Preparation and application of novel tuberculosis subunit vaccine containing different truncated PstS1 fusion protein AP

Abstract

The present application provides a fusion protein containing PstS1 fragment and Ag85A fragment and a tuberculosis subunit vaccine. The fusion protein comprises a first peptide segment containing Ag85A fragment, a second peptide segment containing PstS1 fragment, and the first peptide segment is connected with the second peptide segment; the subunit vaccine comprises the fusion protein. The fusion protein can stimulate cellular immunity and humoral immunity of the body at the same time, and improve the ability of the body to kill Mycobacterium tuberculosis, and the subunit vaccine can effectively prevent tuberculosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular, the present application relates to the preparation and application of a novel tuberculosis subunit vaccine containing different truncated PstS1 fusion protein AP, more particularly, the present application relates to a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell and a subunit vaccine and the application thereof. BACKGROUND

[0002] Tuberculosis (TB) is a chronic and debilitating infectious disease caused by Mycobacterium tuberculosis (Mtb), with more than 9 million new cases and about 1.4 million deaths each year. With the emergence of drug-resistant Mtb and HIV-TB co-infection, the situation has become more complex, making the prognosis and treatment of tuberculosis worse. Bovine tuberculosis (BTB) is a chronic infectious disease caused by Mycobacterium bovis (M.bovis), which is characterized by tuberculous nodular granuloma and caseous, calcified necrotic lesions in organs. It is more common in dairy cows and seriously affects the development of animal husbandry and human health.

[0003] BCG is the only vaccine approved by WHO to prevent tuberculosis, which has a protective effect on infants and adolescents, but its protective effect on adults is not clear. Therefore, there is an urgent need to develop an effective tuberculosis vaccine. SUMMARY

[0004] The present application aims to at least solve one of the technical problems in the prior art. To this end, the present application provides a tuberculosis subunit vaccine containing PstS1 fragment and Ag85A fragment, which can simultaneously stimulate and improve cellular immunity and humoral immunity, and has good preventive effect on tuberculosis.

[0005] The present application is based on the following findings of the inventors:

[0006] There are many vaccines for tuberculosis at present, and the new tuberculosis vaccines being developed include recombinant BCG (rBCG) vaccine, attenuated M.tb vaccine, adjuvant subunit protein vaccine, viral vector vaccine, whole cell vaccine, DNA vaccine, RNA vaccine, etc.

[0007] Subunit vaccine gradually enters people's vision due to its easy production, good safety, strong specificity, good quality control and other characteristics. In the screening of recombinant subunit vaccine antigens, more preference is given to the selection of antigens capable of inducing strong cellular immunity, but researches prove that the accumulation of multiple cellular antigens is not obvious for the improvement of immune protection effect.

[0008] Therefore, the inventors screened recombinant subunit vaccine antigens through a large number of experiments, and finally determined two proteins, Ag85A and phosphate transport system protein 1 (PstS1). The inventors found that Ag85A is the main component of M.tb culture filtrate protein and is also a T cell antigen of M.tb, which can induce extremely strong Th1 type immune response and is important for regulating intracellular infection; PstS1 is an important protein in the cell wall of Mycobacterium tuberculosis and has immunogenicity, which can stimulate the proliferation of B cells and T cells and is an important immunoprotective antigen. The inventors found that the fusion protein prepared by selecting the above two proteins can stimulate cellular immunity and humoral immunity at the same time, and improve the ability of the body to kill Mycobacterium tuberculosis. The inventors prepared a tuberculosis subunit vaccine by mixing the fusion protein with an adjuvant, and injected the tuberculosis subunit vaccine into mice, and then attacked the mice with Mycobacterium tuberculosis. As a result, the load of Mycobacterium tuberculosis in the organs of the mice was significantly reduced, and almost reached the same immune protection as BCG, indicating that the tuberculosis subunit vaccine can greatly improve the ability of the body to resist Mycobacterium tuberculosis and can effectively prevent and treat tuberculosis. Moreover, the inventors found through experiments that the tuberculosis subunit vaccine can effectively enhance the immune effect of BCG.

[0009] In one aspect of the present application, a fusion protein is provided. According to embodiments of the present application, the fusion protein comprises: a first peptide segment comprising an Ag85A fragment; a second peptide segment comprising a PstS1 fragment, the first peptide segment being connected to the second peptide segment. The fusion protein according to embodiments of the present application can stimulate cellular immunity and humoral immunity of the body at the same time, and improve the ability of the body to kill Mycobacterium tuberculosis.

[0010] In another aspect of the present application, a nucleic acid molecule is provided. According to embodiments of the present application, the nucleic acid molecule encodes the aforementioned fusion protein. The nucleic acid molecule according to embodiments of the present application can effectively express the aforementioned fusion protein.

[0011] In yet another aspect of the present application, an expression vector is provided. According to embodiments of the present application, the expression vector carries the aforementioned nucleic acid molecule. The expression vector according to embodiments of the present application can effectively express the aforementioned fusion protein.

[0012] In yet another aspect of the present application, the present application provides a recombinant cell. According to embodiments of the present application, the recombinant cell comprises: carrying the aforementioned nucleic acid molecule or the aforementioned expression vector; or, expressing the aforementioned fusion protein. The recombinant cell according to embodiments of the present application can be used for in vitro expression and mass acquisition of the aforementioned fusion protein.

