact on the biological activity of the protein, and thus can basically retain the biological function of the original protein. In another preferred embodiment, the Mycobacterium tuberculosis fusion antigen provided by the present invention has a sequence homology of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of the fusion antigen as shown above, more preferably at least more than 95% homology, and most preferably at least more than 99% homology.

[0023] The present invention also provides an isolated polynucleotide encoding the Mycobacterium tuberculosis fusion antigen. The nucleic acid molecule of the present invention can be DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0024] In another preferred embodiment, the present invention provides a polynucleotide encoding the Mycobacterium tuberculosis fusion antigen as shown in SEQ ID NO: 17 - SEQ ID NO: 26. More preferably, the present invention provides the gene sequences encoding the Mycobacterium tuberculosis fusion antigens BM - 13 (Rv3875 + Rv3874 + Rv1174c), BM - 20 (Rv3875 + Rv3874 + Rv1793) and BM - 31 (Rv3875 + Rv3874 + Rv2290), and their sequences are shown as SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29 respectively.

[0025] In another preferred embodiment, the gene sequence encoding the Mycobacterium tuberculosis fusion antigen provided by the present invention has a sequence homology of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the gene sequence as shown above, more preferably at least more than 95% homology, and most preferably at least more than 99% homology.

[0026] Using standard gene cloning techniques, the gene encoding the Mycobacterium tuberculosis fusion antigen can be ligated to a suitable vector and transformed or transfected into a prokaryotic or eukaryotic host for the expression and preparation of the Mycobacterium tuberculosis fusion antigen. Suitable prokaryotic hosts include various bacteria such as E. coli, etc., and suitable eukaryotic hosts include yeast (such as Pichia pastoris) and mammalian cells (such as Chinese hamster ovary cells), etc. Preferably, a eukaryotic expression system host is used. For convenient purification, a His tag can be added to the N-terminus or C-terminus of the Mycobacterium tuberculosis fusion antigen.

[0027] Suitable vectors are various commercially available prokaryotic or eukaryotic expression vectors well-known to those skilled in the art. Prokaryotic expression vectors such as pET series vectors, pQE series vectors; yeast expression vectors pPICZ-α-A, pHIL-D2, pPIC9, pHIL-S1 (Invitrogen Corp., San Diego, California, USA), etc.; mammalian cell expression vectors pJW4303, pcDNA3.1, pVAX1, pLenti6.3, pIRES, pSVK3 or pMSG (Amersham Pharmacia Biotech Inc., USA), etc. A preferred example is to ligate the gene encoding the Mycobacterium tuberculosis fusion antigen of the present invention to the eukaryotic expression vector pcDNA3.1 and transform it into 293F cells, zebrafish or mice for the expression of the fusion antigen.

[0028] In another embodiment, the present invention provides a method for preparing a Mycobacterium tuberculosis fusion antigen, which comprises the following steps:

[0029] (a) Introducing a recombinant expression vector carrying the gene encoding the Mycobacterium tuberculosis fusion antigen into a host cell and culturing the recombinant host cell under conditions conducive to the production of the fusion antigen protein; and

[0030] (b) Recovering the Mycobacterium tuberculosis fusion antigen.

[0031] In the production method of the present invention, cells are cultured in a nutrient medium known in the art suitable for the production of the Mycobacterium tuberculosis fusion antigen. For example, cells can be cultured by shake flask culture carried out in a suitable medium and under conditions allowing the expression and / or isolation of the protein, and small-scale or large-scale fermentation (including continuous, batch, fed-batch or solid-state fermentation) in a laboratory or industrial fermenter.

[0032] If the protein is secreted into the nutrient medium, the protein can be recovered from the medium using methods known in the art. For example, it can be recovered from the nutrient medium by conventional methods, which include but are not limited to centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. It can be purified by a variety of methods known in the art, including but not limited to chromatography (such as nickel affinity column, ion exchange, hydrophobic, chromatofocusing, and size exclusion) or any combination thereof and the like.

[0033] In another embodiment, the present invention provides a pharmaceutical composition comprising the Mycobacterium tuberculosis fusion antigen and a pharmaceutically acceptable carrier. The present invention provides a pharmaceutical composition which comprises a Mycobacterium tuberculosis fusion antigen formulated together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and other physiologically compatible carriers. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion).

[0034] The present invention also provides the use of the Mycobacterium tuberculosis fusion antigen in the preparation of a subunit vaccine for preventing acute tuberculosis infection or treating latent tuberculosis infection diseases.

[0035] The Mycobacterium tuberculosis fusion antigen or its pharmaceutical composition provided by the present invention as a subunit vaccine combined with Freund's adjuvant can significantly improve the level of specific immune response in mice, achieving a better anti-tuberculosis effect. In the most preferred embodiment, the Mycobacterium tuberculosis fusion antigens BM-13 (Rv3875+Rv3874+Rv1174c), BM-31 (Rv3875+Rv3874+Rv2290) and BM-20 (Rv3875+Rv3874+Rv1793) prepared by the present invention are expected to become novel subunit vaccines for preventing acute tuberculosis infection or treating latent tuberculosis infection diseases. The fusion antigen construction strategy provided by the present invention is also expected to be applied to the vaccine design of tuberculosis and even other diseases.

