Method for constructing, expressing and purifying mycobacterium tuberculosis fusion protein LT57 and application of mycobacterium tuberculosis fusion protein LT57

Abstract

The invention belongs to the technical field of medicines, and discloses a mycobacterium tuberculosis fusion protein LT57 construction and expression purification method, a mycobacterium tuberculosis cell wall antigen Rv0518 and mycobacterium tuberculosis PE / PPE family antigens PE25 and PPE41 are fused, and the fusion protein PE25-linker-PPE41-Rv0518 is constructed by connecting the fusion protein Rv0518 and the fusion protein PPE41 through flexible peptide fragments. The fusion protein is expressed in a prokaryotic expression vector E.coli BL21 (DE3), and is obtained through column chromatography purification. The fusion protein LT57 and an adjuvant DP are combined to construct a tuberculosis subunit vaccine, a relatively strong specific cellular immune response aiming at a specific antigen of mycobacterium tuberculosis can be induced in a mouse body, the long-term generation of memory T cells is promoted, and the tuberculosis subunit vaccine has relatively strong immunogenicity. The novel fusion protein LT57 is successfully constructed, expressed and purified, and antigen-specific cellular immunity and humoral immunity can be induced.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a construction, expression and purification method of a Mycobacterium tuberculosis fusion protein LT57 and an application thereof. Background Art

[0002] Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis (MTB) and remains one of the world's major deadly infectious diseases. According to the latest data from the World Health Organization (WHO), there will be approximately 10.6 million new cases of tuberculosis worldwide in 2022, of which approximately 1.3 million people will lose their lives due to tuberculosis. Timely and accurate diagnosis and treatment are essential to curb the spread of tuberculosis. In order to achieve the goal of further reducing the incidence of tuberculosis by 2030 and 2035, we need to develop shorter-course and less toxic treatments, improve diagnostic techniques, and develop more effective vaccines. Vaccination with tuberculosis vaccines is not only the most cost-effective means of controlling the disease, but also can fundamentally prevent the outbreak of tuberculosis, and is a key link in achieving the global strategy to eliminate the burden of tuberculosis.

[0003] Bacillus Calmette-Guérin (BCG) is the only vaccine approved to prevent tuberculosis so far. BCG vaccine is mainly effective in preventing tuberculous meningitis and miliary tuberculosis in children, while its protective efficacy in adults is relatively weak. At present, new tuberculosis vaccines under development mainly include whole-cell live attenuated vaccines, whole-cell inactivated vaccines, viral vector vaccines and subunit vaccines.

[0004] Subunit vaccines are currently a hot topic of research. They are composed of immunologically active components such as proteins, peptides, glycolipids, and adjuvants of MTB. Compared with live vaccines, they have the advantages of clear ingredients, low cost, high yield, good safety, easy preparation, and good quality control. The adjuvant in the tuberculosis subunit vaccine can promote the slow release of antigens, help induce the formation of central memory T cells (TCM), and provide long-term protection for the body. In addition, after BCG priming, the regimen of subunit vaccine boosting can induce memory T cells (TM)-mediated immune protection. Our study found that MH constructed with BCG antigens Mtb10.4 and HspX can induce long-lived memory T cell-mediated immunity after two boosters after BCG priming. The Chinese population is generally vaccinated with BCG, and booster immunization of subunit vaccines has huge market potential.

[0005] Currently, a variety of subunit vaccines have entered the clinical trial stage, including M72 / AS01E, Gam TBvac, H56: IC31 (AERAS-456), H4: IC31 (AERAS-404), ID93+GLA-SE and AEC / BC02. Most of these vaccine antigens are concentrated in the MTB cell wall, secreted antigens, etc., and less attention is paid to the PE / PPE family antigens of MTB. Therefore, in order to improve the immune effect and protection range of subunit vaccines, we screened the PE / PPE family antigens of MTB and constructed the fusion protein PE25-PPE41-Rv0518 (molecular weight is about 57KDa, abbreviated as LT57) in combination with the MTB cell wall antigen.

