CN116082520A

CN116082520A - Preparation and application of novel tuberculosis subunit vaccine containing different truncated PstS1 fusion proteins AP

Info

Publication number: CN116082520A
Application number: CN202211192711.4A
Authority: CN
Inventors: 李�浩; 曾令媛
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-09-28
Filing date: 2022-09-28
Publication date: 2023-05-09

Abstract

The invention provides a fusion protein containing PstS1 fragment and Ag85A fragment and tuberculosis subunit vaccine. The fusion protein comprises: a first peptide fragment comprising an Ag85A fragment; a second peptide fragment comprising a PstS1 fragment, the first peptide fragment being linked to the second peptide fragment; subunit vaccines include the fusion proteins described above. The fusion protein can excite cell immunity and humoral immunity of organism, raise the ability of organism to kill mycobacterium tuberculosis, and prevent tuberculosis effectively.

Description

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

Technical Field

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

Background

Tuberculosis (TB) is a very harmful chronic wasting infectious disease caused by mycobacterium Tuberculosis (Mycobacterium Tuberculosis, mtb), with 900 or more new cases per year, and about 140 thousands of deaths. With the advent of drug resistant Mycobacterium tuberculosis and HIV-Mycobacterium Tuberculosis (TB) combined infections, this situation becomes more complex, making the prognosis and treatment of tuberculosis worse. Bovine tuberculosis (Bovine Tuberculosis) is a human and veterinary co-morbid chronic infectious disease caused by mycobacterium bovis (Mycobacterium bovis, m.bovis) and is characterized by tuberculous granuloma and cheesy, calcified necrotic lesions of tissue and organs. Wherein, cows are multiple, which seriously affects the development of animal husbandry and human health.

BCG is currently the only vaccine approved by WHO to prevent tuberculosis, and has protective effects on infants and teenagers, but the protective effects on adults are not clear, so development of an effective tuberculosis vaccine is highly demanded.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides a tuberculosis subunit vaccine containing PstS1 fragments and Ag85A fragments, which can excite and improve cellular immunity and humoral immunity at the same time and has good effect of preventing tuberculosis.

The present invention has been completed based on the following findings by the inventors:

there are many kinds of vaccines against tuberculosis at present, and the novel tuberculosis vaccines under development include recombinant BCG (rBCG) vaccine, attenuated m.tb vaccine, adjuvant subunit protein vaccine, viral vector vaccine, whole cell vaccine, DNA vaccine, RNA vaccine, etc.

Subunit vaccines gradually enter the field of vision of people due to the characteristics of easy production, good safety, strong specificity, good quality control and the like. In the screening of recombinant subunit vaccine antigens, it is preferable to select antigens that induce strong cellular immunity, but studies have shown that accumulation of multiple cellular antigens does not significantly improve their immunoprotection.

Based on this, the inventors screened recombinant subunit vaccine antigens through a number of experiments, and finally determined two proteins, ag85A and phosphate-specific transport system protein 1 (Phosphate transport system protein l, pstS 1). The inventor finds that Ag85A is a main constituent component of M.tb culture filtrate protein and is also a T cell antigen of M.tb, can induce extremely strong Th1 type immune response and is important for regulating intracellular infection; pstS1 is an important protein of the cell wall of mycobacterium tuberculosis, has immunogenicity, can stimulate proliferation of B cells and T cells, and is an important immunoprotection antigen. The inventor finds that the fusion protein prepared by selecting the two proteins can excite cell immunity and humoral immunity at the same time, and improve the capability of the organism to kill mycobacterium tuberculosis. The inventor prepares the fusion protein and the adjuvant into a tuberculosis subunit vaccine, injects the tuberculosis subunit vaccine into a mouse body, then adopts mycobacterium tuberculosis to attack the mouse, and discovers that the load of the mycobacterium tuberculosis in the viscera of the mouse is obviously reduced, almost achieves the same immunoprotection as BCG, and shows that the tuberculosis subunit vaccine can greatly improve the capability of the organism for resisting the mycobacterium tuberculosis and can effectively prevent and treat tuberculosis. Furthermore, the inventor also finds that the tuberculosis subunit vaccine can effectively strengthen the immune effect of the BCG through experiments.

In one aspect of the invention, the invention provides a fusion protein. According to an embodiment of the invention, the fusion protein comprises: a first peptide fragment comprising an Ag85A fragment; a second peptide fragment comprising a PstS1 fragment, the first peptide fragment being linked to the second peptide fragment. The fusion protein provided by the embodiment of the invention can excite the cellular immunity and the humoral immunity of the organism at the same time, and the capability of the organism in killing mycobacterium tuberculosis is improved.

In another aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the aforementioned fusion protein. The nucleic acid molecules according to embodiments of the present invention may be effective in expressing the aforementioned fusion proteins.

In yet another aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the aforementioned nucleic acid molecules are carried. The expression vector according to the embodiment of the invention can effectively express the fusion protein.

In yet another aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises: carrying the nucleic acid molecule as described above or the expression vector as described above; alternatively, the fusion proteins described above are expressed. Recombinant cells according to embodiments of the present invention may be used for in vitro expression and bulk acquisition of the aforementioned fusion proteins.

In a further aspect of the invention, the invention provides the use of a fusion protein as defined above, a nucleic acid molecule as defined above, an expression vector as defined above or a recombinant cell as defined above for the preparation of a subunit vaccine.

In yet another aspect of the invention, the invention provides a subunit vaccine. According to an embodiment of the invention, the subunit vaccine comprises: the fusion protein. The subunit vaccine provided by the embodiment of the invention can improve the capability of resisting mycobacterium tuberculosis of organisms and effectively prevent and treat tuberculosis; and, the immune effect of BCG can be effectively enhanced.

In a further aspect of the invention, the invention proposes the use of a subunit vaccine as described above for the preparation of a medicament for boosting the immune effect of BCG or for preventing and/or treating tuberculosis.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a graph showing the results of gel electrophoresis of SOE-PCR amplification of AP1, AP2, and AP3 in example 1 of the present invention;

FIG. 2 is a schematic diagram of the construction of pET-21a-Ag85A-tnPstS1 vector in example 1 of the present invention;

FIG. 3 shows SDS-PAGE Coomassie staining (left) and WB detection (right) after purification of AP1, AP2 and AP3 in example 1 of the present invention;

FIG. 4 is a flow chart of an animal protection test in example 3 of the present invention;

fig. 5 is a graph showing the results of the bacterial load detection of the viscera of each group of mice after the challenge in the embodiment 3 of the present invention, wherein P <0.05 and P <0.01 and P <0.0001 respectively;

FIG. 6 shows the results of lung pathological changes detection after four weeks of bacterial challenge in each group of mice in example 3 of the present invention;

FIG. 7 shows the results of the change in serum antibody titers of mice in each group after three immunizations in example 4 of the present invention;

FIG. 8 shows the results of the third immunization in example 4.