[0036] The second object of the present invention is to provide a nucleic acid vaccine comprising a Mycobacterium tuberculosis fusion antigen, which can be used for preventing acute tuberculosis infection and treating latent tuberculosis infection.

[0037] The present invention provides a nucleic acid vaccine, which is a eukaryotic vector comprising a polynucleotide encoding a Mycobacterium tuberculosis fusion antigen.

[0038] Using standard gene cloning techniques, the polynucleotide encoding the Mycobacterium tuberculosis fusion antigen can be ligated to a suitable eukaryotic vector, which can transform or transfect eukaryotic host cells to express the Mycobacterium tuberculosis fusion antigen.

[0039] In another embodiment, the eukaryotic vector contains a polynucleotide sequence as shown in SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.

[0040] In another embodiment, the eukaryotic host includes zebrafish, rabbit, human, rat, dog, cat, guinea pig or mouse.

[0041] In another embodiment, the eukaryotic vector can be selected from pJW4303, pcDNA3.1(-), pcDNA3.1(+), pVAX1, pLenti6.3 and pIRES, etc., and the more preferred eukaryotic vector can be the pcDNA3.1(+) vector.

[0042] In another embodiment, the present invention provides a nucleic acid vaccine, which is a eukaryotic vector containing a polynucleotide encoding a Mycobacterium tuberculosis fusion antigen, and further includes a promoter operably coupled to the polynucleotide of the Mycobacterium tuberculosis fusion antigen.

[0043] In another embodiment, the present invention provides a nucleic acid vaccine, which is a eukaryotic vector containing a polynucleotide encoding a Mycobacterium tuberculosis fusion antigen, and further includes a terminator operably coupled to the polynucleotide of the Mycobacterium tuberculosis fusion antigen.

[0044] In another embodiment, the nucleic acid vaccine provided by the present invention is a DNA vaccine or an mRNA vaccine.

[0045] In this invention, 72 Mycobacterium tuberculosis antigens were screened as potential candidate antigens. Through the prediction of B-cell and T-cell epitopes of the 72 antigens, each antigen sequence was truncated, and only the dominant epitope sequences were retained. Subsequently, the 72 antigen dominant epitopes obtained were co-constructed with the full-length sequences of Rv3875 and Rv3874 in the pcDNA 3.1(+) plasmid. The plasmids of the 72 fusion antigens constructed were transfected and expressed in eukaryotic cells respectively, and 52 fusion antigen plasmids with good expression effects in the eukaryotic expression system were screened out. Subsequently, we renamed the 52 fusion antigen plasmids with BM-01 to BM-52 and evaluated their anti-tuberculosis protective effects in model animals as anti-tuberculosis candidate nucleic acid vaccines. In the zebrafish model infected with Mycobacterium marinum, we evaluated the survival rate of zebrafish and the bacterial load in vivo. Combining with the specific situation of the fusion antigens, we screened out 10 nucleic acid vaccines with anti-tuberculosis protective potential from the 52 candidate anti-tuberculosis nucleic acid vaccines. Among them, the 3 nucleic acid vaccines with the best anti-tuberculosis protective potential were BM-13 nucleic acid vaccine (Rv3875 + Rv3874 + Rv1174c), BM-20 nucleic acid vaccine (Rv3875 + Rv3874 + Rv1793) and BM-31 nucleic acid vaccine (Rv3875 + Rv3874 + Rv2290). Finally, in the mouse model, the anti-tuberculosis protective effects of the three nucleic acid vaccines were verified using two forms of Mtb aerosol challenge.

[0046] This invention provides the construction of fusion antigens BM-13, BM-20 and BM-31 and the evaluation of the effects of their nucleic acid vaccines. Using the nucleic acid vaccines provided by this invention, effective antigen-specific cellular and humoral immune responses can be induced in mice. At the same time, it can mediate the protection of mice against large-dose Mtb infection. These three fusion antigens are expected to become new nucleic acid vaccines. The fusion antigen construction strategy provided by this invention is expected to be applied to the vaccine design of tuberculosis and even other diseases. The nucleic acid vaccine containing Mycobacterium tuberculosis fusion antigen provided by this invention can be used as a pharmaceutical preparation for the prevention of acute tuberculosis infection and the treatment of latent tuberculosis infection.

[0047] The Mycobacterium tuberculosis fusion antigen described in this invention, or a nucleic acid vaccine containing a gene sequence encoding the Mycobacterium tuberculosis fusion antigen, or its pharmaceutical preparation form is administered to a subject in need of treatment or prevention. The preferred subject or individual to be administered is a mammal, such as a mouse, monkey, dog, cow, horse or human, and more preferably a human. The optimal dose for treatment can be determined by conventional experiments.