[0013] In yet another aspect of the present application, the present application provides a use of the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector or the aforementioned recombinant cell in the preparation of a subunit vaccine.

[0014] In yet another aspect of the present application, the present application provides a subunit vaccine. According to embodiments of the present application, the subunit vaccine comprises: the aforementioned fusion protein. The subunit vaccine according to embodiments of the present application can improve the body's ability to resist Mycobacterium tuberculosis, effectively prevent and treat tuberculosis; and, can also effectively enhance the immune effect of BCG.

[0015] In yet another aspect of the present application, the present application provides a use of the aforementioned subunit vaccine in the preparation of a medicament for enhancing the immune effect of BCG or for preventing and / or treating tuberculosis.

[0016] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0017] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0018] Figure 1 A gel electrophoresis result diagram of SOE-PCR amplification of AP1, AP2 and AP3 in Example 1 of the present application;

[0019] Figure 2 A mode diagram of pET-21a-Ag85A-tnPstS1 vector construction in Example 1 of the present application;

[0020] Figure 3 A SDS-PAGE Coomassie staining diagram (left) and a WB detection result diagram (right) of AP1, AP2 and AP3 after purification in Example 1 of the present application;

[0021] Figure 4 A flowchart of animal protection test in Example 3 of the present application;

[0022] Figure 5Fig. 3 is a graph showing the results of detection of bacterial load in the organs of mice in each group after challenge in Example 3 of the present application, wherein * indicates P<0.05, ** indicates P<0.01, and **** indicates P<0.0001;

[0023] Figure 6 Fig. 4 is a graph showing the results of detection of lung pathological changes in mice in each group after challenge for four weeks in Example 3 of the present application;

[0024] Figure 7 Fig. 5 is a graph showing the results of changes in serum antibody titers in mice in each group after three immunizations in Example 4 of the present application;

[0025] Figure 8 Fig. 6 is a graph showing the results of titer detection after the third immunization in Example 4 of the present application. DETAILED DESCRIPTION

[0026] Embodiments of the present application are described in detail below. The embodiments described below are examples only and are not to be construed as limiting the present application.

[0027] It should be noted that the terms "first", "second", and the like do not denote any quantity or order but are used only to distinguish one element from another. Thus, a feature defined with "first", "second" may include one or more of the features. Further, in the description of the present application, the meaning of "plurality" is two or more unless otherwise specified.

[0028] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and any values are provided as approximate descriptions of the ranges and are to be construed as being near the reported values within normal experimental error. For numeric ranges, the endpoints of the ranges, the endpoints of the ranges and individual points between them, and individual points between them can be combined with each other to form one or more new numeric ranges, which should be considered as specifically disclosed herein.

[0029] In this document, the terms "comprises", "comprising", "includes", "including" or "contains", "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0030] In this document, the terms "optionally", "optional" or "optional" generally mean that the subsequent event or circumstance can or can not occur, and the description includes cases where the event or circumstance occurs, and cases where the event or circumstance does not occur.

[0031] In the present context, the terms "identity", "homology" or "similarity" are used when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, in terms of the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences determined by conventional means, see, e.g., Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are a number of algorithms that can be used to align sequences and determine sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLAST family of algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs are available that use these algorithms to perform the comparisons, including, but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0032] In the present context, the term "at least 90% similarity" means at least 95%, possibly 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% similarity to a reference sequence.

[0033] In the present context, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers nucleic acid molecules into and / or between host cells. The expression vector can include a vector that is primarily used for inserting DNA or RNA into a cell, a vector that is primarily used for replication of DNA or RNA, and a vector that is primarily used for expression of transcription and / or translation of DNA or RNA. The expression vector also includes a vector having a plurality of the above-described functions. The expression vector can be a polynucleotide that is capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0034] In the present context, the term "recombinant cell" generally refers to a cell having a unique trait stably inherited by modification or recombination of the genetic material of the host cell using genetic engineering techniques or cell fusion techniques. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell into which a recombinant expression vector can be introduced. The term "transformed" or "transfected" used herein means introducing a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. A suitable host cell can be transformed or transfected with a DNA sequence of the present application and can be used for expression and / or secretion of a target protein.

[0035] In the present context, the term "fragment" refers to a protein fragment, which can include a full-length fragment of a protein, and can also include a partial fragment of a protein. Illustratively, an Ag85A fragment can be a full-length fragment of Ag85A protein, and can also be a partial fragment of Ag85A protein; a PstS1 fragment can be a full-length fragment of PstS1 protein, and can also be a partial fragment of PstS1 protein, such as a PstS1 antigenic epitope peptide.

[0036] In the present context, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" as used herein means approved or approvable by a regulatory agency of the Federal or a state government or the United States Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0037] In the present context, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, and the like, suitable for a particular target dosage form. Except insofar as any conventional excipient is incompatible with the compound of the present application, such as by producing any adverse biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this application.

[0038] As used herein, the term "treatment" refers to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of the disease. As used herein, "treatment" covers any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce the symptoms of, or inhibit the disease in the individual, including, but not limited to, administering a drug comprising a compound described herein to an individual in need thereof.

[0039] The present application provides a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell and a subunit vaccine and uses thereof, which will be described in detail below.