Detailed Description

Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.

In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

In this context, the terms "identity", "homology" or "similarity" are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., see Ausubel et al, et al (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: support.3 (National Biomedical Research Foundation, washington, D.C.), there are many algorithms for alignment and determination of sequence identity, including homology alignment algorithms of needle et al (1970) J.mol.biol.48:443, computer programs using these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or the programs of Pearson et al (1988) Proc.Natl.Acad.Sci.85:2444, the Smith-Waterman algorithm (Meth.mol.70:173-187 (1997), and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J.Mol.biol.215:403-410), and include but are also available in the programs of ALIGN or Megalign (DNASTAR), or the programs of BLAST-2, and the programs of Abelson.G.35:266, and the programs of Abelson.35:266, respectively.

As used herein, the term "at least 90% similarity" refers to a similarity of at least 95%, and may be 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% with each reference sequence.

In this context, the term "expression vector" generally refers to a nucleic acid molecule capable of insertion into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells. The expression vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The expression vector also includes vectors having a plurality of the above functions. The expression vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the expression vector will produce the desired expression product by culturing a suitable host cell containing the expression vector.

As used herein, the term "recombinant cell" generally refers to a cell that has been modified or recombined with genetic material of a host cell using genetic engineering techniques or cell fusion techniques to obtain a unique trait that is stably inherited. Wherein the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector may be introduced. The term "transformed" or "transfected" as used herein refers to the introduction of a nucleic acid (e.g., vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the invention and can be used for expression and/or secretion of a target protein.

As used herein, the term "fragment" refers to a fragment of a protein, which may comprise a full-length fragment of the protein, or may comprise a partial fragment of the protein. Illustratively, the Ag85A fragment may be a full length fragment of an Ag85A protein, or may be a partial fragment of an Ag85A protein; the PstS1 fragment may be a full-length fragment of the PstS1 protein, or may be a partial fragment of the PstS1 protein, such as a PstS1 epitope peptide.

As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. Preferably, the term "pharmaceutically acceptable" as used herein refers to use in animals, particularly humans, approved by the federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia.

As used herein, the term "pharmaceutically acceptable excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular dosage form of interest. In addition to the extent to which any conventional adjuvant is incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.

In this context, the term "treatment" refers to the use to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising a compound described herein to an individual in need thereof.

The invention provides a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell and subunit vaccine and application thereof, and the detailed description of the fusion protein, the nucleic acid molecule, the expression vector, the recombinant cell and the subunit vaccine is respectively provided below.

Fusion proteins

According to an embodiment of the invention, the C-terminus of the first peptide fragment is linked to the N-terminus of the second peptide fragment; alternatively, the C-terminus of the second peptide is linked to the N-terminus of the first peptide.

According to an embodiment of the invention, the Ag85A fragment is an Ag85A full-length peptide chain.

According to an embodiment of the invention, 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.

MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA(SEQ ID NO:1)。

According to an embodiment of the invention, the PstS1 fragment comprises at least one selected from the group consisting of a PstS1 full-length peptide chain and a PstS1 epitope peptide.

According to an embodiment of the present invention, the PstS1 epitope peptide comprises at least one selected from the group consisting of tnPstS1 (250-501), tnPstS1 (550-852) and tnPstS1 (250-852). Wherein, tnPstS1 (250-501) has an amino acid sequence shown in any one of SEQ ID NO:2, tnPstS1 (550-852) has an amino acid sequence shown in any one of SEQ ID NO:3, and tnPstS1 (250-852) has an amino acid sequence shown in any one of SEQ ID NO: 4. The inventor finds through experiments that the amino acid sequence is an epitope peptide in PstS1 protein, which can stimulate proliferation of B cells in a body and generate a large amount of antibodies for killing mycobacterium tuberculosis, so that the capability of the body for resisting the mycobacterium tuberculosis is improved.

According to an embodiment of the invention, the PstS1 fragment has an amino acid sequence as shown in any one of SEQ ID NOS.2 to 4 or an amino acid sequence having at least 95% similarity to any one of SEQ ID NOS.2 to 4.

QGTGSGAGIAQAAAGTVNIGASDAYLSEGDMAAHKGLMNIALAISAQQVNYNLPGVSEHLKLNGKVLAAMYQGTIKTWDDPQIA(SEQ ID NO:2)。

HRSDGSGDTFLFTQYLSKQDPEGWGKSPGFGTTVDFPAVPGALGENGNGGMVTGCAETPGCVAYIGISFLDQASQRGLGEAQLGNSSGNFLLPDAQSIQAA(SEQ ID NO:3)。

QGTGSGAGIAQAAAGTVNIGASDAYLSEGDMAAHKGLMNIALAISAQQVNYNLPGVSEHLKLNGKVLAAMYQGTIKTWDDPQIAALNPGVNLPGTAVVPLHRSDGSGDTFLFTQYLSKQDPEGWGKSPGFGTTVDFPAVPGALGENGNGGMVTGCAETPGCVAYIGISFLDQASQRGLGEAQLGNSSGNFLLPDAQSIQAA(SEQ ID NO:4)。

According to an embodiment of the invention, the fusion protein further comprises a connecting peptide.

According to an embodiment of the present invention, the N-terminus of the connecting peptide is connected to the C-terminus of the first peptide fragment, and the C-terminus of the connecting peptide is connected to the N-terminus of the second peptide fragment; alternatively, the N-terminus of the linker peptide is linked to the C-terminus of the second peptide fragment, and the C-terminus of the linker peptide is linked to the N-terminus of the first peptide fragment.

According to an embodiment of the invention, 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. Therefore, the structure of the fusion protein can be more stable by adopting the connecting peptide, and the epitope of the fusion protein is fully exposed.

According to an embodiment of the invention, the connecting peptide comprises the amino acid sequence shown in SEQ ID NO. 5.