[0006] The PE / PPE protein family is mainly found in pathogenic mycobacteria, with significant sequence homology and specific peptide epitopes that can strongly activate T cell responses. Some PE / PPE proteins are located on the MTB cell wall and play a key role in MTB adhesion and invasion of host macrophages and regulation of immune responses to host cells. These characteristics make the PE / PPE protein family have great potential in vaccine development. PE25 and PPE41 are paired secreted PE / PPE family antigens that can induce activation and maturation of dendritic cells in in vitro cell models. In mouse models, they can induce significant B cell responses and promote the secretion of cytokines such as IFN-γ, TNF-α and IL-2 by mouse lymphocytes.

[0007] The cell wall of MTB has complex antigens, which contain a large number of polysaccharides, glycolipids and proteins. These antigens play a vital role in the process of MTB invading host cells and are of great significance in inducing humoral and cellular immune responses. Screening out advantageous MTB cell wall antigens can lay a solid foundation for the construction of an effective tuberculosis vaccine. Rv0518 is a MTB cell wall-related GDSL lipase that helps bacteria utilize glycerol / lipids for growth and regulates resistance to various intracellular stresses through the cell wall.

[0008] The present invention utilizes genetic engineering methods to join PE / PPE family antigens PE25, PPE41 and cell wall antigen Rv0518 at the gene level to construct a new fusion protein LT57. The fusion protein and adjuvant are combined to construct a new tuberculosis subunit vaccine, which is expected to become an effective tuberculosis candidate vaccine.

[0009] Through the above analysis, the problems and defects of the prior art are as follows:

[0010] BCG vaccine mainly has a preventive effect on tuberculous meningitis and miliary tuberculosis in children, but its protective efficacy in adults is relatively weak, and new tuberculosis vaccines need to be developed.

[0011] The various subunit vaccine antigens that have entered the clinical trial stage are mostly concentrated on the MTB cell wall, secretory expressed antigens, etc., and less attention is paid to the PE / PPE family antigens of MTB. Summary of the invention

[0012] In view of the problems existing in the prior art, the present invention provides a method for constructing a Mycobacterium tuberculosis fusion protein LT57 and its expression and purification as well as its application.

[0013] The present invention is achieved by: a method for obtaining Mycobacterium tuberculosis fusion protein LT57:

[0014] Genes PE25, PPE41 and Rv0518 were recombined into a prokaryotic expression vector through genetic engineering technology to obtain the fusion protein LT57.

[0015] Further, the expression plasmid is pET30a, and the plasmid for recombinant expression of fusion protein is:

[0016] pET30a(+)-LT57(PE25-linker-PPE41-Rv0518).

[0017] Furthermore, a method for constructing the Mycobacterium tuberculosis fusion protein LT57 and expressing and purifying the same is provided:

[0018] Step 1: The fusion protein LT57 is expressed in both the supernatant and the inclusion body, but mainly in the inclusion body. First, the fusion protein expressed in the inclusion body is denatured and dissolved with urea, and then preliminarily purified using a molecular sieve column refolding method;

[0019] Step 2: After the initial purification by molecular sieve, the sample containing the target protein is further purified using an ion exchange chromatography QHP column, the impurities are eluted with a 0-40Ms / cm high-salt PB buffer, and the target protein is collected with a 40-48Ms / cm high-salt PB buffer.

[0020] Further, the use of the fusion protein: LT57 alone.

[0021] Furthermore, the fusion protein and adjuvant together constitute the application of tuberculosis subunit vaccine: DP adjuvant is used.

[0022] Furthermore, recombinant protein vaccines are used in anti-tuberculosis prevention.

[0023] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0024] First, the present invention provides a method for constructing, preparing and using a novel tuberculosis subunit vaccine fusion protein LT57. The tuberculosis subunit vaccine is formed by combining the fusion protein provided by the present invention with the adjuvant DP, 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 infection with the attenuated strain H37Ra. The vaccine is expected to become a new type of tuberculosis subunit vaccine.