[0040] Fusion protein

[0041] In one aspect of the present application, a fusion protein is provided. According to embodiments of the present application, the fusion protein comprises: a first peptide segment comprising an Ag85A fragment; a second peptide segment comprising a PstS1 fragment, the first peptide segment being connected to the second peptide segment. The fusion protein according to embodiments of the present application can stimulate both cellular immunity and humoral immunity of the body, and improve the ability of the body to kill Mycobacterium tuberculosis.

[0042] According to embodiments of the present application, the C-terminus of the first peptide segment is connected to the N-terminus of the second peptide segment; or the C-terminus of the second peptide segment is connected to the N-terminus of the first peptide segment.

[0043] According to embodiments of the present application, the Ag85A fragment is a full-length peptide chain of Ag85A.

[0044] According to embodiments of the present application, the Ag85A fragment has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 95% similarity to SEQ ID NO: 1.

[0045] MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA (SEQ ID NO: 1).

[0046] According to an embodiment of the present application, the PstS1 fragment comprises at least one selected from a PstS1 full-length peptide chain and a PstS1 antigen epitope peptide.

[0047] According to an embodiment of the present application, the PstS1 antigen epitope peptide comprises at least one selected from tnPstS1 (250-501), tnPstS1 (550-852) and tnPstS1 (250-852). The tnPstS1 (250-501) has an amino acid sequence as shown in any one of SEQ ID NO: 2, the tnPstS1 (550-852) has an amino acid sequence as shown in any one of SEQ ID NO: 3, and the tnPstS1 (250-852) has an amino acid sequence as shown in any one of SEQ ID NO: 4. The inventors have found through experiments that the above-mentioned amino acid sequences are antigen epitope peptides in the PstS1 protein, which can stimulate the proliferation of B cells in the body and produce a large amount of antibodies that kill Mycobacterium tuberculosis, thereby improving the ability of the body to resist Mycobacterium tuberculosis.

[0048] According to an embodiment of the present application, the PstS1 fragment has an amino acid sequence as shown in any one of SEQ ID NO: 2-4 or an amino acid sequence having at least 95% similarity with any one of SEQ ID NO: 2-4.

[0049] QGTGSGAGIAQAAAGTVNIGASDAYLSEGDMAAHKGLMNIALAISAQQVNYNLPGVSEHLKLNGKVLAAMYQGTIKTWDDPQIA (SEQ ID NO: 2).

[0050] HRSDGSGDTFLFTQYLSKQDPEGWGKSPGFGTTVDFPAVPGALGENGNGGMVTGCAETPGCVAYIGISFLDQASQRGLGEAQLGNSSGNFLLPDAQSIQAA (SEQ ID NO: 3).

[0051] QGTGSGAGIAQAAAGTVNIGASDAYLSEGDMAAHKGLMNIALAISAQQVNYNLPGVSEHLKLNGKVLAAMYQGTIKTWDDPQIAALNPGVNLPGTAVVPLHRSDGSGDTFLFTQYLSKQDPEGWGKSPGFGTTVDFPAVPGALGENGNGGMVTGCAETPGCVAYIGISFLDQASQRGLGEAQLGNSSGNFLLPDAQSIQAA (SEQ ID NO: 4).

[0052] According to an embodiment of the present application, the fusion protein further comprises a connecting peptide.

[0053] According to an embodiment of the present application, the N terminus of the connecting peptide is connected to the C terminus of the first peptide segment, and the C terminus of the connecting peptide is connected to the N terminus of the second peptide segment; or the N terminus of the connecting peptide is connected to the C terminus of the second peptide segment, and the C terminus of the connecting peptide is connected to the N terminus of the first peptide segment.

[0054] According to an embodiment of the present application, the amino acid sequence of the connecting peptide is (GGGGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 2 or 3. Thus, the use of the connecting peptide described above can make the structure of the fusion protein more stable, and the antigen epitope of the fusion protein is fully exposed.

[0055] According to an embodiment of the present application, the connecting peptide comprises the amino acid sequence shown in SEQ ID NO: 5.

[0056] GGGGSGGGGSGGGGS (SEQ ID NO: 5).

[0057] According to an embodiment of the present application, the fusion protein further comprises a tag.

[0058] According to an embodiment of the present application, the C terminus of the first peptide segment is connected to the N terminus of the second peptide segment, and the tag is connected to the C terminus of the second peptide segment or the N terminus of the first peptide segment; or the C terminus of the second peptide segment is connected to the N terminus of the first peptide segment, and the tag is connected to the C terminus of the first peptide segment or the N terminus of the second peptide segment.

[0059] According to an embodiment of the present application, the tag is at least one of a HIS tag, a FLAG tag, an HA tag, a GST tag, a Strep II tag and an MBP tag. Thus, the above-mentioned tags can further improve the accuracy of the fusion protein structure space.

[0060] According to an embodiment of the present application, the HIS tag has an amino acid sequence as shown in SEQ ID NO: 6.

[0061] HHHHHH (SEQ ID NO: 6).

[0062] According to an embodiment of the present application, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 7-9. The inventors have found through experiments that the above-mentioned fusion protein can simultaneously activate the humoral immunity and the cellular immunity in the body, thereby further improving the ability of the body to kill Mycobacterium tuberculosis.

[0063] MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGAGGGGSGGGGSGGGGSQGTGSGAGIAQAAAGTVNIGASDAYLSEGDMAAHKGLMNIALAISAQQVNYNLPGVSEHLKLNGKVLAAMYQGTIKTWDDPQIAHHHHHH (SEQ ID NO: 7).