[0048] In summary, the present invention provides a Mycobacterium tuberculosis fusion antigen, which can be used as a subunit vaccine to prevent acute tuberculosis infection and treat latent tuberculosis infection. On this basis, the present invention also constructs a nucleic acid vaccine to provide effective anti-tuberculosis protection. The Mycobacterium tuberculosis fusion antigen and its nucleic acid vaccine involved in the present invention are expected to become a new type of anti-tuberculosis vaccine.

[0049] The technical solutions of the present invention are summarized as follows:

[0050] 1. A Mycobacterium tuberculosis fusion antigen, whose structural form is A-linker-B-linker-C, where A and B are each independently selected from the sequences shown in SEQ ID NO:1 or SEQ ID NO:2; linker is a linker peptide; C is selected from any one of the sequences shown in SEQ ID NO:6 - SEQ ID NO:15.

[0051] 2. The Mycobacterium tuberculosis fusion antigen according to Technical Solution 1, characterized in that: the structural form of the fusion antigen is A-linker-B-linker-C, where A has the sequence shown in SEQ ID NO:1; B has the sequence shown in SEQ ID NO:2; linker is a linker peptide; C is selected from any one of the sequences shown in SEQ ID NO:6 - SEQ ID NO:15.

[0052] 3. The Mycobacterium tuberculosis fusion antigen according to Technical Solution 1, characterized in that: the linker in the fusion antigen is a linker peptide with 5 - 25 amino acids.

[0053] 4. The Mycobacterium tuberculosis fusion antigen according to Technical Solution 3, characterized in that: the linker in the fusion antigen is a short peptide with 10 - 15 amino acids.

[0054] 5. The Mycobacterium tuberculosis fusion antigen according to Technical Solution 3, characterized in that: the linker in the fusion antigen has a sequence general formula of (GSGGS) n , (G4S) n , (SG4) n or G4(SG4) n , and n is a number between 1 and 4.

[0055] 6. The Mycobacterium tuberculosis fusion antigen according to Technical Solution 5, characterized in that: n in the sequence general formula is 2.

[0056] 7. The Mycobacterium tuberculosis fusion antigen according to Technical Solution 6, characterized in that: the linker peptide is GSGGSGSGGS (SEQ ID NO:16).

[0057] 8. The Mycobacterium tuberculosis fusion antigen according to Technical Solution 1, characterized in that: the structural form of the fusion antigen is A-linker-B-linker-C, wherein A has the sequence shown in SEQ ID NO:1; B has the sequence shown in SEQ ID NO:2; linker is a linking peptide and has the sequence shown in SEQ ID NO:16; C is selected from any one of the sequences shown in SEQ ID NO:6 - SEQ ID NO:15.

[0058] 9. The Mycobacterium tuberculosis fusion antigen according to Technical Solution 8, characterized in that: the fusion antigen has any one of the sequences shown in SEQ ID NO:17 - SEQ ID NO:26.

[0059] 10. The Mycobacterium tuberculosis fusion antigen according to Technical Solution 9, characterized in that: the fusion antigen has the sequence shown in SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

[0060] 11. A Mycobacterium tuberculosis fusion antigen, characterized in that: the amino acid sequence of the fusion antigen has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with the fusion antigen described in any one of Technical Solutions 1 - 10.

[0061] 12. An isolated polynucleotide encoding the Mycobacterium tuberculosis fusion antigen described in any one of Technical Solutions 1 - 11.

[0062] 13. The polynucleotide according to Technical Solution 12, characterized in that: the polynucleotide molecule is DNA or RNA, and may or may not contain intron sequences.

[0063] 14. The polynucleotide according to Technical Solution 13, characterized in that: the polynucleotide molecule is a cDNA molecule.

[0064] 15. A polynucleotide encoding the Mycobacterium tuberculosis fusion antigen shown in any one of SEQ ID NO:17 - SEQ ID NO:26.

[0065] 16. The polynucleotide according to Technical Solution 15, characterized in that: the polynucleotide sequence is as shown in SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.

[0066] 17. A polynucleotide having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with the polynucleotide according to any one of claims 12 - 16.

[0067] 18. A vector carrying the polynucleotide according to any one of technical solutions 12 - 16.

[0068] 19. The vector according to technical solution 18, characterized in that: the vector is selected from prokaryotic expression vectors such as pET series vectors, pQE series vectors; yeast expression vectors pPICZ-α-A, pHIL-D2, pPIC9, pHIL-S1; mammalian cell expression vectors pJW4303, pcDNA3.1, pVAX1, pLenti6.3, pIRES, pSVK3 or pMSG.

[0069] 20. The vector according to technical solution 19, characterized in that: the vector is pcDNA3.1.

[0070] 21. A host obtained by transforming or transfecting a prokaryotic or eukaryotic host with the vector according to any one of technical solutions 18 - 20.

[0071] 22. The host according to technical solution 21, which is a bacterium, yeast or mammalian cell.

[0072] 23. The host according to technical solution 13, which is E.coli, 293F cells, zebrafish or mouse cells.

