A tuberculosis subunit vaccine and preparation method thereof

Abstract

The present invention provides a tuberculosis subunit vaccine, which comprises a fusion protein LT33, a fusion protein LT28 and an adjuvant; the fusion protein LT33 is prepared by sequentially connecting ESAT6, CFP10 and Rv1738; the fusion protein LT28 is prepared by sequentially connecting MPT64 61‑170 、Rv1978 8‑60 and Rv2645 21‑80 The invention also provides a preparation method thereof. The tuberculosis subunit vaccine of the invention can effectively induce tuberculosis antigen-specific cellular and humoral immune responses in mice, and at the same time, the vaccine can mediate the protection of mice against Mycobacterium tuberculosis H37Rv infection. The vaccine is expected to become a new type of tuberculosis subunit vaccine. It has good application development prospects.

Description

Technical Field

[0001] The invention relates to a tuberculosis subunit vaccine and a preparation method thereof. Background Art

[0002] Mycobacterium tuberculosis (MTB) is the pathogen of tuberculosis. The existing tuberculosis vaccine Bacillus Calmette-Guérin (BCG) can effectively prevent severe meningeal tuberculosis and systemic miliary tuberculosis in newborns and children, but it cannot effectively prevent the occurrence of pulmonary tuberculosis in adults. In order to completely eliminate tuberculosis worldwide, we urgently need to develop a more effective new tuberculosis vaccine.

[0003] At present, new tuberculosis vaccines are mainly divided into two categories. One is replacement vaccines, such as recombinant BCG, attenuated tuberculosis strains and non-pathogenic mycobacterium vaccines; the other is enhanced subunit vaccines, such as protein vaccines and DNA vaccines. Subunit vaccines are currently a hot topic in research, and several subunit vaccines have entered clinical trials, such as M72, H4 / IC31 and H1 / H56 / IC31, which have entered clinical phase II. However, the subunit candidate vaccines currently under clinical development are basically composed of antigens during the growth period of Mycobacterium tuberculosis, such as Ag85B, ESAT-6, TB10.4, etc., and there is a lack of tuberculosis subunit vaccines that induce immunity independent of BCG. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a tuberculosis subunit vaccine and a preparation method thereof.

[0005] BCG priming and subunit vaccine boosting regimens usually induce T EM Mediate short-term immune protection. The inventors of the present application found that subunit vaccines composed of missing region antigens and BCG antigens require different booster immunization schemes after BCG priming. The inventors of the present application used the fusion protein EC constructed by the missing region antigens ESAT-6 and CFP10, and the MH constructed by the BCG antigens Mtb10.4 and HspX. MH can induce long-lived memory T cell-mediated immunity when boosted twice after BCG priming, and EC can induce the same immune response when boosted three times after BCG priming. The Chinese population is generally vaccinated with BCG. In order to better enhance the immune protection effect of BCG in the population, the inventors of the present application screened BCG missing antigens and latent antigens and constructed a new subunit vaccine.

[0006] The tuberculosis subunit vaccine constructed and prepared by the inventors of the present application contains missing region antigens ESAT6, CFP10, MPT64, Rv1978 and Rv2645, and latent antigens Rv1738 and Rv1978. ESAT-6 is one of the virulence factors of tuberculosis secreted by MTB in the early growth stage. This gene is only present in pathogenic mycobacteria and is missing in BCG and non-pathogenic mycobacteria. ESAT-6 has multiple CD4 + T cell epitopes and CD8 + T cell epitope, which induces a strong Th1 immune response in the human body and produces specific cytotoxic T cells. CFP10 (culture filtrate protein-10) is one of the antigens secreted in the early stage of MTB growth. After infection with Mycobacterium tuberculosis, a large number of CFP10-specific T cells are recruited to the lungs to induce the body's immune response and protection. Rv1738 is a latent antigen that can induce a strong IFN-γ response in latent infection and has good immunogenicity. Rv1978 is upregulated during latent infection of the host and can induce tuberculosis-specific IFN-γ response in latently infected people. MPT64 is encoded by the gene Rv1980c, which can induce IFN-γ in mouse macrophages and tuberculosis patients, and reduce apoptosis by increasing the expression of TGF-β and reducing inflammation. After Rv2645 is complemented with the BCG strain, it can improve the antigen presentation ability of dendritic cells, trigger stronger Th1 and Th17 responses, and mediate the protection of mice and macaques against infection with the Mycobacterium tuberculosis H37Rv strain.

[0007] The present invention utilizes a genetic engineering method to combine the above proteins at the gene level to construct new fusion proteins LT33 and LT28. Fusion proteins LT33 and LT28 can be expressed in large quantities in a prokaryotic expression system, purified by ion exchange chromatography and gel filtration chromatography, and preparation research has been conducted. The fusion protein and adjuvant are combined to construct a new tuberculosis subunit vaccine, which is expected to become an effective candidate vaccine for tuberculosis.

[0008] The present invention provides a tuberculosis subunit vaccine, which comprises a fusion protein LT33, a fusion protein LT28 and an adjuvant; the fusion protein LT33 is prepared by sequentially connecting ESAT6, CFP10 and Rv1738; the fusion protein LT28 is prepared by sequentially connecting MPT64 61-170 、Rv1978 8-60 and Rv2645 21-80 Preferably, in the tuberculosis subunit vaccine, the weight ratio of fusion protein LT33 to fusion protein LT28 is 1:1.