[0064] MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGAGGGGSGGGGSGGGGSGGGGSHRSDGSGDTFLFTQYLSKQDPEGWGKSPGFGTTVDFPAVPGALGENGNGGMVTGCAETPGCVAYIGISFLDQASQRGLGEAQLGNSSGNFLLPDAQSIQAAHHHHHH (SEQ ID NO: 8).

[0065] MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGAGGGGSGGGGSGGGGSQGTGSGAGIAQAAAGTVNIGASDAYLSEGDMAAHKGLMNIALAISAQQVNYNLPGVSEHLKLNGKVLAAMYQGTIKTWDDPQIAALNPGVNLPGTAVVPLHRSDGSGDTFLFTQYLSKQDPEGWGKSPGFGTTVDFPAVPGALGENGNGGMVTGCAETPGCVAYIGISFLDQASQRGLGEAQLGNSSGNFLLPDAQSIQAAHHHHHH (SEQ ID NO: 9).

[0066] Nucleic acid molecule, nucleic acid molecule and recombinant cell

[0067] In the process of preparing or obtaining these fusion proteins, nucleic acid molecules expressing these fusion proteins can be used, which are linked with different vectors and then expressed in different cells to obtain the corresponding fusion proteins.

[0068] In another aspect of the present application, the present application provides a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the aforementioned fusion protein. The nucleic acid molecule can be effectively used to express the aforementioned fusion protein, especially in prokaryotic or lower eukaryotic expression systems.

[0069] According to an embodiment of the present application, the nucleic acid molecule is DNA.

[0070] The nucleotide sequence shown in SEQ ID NO: 10 is used to encode SEQ ID NO: 7.

[0071] The nucleotide sequence set forth in SEQ ID NO: 11 is used to encode SEQ ID NO: 8.

[0072] The nucleotide sequence set forth in SEQ ID NO: 12 is used to encode SEQ ID NO: 9.

[0073]

[0074]

[0075]

[0076] In yet another aspect of the present application, a nucleic acid molecule is provided. According to embodiments of the present application, the nucleic acid molecule carries the aforementioned fusion protein. The expression vector can be effectively used to express the aforementioned fusion protein, especially in prokaryotic or lower eukaryotic expression systems.

[0077] According to embodiments of the present application, the expression vector is a eukaryotic expression vector.

[0078] According to embodiments of the present application, the expression vector is a lentiviral vector.

[0079] In yet another aspect of the present application, a recombinant cell is provided. According to embodiments of the present application, the recombinant cell comprises: the aforementioned nucleic acid molecule or the aforementioned expression vector; or, expresses the aforementioned fusion protein. The recombinant cell of embodiments of the present application can be used for in vitro expression and mass production of the aforementioned fusion protein.

[0080] According to embodiments of the present application, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0081] According to embodiments of the present application, the recombinant cell comprises a eukaryotic cell or a prokaryotic cell. Illustratively, the recombinant cell is a BL21(DE3) competent cell.

[0082] It is understood by those skilled in the art that the features and advantages described above for the fusion protein are equally applicable to the nucleic acid molecule, the expression vector and the recombinant cell, and will not be repeated here.

[0083] Subunit vaccine

[0084] In yet another aspect of the present application, a subunit vaccine is provided. According to embodiments of the present application, the subunit vaccine comprises: the aforementioned fusion protein. The subunit vaccine of embodiments of the present application can improve the body's ability to resist Mycobacterium tuberculosis, effectively preventing and treating tuberculosis. Moreover, it can effectively enhance the immune effect of BCG.

[0085] According to embodiments of the present application, the subunit vaccine further comprises a pharmaceutically acceptable excipient.

[0086] According to embodiments of the present application, the excipient comprises an adjuvant.

[0087] According to embodiments of the present application, the adjuvant comprises at least one selected from the group consisting of Poly IC adjuvant, aluminum adjuvant, Freund's adjuvant, CpG ODN, DDA, MPLA, IC31, AS01 and AS03, preferably aluminum adjuvant. The inventors have found through experiments that the use of the above adjuvant can improve the immunogenicity of the subunit vaccine.

[0088] It should be noted that the "aluminum adjuvant" generally refers to an immunological adjuvant comprising aluminum, which at least includes but is not limited to one or more of aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum and potassium alum. For example, it can be a suspension of aluminum hydroxide and magnesium hydroxide.

[0089] According to embodiments of the present application, the adjuvant is an aluminum hydroxide adjuvant.

[0090] According to embodiments of the present application, the volume ratio of the fusion protein to the adjuvant is 3:1. In this way, the immunogenicity of the subunit vaccine can be further improved.

[0091] It can be understood by those skilled in the art that the features and advantages described above for the fusion protein, the nucleic acid molecule, the expression vector and the recombinant cell also apply to the subunit vaccine, which will not be repeated here.

[0092] Use

[0093] In another aspect of the present application, the present application provides a use of the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector or the aforementioned recombinant cell in the preparation of a subunit vaccine.

[0094] In another aspect of the present application, the present application provides a use of the aforementioned subunit vaccine in the preparation of a medicament for enhancing the immunological effect of BCG or for preventing and / or treating tuberculosis.