[0073] 24. A method for preparing the Mycobacterium tuberculosis fusion antigen according to any one of technical solutions 1 - 11, comprising the following steps:

[0074] (a) Introducing a recombinant expression vector carrying the gene encoding the Mycobacterium tuberculosis fusion antigen into a host cell and culturing the recombinant host cell under conditions conducive to the production of the fusion antigen protein;

[0075] (b) Recovering the Mycobacterium tuberculosis fusion antigen.

[0076] 25. The method according to technical solution 24, characterized in that: the recovery method includes centrifugation, filtration, extraction, spray drying, evaporation or precipitation.

[0077] 26. The method according to technical solution 24, characterized in that: it is recovered by purification methods such as nickel affinity column, ion exchange, hydrophobic, chromatofocusing, size exclusion or any combination thereof.

[0078] 27. A pharmaceutical composition comprising the Mycobacterium tuberculosis fusion antigen according to any one of Technical Solutions 1-11 and a pharmaceutically acceptable carrier.

[0079] 28. The pharmaceutical composition according to Technical Solution 27, wherein: the pharmaceutically acceptable carrier includes any and all physiologically compatible carriers such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc.

[0080] 29. The pharmaceutical composition according to Technical Solution 27, wherein: the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration.

[0081] 30. Use of the Mycobacterium tuberculosis fusion antigen according to any one of Technical Solutions 1-11 in the preparation of a subunit vaccine for preventing acute tuberculosis infection or treating latent tuberculosis infection diseases.

[0082] 31. A nucleic acid vaccine, which is a eukaryotic vector containing a polynucleotide encoding the Mycobacterium tuberculosis fusion antigen according to any one of Technical Solutions 1-11.

[0083] 32. The nucleic acid vaccine according to Technical Solution 31, wherein: the polynucleotide encoding the Mycobacterium tuberculosis fusion antigen according to any one of Technical Solutions 1-11 is ligated to a suitable eukaryotic vector, which can transform or transfect a eukaryotic host to express the Mycobacterium tuberculosis fusion antigen.

[0084] 33. The nucleic acid vaccine according to Technical Solution 31, which includes a promoter operably coupled to the polynucleotide of the Mycobacterium tuberculosis fusion antigen.

[0085] 34. The nucleic acid vaccine according to Technical Solution 31, which includes a terminator operably coupled to the polynucleotide of the Mycobacterium tuberculosis fusion antigen.

[0086] 35. The nucleic acid vaccine according to Technical Solution 31, wherein: the eukaryotic vector is selected from pJW4303, pcDNA3.1(-), pcDNA3.1(+), pVAX1, pLenti6.3 and pIRES.

[0087] 36. The nucleic acid vaccine according to Technical Solution 31, wherein: the eukaryotic vector is the pcDNA3.1 vector.

[0088] 37. The nucleic acid vaccine according to Technical Solution 31, wherein: the eukaryotic vector contains a polynucleotide sequence shown in SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.

[0089] 38. Use of the nucleic acid vaccine according to any one of Technical Solutions 31-37 in the preparation of a pharmaceutical preparation for preventing acute tuberculosis infection or treating latent tuberculosis infection diseases.

[0090] 39. The use according to Technical Solution 30 or 38, which is to administer a Mycobacterium tuberculosis fusion antigen or a nucleic acid vaccine in the form of a pharmaceutical preparation containing a gene sequence encoding a Mycobacterium tuberculosis fusion antigen to a subject in need of treatment or prevention.

[0091] 40. The use according to Technical Solution 39, characterized in that: the subject is a mammal.

[0092] 41. The use according to Technical Solution 39, characterized in that: the subject is a mouse, a monkey, a dog, a cow, a horse or a human.

[0093] 42. The use according to Technical Solution 39, characterized in that: the optimal dose for treatment can be determined by conventional experiments. Description of the Drawings

[0094] Figure 1 It is the construction strategy of 72 fusion antigens of the present invention and the construction of the corresponding nucleic acid vaccines.

[0095] Figure 2 It is the expression of 72 fusion antigens in eukaryotic cells. In the figure, the black numbers above mark the Rv number as the gene number corresponding to the recombinant immunogen plasmid, the black numbers mark the MW as the predicted molecular weight size of the recombinant immunogen plasmid-expressed protein, and the red arrow indicates the position of the target band. The positive control (+) expresses only two fixed proteins, Rv3875 and Rv3874. The negative control (-) is the pcDNA3.1(+) empty plasmid. Actin is the internal reference for this experiment. M is the protein molecular weight marker.

[0096] Figure 3 shows the evaluation of the immune protection induced by 52 fusion antigen plasmids through the zebrafish model. Figure 3a It is the experimental protocol for evaluating the protective effect of the recombinant immunogen candidate vaccine in the zebrafish model. Figure 3b It is the effect of 52 fusion antigen plasmids on the survival rate of zebrafish. Figure 3c It is the effect of the fusion antigen plasmid on the bacterial load in zebrafish.