[0009] Preferably, the tuberculosis subunit vaccine also includes fusion protein MH; the fusion protein MH is prepared by sequentially connecting Mtb10.4 and HspX; preferably, in the tuberculosis subunit vaccine, the weight ratio of fusion protein LT33, fusion protein LT28 and fusion protein MH is 1:1:1.

[0010] Preferably, the amino acid sequence of the fusion protein LT33 is SEQ ID No. 2 in the sequence listing; and / or

[0011] The amino acid sequence of the fusion protein LT28 is SEQ ID No. 4 in the sequence listing.

[0012] Preferably, the purification method of the fusion protein LT33 is as follows: first, the bacteria of the fusion protein LT33 are crushed and centrifuged, and the supernatant is collected, and then the supernatant is salted out using a saturated ammonium sulfate solution, and finally, an ion exchange chromatography column is used for purification, and impurities are eluted with a 0-15% high salt PB buffer, and the target protein is collected with a 17-23% high salt PB buffer, and the purified protein is obtained by electrophoresis; and / or

[0013] The purification method of the fusion protein LT28 is as follows: firstly, the bacteria of the fusion protein LT33 are crushed and centrifuged to collect the precipitate, then washed with a washing buffer and centrifuged to collect the precipitate; then, urea and Tris are used for dilution and renaturation, and finally, the target protein is purified by gel filtration chromatography.

[0014] Preferably, the adjuvant consists of PolyI:C and DDA; the weight concentration ratio of PolyI:C to DDA is 1:5; or

[0015] The adjuvant consists of PolyI:C, DDA and cholesterol; the weight concentration ratio of PolyI:C, DDA and cholesterol is 1:5:1.5.

[0016] The present invention also provides a method for preparing the above-mentioned tuberculosis subunit vaccine, comprising diluting the fusion protein and PolyI:C with PBS respectively; diluting the cationic liposome dimethyl tricetylammonium with PBS, placing it in a water bath at 80°C for 10 minutes, and cooling it to room temperature; dissolving cholesterol with anhydrous ethanol and evaporating it, and then diluting it with PBS; taking the PolyI:C solution and the fusion protein and mixing them thoroughly, adding the cholesterol solution and the DDA solution, and then fully emulsifying them to obtain the vaccine.

[0017] The present invention also provides the use of the tuberculosis subunit vaccine in preparing medicine for preventing tuberculosis.

[0018] The present invention also provides gene molecules encoding the above-mentioned fusion protein LT33 and fusion protein LT28;

[0019] Preferably, the gene sequence encoding the fusion protein LT33 is SEQ ID No. 1 in the sequence list;

[0020] Preferably, the gene sequence encoding the fusion protein LT28 is SEQ ID No. 3 in the sequence listing.

[0021] The present invention also provides an expression vector comprising the above gene; preferably, the expression vector is a prokaryotic expression vector; further preferably, the prokaryotic expression vector is pET30a.

[0022] The present invention also provides a host cell comprising the above-mentioned vector; preferably, the host cell is Escherichia coli BL21 (DE3).

[0023] The present invention uses genetic engineering technology to successfully construct, express and purify Mycobacterium tuberculosis fusion proteins LT33 and LT28 without tags. The fusion protein LT33 is effectively purified by a combination of salting out and ion chromatography; the fusion protein LT28 is effectively purified by inclusion body column renaturation. The fusion protein is stably expressed and can be prepared on a large scale. The fusion protein provided by the present invention is combined with the adjuvant DPC to form a tuberculosis subunit vaccine, which can effectively induce tuberculosis antigen-specific cellular and humoral immune responses in mice. At the same time, the vaccine can mediate the protection of mice against Mycobacterium tuberculosis H37Rv infection. The vaccine is expected to become a new type of tuberculosis subunit vaccine. It has good application development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0025] Figure 1 The figure shows the expression and purification results of the fusion protein LT33 in E. coli BL21 (DE3).

[0026] Figure 2 The figure shows the expression and purification results of the fusion protein LT28 in E. coli BL21 (DE3).

[0027] Figure 3 The immunogenicity of the fusion protein LT33 / DP.

[0028] Figure 4 Immunogenicity of fusion protein LT28 / DP

[0029] Figure 5 This is the serum antibody level after mice were immunized with the fusion protein LT33 / DP.

[0030] Figure 6 This is the immune protection effect after tail vein injection of the H37Ra attenuated strain.

[0031] Figure 7 This is the immune protection effect after aerosol attack of H37Rv strain. DETAILED DESCRIPTION

[0032] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0033] The present invention first needs to fuse the active phase antigens ESAT6 and CFP10 of Mycobacterium tuberculosis at the gene level. Considering the influence of the protein spatial structure, a linker (GGGGS) 3 sequence is added between ESAT6 and CFP10 to assist the folding and expression of the fusion protein and reduce the mutual influence of the spatial structure. The spatial structure of Rv1738 shows that its N segment has a partial flexible fragment, which can be used as a protein fusion linker, so there is no need to add a linker sequence between CFP10 and Rv1738. Based on this, the recombinant plasmid pET30a(+)-LT33 (ESAT6-linker-CFP10-Rv1738) is constructed.