[0095] It can be understood by those skilled in the art that the features and advantages described above for the fusion protein, the nucleic acid molecule, the expression vector, the recombinant cell and the subunit vaccine also apply to the use, which will not be repeated here.

[0096] Method

[0097] In another aspect of the present application, the present application provides a method for preventing and / or treating tuberculosis. According to embodiments of the present application, a pharmaceutically acceptable amount of the aforementioned fusion protein or subunit vaccine is administered to a subject. The method according to embodiments of the present application can improve the body's ability to resist Mycobacterium tuberculosis, effectively preventing and treating tuberculosis.

[0098] According to embodiments of the present application, the dose of the fusion protein or subunit vaccine administered is 20 μg.

[0099] According to an embodiment of the present application, the fusion protein or subunit vaccine is administered 3 times, with an interval of 1-2 weeks.

[0100] According to an embodiment of the present application, the administration route of the method comprises subcutaneous injection or inhalation immunization.

[0101] It is understood by those skilled in the art that the features and advantages described above for the subunit vaccine also apply to the method, which will not be repeated here.

[0102] The schemes of the present application will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0103] It should be noted that the BCG of the present application is obtained by screening by conventional methods, and its immunization effect can be seen in Figure 5 .

[0104] Example 1: Preparation of fusion protein

[0105] 1. Amplification of Ag85A-PstS1 gene

[0106] The inventors amplified Ag85A(amino acid sequence of SEQ ID NO: 1) + connecting peptide(amino acid sequence of SEQ ID NO: 5) using H37Rv as a template and P1 and P2 as upstream and downstream primers, i.e., the amplified fragment 1(nucleotide sequence of SEQ ID NO: 19). In addition, the inventors selected three truncated sequences of PstS1(tnPstS1), which were named as tnPstS1(250-501), tnPstS1(550-852) and tnPstS1(250-852), and were abbreviated as Ps1, Ps2 and Ps3, respectively. Then, the inventors amplified connecting peptide(amino acid sequence of SEQ ID NO: 5) + Ps1(amino acid sequence of SEQ ID NO: 2) using H37Rv as a template and P3 and P5 as upstream and downstream primers, i.e., the amplified fragment 2(nucleotide sequence of SEQ ID NO: 20); amplified connecting peptide(amino acid sequence of SEQ ID NO: 5) + Ps2(amino acid sequence of SEQ ID NO: 3) using P4 and P6 as upstream and downstream primers, i.e., the amplified fragment 3(nucleotide sequence of SEQ ID NO: 21); and amplified connecting peptide(amino acid sequence of SEQ ID NO: 5) + Ps3(amino acid sequence of SEQ ID NO: 4) using P3 and P6 as upstream and downstream primers, i.e., the amplified fragment 4(nucleotide sequence of SEQ ID NO: 22). The nucleotide sequences of P1, P2, P3, P4, P5 and P6 primers are shown in Table 1; the PCR reaction system is shown in Table 2; and the PCR reaction procedure is as follows: 98℃ 3min; 98℃ 10s, 55℃ 5s, 72℃ 1min / kb, 30 cycles; and 72℃ 5min.

[0107] The three truncated fragments Ps1, Ps2, Ps3 containing Ag85A and PstS1 were first amplified into amplification fragments 1-4 according to the above reaction system, and then the nucleotide sequence of amplification fragment 1 containing Ag85A, one of the amplification fragments 2-4 containing the truncated fragment of PstS1 and HIS tag (amino acid sequence is SEQ ID NO: 6; nucleotide sequence is SEQ ID NO: 23) were connected by SOE-PCR method to obtain the fusion gene Ag85A-tnPstS1 fragments of the three truncated fragments Ps1, Ps2, Ps3 of Ag85A and PstS1, respectively, which were named as AP1 (amino acid sequence is SEQ ID NO: 7, nucleotide sequence is SEQ ID NO: 10), AP2 (amino acid sequence is SEQ ID NO: 8, nucleotide sequence is SEQ ID NO: 11), AP3 (amino acid sequence is SEQ ID NO: 9, nucleotide sequence is SEQ ID NO: 12). The reaction system of SOE-PCR is shown in Table 3 and Table 4, and the reaction procedure is as follows: 98℃ 3min; 98℃ 10s, 55℃ 5s, 72℃ 1min / kb, 5 cycles. Then the above AP1, AP2 and AP3 products were mixed with the reagents in Table 4, and the reaction procedure was as follows: 98℃ 3min; 98℃ 10s, 55℃ 5s, 72℃ 1min / kb, 30 cycles; 72℃ 5min. The target gene was obtained. After PCR, the AP1, AP2 and AP3 obtained in the above step were subjected to 1% agarose gel electrophoresis, and the electrophoresis results are shown in Figure 1

[0108] It should be noted that the high-fidelity enzyme (PrimeSTAR HS DNA Polymerase) required in the amplification process of this example was purchased from TaKaRa (Beijing) Co., Ltd.