[0097] Figure 4 shows the spatial simulation of the three candidate fusion antigens, BM-13, BM-20 and BM-31, screened. Figure 4a It is the fusion antigen sequence of BM-13 and the prediction result of the protein structure. Figure 4b It is the fusion antigen sequence of BM-20 and the prediction result of the protein structure. Figure 4c It is the fusion antigen sequence of BM-31 and the prediction result of the protein structure.

[0098] Figure 5 shows the evaluation of the immune protection induced by three nucleic acid vaccines, BM-13, BM-20, and BM-31, through a mouse infection model. Figure 5a It is the statistical result of the bacterial load in the lungs and spleens of the mouse infection model. Figure 5b It is the H&E staining result of the lung pathological sections of the mouse infection model. Figure 5c It is the H&E staining result of the spleen pathological sections of the mouse infection model.

[0099] Figure 6 shows the expression and purification results of the in vitro fusion antigens of BM-13, BM-20, and BM-31. Figure 6a It is the result of Ni affinity chromatography and gel filtration chromatography of the BM-13 fusion antigen protein. Figure 6b It is the result of Ni affinity chromatography and gel filtration chromatography of the BM-31 fusion antigen protein. Figure 6c It is the result of Ni affinity chromatography and gel filtration chromatography of the BM-20 fusion antigen protein.

[0100] Figure 7 shows the detection of T cells and intracellular cytokines in mouse spleen lymphocytes by flow cytometry. Figure 7a It is the gating logic diagram for ICS flow cytometry detection. Figure 7b Results of the sorting of T cells in mouse spleen lymphocytes and the detection of intracellular factors IFN-γ, TNF-α, and IL-2. Detailed implementation manners

[0101] The following describes the preferred embodiments of the present invention. It should be understood that the preferred embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not used to limit the present invention.

[0102] Example 1: Screening of candidate antigens for latent tuberculosis infection according to literature reports or predictions

[0103] After preliminary literature research and related bioinformatics analysis, a total of 72 Mycobacterium tuberculosis proteins were screened in this project as potential immunogens for the construction of candidate anti-tuberculosis vaccines. The specific gene information, functions of the encoded proteins, number of amino acids, and subcellular localization are shown in Table 1. The data refer to the UniProt protein database (https: / / www.uniprot.org). The common feature of these 72 potential immunogens is that they have abundant T cell epitopes, potential immunogenicity, and most of them are secreted proteins. In the present invention, 72 Mycobacterium tuberculosis antigens were screened as candidate antigens according to literature reports and predictions (candidate antigen information is shown in Table 1, and some of the sequences are shown in Table 2)

[0104] 1. Prediction of human B cell and T cell epitopes for 72 Mycobacterium tuberculosis antigens

[0105] (1) We selected the SEPPA 3.0 (http: / / bidd2.nus.edu.sg / SEPPA3) B-cell epitope prediction platform to predict 72 antigens. When predicting, the structural data of the corresponding antigen needs to be provided. If the antigen has the structural data of the full-length or core region and has been uploaded to the PDB (Protein Data Bank) database, the PDB ID can be directly input. If the antigen structure has not been resolved, first use the AlphaFold2 protein structure prediction platform (https: / / cloud.zelixir.com / fastaf2 / # / fast-af2) to predict its structure. After obtaining the predicted structure, evaluate the structural data, delete or adjust the parts with low confidence in the predicted structure, and input the result file into the prediction website. Then select the type of the corresponding antigen and humans as the immune host, and the complete B-cell epitope prediction results can be obtained after submission.

[0106] (2) We selected the IEDB database (www.iedb.org) T-cell epitope prediction platform to predict 72 antigens. Select the T-cell epitope prediction function in IEDB (T Cell Epitope Prediction) to predict the potential binding sites of Mycobacterium tuberculosis antigens with human MHC I and MHC II respectively. Input the amino acid sequence of the target antigen into the system, select the predicted peptide length and perform the prediction. After submission, different predicted peptide sequences in the sequence and their corresponding scores will be obtained. The higher the score, the higher the matching degree of the prediction result. Select the best T-cell epitope according to the score.

[0107] Table 1 Summary table of 72 Mycobacterium tuberculosis candidate immunogens

[0108]

[0109]

[0110]

[0111]

[0112] The present invention screened 72 Mycobacterium tuberculosis antigens as potential candidate antigens. Through the prediction of B-cell and T-cell epitopes of 72 antigens, the preferred candidate sequences Rv1174c, Rv2290 and

[0113] Rv1793 (the sequences are shown in SEQ ID NO:3 - SEQ ID NO:5).

[0114] Table 2 Sequences of some candidate immune antigens

[0115]

[0116] Using AI design, the candidate original Rv1174c, Rv2290, and Rv1793 antigen sequences were truncated, and only the dominant epitope sequences were retained. After modification, they have the sequences shown in SEQ ID NO:6 - SEQ ID NO:8 respectively. Similarly, other candidate sequences were optimized through AI design, and the obtained dominant epitope sequences are shown in Table 3.