[0034] Then, the pET30a(+)-LT33 plasmid vector was transformed into E. coli BL21(DE3) to express the fusion protein LT33. Finally, according to the protein characteristics of the protein, the fusion protein was successfully purified by combining salting out and ion chromatography. The specific steps are as follows:

[0035] 1. Construction of plasmid pET30a(+)-LT33 (ESAT6-linker-CFP10-Rv1738) by full gene synthesis

[0036] The single antigens ESAT6, CFP10, and Rv1738 were sequentially linked to the vector pET30a by full gene synthesis, with the 5' end restriction site being EcoRⅠ (CATATG) and the 3' end restriction site being HindⅢ (AAGCTT). At the same time, a linker sequence was added between the single antigens ESAT6 and CFP10 to promote protein folding and increase stability (GGTGGTGGTGGTTCT) 3, completing the construction of the recombinant gene vector pET30a(+)-LT33 (ESAT6-linker-CFP10-Rv1738). The following is the nucleotide sequence and amino acid sequence of the fusion protein LT33 (ESAT6-linker-CFP10-Rv1738):

[0037] The italic part is the restriction site. "The underlined part is the nucleic acid sequence of ESAT6;" " is the nucleic acid sequence of CFP10," " is the nucleic acid sequence of Rv1738; the nucleic acid sequence of linker (GGGGS) 3 is in bold:

[0038] CATATG ATGACAGAGCAGCAGTGGAATTTCGCGGGTATCGAGGCCGCGGCAAGCGCAATCCAGGGAAAT GTCACGTCCATTCATTCCCTCCTTGACGAGGGGAAGCAGTCCCTGACCAAGCTCGCAGCGGCCTGGGGCGGTAGCGG TTCGGAGGCGTACCAGGGTGTCCAGCAAAAATGGGACGCCACGGCTACCGAGCTGAACAACGCGCTGCAGAACCTGG CGCGGACGATCAGCGAAGCCGGTCAGGCAATGGCTTCGACCGAAGGCAACGTCACTGGGATGTTCGCA GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCT ATGGCAGAGATGAAGACCGATGCCGCTACCCTCGCGCAGGA GGCAGGTAATTTCGAGCGGATCTCCGGCGACCTGAAAACCCAGATCGACCAGGTGGAGTCGACGGCAGGTTCGTTGC AGGGCCAGTGGCGCGGCGCGGCGGGGACGGCCGCCCAGGCCGCGGTGGTGCGCTTCCAAGAAGCAGCCAATAAGCAG AAGCAGGAACTCGACGAGATCTCGACGAATATTCGTCAGGCCGGCGTCCAATACTCGAGGGCCGACGAGGAGCAGCA GCAGGCGCTGTCCTCGCAAATGGGCTTC GAG CTGTGCGGCGACCAGTCGGATCACGTGCTGCAGCACTGGACCGTCG ACATATCGATCGACGAACACGAAGGATTGACTCGGGCGAAGGCACGGCTGCGTTGGCGGGAAAAGGAATTGGTGGGT GTTGGCCTGGCAAGGCTCAATCCGGCCGACCGCAACGTCCCCGAGATCGGCGATGAACTCTCGGTCGCCCGAGCCTT GTCCGACTTGGGGAAGCGAATGTTGAAGGTGTCGACCCACGACATCGAAGCTGTTACCCATCAGCCGGCGCGATTGT TGTATTGA AAGCTT

[0039] The italic part is the restriction site. "The amino acid sequence of ESAT6 is underlined;" " is the amino acid sequence of CFP10," " is the amino acid sequence of Rv1738; the bold is the amino acid sequence of linker (GGGGS) 3:

[0040] HM MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELN NALQNLARTISEAGQAMASTEGNVTGMFA GGGGSGGGGSGGGGS MAEMKTDAATLAQEAGNFERISGDLKTQIDQV ESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF E LCGDQSD HVLQHWTVDISIDEHEGLTRAKARLRWREKELVGVGLARLNPADRNVPEIGDELSVARALSDLGKRMLKVSTHDIE AVTHQPARLLY * KL

[0041] 2. Expression and purification of fusion protein LT33

[0042] Expression of fusion protein LT33

[0043] The successfully constructed pET30a(+)-LT33 (ESAT6-linker-CFP10-Rv1738) vector was transformed into the expression vector Escherichia coli BL21 (DE3). Activate the strain of E. coli BL21 (DE3) expressing LT33, take 1 ml of the activated bacterial solution and add it to 200 ml of LB medium and culture it at 37℃ for 4 hours; add 100ul of protein inducer IPTG (1.0mmol / L) and induce shaking culture at 37℃ for 4 hours; collect the bacteria by centrifugation at 4℃ 10000G for 10 minutes; resuspend them in a ratio of 1g wet bacteria to 10ml PB buffer (Na2HPO4·12H2O 20mmol / L, NaH2PO4·2H2O 20mmol / L, pH7.4), and disrupt the bacteria by ultrasound for 0.5h in an ice bath (180-200W, ultrasound for 3s and stop for 3s); collect the supernatant and precipitate after centrifugation at 10000G for 15 minutes, and perform polyacrylamide gel electrophoresis on the supernatant and precipitate respectively; analyze them by SDS-PAGE electrophoresis, and the results showed that the supernatant and precipitate were different from those of E. coli Compared with BL21(DE3) empty bacteria, there is an obvious specific protein expression band with a molecular weight of about 33KD, which is mainly expressed in the supernatant, and there is relatively little protein in the precipitate. Figure 1 a】.