[0109] The nucleotide sequence of amplification fragment 1 is as follows:

[0110] ​

[0111] The nucleotide sequence of amplified fragment 2 is:

[0112] GGTGGAGGCGGTTCAGGTGGAGGCGGTTCAGGTGGAGGCGGTTCACAGGGCACCGGTTCTGGTGCCGGGATCGCGCAGGCCGCCGCCGGGACGGTCAACATTGGGGCCTCCGACGCCTATCTGTCGGAAGGTGATATGGCCGCGCACAAGGGGCTGATGAACATCGCGCTAGCCATCTCCGCTCAGCAGGTCAACTACAACCTGCCCGGAGTGAGCGAGCACCTCAAGCTGAACGGAAAAGTCCTGGCGGCCATGTACCAGGGCACCATCAAAACCTGGGACGACCCGCAGATCGCTCACCACCACCACCACCACCACATG (SEQ ID NO: 20);

[0113] The nucleotide sequence of amplified fragment 3 is:

[0114] GGTGGAGGCGGTTCAGGTGGAGGCGGTTCAGGTGGAGGCGGTTCACACCGCTCCGACGGGTCCGGTGACACCTTCTTGTTCACCCAGTACCTGTCCAAGCAAGATCCCGAGGGCTGGGGCAAGTCGCCCGGCTTCGGCACCACCGTCGACTTCCCGGCGGTGCCGGGTGCGCTGGGTGAGAACGGCAACGGCGGCATGGTGACCGGTTGCGCCGAGACACCGGGCTGCGTGGCCTATATCGGCATCAGCTTCCTCGACCAGGCCAGTCAACGGGGACTCGGCGAGGCCCAACTAGGCAATAGCTCTGGCAATTTCTTGTTGCCCGACGCGCAAAGCATTCAGGCCGCGCACCACCACCACCACCACATG (SEQ ID NO: 21);

[0115] The nucleotide sequence of amplified fragment 4 is:

[0116] GGTGGAGGCGGTTCAGGTGGAGGCGGTTCAGGTGGAGGCGGTTCACAGGGCACCGGTTCTGGTGCCGGGATCGCGCAGGCCGCCGCCGGGACGGTCAACATTGGGGCCTCCGACGCCTATCTGTCGGAAGGTGATATGGCCGCGCACAAGGGGCTGATGAACATCGCGCTAGCCATCTCCGCTCAGCAGGTCAACTACAACCTGCCCGGAGTGAGCGAGCACCTCAAGCTGAACGGAAAAGTCCTGGCGGCCATGTACCAGGGCACCATCAAAACCTGGGACGACCCGCAGATCGCTGCGCTCAACCCCGGCGTGAACCTGCCCGGCACCGCGGTAGTTCCGCTGCACCGCTCCGACGGGTCCGGTGACACCTTCTTGTTCACCCAGTACCTGTCCAAGCAAGATCCCGAGGGCTGGGGCAAGTCGCCCGGCTTCGGCACCACCGTCGACTTCCCGGCGGTGCCGGGTGCGCTGGGTGAGAACGGCAACGGCGGCATGGTGACCGGTTGCGCCGAGACACCGGGCTGCGTGGCCTATATCGGCATCAGCTTCCTCGACCAGGCCAGTCAACGGGGACTCGGCGAGGCCCAACTAGGCAATAGCTCTGGCAATTTCTTGTTGCCCGACGCGCAAAGCATTCAGGCCGCGCACCACCACCACCACCACATG (SEQ ID NO:22);

[0117] The nucleotide sequence of the HIS tag is:

[0118] CACCACCACCACCACCAC (SEQ ID NO: 23).

[0119] Table 1: Nucleotide sequences of P1-P6 primers

[0120]

[0121]

[0122] Table 2: PCR reaction system (50 μL)

[0123] Reagent Dosage 5x PrimeSTAR Buffer (Mg 2+ Plus) 10 μL dNTP Mixture (2.5 mM each) 4 μL Primer 1 2 μL Primer 2 2 μL PrimeSTAR HS DNA Polymerase (2.5 U / μl) 0.5 μL Template (H37Rv genome) 2 μL ddH2O 39.5 μL

[0124] Table 3: SOE-PCR reaction system

[0125] Reagent Dosage 5x PrimeSTAR Buffer (Mg2+ Plus) 10 μL dNTP Mixture (2.5 mM each) 4 μL Ag85A 2 μL Ps1 / Ps2 / Ps3 2 μL ddH2O 27.5 μL

[0126] Table 4: Amplification of AP1, AP2 and AP3 fragments

[0127]

[0128]

[0129] 2. Construction of pET-21a-AP1, pET-21a-AP2 and pET-21a-AP3 expression vectors

[0130] The amplified target gene (AP1, AP2 or AP3) was double-digested with the vector pET-21a, and then ligated with T4 DNA ligase to construct the pET-21a-Ag85A-tnPstS1 vector (see Figure 2 ). After being introduced into DH5a competent cells, the correct recombinant vector was obtained after screening and sequencing. The specific steps are as follows:

[0131] The amplified target gene (AP1, AP2 or AP3) was recovered using a rapid agarose gel DNA recovery kit (CWBIO, item number: CW2302) according to the product instructions. After recovery, the concentration was detected using TECAN.

[0132] The pET-21a strain was cultured overnight at 37°C and 200 rpm (transferred at 1:1000 into 5 mL LB medium containing 100 μg / mL ampicillin). The next day, the bacterial solution was centrifuged to obtain the bacterial body, and the plasmid was extracted using a high-purity plasmid extraction kit (CWBIO, item number: CW0500) according to the product instructions for standby use, and the concentration was detected using TECAN.