[0117] Table 3 Dominant epitope sequences of optimized immunogenic antigens

[0118]

[0119] 2. Construction and screening of nucleic acid vaccines corresponding to 72 fusion antigens

[0120] (1) According to the Figure 1 fusion antigen construction strategy, the optimal B cell and T cell sequences screened for each Mycobacterium tuberculosis antigen were linked to Rv3875 and Rv3874 through linker (GSGGSGSGGS) (SEQ ID NO:16) to recombine into 72 fusion antigens.

[0121] (2) The 72 fusion antigens were constructed into the expression vector pcDNA 3.1(+) plasmid.

[0122] (3) The 72 fusion antigen plasmids were transfected into 293T cells. The amount of transfected recombinant immunogen plasmid was 1 μg, the transfection time was 36 h, and PolyJet transfection reagent was used. After transfection, the cells were harvested, the supernatant was discarded, and the cells were lysed with cell lysis buffer in RIPA, and then the cell lysate was collected for verification of the expression level by Western Blot. The results are as Figure 2 shown.

[0123] (4) According to the Western Blot results, 52 fusion antigen plasmids with good expression effects in the eukaryotic expression system were screened, and the screening results are shown in Table 2.

[0124] Table 4 Information of 52 recombinant immunogen plasmids and expression in 293T cells

[0125]

[0126]

[0127]

[0128] *When constructing the partial fusion antigen, the introduction of a promoter (M), restriction sites, and / or tags will increase the theoretical molecular weight; 3. Evaluation of the immunoprotective effect induced by 52 fusion antigen plasmids in a zebrafish model

[0129] (1) Experimental animals and materials: Wild-type zebrafish; 52 fusion antigen plasmids; Mycobacterium marinum M.marinum 535.

[0130] (2) Experimental groups: Each batch included the fusion antigen plasmid as the experimental group, the pcDNA-E6C10 (ESAT-6 + CFP-10) vaccine as the positive control group, the pcDNA empty plasmid as the negative control group, and the environmental blank control group. A total of 64 groups of zebrafish in four batches, with the number of zebrafish in each group n = 17. The zebrafish in each group were cultured in independent breeding fish tanks.

[0131] (3) Experimental procedure: The specific experimental protocol is as Figure 3a shown. There were a total of two DNA intramuscular injections in the back for immunization and supplemented with electroshock, with an interval of 2 weeks. Two weeks after the last immunization, Mycobacterium marinum 535 was used for intraperitoneal challenge.

[0132] (4) Detection indicators: The survival rate of zebrafish in each group and the bacterial load CFU in vivo.

[0133] (5) Detection method: Within 60 days after the challenge, observe the survival of zebrafish every day and count ( Figure 3b ). Four weeks after the challenge, randomly select 4 zebrafish from each group for grinding and plating, count the number of colonies, and calculate the bacterial load in zebrafish. Evaluate the anti-tuberculosis protective effect of the recombinant immunogen candidate vaccine based on the survival rate of zebrafish and the bacterial load in vivo ( Figure 3c ).

[0134] (6) Results: Considering the survival rate of zebrafish, the bacterial load CFU in vivo, and the properties of the corresponding fusion antigens, we found that BM-13 (Rv3875 + Rv3874 + Rv1174c), BM-31

[0135] (Rv3875+Rv3874+Rv2290), BM-20 (Rv3875+Rv3874+Rv1793), BM-14 (Rv3875+Rv3874+Rv1184c), BM-30 (Rv3875+Rv3874+Rv2223c), BM-08 (Rv3875+Rv3874+Rv0667), BM-07 (Rv3875+Rv3874+Rv0309), BM-36 (Rv3875+Rv3874+Rv2666), BM-10 (Rv3875+Rv3874+Rv0934), BM-46 (Rv3875+Rv3874+Rv3418c) have good anti-tuberculosis protection effects. For the fusion antigen sequences mentioned above, see Table 5.

[0136] Table 5 Fusion Antigen Sequences

[0137]

[0138]

[0139] Preferably, we selected BM-13, BM-20 and BM-31 as candidate anti-tuberculosis nucleic acid vaccines for subsequent verification. These three fusion antigen plasmids can significantly improve the survival rate of zebrafish and effectively reduce the bacterial load in zebrafish.

[0140] Figure 4 shows the sequences of three fusion antigens and the predicted protein structure diagrams. BM-13 is a fusion antigen composed of Rv3875+Rv3874+Rv1174c, where Rv3875 and Rv3874 are full-length sequences, and Rv1174 is a partial sequence after epitope screening ( Figure 4a ). BM-20 is

[0141] a fusion antigen composed of Rv3875+Rv3874+Rv1793, where Rv3875 and Rv3874 are full-length sequences, and Rv1793 is a partial sequence after epitope screening ( Figure 4b ). BM-31 is a fusion antigen composed of Rv3875+Rv3874+Rv2290, where Rv3875 and Rv3874 are full-length sequences, and Rv2290 is a partial sequence after epitope screening ( Figure 4c ).

[0142] Example 2: Evaluation of the immune protection induced by three candidate nucleic acid vaccines BM-13, BM-20 and BM-31 in a mouse infection model.