[0044] (2) Purification of fusion protein LT33

[0045] The fusion protein LT33 was expressed in large quantities. The collected bacteria were resuspended in 20 mM PB buffer, ultrasonically disrupted for about 0.5 h in an ice bath, centrifuged at 4°C / 10000G for 10 min, and the supernatant containing LT33 protein was collected after centrifugation; the supernatant was filtered with a 0.45 um filter. First, the fusion protein was salted out with a 25% saturated ammonium sulfate solution; then, the salted out protein (supernatant) was resuspended in PB buffer (Na2HPO4·12H2O 20mmol / L, NaH2PO4·2H2O 20mmol / L, pH7.4); subsequently, the collected fusion protein was subjected to ion exchange chromatography, and the ion chromatography material Q Sepharose High Performance (GE Healthcare) was used for purification, and the sample was loaded with low-salt PB buffer (Na2HPO4·12H2O 20mmol / L, NaH2PO4·2H2O20mmol / L, pH7.4), and the sample was washed with 0-15% high-salt PB buffer (Na2HPO4·12H2O 20mmol / L, NaH2PO4·2H2O20mmol / L, NaCl 1mol / L, pH7.4) to wash the impurities, 17-23% high salt PB buffer (Na2HPO4·12H2O20mmol / L, NaH2PO4·2H2O 20mmol / L, NaCl 1mol / L, pH7.4) to collect the sample containing the target protein, and perform polyacrylamide gel (SDS-PAGE) electrophoresis analysis to obtain the final purified sample. Figure 1 b, c].

[0046] Figure 1 The expression and purification results of fusion protein LT33 in E. coli BL21 (DE3). Among them, (a) the expression result of fusion protein LT33. M, marker; 1, BL21 empty bacteria; 2, whole bacterial liquid after bacterial cell crushing; 3, supernatant after bacterial cell crushing; 4, precipitate after bacterial cell crushing. (b) the salting-out purification result of fusion protein LT33. M, marker; 1, supernatant after bacterial cell crushing; 2, supernatant after LT33 salting-out and re-dissolution; 3, precipitate after LT33 salting-out and re-dissolution. (c) the ion exchange chromatography result of fusion protein LT33. M, marker; 1, sample (supernatant) after LT33 salting-out; 2-4, samples eluted with 17%-23% high salt PB buffer.

[0047] 1. Construction of plasmid pET30a(+)-LT28 by whole gene synthesis

[0048] The single antigen MPT64 was synthesized by whole gene synthesis 61-170 、Rv1978 8-60 and Rv2645 21-80The constructs were sequentially connected to the vector pET30a, with the 5' end restriction site being EcoRⅠ (CATATG) and the 3' end restriction site being HindⅢ (AAGCTT). At the same time, linker sequences were added between the single antigens MPT64 and Rv1978, and between Rv1978 and Rv2645 to promote protein folding and increase stability (GGTGGTGGTGGTTCT) 3, completing the construction of the recombinant gene vector pET30a(+)-LT28 (MPT64-linker-Rv1978-linker-Rv2645). The following is the nucleotide sequence and amino acid sequence of the fusion protein LT28 (MPT64-linker-Rv1978-linker-Rv2645):

[0049] The italic part is the restriction site. "The one marked out is MPT64 61-170 The nucleic acid sequence of "For Rv1978 8-60 The nucleic acid sequence of " for Rv2645 21-80 The nucleic acid sequence of linker (GGGGS) 3 is in bold:

[0050] CATATG CCCGACCAGAAGTCGCTGGAAAATTACATCGCCCAGACGCGCGACAAGTTCCTCAGCGCGGC CACATCGTCCACTCCACGCGAAGCCCCCTACGAATTGAATATCACCTCGGCCACATACCAGTCCGCGATACCGCCG CGTGGTACGCAGGCCGTGGTGCTCAAGGTCTACCAGAACGCCGGCGGCACGCACCCAACGACCACGTACAAGGCCT TCGATTGGGACCAGGCCTATCGCAAGCCAATCACCTATGACACGCTGTGGCAGGCTGACACCGATCCGCTGCCAGT CGTCTTCCCCATTGTGCAAGGTGAACTGAGCAAGCAGACC GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCT GAGCAGGCGATCGCCACGATGCCACGGGGTGGCCCCGACGCGTCTTGGCTGGATCGTCGATTCCAGA CCGACGCACTGGAGTACCTCGACCGCGACGATGTGCCCGATGAGGTCAAACAGAAGATCATCGGGGTGCTCGACCG GGTGGGCACCCTGACC GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCT GGCCGCTGCGCCTGC GGCCAGCAGCTCGCCGACGTCGGCCCGGCCACCCCGCCACCGCCCTGGTGCGAACCGGGCACCGAACCCATCTGGG AGCAGCTCACCGAACGATACGGCGGCGTCACAATCTGCCAGTGGACACGATATTTTCCGGCCGGCGACCCGGTGGC TGCCGACGTGTGGTGA AAGCTT