[0133] The target gene (AP1, AP2 or AP3) was double-digested with the vector pET-21a. The enzymes used for double digestion were BamHI-HF (star enzyme) (NEB, item number #R3136V) and EcoRI-HF (star enzyme) (NEB, item number #R3101V). The double digestion experiment was performed using the instructions, and the addition amounts of each reagent are shown below:

[0134] Reagent Dosage BamHI-HF 1 μL EcoRI-HF 1 μL Gene of interest / pET-21a vector 1000 ng CutSmart Buffer 5 μL ddH2O Make up to 50 μL

[0135] After enzyme digestion using double enzyme, enzyme digestion fragments were recovered using a rapid agarose gel DNA recovery kit (CWBIO, item number: CW2302) according to the product instructions. The concentration was detected using TECAN after recovery. The recovered fragments were connected using T4 DNA ligase (NEB, item number #M0202V), and the addition amounts of each reagent are as follows:

[0136] Reagent Dosage AP1 / AP2 / AP3 after enzyme digestion 1 μL (70 ng) pET-21a vector after enzyme digestion 1 μL (50 ng) T4 DNA ligase 1 μL T4 DNA ligase reaction buffer 2 μL ddH2O 15 μL

[0137] After connection, 5 μL of the connected fragments were introduced into DH5a competent cells (CWBIO, item number: CW0808) according to the product instructions and plated.

[0138] The next day, colonies were randomly selected from the plate, and after positive colony PCR identification, the company Sequencing was performed. After successful sequencing, the bacterial solution was amplified, and high-purity plasmid extraction kit (CWBIO, item number: CW0500) was used to extract the plasmid according to the product instructions for standby, and the concentration was detected using TECAN.

[0139] 3. Induced expression and purification of AP1, AP2 and AP3

[0140] The plasmids of AP1, AP2 and AP3 with correct sequencing were introduced into BL21 (DE3) competent cells (CWBIO, CW0809) according to the product instructions, and single colonies were selected for culture and freezing. When expressing, the cells were thawed and transferred to LB liquid medium containing ampicillin (Amp, 100 μg / mL) for 37 °C shaking culture, and the OD 600 When the OD value was 0.6-0.8, 1 mM IPTG was added to induce, and the bacterial cells were obtained by centrifugation after 4 hours of induction. The bacterial cells were resuspended with PBS and broken by ultrasonic, and the supernatant and precipitate were obtained by centrifugation. The precipitate was completely dissolved with 8M urea at 4 °C, and the supernatant was obtained by centrifugation. The obtained AP1, AP2 and AP3 were purified using affinity chromatography nickel column. The purified AP1, AP2 and AP3 were identified by SDS-PAGE Coomassie blue staining and WB (Western Bloting), and it was found that the AP1, AP2 and AP3 proteins were all in inclusion bodies. The SDS-PAGE Coomassie blue staining detection result and the WB detection result are shown in Figure 3 Figure 3 Figure 3

[0141] Example 2: Preparation of subunit vaccine

[0142] The AP1, AP2 and AP3 proteins obtained after purification in Example 1 and aluminum salt adjuvant (Thermo Scientific TM ​​​(Catalyst No.: 77161) was mixed uniformly at a volume ratio of 3:1 at 4°C and 500 rpm for 30 min. These were named subunit vaccine 1 (Vaccine 1), subunit vaccine 2 (Vaccine 2), and subunit vaccine 3 (Vaccine 3), respectively.

[0143] Example 3: Animal protective experiment

[0144] Six- to eight-week-old female SPF C57BL / 6J mice (purchased from Spiford (Beijing) Biotechnology Co., Ltd.) were used for the experiment. For detailed experimental procedures, please refer to [link to relevant documentation]. Figure 4 The experiment consisted of five groups: the PBS group (immunized three times, with each mouse receiving 100 μL of PBS), the BCG group (initial immunization once, with each mouse receiving 10 μL of PBS), and the BCG group (immunized once, with each mouse receiving 10 μL of PBS). 6 The mice were immunized with CFU (carbohydrate, bismuth subunit), BCG, or PBS. The subunit vaccines, BCG, or PBS were administered subcutaneously (sc). Initial immunization was performed at week 0, followed by three immunizations at weeks 0, 2, and 4. Cytokine and antibody titers were measured two weeks after immunization. Following these measurements, mice were challenged with 200 CFU of *M. bovis* C68004 via aerosol infection. Four weeks later, mice were anesthetized and euthanized. Bacterial load and histopathological changes in the lungs and spleen were measured. Results are as follows: Figure 5 and Figure 6 As shown in the figure. The results showed that, compared with the PBS group, the bacterial load in the lungs and spleen of mice in the AP1, AP2, and AP3 groups was reduced. In particular, the inhibitory ability of the AP2 and AP3 groups against *M. bovis* reached a level comparable to that of the BCG group. Therefore, these results further demonstrate that vaccines 1, 2, and 3 can effectively prevent tuberculosis.