[0143] 1. Experimental animals and materials: Female Balb / c mice; Three candidate nucleic acid vaccines, BM-13, BM-20 and BM-31; Mycobacterium tuberculosis H37Rv strain.

[0144] 2. Experimental groups: The experimental groups were the BM-13, BM-20 and BM-31 immunization groups, the positive control group was the pcDNA-E6C10 (ESAT-6 + CFP-10) immunization group, and the negative control group was the pcDNA empty plasmid immunization group. There were 6 mice in each group.

[0145] 3. Experimental procedures: A total of three intramuscular injections of nucleic acid vaccines were given at two-week intervals. Then, 4 weeks after the last immunization, the mice were challenged with Mtb H37Rv by aerosol. The mice were sacrificed 3 weeks after the challenge, and the lung and spleen tissues of the mice were taken.

[0146] 4. Detection indicators: The bacterial loads in the lungs and spleens of the mice, as well as the pathological sections of the lungs and spleens.

[0147] 5. Detection methods: All the mice in each group were sacrificed, and the lung and spleen tissues were taken out. After washing the lungs and spleens of the mice with sterile PBS, the smallest lobe of the lung and part of the spleen tissue were taken for making pathological sections. The remaining spleen and lung tissues of the mice were ground, and the tissue homogenates were serially diluted and plated. Colony counting was performed after culturing at 37°C for two weeks. The pathological sections were stained with H&E.

[0148] 6. Detection results: Compared with the negative control, the BM-13, BM-20 and BM-31 experimental groups could effectively reduce the bacterial loads in the lungs and spleens of the mice ( Figure 5a ). At the same time, the results of the pathological sections of the lungs showed that, compared with the negative control group, the BM-13, BM-20 and BM-31 experimental groups had higher alveolar integrity, less aggregation of inflammatory cells, and fewer inflammatory foci ( Figure 5b ). The pathological sections of the spleens showed that the BM-13, BM-20 and BM-31 experimental groups had reduced inflammation and clear splenic corpuscle structure compared with the negative control group ( Figure 5c ). The results showed that BM-13, BM-20 and BM-31, as candidate anti-tuberculosis nucleic acid vaccines, could provide strong immune protection for mice.

[0149] Example 3: Expression and purification of the corresponding proteins of BM-13, BM-20 and BM-31

[0150] 1. Clone the gene encoding the BM-13 and BM-31 fusion antigen proteins onto the corresponding vector pcDNA3.1 using standard gene cloning techniques (for ease of purification, a histidine tag can be designed and added to the C-terminus of the fusion antigen, but this process is not necessary). Transfect the pcDNA3.1-BM-13 and pcDNA3.1-BM-31 plasmids into 293F cells to express the fusion proteins BM-13 and BM-31. According to nickel affinity chromatography and gel filtration chromatography (superdex 200 10 / 300), the fusion proteins BM-13 and BM-31 were successfully obtained ( Figure 6a , b).

[0151] 2. Clone the gene encoding the BM-20 fusion antigen protein onto the corresponding vector using standard gene cloning techniques (for ease of purification, a histidine tag can be designed and added to the C-terminus of the fusion antigen, but this process is not necessary). Transform the PETDuet-BM-20 plasmid into Escherichia coli BL21(DE3) to express the fusion protein BM-20. According to nickel affinity chromatography, anion exchange chromatography and gel filtration chromatography (superdex 200 10 / 300), the fusion protein BM-20 was successfully obtained ( Figure 6c ).

[0152] The coding gene sequences of the BM-13, BM-20 and BM-31 fusion antigen proteins are shown in Table 6.

[0153]

[0154]

[0155] Example 4: Detection of T cells and intracellular cytokines in mouse spleen lymphocytes by flow cytometry.

[0156] 1. Experimental animals and materials: C57 female mice; protein subunit vaccines corresponding to BM-13, BM-20 and BM-31; Freund's complete adjuvant; Freund's incomplete adjuvant; PBS.

[0157] 2. Experimental grouping: The experimental groups were the BM-13, BM-20 and BM-31 protein subunit vaccine immunization groups, the positive control group was the E6C10 (ESAT-6 + CFP-10) protein subunit vaccine immunization group, and the negative control group was the PBS + adjuvant immunization group. Each group had 6 mice.

[0158] 3. Experimental procedure: A total of three subunit vaccines combined with Freund's adjuvant were used for intramuscular injection immunization at two-week intervals. Subsequently, the mice were sacrificed 3 weeks after the last immunization, and spleen lymphocytes were isolated. All experimental groups and the PBS group were dissected to extract spleen lymphocytes, at 2×10 6 / 200 μL was seeded into 24-well plates. Two sets were set for each sample. One set was stimulated with the corresponding protein of the subunit vaccine, and the other set was not stimulated. After culturing for 20 - 30 h, 1 μL of Monensin Solution was added and stimulation continued for 4 - 6 h. For the positive control group, 50 ng / mL PMA and 500 ng / mL ionomycin were added, and Monensin Solution was added simultaneously for stimulation for 4 - 6 h. Cells and cell supernatants were collected for flow cytometry detection. The cell surface staining indicators were CD3(APC / Fire TM 750), CD4(Alexa 700), CD8(PerCP / Cyanine5.5), CD44(PE), CD62L(FITC), and the indicators for intracellular cytokine staining were IFN-γ(PE / Dazzle TM 594), TNF(APC), IL-2(Brilliant Violet 421 TM ).