[0051] The italic part is the restriction site. "The one marked out is MPT64 61-170 The amino acid sequence of "For Rv1978 8-60 The amino acid sequence of " for Rv2645 21-80 The amino acid sequence of linker (GGGGS) 3 is in bold:

[0052] HM PDQKSLENYIAQTRDKFLSAATSSTPREAPYELNITSATYQSAIPPRGTQAVVLKVYQNAGGTHPT TTYKAFDWDQAYRKPITYDTLWQADTDPLPVVFPIVQGELSKQTGGGGSGGGGSGGGGS EQAIATMPRGGPDASWL DRRFQTDALEYLDRDDVPDEVKQKIIGVLDRVGTLT GGGGSGGGGSGGGGS GRCACGQQLADVGPATPPPPWCEPG TEPIWEQLTERYGGVTICQWTRYFPAGDPVAADVW * KL

[0053] 2. Expression and purification of fusion protein LT28

[0054] (1) Expression of fusion protein LT28

[0055] The successfully constructed pET30a(+)-LT28(MPT64-Rv1978-Rv2645) vector was transformed into the expression vector Escherichia coli BL21(DE3). Activate the strain of E. coli BL21 (DE3) expressing LT28, take 1 ml of the activated bacterial solution and add it to 200 ml of LB medium and culture it at 37℃ for 4 hours; add 100ul of protein inducer IPTG (1.0mmol / L) and induce shaking culture at 37℃ for 4 hours; collect the bacteria by centrifugation at 4℃ 10000G for 10 minutes; resuspend them in a ratio of 1g wet bacteria to 10ml PB buffer (Na2HPO4·12H2O 20mmol / L, NaH2PO4·2H2O 20mmol / L, pH7.4), and disrupt the bacteria by ultrasound for 0.5h in an ice bath (180-200W, ultrasound for 3s and stop for 3s); collect the supernatant and precipitate after centrifugation at 10000G for 15 minutes, and perform polyacrylamide gel electrophoresis on the supernatant and precipitate respectively; analyze them by SDS-PAGE electrophoresis, and the results are consistent with those of E. coli Compared with BL21(DE3) empty bacteria, there is an obvious specific protein expression band with a molecular weight of about 28KD, which is mainly expressed in the form of precipitation, and there is relatively little protein in the supernatant. Figure 2 a】.

[0056] (2) Purification of fusion protein LT28

[0057] The fusion protein LT28 was expressed in large quantities. The collected bacteria were resuspended in 20mM PB buffer, ultrasonically disrupted for about 0.5h in an ice bath, centrifuged at 4℃ / 10000G for 10min, and the precipitate containing LT28 protein was collected after centrifugation. First, the inclusion bodies were resuspended in washing buffer (NaCl, 20 mmol / L; EDTA, 20 mmol / L; Triton-100, 20 mmol / L; urea, 8 mol / L, pH7.4), centrifuged (10000rpm, 10min) to collect the precipitate, and treated three times. The sample collected by centrifugation was dissolved using 8M urea + 50mM Tris (pH8.0-8.5) at 4℃. The 8M urea solution containing the target protein was diluted to 7M urea using 50mM Tris (pH8.0-8.5) and treated at 4℃ for 4h. Use 50mM Tris (pH8.0-8.5) to dilute the 7M urea solution containing the target protein to 6M urea and treat at 4℃ for 4h. Use 50mM Tris (pH8.0-8.5) to dilute the 6M urea solution containing the target protein to 4M urea and treat at 4℃ for 4h. Use 50mM Tris (pH8.0-8.5) to dilute the 4M urea solution containing the target protein to 3M urea and treat at 4℃ for 12h. The treated sample was further purified by HiLoad 16 / 600 Superdex 75 pg gel filtration chromatography (molecular sieve) to obtain the fusion protein LT28 with a purity of 95% [ Figure 2 b】.

[0058] Figure 2 The expression and purification results of fusion protein LT28 in E. coli BL21 (DE3). (a) Expression results of fusion protein LT28. M, marker; 1, whole bacterial liquid after bacterial cell crushing; 2, supernatant after bacterial cell crushing; 3, precipitate after bacterial cell crushing. (b) Purification results of fusion protein LT28. M, marker; 1, sample after molecular sieve purification of fusion protein LT28.

[0059] The fusion proteins LT33 and LT28 were diluted to 0.2 mg / ml with PBS buffer respectively; PolyI:C was dissolved to 1 mg / ml with PBS buffer; the cationic liposome dimo-thylidioctyl ammoniumbromide (DDA) was prepared to 5 mg / ml with PBS, incubated in an 80°C water bath for 10 min (the phase transition temperature of DDA is 80°C, and it can only be dissolved after the phase transition), and cooled to room temperature; cholesterol was dissolved in anhydrous ethanol and then the anhydrous ethanol was evaporated and prepared to 1.5 mg / ml with PBS.