[0025] The present invention utilizes a genetic engineering method to join PE / PPE family antigens PE25, PPE41 and cell wall antigen Rv0518 at the gene level, successfully constructs, expresses and purifies the untagged Mycobacterium tuberculosis fusion protein LT57, and effectively purifies the fusion protein LT57 by combining molecular sieve column renaturation and ion chromatography. The fusion protein is stably expressed, and the purification method can be prepared and amplified on a large scale. The fusion protein and adjuvant are combined to construct a new tuberculosis subunit vaccine. In the use of the vaccine, the fusion protein LT57 achieves a good immune protection effect and has a good application development prospect.

[0026] Second, the expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0027] The purification method of the fusion protein LT57 constructed by the present invention is relatively simple, has the potential for scale-up production, is highly safe, and has good prospects for industrial development. In addition, the fusion protein LT57 combined with laboratory DP against H37Ra infection can induce mice to produce a protection efficiency similar to that of BCG, thereby achieving the purpose of improving the immune protection efficiency. It has important social and economic value in the prevention and treatment of tuberculosis, and also has important inspiration for the vaccine research of other complex pathogens.

[0028] The technical solution of the present invention fills the technical gap in the industry at home and abroad: various subunit vaccines such as M72 / AS01E, Gam TBvac, H56:IC31 (AERAS-456), H4:IC31 (AERAS-404), ID93+GLA-SE and AEC / BC02 that have entered the clinical trial stage, most of their antigens are concentrated in the MTB cell wall and secretory expressed antigens, etc., and less attention is paid to the PE / PPE family antigens of MTB.

[0029] The present invention uses the PE / PPE family antigens of MTB as the main framework to construct the fusion protein LT57, which expands the antigen abundance in the subunit vaccine and is expected to become an effective candidate vaccine for tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The present invention provides a flowchart of the construction, expression and purification method and application of the Mycobacterium tuberculosis fusion protein LT57.

[0031] Figure 2 The expression and purification results of the fusion protein LT57 provided in the embodiment of the present invention in E. coli BL21 (DE3) are shown. (A) Expression results of fusion protein LT57; M: marker; 1: whole bacterial liquid after bacterial cell crushing; 2: supernatant after bacterial cell crushing; 3: precipitate after bacterial cell crushing; 4: BL21 empty bacteria. (B) Molecular sieve purification results of fusion protein LT57; M: marker; 1: sample eluted with 50mM Tris buffer. (C) Ion exchange chromatography results of fusion protein LT57; M: marker; 1: sample eluted with 40-48Ms / cm high salt PB buffer. (D) Gel Doc TM XR+ analyzes the purity of fusion protein LT57.

[0032] Figure 3 The cytokine secretion level of mouse lymphocytes stimulated by Rv0518 antigen was detected by flow cytometry 6 weeks after the last immunization of LT57 provided in the embodiment of the present invention. (A) CD4 + Statistical analysis of the proportion of T cells; (B) IL-2 secreting CD4 + Statistical analysis of the proportion of T cells; (C) CD4 + Statistical analysis of the proportion of T cells; (D) CD4 + Statistical analysis of T cell numbers; (E) IL-2 secreting CD4 + Statistical analysis of T cell numbers; (F) IL-17A secreting CD4 + Statistical analysis of T cell numbers; (G) CD8 + Statistical analysis of T cell granzyme B secretion levels. n=5, *p<0.05, p<0.01, *p<0.001.

[0033] Figure 4 The cytokine secretion level of mouse lymphocytes stimulated by PE25-PPE41 antigen was detected by flow cytometry 6 weeks after the last immunization of LT57 provided in the embodiment of the present invention. (A) CD4 + Statistical analysis of the proportion of T cells; (B) CD4 + Statistical analysis of the proportion of T cells; (C) CD4 + Statistical analysis of the proportion of T cells; (D) CD4 + Statistical analysis of T cell numbers; (E) IL-2 secreting CD4 + Statistical analysis of T cell numbers; (F) IL-17A secreting CD4 +Statistical analysis of T cell numbers; (G) CD8 + Statistical analysis of T cell granzyme B secretion levels. n=5, *p<0.05, p<0.01, *p<0.001.