[0159] 4. Detection method: Flow cytometry.

[0160] 5. Detection indicators: Lymphocyte population gating, de-adhesion, live cell population gating, CD3 + T cell population gating, CD4 + T cell gating, CD8 + T cell gating, CD44 + T cell gating, central memory T cell population gating, and IFN-γ, TNF-α, IL-2 intracellular cytokine positive T cell population gating in CD4 + cells.

[0161] 6. Detection results: Figure 7a It is the gating logic diagram for ICS flow cytometry detection. Sequentially perform lymphocyte population gating, de-adhesion, live cell population gating, CD3 + T cell population gating, CD4 + T cell gating, CD8 + T cell gating, CD44 + T cell gating, central memory T cell population gating, and IFN-γ, TNF-α, IL-2 intracellular cytokine positive T cell gating in CD4 + cells. The results showed that gated by the negative control tube, single-stained tube, and FMO tube, after removing adherent cells and dead cells, CD3 + CD4 + cells were mainly CD4 + T cells (helper T cells), and CD3 + CD8 + cells were mainly CD8 +T cells (cytotoxic T cells). Among them, there were no significant differences in the numbers of CD4 + , CD8 + T cells and memory T cells among the groups stimulated by the corresponding proteins BM-13, BM-20, and BM-31, indicating that the stimulation of these three types of proteins did not change the T cell subsets. In the positive control group, CD3 + CD4 + slightly decreased, and the proportion of CD3 + CD8 + cells increased, indicating that the positive control (E6C10) could lead to a decrease in the CD4 + / CD8 + ratio, which might cause certain damage to the immune system ( Figure 7b ).

[0162] CD3 + CD44 + CD62L + cells were mainly central memory T cells. After stimulation with the corresponding proteins BM-13, BM-20, and BM-31 and in the positive control, the proportion of the corresponding CD3 + CD44 + CD62L + cells increased significantly, indicating that more central memory T cells could respond rapidly after the next antigen stimulation, and the vaccine could elicit long-term memory immunity to the same antigen in the organism ( Figure 7b ). In addition, after immunization of mice spleen cells with these three subunit vaccines and stimulation with the corresponding proteins, the cytokines IFN-γ, TNFα, and IL-2 secreted by CD4 + cells were significantly increased. The secretion of these cytokines could activate immune cells or play other immune regulatory roles, and it was one of the important indicators for whether the tuberculosis vaccine exerted T cell immune effects.

[0163] The above invention results show that BM-13, BM-20, and BM-31 are fusion antigens with good immunogenicity. The nucleic acid vaccine or subunit vaccine formed based on this can elicit a good immune response in mice and effectively reduce the infection symptoms of Mycobacterium tuberculosis, having good development prospects.

[0164] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Mycobacterium tuberculosis fusion antigen with a structural form of A-linker-B-linker-C, wherein A and B are each independently selected from the sequences shown in SEQ ID NO:1 or SEQ ID NO:2; linker is a linker peptide; C is selected from the sequences shown in any one of SEQ ID NO:6 - SEQ ID NO:

15.

2. The Mycobacterium tuberculosis fusion antigen according to claim 1, wherein: The fusion antigen has a structural form of A-linker-B-linker-C, wherein A has the sequence shown in SEQ ID NO:1; B has the sequence shown in SEQ ID NO:2; linker is a linker peptide; C is selected from the sequences shown in any one of SEQ ID NO:6 - SEQ ID NO:

15.

3. The Mycobacterium tuberculosis fusion antigen according to claim 1, characterized in that: The linker in the fusion antigen is a linker peptide having 5 - 25 amino acids.

4. The Mycobacterium tuberculosis fusion antigen according to claim 1, wherein: The fusion antigen has a sequence shown in any one of SEQ ID NO:17 - SEQ ID NO:

26.

5. An isolated polynucleotide encoding the Mycobacterium tuberculosis fusion antigen according to any one of claims 1 - 4.

6. A vector carrying the polynucleotide according to claim 5.

7. A host obtained by transforming or transfecting a prokaryotic or eukaryotic host with the vector according to claim 6.

8. A pharmaceutical composition comprising the Mycobacterium tuberculosis fusion antigen according to any one of claims 1 - 4 and a pharmaceutically acceptable carrier.

9. A nucleic acid vaccine, which is a eukaryotic vector comprising a polynucleotide encoding the Mycobacterium tuberculosis fusion antigen according to any one of claims 1 - 4.

10. Use of the Mycobacterium tuberculosis fusion antigen according to any one of claims 1 - 4 or the nucleic acid vaccine according to claim 9 in the preparation of a pharmaceutical preparation for preventing acute tuberculosis infection or treating latent tuberculosis infection diseases.

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