GGGGSGGGGSGGGGS(SEQ ID NO:5)。

According to an embodiment of the invention, the fusion protein further comprises a tag.

According to an embodiment of the invention, the C-terminal of the first peptide fragment is connected to the N-terminal of the second peptide fragment, the tag is connected to the C-terminal of the second peptide fragment or the tag is connected to the N-terminal of the first peptide fragment; alternatively, the C-terminus of the second peptide fragment is linked to the N-terminus of the first peptide fragment, and the tag is linked to the C-terminus of the first peptide fragment or the tag is linked to the N-terminus of the second peptide fragment.

According to an embodiment of the present invention, 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. Therefore, the accuracy of the structural space of the fusion protein can be further improved by adopting the tag.

According to an embodiment of the invention, the HIS tag has the amino acid sequence shown in SEQ ID NO. 6.

HHHHHH(SEQ ID NO:6)。

According to an embodiment of the invention, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs 7 to 9. Experiments show that the fusion protein can activate humoral immunity and cellular immunity in a body at the same time, so that the capability of the body for killing mycobacterium tuberculosis is further improved.

MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGAGGGGSGGGGSGGGGSQGTGSGAGIAQAAAGTVNIGASDAYLSEGDMAAHKGLMNIALAISAQQVNYNLPGVSEHLKLNGKVLAAMYQGTIKTWDDPQIAHHHHHH(SEQ ID NO:7)。

MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGAGGGGSGGGGSGGGGSHRSDGSGDTFLFTQYLSKQDPEGWGKSPGFGTTVDFPAVPGALGENGNGGMVTGCAETPGCVAYIGISFLDQASQRGLGEAQLGNSSGNFLLPDAQSIQAAHHHHHH(SEQ ID NO:8)。

MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGAGGGGSGGGGSGGGGSQGTGSGAGIAQAAAGTVNIGASDAYLSEGDMAAHKGLMNIALAISAQQVNYNLPGVSEHLKLNGKVLAAMYQGTIKTWDDPQIAALNPGVNLPGTAVVPLHRSDGSGDTFLFTQYLSKQDPEGWGKSPGFGTTVDFPAVPGALGENGNGGMVTGCAETPGCVAYIGISFLDQASQRGLGEAQLGNSSGNFLLPDAQSIQAAHHHHHH(SEQ ID NO:9)。

Nucleic acid molecules, nucleic acid molecules and recombinant cells

In preparing or obtaining these fusion proteins, nucleic acid molecules expressing these fusion proteins may be used, linked to different vectors, and then expressed in different cells to obtain the corresponding fusion proteins.

In another aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the aforementioned fusion protein. The nucleic acid molecule can be effectively used for expressing the fusion protein, and particularly the fusion protein can be effectively expressed in a prokaryotic or lower eukaryote expression system.

According to an embodiment of the invention, the nucleic acid molecule is DNA.

The nucleotide sequence shown as SEQ ID NO. 10 is used for encoding SEQ ID NO. 7.

The nucleotide sequence shown as SEQ ID NO. 11 is used for encoding SEQ ID NO. 8.

The nucleotide sequence shown as SEQ ID NO. 12 is used for encoding SEQ ID NO. 9.

ATGCAGCTTGTTGACAGGGTTCGTGGCGCCGTCACGGGTATGTCGCGTCGACTCGTGGTCGGGGCCGTCGGCGCGGCCCTAGTGTCGGGTCTGGTCGGCGCCGTCGGTGGCACGGCGACCGCGGGGGCATTTTCCCGGCCGGGCTTGCCGGTGGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGTGACATCAAGGTCCAATTCCAAAGTGGTGGTGCCAACTCGCCCGCCCTGTACCTGCTCGACGGCCTGCGCGCGCAGGACGACTTCAGCGGCTGGGACATCAACACCCCGGCGTTCGAGTGGTACGACCAGTCGGGCCTGTCGGTGGTCATGCCGGTGGGTGGCCAGTCAAGCTTCTACTCCGACTGGTACCAGCCCGCCTGCGGCAAGGCCGGTTGCCAGACTTACAAGTGGGAGACCTTCCTGACCAGCGAGCTGCCGGGGTGGCTGCAGGCCAACAGGCACGTCAAGCCCACCGGAAGCGCCGTCGTCGGTCTTTCGATGGCTGCTTCTTCGGCGCTGACGCTGGCGATCTATCACCCCCAGCAGTTCGTCTACGCGGGAGCGATGTCGGGCCTGTTGGACCCCTCCCAGGCGATGGGTCCCACCCTGATCGGCCTGGCGATGGGTGACGCTGGCGGCTACAAGGCCTCCGACATGTGGGGCCCGAAGGAGGACCCGGCGTGGCAGCGCAACGACCCGCTGTTGAACGTCGGGAAGCTGATCGCCAACAACACCCGCGTCTGGGTGTACTGCGGCAACGGCAAGCCGTCGGATCTGGGTGGCAACAACCTGCCGGCCAAGTTCCTCGAGGGCTTCGTGCGGACCAGCAACATCAAGTTCCAAGACGCCTACAACGCCGGTGGCGGCCACAACGGCGTGTTCGACTTCCCGGACAGCGGTACGCACAGCTGGGAGTACTGGGGCGCGCAGCTCAACGCTATGAAGCCCGACCTGCAACGGGCACTGGGTGCCACGCCCAACACCGGGCCCGCGCCCCAGGGCGCCGGTGGAGGCGGTTCAGGTGGAGGCGGTTCAGGTGGAGGCGGTTCACAGGGCACCGGTTCTGGTGCCGGGATCGCGCAGGCCGCCGCCGGGACGGTCAACATTGGGGCCTCCGACGCCTATCTGTCGGAAGGTGATATGGCCGCGCACAAGGGGCTGATGAACATCGCGCTAGCCATCTCCGCTCAGCAGGTCAACTACAACCTGCCCGGAGTGAGCGAGCACCTCAAGCTGAACGGAAAAGTCCTGGCGGCCATGTACCAGGGCACCATCAAAACCTGGGACGACCCGCAGATCGCTCACCACCACCACCACCACTAG(SEQ ID NO:10)。