[0034] Figure 5 The cytokine secretion level of mouse lymphocytes stimulated by Rv0518 antigen was detected by flow cytometry 12 weeks after the last immunization of LT57 provided in the embodiment of the present invention. (A) CD4 + Statistical analysis of the proportion of T cells; (B) IL-2 secreting CD4 + Statistical analysis of the proportion of T cells; (C) CD4 + Statistical analysis of the proportion of T cells; (D) CD4 + Statistical analysis of T cell numbers; (E) IL-2 secreting CD4 + Statistical analysis of T cell numbers; (F) IL-17A secreting CD4 + Statistical analysis of T cell numbers; (G) CD8 + Statistical analysis of T cell granzyme B secretion levels. n=5, *p<0.05, p<0.01, *p<0.001.

[0035] Figure 6 The cytokine secretion level of PE25-PPE41 antigen-stimulated lymphocytes was detected by flow cytometry 12 weeks after the last immunization of LT57 provided in the embodiment of the present invention. (A) CD4 + Statistical analysis of the proportion of T cells; (B) IL-2 secreting CD4 + Statistical analysis of the proportion of T cells; (C) CD4 + Statistical analysis of the proportion of T cells; (D) CD4 + Statistical analysis of T cell numbers; (E) IL-2 secreting CD4 + Statistical analysis of T cell numbers; (F) IL-17A secreting CD4 + Statistical analysis of T cell numbers; (G) CD8 + Statistical analysis of T cell granzyme B secretion levels. n=5, *p<0.05, p<0.01, *p<0.001.

[0036] Figure 7 The flow cytometry test of the proliferation of mouse lymphocytes stimulated by Rv0518 antigen 12 weeks after the last immunization of LT57 provided in the embodiment of the present invention. (A) Proliferated CD4 + T cell statistical analysis; (B) Proliferation of CD8+ T cell statistical analysis. n=5, *p<0.05, p<0.01, *p<0.001.

[0037] Figure 8 The flow cytometry test of the proliferation of mouse lymphocytes stimulated by PE25-PPE41 antigen was performed 12 weeks after the last immunization of LT57 provided in the embodiment of the present invention. (A) Proliferated CD4 + T cell statistical analysis; (B) Proliferation of CD8 + T cell statistical analysis. n=5, *p<0.05, p<0.01, *p<0.001.

[0038] Fig. 9 The ELISA test of Rv0518-specific antibody level in mouse serum 6 weeks after the last immunization of LT57 provided in the embodiment of the present invention. (AC) Rv0518 antigen coating was used to detect IgG, IgG1 and IgG2c antibody levels respectively; (D) Antibody titers of different groups. The results are expressed as mean ± standard deviation, n = 5, *p < 0.05, p < 0.01, *p < 0.001.

[0039] Fig.10 The ELISA test of PE25-PPE41 specific antibody levels in mouse serum 6 weeks after the last immunization of LT57 provided in the embodiment of the present invention. (AC) PE25-PPE41 antigen coating was used to detect IgG, IgG1 and IgG2c antibody levels respectively; (D) Antibody titers of different groups. The results are expressed as mean ± standard deviation, n = 5, *p < 0.05, p < 0.01, *p < 0.001.

[0040] Fig.11 The ELISA test of the Rv0518-specific antibody level in the mouse serum 12 weeks after the last immunization of LT57 provided in the embodiment of the present invention. (AC) Rv0518 antigen coating was used to detect the IgG, IgG1 and IgG2c antibody levels respectively; (D) Antibody titers of different groups. n=5, *p<0.05, p<0.01, *p<0.001.

[0041] Fig.12 The ELISA test of PE25-PPE41 specific antibody levels in mouse serum 12 weeks after the last immunization of LT57 provided in the embodiment of the present invention. (AC) PE25-PPE41 antigen coating was used to detect IgG, IgG1 and IgG2c antibody levels respectively; (D) Antibody titers of different groups. n=5, *p<0.05, p<0.01, *p<0.001.

[0042] Fig.13This is the immune protection effect after attack by the H37Ra attenuated strain provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] A method for obtaining the Mycobacterium tuberculosis fusion protein LT57 provided in an embodiment of the present invention is as follows:

[0045] Genes PE25, PPE41 and Rv0518 were recombined into a prokaryotic expression vector through genetic engineering technology to obtain the fusion protein LT57.