[0060] Take 50μl of PolyI:C solution and mix it with an equal volume of protein solution, and place it at room temperature for 1 minute. Add 50μL of cholesterol solution and 50μL of DDA solution dropwise to the mixed solution, and then fully emulsify it to make the vaccine uniform emulsion to obtain subunit vaccines LT33-DDA / PolyI:C / Cholesterol (LT33 / DPC), LT28-DDA / PolyI:C / Cholesterol (LT28 / DPC) and LT (33+28)-DDA / PolyI:C / Cholesterol ((LT33+LT28) / DPC)) (wherein the weight ratio of fusion protein LT33 and LT28 is 1:1). When the vaccine is used for immunization, the dosage is 200μL / mouse.

[0061] 1. Implementation materials: fusion protein LT33, fusion protein LT28, subunit vaccine MPT64-DDA / PolyI:C (abbreviated as "MPT64 / DP"); subunit vaccine Rv1978-DDA / PolyI:C (abbreviated as "Rv1978 / DP"); subunit vaccine LT33-DDA / PolyI:C (abbreviated as "LT33 / DP"); subunit vaccine LT33-DDA / PolyI:C / Cholesterol (abbreviated as "LT33 / DPC"); subunit vaccine LT28-DDA / PolyI:C (abbreviated as "LT28 / DP"); subunit vaccine LT28-DDA / PolyI:C / Cholesterol (abbreviated as "LT28 / DPC"); subunit vaccine LT (33+28)-DDA / PolyI:C / Cholesterol (abbreviated as "(LT33+LT28) / DPC"); Bacillus Calmette-Guérin (BCG); phosphate buffered saline (PBS).

[0062] 2. Experimental animals: C57BL / 6 Mouse.

[0063] 3. Immune index determination method

[0064] (1) Intracellular factor staining was used to detect the secretion levels of IFN-γ and IL-2 by mouse splenic T lymphocytes.

[0065] Splenic lymphocytes were isolated 6-8 weeks after the last immunization of mice, stimulated in vitro with the corresponding single antigen (5ug / ml) for 4h, and 1ul of protein transport inhibitor (Brefeldin A (BFA, Brefeldin A) was added at a concentration of 2μL / ml). After incubation for 16h, CD4 + T cells and CD8 + IFN-γ and IL-2 secretion levels of T cells Figure 3, 4 】.

[0066] Figure 3 The immunogenicity of the fusion protein LT33 / DP.

[0067] Figure 3 In the experiment, the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT33 / DP group was immunized three times at weeks 0, 2, and 4 in C57BL / 6 mice. Six weeks after the last immunization, the mouse spleen lymphocytes were isolated and incubated with antigens ESAT6, CFP10, and Rv1738 (5 μg / mL) for 20 h, respectively, and the expression levels of IFN-γ and IL-2 were detected by flow cytometry. + Statistical analysis of IFN-γ production by T cells; (B) CD8 + Statistical analysis of IFN-γ production by T cells; (C) CD4 + Statistical analysis of IL-2 production by T cells; (D) CD8 + Statistical analysis of IL-2 production by T cells. N ≥ 3. a, compared with the PBS group, p <0.05; b, compared with the BCG group, p <0.05; c, compared with the LT33 group, p <0.05.

[0068] Depend on Figure 3 It can be seen that the fusion protein LT33 / DP group can induce the production of multifunctional T cells after being stimulated by single antigens ESAT6, CFP10 and Rv1738, indicating that it has good immunogenicity.

[0069] Figure 4 Immunogenicity of fusion protein LT28 / DP

[0070] Figure 4 In the study, the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT28 / DP group was immunized three times at weeks 0, 4, and 12 in C57BL / 6 mice. Eight weeks after the last immunization, the mouse spleen lymphocytes were isolated and incubated with antigens MPT64, Rv1978, and Rv2645-P peptides (5 μg / mL) for 20 h, and the expression levels of IFN-γ and IL-2 were detected by flow cytometry. N ≥ 3. a, Compared with the BCG group, p <0.05; b, compared with the BCG group, p <0.05; c, compared with the BCG group, p <0.05.

[0071] Depend on Figure 4It can be seen that the antigen-specific T cells induced by mice in the single antigen immunization group were less than those in the fusion protein group, indicating that MPT64 61-170 、Rv1978 8-60 and Rv2645 21-80 The composed fusion protein has good immunogenicity and the single antigen epitope is well displayed.

[0072] (2) The ELISA method was used to detect the levels of mouse serum specific antibodies IgG, IgG1 and IgG2c.

[0073] Serum was collected 6 weeks after the last immunization of mice. 96-well plates were coated with the corresponding single antigens (5ug / ml) at 4°C overnight; the plates were washed 5 times with 300u1 / we11 of PBST solution; the plates were blocked with 5% skimmed milk powder and washed. 100u1 of serum samples diluted in multiples were added and placed at 37°C for 1h. After washing the plates, 100u1 / we11 of 1:5000 diluted goat anti-mouse IgG was added and placed at 37°C for 1h. After washing the plates, 100u1 / we11 of TMB colorimetric solution was added and reacted at room temperature in the dark for 15 minutes. After color development, 50u1 / we11 of stop solution (2M H2SO4) was added to stop the reaction; the OD value was detected at 450nm. The antibody levels of IgG1 and IgG2c were detected in the same way. Figure 5 and Table 1].