ATGCAGCTTGTTGACAGGGTTCGTGGCGCCGTCACGGGTATGTCGCGTCGACTCGTGGTCGGGGCCGTCGGCGCGGCCCTAGTGTCGGGTCTGGTCGGCGCCGTCGGTGGCACGGCGACCGCGGGGGCATTTTCCCGGCCGGGCTTGCCGGTGGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGTGACATCAAGGTCCAATTCCAAAGTGGTGGTGCCAACTCGCCCGCCCTGTACCTGCTCGACGGCCTGCGCGCGCAGGACGACTTCAGCGGCTGGGACATCAACACCCCGGCGTTCGAGTGGTACGACCAGTCGGGCCTGTCGGTGGTCATGCCGGTGGGTGGCCAGTCAAGCTTCTACTCCGACTGGTACCAGCCCGCCTGCGGCAAGGCCGGTTGCCAGACTTACAAGTGGGAGACCTTCCTGACCAGCGAGCTGCCGGGGTGGCTGCAGGCCAACAGGCACGTCAAGCCCACCGGAAGCGCCGTCGTCGGTCTTTCGATGGCTGCTTCTTCGGCGCTGACGCTGGCGATCTATCACCCCCAGCAGTTCGTCTACGCGGGAGCGATGTCGGGCCTGTTGGACCCCTCCCAGGCGATGGGTCCCACCCTGATCGGCCTGGCGATGGGTGACGCTGGCGGCTACAAGGCCTCCGACATGTGGGGCCCGAAGGAGGACCCGGCGTGGCAGCGCAACGACCCGCTGTTGAACGTCGGGAAGCTGATCGCCAACAACACCCGCGTCTGGGTGTACTGCGGCAACGGCAAGCCGTCGGATCTGGGTGGCAACAACCTGCCGGCCAAGTTCCTCGAGGGCTTCGTGCGGACCAGCAACATCAAGTTCCAAGACGCCTACAACGCCGGTGGCGGCCACAACGGCGTGTTCGACTTCCCGGACAGCGGTACGCACAGCTGGGAGTACTGGGGCGCGCAGCTCAACGCTATGAAGCCCGACCTGCAACGGGCACTGGGTGCCACGCCCAACACCGGGCCCGCGCCCCAGGGCGCCGGTGGAGGCGGTTCAGGTGGAGGCGGTTCAGGTGGAGGCGGTTCACACCGCTCCGACGGGTCCGGTGACACCTTCTTGTTCACCCAGTACCTGTCCAAGCAAGATCCCGAGGGCTGGGGCAAGTCGCCCGGCTTCGGCACCACCGTCGACTTCCCGGCGGTGCCGGGTGCGCTGGGTGAGAACGGCAACGGCGGCATGGTGACCGGTTGCGCCGAGACACCGGGCTGCGTGGCCTATATCGGCATCAGCTTCCTCGACCAGGCCAGTCAACGGGGACTCGGCGAGGCCCAACTAGGCAATAGCTCTGGCAATTTCTTGTTGCCCGACGCGCAAAGCATTCAGGCCGCGCACCACCACCACCACCACTAG(SEQ ID NO:11)。

ATGCAGCTTGTTGACAGGGTTCGTGGCGCCGTCACGGGTATGTCGCGTCGACTCGTGGTCGGGGCCGTCGGCGCGGCCCTAGTGTCGGGTCTGGTCGGCGCCGTCGGTGGCACGGCGACCGCGGGGGCATTTTCCCGGCCGGGCTTGCCGGTGGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGTGACATCAAGGTCCAATTCCAAAGTGGTGGTGCCAACTCGCCCGCCCTGTACCTGCTCGACGGCCTGCGCGCGCAGGACGACTTCAGCGGCTGGGACATCAACACCCCGGCGTTCGAGTGGTACGACCAGTCGGGCCTGTCGGTGGTCATGCCGGTGGGTGGCCAGTCAAGCTTCTACTCCGACTGGTACCAGCCCGCCTGCGGCAAGGCCGGTTGCCAGACTTACAAGTGGGAGACCTTCCTGACCAGCGAGCTGCCGGGGTGGCTGCAGGCCAACAGGCACGTCAAGCCCACCGGAAGCGCCGTCGTCGGTCTTTCGATGGCTGCTTCTTCGGCGCTGACGCTGGCGATCTATCACCCCCAGCAGTTCGTCTACGCGGGAGCGATGTCGGGCCTGTTGGACCCCTCCCAGGCGATGGGTCCCACCCTGATCGGCCTGGCGATGGGTGACGCTGGCGGCTACAAGGCCTCCGACATGTGGGGCCCGAAGGAGGACCCGGCGTGGCAGCGCAACGACCCGCTGTTGAACGTCGGGAAGCTGATCGCCAACAACACCCGCGTCTGGGTGTACTGCGGCAACGGCAAGCCGTCGGATCTGGGTGGCAACAACCTGCCGGCCAAGTTCCTCGAGGGCTTCGTGCGGACCAGCAACATCAAGTTCCAAGACGCCTACAACGCCGGTGGCGGCCACAACGGCGTGTTCGACTTCCCGGACAGCGGTACGCACAGCTGGGAGTACTGGGGCGCGCAGCTCAACGCTATGAAGCCCGACCTGCAACGGGCACTGGGTGCCACGCCCAACACCGGGCCCGCGCCCCAGGGCGCCGGTGGAGGCGGTTCAGGTGGAGGCGGTTCAGGTGGAGGCGGTTCACAGGGCACCGGTTCTGGTGCCGGGATCGCGCAGGCCGCCGCCGGGACGGTCAACATTGGGGCCTCCGACGCCTATCTGTCGGAAGGTGATATGGCCGCGCACAAGGGGCTGATGAACATCGCGCTAGCCATCTCCGCTCAGCAGGTCAACTACAACCTGCCCGGAGTGAGCGAGCACCTCAAGCTGAACGGAAAAGTCCTGGCGGCCATGTACCAGGGCACCATCAAAACCTGGGACGACCCGCAGATCGCTGCGCTCAACCCCGGCGTGAACCTGCCCGGCACCGCGGTAGTTCCGCTGCACCGCTCCGACGGGTCCGGTGACACCTTCTTGTTCACCCAGTACCTGTCCAAGCAAGATCCCGAGGGCTGGGGCAAGTCGCCCGGCTTCGGCACCACCGTCGACTTCCCGGCGGTGCCGGGTGCGCTGGGTGAGAACGGCAACGGCGGCATGGTGACCGGTTGCGCCGAGACACCGGGCTGCGTGGCCTATATCGGCATCAGCTTCCTCGACCAGGCCAGTCAACGGGGACTCGGCGAGGCCCAACTAGGCAATAGCTCTGGCAATTTCTTGTTGCCCGACGCGCAAAGCATTCAGGCCGCGCACCACCACCACCACCACTAG(SEQ ID NO:12)。

In yet another aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the aforementioned nucleic acid molecules are carried. The expression vector can be effectively used for expressing the fusion protein, and particularly can be effectively expressed in a prokaryotic or lower eukaryote expression system.