[0046] The expression plasmid provided in the embodiment of the present invention is pET30a, and the plasmid for recombinantly expressing the fusion protein is:

[0047] pET30a(+)-LT57(PE25-linker-PPE41-Rv0518).

[0048] like Figure 1 As shown, a method for constructing and expressing and purifying a Mycobacterium tuberculosis fusion protein LT57 provided in an embodiment of the present invention is as follows:

[0049] S101, the fusion protein LT57 was expressed in both the supernatant and inclusion bodies, but mainly in the inclusion bodies; the fusion protein expressed in the inclusion bodies was first denatured and dissolved with urea, and then preliminarily purified using a molecular sieve column renaturation method;

[0050] S102, after preliminary purification by molecular sieve, the sample containing the target protein is further purified using an ion exchange chromatography QHP column, the impurities are eluted with a 0-40Ms / cm high salt PB buffer, and the target protein is collected with a 40-48Ms / cm high salt PB buffer.

[0051] The Mycobacterium tuberculosis fusion protein LT57 construction and expression and purification method provided in the embodiment of the present invention first realizes the expression and preliminary purification of the fusion protein LT57 through step S101. Specifically, after the fusion protein LT57 is expressed in the host cell, it exists both in the cell supernatant and in the inclusion body, and the inclusion body expression is the main one. In order to preliminarily purify the fusion protein, the fusion protein expressed in the inclusion body is denatured and dissolved with urea, and the molecular sieve column refolding method and pore structure are used to achieve the selective adsorption and separation effect of different molecules, thereby achieving the preliminary enrichment and purification of the target protein.

[0052] Next, in step S102, the protein sample after preliminary purification is loaded onto an ion exchange chromatography QHP column for further purification. Ion exchange chromatography is based on the charge characteristics of the surface of protein molecules, and the target protein is separated from the impurity protein by gradient elution using buffers of different salt concentrations. In the present invention, 0-40Ms / cm high-salt PB buffer is first used for elution to remove impurity proteins with different charge characteristics from the target protein, thereby ensuring the purity of the target protein.

[0053] Finally, in the high-salt elution process of ion exchange chromatography, the target protein LT57 was eluted from the chromatography column using a high-salt PB buffer of 40-48Ms / cm. This process ensures the separation of the target protein from other impurities and achieves the recovery of high-purity protein. Through this method, the Mycobacterium tuberculosis fusion protein LT57 finally obtained has high purity and activity and can be used for further research and application.

[0054] The usage of the fusion protein provided in the embodiment of the present invention: LT57 is used alone.

[0055] The fusion protein provided in the embodiment of the present invention and the adjuvant together constitute the application of the recombinant tuberculosis subunit vaccine: DP adjuvant is used.

[0056] The recombinant protein vaccine provided in the embodiment of the present invention is used in anti-tuberculosis prevention.

[0057] Specific embodiments of the present invention:

[0058] Example 1 Construction, expression and purification of fusion protein LT57

[0059] The present invention firstly fuses Mycobacterium tuberculosis PE / PPE family antigens PE25 and PPE41 at the gene level, and considering the influence of the protein spatial structure, adds a linker (GGGGS) 3 sequence between PE25 and PPE41 to assist the fusion protein folding expression and reduce the mutual influence of the spatial structure. The spatial structure of Rv0518 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 PPE41 and Rv0518. Based on this, a recombinant plasmid pET30a (+) -LT57 (PE25-linker-PPE41-Rv0518) is constructed.