[0145] Example 4: Antibody titer detection

[0146] In Example 3, serum was collected from the tail vein of the five groups of mice at the end of the second week after each immunization and before the next immunization, and stored at -80°C for later use. When detecting antibody titers, *M. bovis* was plated, and after autoclaving, the *M. bovis* was resuspended in H2O and the OD was adjusted. 600For 1.0, 100 μL of the M. bovis suspension with OD value of 1.0 was added to each well of the 96-well plate, which was placed in an oven at 60°C to dry, and after drying, fixed with ice-cold methanol for 2 h. The plate was washed with PBS for three times, each time for 5 min. 100 μL of serum was diluted at 1:1000, and added to the 96-well plate, which was incubated at 37°C for 1 h. The plate was washed with PBS for three times, each time for 5 min. 100 μL of HRP-labeled goat anti-mouse IgG, IgG1, IgG2c, IgG3, IgM, IgA (Abeam) was diluted at 1:10000, and added to the 96-well plate, which was incubated at 37°C for 1 h. The plate was washed with PBS for three times, each time for 5 min. 100 μL of TMB (Solebo, Beijing) was added to each well for color development, and after 20 min, 50 μL of 2M H2SO4 was added to each well to terminate the color development. The OD value was read by a microplate reader 450 , and the detection results are shown in Figure 7 , and the titer detection after the third immunization is shown in Figure 8 , wherein if not specified, it means that there is a difference between the PBS group.

[0147] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the different embodiments or examples described in the present specification and the features of the different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0148] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and cannot be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A fusion protein, characterized in that, The fusion protein is connected by a first peptide segment and a second peptide segment; The first peptide segment is an Ag85A full-length peptide chain, and the amino acid sequence is shown as SEQ ID NO: 1; The second peptide segment is a PstS1 fragment, and the amino acid sequence is shown as any one of SEQ ID NO: 2-4.

2. The fusion protein of claim 1, wherein, The C-terminal of the first peptide segment is connected with the N-terminal of the second peptide segment, or the C-terminal of the second peptide segment is connected with the N-terminal of the first peptide segment.

3. The fusion protein of claim 1, wherein, The fusion protein further comprises a connecting peptide.

4. The fusion protein of claim 3, wherein, The N-terminal of the connecting peptide is connected with the C-terminal of the first peptide segment, and the C-terminal of the connecting peptide is connected with the N-terminal of the second peptide segment, or the N-terminal of the connecting peptide is connected with the C-terminal of the second peptide segment, and the C-terminal of the connecting peptide is connected with the N-terminal of the first peptide segment.

5. The fusion protein of claim 3, wherein, The amino acid sequence of the connecting peptide is (GGGGS) n wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

6. The fusion protein of claim 5, wherein, The n is 2 or 3.

7. The fusion protein of claim 3, wherein, The amino acid sequence of the connecting peptide is shown as SEQ ID NO:

5.

8. The fusion protein of claim 1, wherein, The fusion protein further comprises a tag.

9. The fusion protein of claim 8, wherein, The C-terminal of the first peptide segment is connected with the N-terminal of the second peptide segment, and the tag is connected with the C-terminal of the second peptide segment or the N-terminal of the first peptide segment, or the C-terminal of the second peptide segment is connected with the N-terminal of the first peptide segment, and the tag is connected with the C-terminal of the first peptide segment or the N-terminal of the second peptide segment.

10. The fusion protein of claim 8, wherein, The tag is at least one of HIS tag, FLAG tag, HA tag, GST tag, Strep II tag and MBP tag.

11. The fusion protein of claim 10, wherein, The amino acid sequence of the HIS tag is shown as SEQ ID NO:

6.

12. The fusion protein of claim 1, wherein, The amino acid sequence of the fusion protein is shown as any one of SEQ ID NO: 7-9.

13. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein of any one of claims 1-12.

14. The nucleic acid molecule of claim 13, wherein The nucleic acid molecule is DNA.

15. An expression vector comprising the nucleic acid of claim 14. The nucleic acid molecule of claim 13 or 14.

16. The expression vector of claim 15, wherein, The expression vector is a eukaryotic expression vector.

17. The expression vector of claim 15, wherein, The expression vector is a lentiviral vector.

18. A recombinant cell, wherein, Comprising: The nucleic acid molecule of claim 13 or 14 or the expression vector of any one of claims 15-17; or, expressing the fusion protein of any one of claims 1-12.

19. The recombinant cell of claim 18, wherein, The recombinant cell is obtained by introducing the expression vector of any one of claims 15-17 into a host cell.

20. The recombinant cell of claim 18, wherein, The recombinant cell comprises a eukaryotic cell or a prokaryotic cell.

21. Use of the fusion protein of any one of claims 1-12, the nucleic acid molecule of claim 13 or 14, the expression vector of any one of claims 15-17 or the recombinant cell of any one of claims 18-20 in the preparation of a subunit vaccine.

22. A subunit vaccine, characterized in that, Comprising: The fusion protein of any one of claims 1-12.

23. The subunit vaccine of claim 22, characterized in that, Further comprising a pharmaceutically acceptable adjuvant.

24. The subunit vaccine of claim 23, characterized in that, The adjuvant comprises at least one selected from the group consisting of Poly IC adjuvant, aluminum adjuvant, Freund's adjuvant, CpG ODN, DDA, MPLA, IC31, AS01 and AS03.

25. The subunit vaccine of claim 24, characterized in that, The volume ratio of the fusion protein and the adjuvant is 3:

1.

26. The subunit vaccine of claim 24, characterized in that, The adjuvant is an aluminum hydroxide adjuvant.

27. The subunit vaccine of claim 24, characterized in that, ​ 28. Use of a subunit vaccine according to any one of claims 22 to 27 for the preparation of a medicament for boosting the immunizing effect of BCG or for the prophylaxis and / or treatment of tuberculosis.

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