According to an embodiment of the invention, the expression vector is a eukaryotic expression vector.

According to an embodiment of the invention, the expression vector is a lentiviral vector.

In yet another aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises: carrying the nucleic acid molecule as described above or the expression vector as described above; alternatively, the fusion proteins described above are expressed. The recombinant cells of the embodiments described herein can be used for in vitro expression and bulk acquisition of the aforementioned fusion proteins.

According to an embodiment of the invention, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

According to an embodiment of the invention, the recombinant cells comprise eukaryotic cells or prokaryotic cells. Illustratively, the recombinant cell is a BL21 (DE 3) competent cell.

Those skilled in the art will appreciate that the features and advantages described above for fusion proteins are equally applicable to such nucleic acid molecules, expression vectors and recombinant cells and will not be described in detail herein.

Subunit vaccine

In yet another aspect of the invention, the invention provides a subunit vaccine. According to an embodiment of the invention, the subunit vaccine comprises: the fusion protein. The subunit vaccine of the embodiment of the invention can improve the capability of resisting mycobacterium tuberculosis of organisms and effectively prevent and treat tuberculosis. And, the immune effect of BCG can be effectively enhanced.

According to an embodiment of the invention, the subunit vaccine further comprises pharmaceutically acceptable excipients.

According to an embodiment of the invention, the adjuvant comprises an adjuvant.

According to an embodiment of the invention, the adjuvant comprises at least one selected from the group consisting of Poly IC adjuvant, aluminium adjuvant, freund's adjuvant, cpG ODN, DDA, MPLA, IC, AS01 and AS03, preferably aluminium adjuvant. The inventor finds that the immunogenicity of subunit vaccines can be improved by adopting the adjuvant through experiments.

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

According to an embodiment of the invention, the adjuvant is an aluminium hydroxide adjuvant.

According to an embodiment of the invention, the volume ratio of the fusion protein and the adjuvant is 3:1. thus, the immunogenicity of the subunit vaccine can be further improved.

Those skilled in the art will appreciate that the features and advantages described above for fusion proteins, nucleic acid molecules, expression vectors and recombinant cells are equally applicable to the subunit vaccine and will not be described in detail herein.

Use of the same

Those skilled in the art will appreciate that the features and advantages described above for fusion proteins, nucleic acid molecules, expression vectors, recombinant cells and subunit vaccines are equally applicable for this purpose and will not be described in detail herein.

Method

In yet another aspect of the invention, the invention provides a method of preventing and/or treating tuberculosis. According to an embodiment of the invention, a pharmaceutically acceptable amount of the fusion protein or subunit vaccine described previously is administered to a subject. The method according to the embodiment of the invention can improve the capability of the organism for resisting the mycobacterium tuberculosis and effectively prevent and treat tuberculosis.

According to an embodiment of the invention, the fusion protein or subunit vaccine is administered at a dose of 20 μg.

According to an embodiment of the invention, the fusion protein or subunit vaccine is administered 3 times at 1-2 week intervals.

According to an embodiment of the invention, the route of administration of the method comprises subcutaneous injection or inhalation immunization.

Those skilled in the art will appreciate that the features and advantages described above for subunit vaccines are equally applicable to this method and will not be described in detail here.

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The BCG of the present invention was obtained by screening by a conventional method, and the immune effect thereof is shown in fig. 5.

Example 1: preparation of fusion proteins

1. Amplification of Ag85A-PstS1 Gene

The inventor uses H37Rv as a template and P1 and P2 as upstream and downstream primers to carry out PCR amplification on Ag85A (the amino acid sequence is SEQ ID NO: 1) +connecting peptide (the amino acid sequence is SEQ ID NO: 5), namely the amplified fragment 1 (the nucleotide sequence is SEQ ID NO: 19). The inventors selected three truncated sequences of PstS1 (tnPstS 1), named as tnPstS1 (250-501), tnPstS1 (550-852) and tnPstS1 (250-852), abbreviated as Ps1, ps2 and Ps3, respectively; then, PCR amplification of the connecting peptide (the amino acid sequence is SEQ ID NO: 5) +Ps1 (the amino acid sequence is SEQ ID NO: 2) is carried out by taking H37Rv as a template and P3 and P5 as upstream and downstream primers, namely the amplified fragment 2 (the nucleotide sequence is SEQ ID NO: 20); PCR amplification of the connecting peptide (the amino acid sequence is SEQ ID NO: 5) +Ps2 (the amino acid sequence is SEQ ID NO: 3) is carried out by taking P4 and P6 as upstream and downstream primers, namely the amplified fragment 3 (the nucleotide sequence is SEQ ID NO: 21); PCR amplification of the connecting peptide (SEQ ID NO: 5) +Ps3 (SEQ ID NO: 4) was performed using P3 and P6 as the upstream and downstream primers, to obtain amplified fragment 4 (SEQ ID NO: 22). Wherein, the nucleotide sequences of the P1, P2, P3, P4, P5 and P6 primers are shown in Table 1; see table 2 for PCR reaction systems, PCR reaction procedure: 98 ℃ for 3min;98℃for 10s,55℃for 5s,72℃for 1min/kb for 30 cycles; and at 72℃for 5min.