[0074] Table 1 Mouse serum specific antibody titer

[0075]

[0076] Figure 5 This is the serum antibody level after mice were immunized with the fusion protein LT33 / DP.

[0077] Figure 5 In the experiment, the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT33 / DP group was immunized three times at weeks 0, 2, and 4. Six weeks after the last immunization, the mouse sera were collected to detect the titers of IgG, IgG1, and IgG2c antibodies specific to ESAT6, CFP10, and Rv1738 antigens.

[0078] Depend on Figure 5 It can be seen that the individual antigens ESAT6, CFP10 and Rv1738 epitopes constituting the fusion protein LT33 are well displayed and can induce a good antibody response.

[0079] (3) Bacterial load index determination method

[0080] Twelve weeks after the last immunization, the experimental mice were infected with H37Rv and H37Ra strains, respectively. H37Ra strain was injected into the tail vein, and H37Rv strain was infected by aerosol. Afterwards, the mice were killed, and the lungs and spleens were taken, ground and smeared on plates to count CFU for tuberculosis load detection. Figure 6 , 7 】.

[0081] Figure 6 This is the immune protection effect after tail vein injection of the H37Ra attenuated strain.

[0082] Figure 6 In the experiment, the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine group was immunized three times at weeks 0, 4, and 12. Twelve weeks after the last immunization, the mice were infected with the H37Ra strain via tail vein injection, with an infection dose of 5×10 6 CFU, the bacterial load of the lungs and spleens of mice in each experimental group was detected 3 weeks after infection. In the figure, (A) bacterial load of the lungs of mice in the experimental group; (B) bacterial load of the spleens of mice in the experimental group. Each group of mice n = 3-6, * p <0.05,** p <0.01 and *** p <0.001.

[0083] Depend on Figure 6 It can be seen that BCG has a good immune protection effect. The immune protection effect of single fusion proteins LT33 and LT28 is not good, and the protection effect is improved after combining them, but its protection effect against high-dose H37Ra strain blood infection is still weaker than that of the BCG group.

[0084] Figure 7 This is the immune protection effect after aerosol attack of H37Rv strain.

[0085] Figure 7 In the experiment, the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine group was immunized three times at weeks 0, 4, and 12 in C57BL / 6 mice. 12 weeks after the last immunization, the mice were infected with the H37Rv strain by aerosol, and the infection dose was 100-1000 CFU / mouse. The bacterial load in the lungs and spleens of mice in each experimental group was detected 6 weeks after infection. In the experiment, all mice in the PBS group died during the attack of the H37Rv strain, so they were not included in the statistics. No deaths were observed in mice in the vaccine group and the adjuvant group during the experiment. In the figure, (A) bacterial load in the lungs of mice in the experimental group; (B) bacterial load in the spleens of mice in the experimental group. n=3-6 mice in each group, * p <0.05,** p <0.01 and *** p <0.001.

[0086] Depend on Figure 7 It can be seen that the fusion proteins LT33 and LT28, which are mainly based on the missing region antigen, can significantly prolong the survival time of mice after aerosol attack of Mycobacterium tuberculosis strain H37Rv with the aid of adjuvant DDA+PolyI:C+cholesterol (DPC); compared with the adjuvant group DPC, the (LT33+LT28) / DPC group can reduce the H37Rv bacterial load in the lungs of mice by about 0.77 log10CFU (P <0.05). The recombinant subunit vaccine (LT33+LT28) / DPC of the present invention has good immunogenicity and protective effect, and can be used for the prevention of Mycobacterium tuberculosis infection.

[0087] 1. Materials: subunit vaccine (LT33+LT28)-DDA / PolyI:C / Cholesterol (abbreviated as “(LT33+LT28) / DPC”); subunit vaccine (LT33+LT28+MH)-DDA / PolyI:C / Cholesterol (abbreviated as “(LT33+LT28+MH) / DPC”); Bacillus Calmette-Guérin (BCG); adjuvant control group DDA / PolyI:C / Cholesterol (abbreviated as “DPC”); phosphate buffered saline (PBS).

[0088] Among them, MH is the fusion protein Mtb10.4-Hspx in the invention patent “CN201010613320.6, Construction, expression and purification method of Mycobacterium tuberculosis fusion protein Mtb10.4-Hsp16.3 and its application”.

[0089] LT29 is the fusion protein in the patent application "CN202110155163.7, Construction of Mycobacterium tuberculosis fusion protein LT29, expression and purification method and application thereof", and its gene sequence and amino acid sequence can be found in paragraphs 94-100 of the patent specification.

[0090] The preparation method of the subunit vaccine (LT29+LT28)-DDA / PolyI:C / Cholesterol is as follows: the two fusion proteins are used in a mass ratio of 1:1, and the subunit vaccine is prepared according to the method of Example 3; each mouse is immunized each time with 200 μL / mouse, and the total dose of the fusion protein is 10 μg.

[0091] The preparation method of the subunit vaccine (LT33+LT28+MH)-DDA / PolyI:C / Cholesterol is as follows: the mass ratio of the three fusion proteins is 1:1:1, and the subunit vaccine is prepared according to the method of Example 3; 200 µL / mouse is used for each immunization, and the total dose of the fusion protein is 10 μg.