According to the reaction system, firstly amplifying an amplified fragment 1-amplified fragment 4 containing three truncated fragments Ps1, ps2 and Ps3 of Ag85A and PstS1, then connecting one of the amplified fragment 1 containing Ag85A, the amplified fragment 2-amplified fragment 4 containing the truncated fragment of PstS1 and the nucleotide sequence of the HIS tag (the amino acid sequence is SEQ ID NO:6; the nucleotide sequence is SEQ ID NO: 23) by a method of overlapping, extending and splicing PCR (SOE-PCR), and respectively obtaining fused target genes Ag85A-tnPstS1 fragments by connecting the three truncated fragments Ps1, ps2 and Ps3 of Ag85A and PstS1, wherein the fragments are respectively named as AP1 (the amino acid sequence is SEQ ID NO:7, the nucleotide sequence is SEQ ID NO: 10), and AP2 (the amino acid sequence is SEQ ID NO:8, the nucleotide sequence is SEQ ID NO: 11) and the nucleotide sequence is AP3 (the amino acid sequence is SEQ ID NO:9 and the nucleotide sequence is SEQ ID NO: 12). Wherein, the reaction system of SOE-PCR is shown in Table 3 and Table 4, the reaction procedure is as follows: 98 ℃ for 3min;98℃for 10s,55℃for 5s,72℃for 1min/kb for 5 cycles. The above AP1, AP2 and AP3 products were then mixed with the reagents in table 4, respectively, reaction procedure: 98 ℃ for 3min;98℃for 10s,55℃for 5s,72℃for 1min/kb for 30 cycles; and at 72℃for 5min. Obtaining the target gene. After the completion of PCR, the AP1, AP2 and AP3 obtained in the above steps were subjected to 1% agarose gel electrophoresis, and the electrophoresis results are shown in FIG. 1. The results showed that the agarose gel electrophoresis band size was consistent with the theoretical size.

The high fidelity enzymes (PrimeSTAR HS DNA Polymerase) required in the amplification process of this example were all purchased from TaKaRa (Beijing) Inc.

Wherein the nucleotide sequence of amplified fragment 1 is:

ATGCAGCTTGTTGACAGGGTTCGTGGCGCCGTCACGGGTATGTCGCGTCGACTCGTGGTCGGGGCCGTCGGCGCGGCCCTAGTGTCGGGTCTGGTCGGCGCCGTCGGTGGCACGGCGACCGCGGGGGCATTTTCCCGGCCGGGCTTGCCGGTGGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGTGACATCAAGGTCCAATTCCAAAGTGGTGGTGCCAACTCGCCCGCCCTGTACCTGCTCGACGGCCTGCGCGCGCAGGACGACTTCAGCGGCTGGGACATCAACACCCCGGCGTTCGAGTGGTACGACCAGTCGGGCCTGTCGGTGGTCATGCCGGTGGGTGGCCAGTCAAGCTTCTACTCCGACTGGTACCAGCCCGCCTGCGGCAAGGCCGGTTGCCAGACTTACAAGTGGGAGACCTTCCTGACCAGCGAGCTGCCGGGGTGGCTGCAGGCCAACAGGCACGTCAAGCCCACCGGAAGCGCCGTCGTCGGTCTTTCGATGGCTGCTTCTTCGGCGCTGACGCTGGCGATCTATCACCCCCAGCAGTTCGTCTACGCGGGAGCGATGTCGGGCCTGTTGGACCCCTCCCAGGCGATGGGTCCCACCCTGATCGGCCTGGCGATGGGTGACGCTGGCGGCTACAAGGCCTCCGACATGTGGGGCCCGAAGGAGGACCCGGCGTGGCAGCGCAACGACCCGCTGTTGAACGTCGGGAAGCTGATCGCCAACAACACCCGCGTCTGGGTGTACTGCGGCAACGGCAAGCCGTCGGATCTGGGTGGCAACAACCTGCCGGCCAAGTTCCTCGAGGGCTTCGTGCGGACCAGCAACATCAAGTTCCAAGACGCCTACAACGCCGGTGGCGGCCACAACGGCGTGTTCGACTTCCCGGACAGCGGTACGCACAGCTGGGAGTACTGGGGCGCGCAGCTCAACGCTATGAAGCCCGACCTGCAACGGGCACTGGGTGCCACGCCCAACACCGGGCCCGCGCCCCAGGGCGCCGGTGGAGGCGGTTCAGGTGGAGGCGGTTCAGGTGGAGGCGGTTCA(SEQ ID NO:19)；

the nucleotide sequence of amplified fragment 2 is:

GGTGGAGGCGGTTCAGGTGGAGGCGGTTCAGGTGGAGGCGGTTCACAGGGCACCGGTTCTGGTGCCGGGATCGCGCAGGCCGCCGCCGGGACGGTCAACATTGGGGCCTCCGACGCCTATCTGTCGGAAGGTGATATGGCCGCGCACAAGGGGCTGATGAACATCGCGCTAGCCATCTCCGCTCAGCAGGTCAACTACAACCTGCCCGGAGTGAGCGAGCACCTCAAGCTGAACGGAAAAGTCCTGGCGGCCATGTACCAGGGCACCATCAAAACCTGGGACGACCCGCAGATCGCTCACCACCACCACCACCACATG(SEQ ID NO:20)；

the nucleotide sequence of amplified fragment 3 is:

GGTGGAGGCGGTTCAGGTGGAGGCGGTTCAGGTGGAGGCGGTTCACACCGCTCCGACGGGTCCGGTGACACCTTCTTGTTCACCCAGTACCTGTCCAAGCAAGATCCCGAGGGCTGGGGCAAGTCGCCCGGCTTCGGCACCACCGTCGACTTCCCGGCGGTGCCGGGTGCGCTGGGTGAGAACGGCAACGGCGGCATGGTGACCGGTTGCGCCGAGACACCGGGCTGCGTGGCCTATATCGGCATCAGCTTCCTCGACCAGGCCAGTCAACGGGGACTCGGCGAGGCCCAACTAGGCAATAGCTCTGGCAATTTCTTGTTGCCCGACGCGCAAAGCATTCAGGCCGCGCACCACCACCACCACCACATG(SEQ ID NO:21)；

the nucleotide sequence of amplified fragment 4 is:

GGTGGAGGCGGTTCAGGTGGAGGCGGTTCAGGTGGAGGCGGTTCACAGGGCACCGGTTCTGGTGCCGGGATCGCGCAGGCCGCCGCCGGGACGGTCAACATTGGGGCCTCCGACGCCTATCTGTCGGAAGGTGATATGGCCGCGCACAAGGGGCTGATGAACATCGCGCTAGCCATCTCCGCTCAGCAGGTCAACTACAACCTGCCCGGAGTGAGCGAGCACCTCAAGCTGAACGGAAAAGTCCTGGCGGCCATGTACCAGGGCACCATCAAAACCTGGGACGACCCGCAGATCGCTGCGCTCAACCCCGGCGTGAACCTGCCCGGCACCGCGGTAGTTCCGCTGCACCGCTCCGACGGGTCCGGTGACACCTTCTTGTTCACCCAGTACCTGTCCAAGCAAGATCCCGAGGGCTGGGGCAAGTCGCCCGGCTTCGGCACCACCGTCGACTTCCCGGCGGTGCCGGGTGCGCTGGGTGAGAACGGCAACGGCGGCATGGTGACCGGTTGCGCCGAGACACCGGGCTGCGTGGCCTATATCGGCATCAGCTTCCTCGACCAGGCCAGTCAACGGGGACTCGGCGAGGCCCAACTAGGCAATAGCTCTGGCAATTTCTTGTTGCCCGACGCGCAAAGCATTCAGGCCGCGCACCACCACCACCACCACATG(SEQ ID NO:22)；

the nucleotide sequence of the HIS tag is:

CACCACCACCACCACCAC(SEQ ID NO:23)。

table 1: nucleotide sequence of P1-P6 primer

/>

Table 2: PCR reaction system (50. Mu.L)

Reagent(s)	Dosage of
		5×PrimeSTAR Buffer(Mg ²⁺ Plus)	10μL
dNTP Mixture(2.5mM each)	4μL
		Primer
1	2μL
		Primer
2	2μL
		PrimeSTAR HS DNA Polymerase(2.5U/μl)	0.5μL
Templite (H37 Rv genome)	2μL
		ddH ₂ O	39.5μL

Table 3: SOE-PCR reaction system

Reagent(s)	Dosage of
		5×PrimeSTAR Buffer(Mg2+Plus)	10μL
dNTP Mixture(2.5mM each)	4μL
		Ag85A	2μL
Ps1/Ps2/Ps3	2μL
		ddH ₂ O	27.5μL

Table 4: amplification of AP1, AP2 and AP3 fragments

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

And (3) respectively carrying out double enzyme digestion on the amplified target genes (AP 1, AP2 or AP 3) and a vector pET-21a, connecting by using T4 DNA ligase, constructing a pET-21a-Ag85A-tnPstS1 vector (see specifically FIG. 2), introducing into DH5 alpha competent cells, screening and sequencing to obtain the correct recombinant vector. The method comprises the following specific steps:

the amplified target gene (AP 1, AP2 or AP 3) was subjected to enzyme fragment recovery using a rapid agarose gel DNA recovery kit (CWBIO, cat# CW 2302) according to the commercial instructions. The concentration was measured after recovery using TECAN.

pET-21a strain was cultured overnight at 37℃at 200rpm (1:1000 switch to 5mL LB medium containing 100. Mu.g/mL ampicillin). The bacterial liquid was centrifuged the next day to obtain bacterial cells, and plasmids were extracted for use according to the commodity instructions using a high purity plasmid miniprep kit (CWBIO, cat# CW 0500) and the concentration was measured using TECAN.

The target gene (AP 1, AP2 or AP 3) and the vector pET-21a were subjected to double digestion, and the enzyme used for double digestion was BamHI-HF (star enzyme) (NEB, cat#R3136V) and EcoRI-HF (star enzyme) (NEB, cat#R3101V), and the double digestion experiments were carried out using the instructions thereof, the amounts of each reagent added being as follows:

reagent(s)	Dosage of
		BamHI-HF	1μL
EcoRI-HF	1μL
		Target gene/pET-21 a vector	1000ng
CutSmart buffer	5μL
		ddH2O	Make up 50 mu L

After cleavage with double cleavage enzymes, the cleavage fragments were recovered using a rapid agarose gel DNA recovery kit (CWBIO, cat# CW 2302) according to the instructions of the commercial product. The concentration was measured after recovery using TECAN. The recovered fragments were ligated using T4 DNA ligase (NEB, cat#M0202V) with the following amounts of reagents:

reagent(s)	Dosage of
		AP1/AP2/AP3 after cleavage	1μL(70ng)
pET-21a vector after enzyme digestion	1μL(50ng)
		T4 DNA ligase	1μL
T4 DNA ligase reaction buffer	2μL
		ddH2O	15μL

After ligation, 5. Mu.L of the ligated fragment was introduced into DH 5. Alpha. Competent cells (CWBIO, cat# CW 0808) according to the commercial instructions and plated.

The next day, colonies in the plates were randomly picked, sequenced by Jin Weizhi company after colony PCR identification positive, and after successful sequencing, bacterial solutions were amplified, plasmids were extracted for later use according to the commodity instructions using a high purity plasmid miniprep kit (CWBIO, cat# CW 0500), and their concentrations were detected using TECAN.

3. Inducible expression and purification of AP1, AP2 and AP3

Plasmids of AP1, AP2 and AP3 which are correctly sequenced are respectively introduced into BL21 (DE 3) competent cells (CWBIO, CW 0809) according to the commodity instruction, and single colony is selected for culture and freezing. Resuscitating and transferring to LB liquid medium containing ampicillin (Amp, 100 μg/mL) at 37deg.C for shake culture until reaching OD ₆₀₀ At the time of 0.6-0.8, adding IPTG with the final concentration of 1mM for induction, and centrifuging after 4 hours of induction to obtain thalli. The cells were resuspended in PBS, sonicated, centrifuged to obtain a supernatant and pellet, and the pellet was completely dissolved in 8M urea at 4℃and the supernatant was centrifuged and purified using an affinity chromatography nickel column. Purified AP1, AP2 and AP3 were identified using SDS-PAGE coomassie blue staining and WB (Western Bloting), and the AP1, AP2 and AP3 proteins were found to be located in inclusion bodies. The SDS-PAGE coomassie staining detection result and the WB detection result are shown in FIG. 3, the SDS-PAGE coomassie staining detection result is a left graph of FIG. 3, and the WB detection result is a right graph of FIG. 3.

Example 2: preparation of subunit vaccine

The AP1, AP2 and AP3 proteins obtained after purification of example 1 and the aluminum salt adjuvant (Siemens Thermo Scientific) ^TM Cargo number 77161) in a volume ratio of 3:1 at a temperature of 4℃and at 500rpmMixing uniformly for 30min. Respectively named as subunit vaccine 1 (abbreviated as vaccine 1), subunit vaccine 2 (abbreviated as vaccine 2) and subunit vaccine 3 (abbreviated as vaccine 3).