[0092] The preparation method of the adjuvant control group DDA / PolyI:C / Cholesterol is as follows: according to the method of Example 3, only three adjuvants are used without adding fusion protein to prepare the adjuvant control group DPC.

[0093] 2. Experimental animals: C57BL / 6 mice.

[0094] 3. Immunization Methods

[0095] The BCG group was immunized once at week 0, and the PBS group, adjuvant group, and vaccine group were immunized three times according to the 0-4-12 week strategy.

[0096] 4. Immunity index determination method (bacterial load index determination method)

[0097] Twelve weeks after the last immunization, all experimental mice were infected with the H37Rv strain by aerosol infection, with an infection dose of 100-1000 CFU / mouse. 42 days after infection, the mice were killed, and the lungs and spleens were removed, ground and smeared on plates to count CFU for tuberculosis load detection. Figure 6 , 7 】.

[0098] Depend on Figure 6 It can be seen that the immune protection effect of adding MH to LT33 and LT28 is equivalent to that of the LT33 and LT28 combination group. The results show that LT33+LT28 and LT33+LT28+MH are both good fusion protein combinations and have the potential to become tuberculosis subunit vaccines.

[0099] Depend on Figure 7 It can be seen that LT33+LT28 combined with MH can further enhance the immune protection effect. The H37Rv bacterial load in the lungs of mice in the (LT33+LT28+MH) / DPC group was 1.09 log10 CFU lower than that in the adjuvant group (P < 0.001), and was 0.65 log10 CFU lower than that in the BCG group (P < 0.05).

[0100] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A tuberculosis subunit vaccine, characterized in that: The tuberculosis subunit vaccine comprises fusion protein LT33, fusion protein LT28 and an adjuvant; the adjuvant consists of PolyI:C, DDA and cholesterol; The amino acid sequence of the fusion protein LT33 is: HM MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAGGGGSGGGGSGGGGSMAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF E LCGDQSDHVLQHWTVDISIDEHEGLTRAKARLRWREKELVGVGLARLNPADRNVPEIGDELSVARALSDLGKRMLKVSTHDIEAVTHQPARLLY* KL ; The amino acid sequence of the fusion protein LT28 is: HM PDQKSLENYIAQTRDKFLSAATSSTPREAPYELNITSATYQSAIPPRGTQAVVLKVYQNAGGTHPTTTYKAFDWDQAYRKPITYDTLWQADTDPLPVVFPIVQGELSKQTGGGGSGGGGSGGGGSEQAIATMPRGGPDASWLDRRFQTDALEYLDRDDVPDEVKQKIIGVLDRVGTLTGGGGSGGGGSGGGGSGRCACGQQLADVGPATPPPPWCEPGTEPIWEQLTERYGGVTICQWTRYFPAGDPVAADVW* KL 。 2. The tuberculosis subunit vaccine according to claim 1, characterized in that: In the tuberculosis subunit vaccine, the mass ratio of fusion protein LT33 to fusion protein LT28 is 1:

1.

3. The tuberculosis subunit vaccine according to claim 1 or 2, characterized in that: The tuberculosis subunit vaccine also includes a fusion protein MH; the fusion protein MH is prepared by sequentially connecting the BCG antigen Mtb10.4 and HspX.

4. The tuberculosis subunit vaccine according to claim 3, characterized in that: In the tuberculosis subunit vaccine, the mass ratio of fusion protein LT33, fusion protein LT28 and fusion protein MH is 1:1:

1.

5. The tuberculosis subunit vaccine according to claim 1 or 2, characterized in that: The weight concentration ratio of PolyI:C, DDA and cholesterol is 1:5:1.

5.

6. The method for preparing the tuberculosis subunit vaccine according to any one of claims 1 to 5, characterized in that: The fusion protein and PolyI:C were diluted with PBS respectively; the cationic liposome dimethyl tricetylammonium was diluted with PBS, placed in a water bath at 80°C for 10 minutes, and cooled to room temperature; cholesterol was dissolved with anhydrous ethanol and evaporated, and then diluted with PBS; the PolyI:C solution and the fusion protein were fully mixed, and the cholesterol solution and DDA solution were added, and then fully emulsified to obtain the product.

7. Use of the tuberculosis subunit vaccine according to any one of claims 1 to 5 in the preparation of a drug for preventing tuberculosis.

8. A gene molecule combination encoding the fusion protein LT33 and the fusion protein LT28 in the tuberculosis subunit vaccine according to claim 1, characterized in that: The gene sequence encoding the fusion protein LT33 is SEQ ID No. 1 in the sequence list; The gene sequence encoding the fusion protein LT28 is SEQ ID No. 3 in the sequence list.

9. An expression vector comprising the gene molecule combination according to claim 8.

10. The expression vector according to claim 9, characterized in that: The expression vector is a prokaryotic expression vector.

11. The expression vector according to claim 10, characterized in that: The prokaryotic expression vector is pET30a.

12. A host cell comprising the vector according to any one of claims 9 to 11.

13. The host cell according to claim 12, characterized in that: The host cell is E. coli BL21 (DE3).

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