Example 3: animal protection test

C57BL/6J mice (available from Si Bei Fu (Beijing) Biotechnology Co., ltd.) of female SPF of 6-8 weeks old were selected for the test, and the specific test flow is shown in FIG. 4. The test was divided into five groups in total, PBS group (three immunizations, 100. Mu.L of PBS per mouse), BCG group (primary immunization, 10 immunizations per mouse) ⁶ CFU BCG), AP1 (three immunizations, 20 μg vaccine 1 per mouse), AP2 (three immunizations, 20 μg vaccine 2 per mouse), AP3 (three immunizations, 20 μg vaccine 3 per mouse). The subunit vaccines, BCG or PBS of the five groups described above were all injected subcutaneously (s.c.), the initial immunization was performed at week 0, and three immunizations were performed at

weeks

0, 2 and 4, respectively. Cytokine and antibody titers are detected two weeks after immunization is completed, and after detection, bacterial attack is performed by using M.bovis C68004 with a bacterial attack dosage of 200CFU, and aerosol infection is adopted for bacterial attack. Four weeks later the mice were euthanized and the bacterial load and histopathological changes of the lungs and spleen were examined and the results are shown in fig. 5 and 6. As a result, it was found that the bacterial load of the lung and spleen of mice in the AP1 group, the AP2 group and the AP3 group was reduced as compared with the PBS group, and in particular, the inhibitory ability of the AP2 group and the AP3 group against m.bovis reached a level comparable to that of the BCG group. Thus, the results further demonstrate that vaccine 1, vaccine 2 and vaccine 3 are effective in preventing tuberculosis.

Example 4: antibody titer detection

Five groups of mice in example 3 were pooled in tail vein and frozen at-80℃until the next weekend after each immunization. Antibody titers were measured using M.bovis plates, which were autoclaved and then incubated with H ₂ O re-suspension, adjust OD ₆₀₀ To 1.0, 100. Mu.L of an M.bovis suspension with an OD of 1.0 was added to each well of a 96-well plate, and the mixture was dried in an oven at 60℃and fixed with ice-cold methanol for 2 hours. The plates were washed three times with PBS for five minutes each. Serum 1: the mixture is diluted by 1000 a and then,mu.L was added to a 96-well plate and incubated at 37℃for 1h. The plates were washed three times with PBS for five minutes each. HRP-labeled goat anti-mouse IgG, igG1, igG2c, igG3, igM, igA (Abcam) at 1:10000 diluted, 100. Mu.L added to 96-well plate and incubated at 37℃for 1h. The plates were washed three times with PBS for five minutes each. Adding 100 μL LTMB (Soxhaust, beijing) to each well for color development, and adding 50 μL 2M H to each well after 20min ₂ SO ₄ The color development was terminated. ELISA reader reading OD ₄₅₀ The results of the assay are shown in fig. 7, and the titers after the third immunization are shown in fig. 8, where if not indicated, this indicates a difference from the PBS group.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A fusion protein comprising:

a first peptide fragment comprising an Ag85A fragment;

a second peptide fragment comprising a PstS1 fragment, the first peptide fragment being linked to the second peptide fragment.

2. The fusion protein of claim 1, wherein the C-terminus of the first peptide is linked to the N-terminus of the second peptide; or alternatively

The C end of the second peptide segment is connected with the N end of the first peptide segment;

optionally, the Ag85A fragment is an Ag85A full-length peptide chain;

optionally, 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;

optionally, the PstS1 fragment comprises at least one selected from the group consisting of a PstS1 full-length peptide chain and a PstS1 epitope peptide;

optionally, the PstS1 fragment has an amino acid sequence as shown in any one of SEQ ID NOS.2-4 or an amino acid sequence having at least 95% similarity to any one of SEQ ID NOS.2-4.

3. The fusion protein of claim 1, further comprising a linker peptide;

optionally, the N-terminus of the linker peptide is linked to the C-terminus of the first peptide fragment, and the C-terminus of the linker peptide is linked to the N-terminus of the second peptide fragment; or alternatively

The N end of the connecting peptide is connected with the C end of the second peptide segment, and the C end of the connecting peptide is connected with the N end of the first peptide segment;

optionally, 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;

optionally, the linker peptide comprises the amino acid sequence shown in SEQ ID NO. 5;

optionally, the fusion protein further comprises a tag;

optionally, the C-terminus of the first peptide fragment is linked to the N-terminus of the second peptide fragment, the tag is linked to the C-terminus of the second peptide fragment or the tag is linked to the N-terminus of the first peptide fragment; or alternatively

The C end of the second peptide fragment is connected with the N end of the first peptide fragment, and the tag is connected with the C end of the first peptide fragment or the tag is connected with the N end of the second peptide fragment;

optionally, the tag is at least one of a HIS tag, FLAG tag, HA tag, GST tag, strep II tag, and MBP tag;

optionally, the HIS tag has an amino acid sequence as shown in SEQ ID NO. 6;

optionally, the fusion protein has an amino acid sequence as set forth in any one of SEQ ID NOs 7 to 9.

4. A nucleic acid molecule encoding the fusion protein of any one of claims 1-3;

optionally, the nucleic acid molecule is DNA.

5. An expression vector carrying the nucleic acid molecule of claim 4;

optionally, the expression vector is a eukaryotic expression vector;

preferably, the expression vector is a lentiviral vector.

6. A recombinant cell comprising:

carrying the nucleic acid molecule of claim 4 or the expression vector of claim 5; or alternatively, the first and second heat exchangers may be,

expressing the fusion protein of any one of claims 1-3;

optionally, the recombinant cell is obtained by introducing the expression vector of claim 5 into a host cell;

optionally, the recombinant cell comprises a eukaryotic cell or a prokaryotic cell.

7. Use of the fusion protein of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the expression vector of claim 5 or the recombinant cell of claim 6 in the preparation of a subunit vaccine.

8. A subunit vaccine comprising:

a fusion protein according to any one of claims 1 to 3.

9. The subunit vaccine of claim 1 further comprising a pharmaceutically acceptable adjuvant;

optionally, the adjuvant comprises an adjuvant;

optionally, 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 AS 03;

optionally, the volume ratio of the fusion protein to the adjuvant is 3:1;

optionally, the adjuvant is an aluminum hydroxide adjuvant.

10. Use of a subunit vaccine according to claim 8 or 9 in the manufacture of a medicament for boosting the immune effect of BCG or for preventing and/or treating tuberculosis.