[0060] Then, the pET30a(+)-LT57 plasmid vector was transformed into E. coli to express the fusion protein LT57. Finally, according to the protein characteristics of the protein, the fusion protein was successfully purified by combining molecular sieve column renaturation and ion chromatography. The specific steps are as follows:

[0061] 1. Construction of plasmid pET30a(+)-LT57 by full gene synthesis

[0062] 2. Expression and purification of fusion protein LT57

[0063] Expression of fusion protein LT57

[0064] The successfully constructed pET30a(+)-LT57(PE25-linker-PPE41-Rv0518) vector was transformed into the expression vector Escherichia coli BL21(DE3). Activate the strain of E.coli BL21 (DE3) expressing LT57, take 2 ml of the activated bacterial solution and add it to 200 ml of LB medium, add 200 ul of kanamycin Kana (10 mmol / L) at the same time, and culture at 37°C with shaking for 4 h; add 100 ul of protein inducer IPTG (1.0 mmol / L), and induce shaking culture at 25°C for 12 h; collect the bacteria by centrifugation at 4°C and 10000G for 10 min; resuspend the bacteria in PB buffer (Na2HPO4·12H2O 20 mMol / L, NaH2PO4·2H2O20 mMol / L, pH7.4) according to the 10 ml / g wet bacteria standard, and disrupt the bacteria by ultrasonication for 0.5 h in an ice bath (180-200 W, ultrasonication for 3 s and stop for 3 s); collect the supernatant and precipitate after centrifugation at 10000G for 15 min, and perform polyacrylamide gel electrophoresis on the supernatant and precipitate respectively; and analyze the supernatant and precipitate by SDS-PAGE electrophoresis. Compared with BL21(DE3) empty bacteria, there is an obvious specific protein expression band with a molecular weight of about 57KD, which is expressed in both the supernatant and the precipitate, and mainly in the form of the precipitate ( Figure 2 A).

[0065] (2) Purification of fusion protein LT57

[0066] The fusion protein LT57 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 LT57 protein was collected after centrifugation; the collected precipitate was added with inclusion body washing buffer (NaCl 11.7g / L, Tris 3.029g, urea 60.06g / L, EDTA0.372g / L, pH8.0) according to the standard of 10mL / g, washed by blowing, centrifuged at 4℃ / 10000G for 10min, washed 3 times, and the supernatant was discarded. Subsequently, 50mM Tris solution (pH8.0) containing 8M urea was added according to the standard of 5mL / g, dissolved at 4℃ for 30min, and 50mM Tris solution was added in sequence to dissolve the precipitate in a gradient manner until the final solution contained 3M urea. The liquid became clear and was filtered through a 0.22μm filter and stored at 4℃. The filler selected was Sepharose TMThe collected fusion protein column was renatured and initially purified by using CL-4B (Cytiva) molecular sieve. The molecular sieve column was rinsed with 50 mM Tris solution (pH 8.0). After the cond value stabilized, 4 mL of sample was drawn and injected into the sample loop. Sample collection began when a peak appeared 40 minutes later ( Figure 2 B).

[0067] After preliminary purification by molecular sieve, the ion chromatography material Q Sepharose High Performance (GE Healthcare) was used for further purification. The sample was loaded with low-salt PB buffer (Na2HPO4·12H2O 20mMol / L, NaH2PO4·2H2O 20mMol / L, pH7.4), and the impurities were washed with 0-40% Ms / cm high-salt PB buffer (Na2HPO4·12H2O 20mMol / L, NaH2PO4·2H2O 20mMol / L, NaCl 1Mol / L, pH7.4). The sample containing the target protein was collected with 40-48Ms / cm high-salt PB buffer (Na2HPO4·12H2O 20mMol / L, NaH2PO4·2H2O 20mMol / L, NaCl 1Mol / L, pH7.4), and polyacrylamide gel (SDS-PAGE) electrophoresis analysis to obtain the final purified sample ( Figure 2 C) Using Gel Doc TM XR+ analysis of fusion protein LT57 protein purity ( Figure 2 D).

[0068] Example 2 Preparation of fusion protein LT57 subunit vaccine

[0069] The fusion protein LT57 was diluted to 0.2 mg / ml with PBS buffer; Poly I:C was dissolved to 1 mg / ml with PBS buffer; the cationic liposome dimethyl trihexadecyl ammonium (DDA) was prepared to 5 mg / ml with PBS, placed in a water bath at 80°C for 10 minutes, and cooled to room temperature; 50 μl of Poly I:C solution was mixed with an equal volume of protein solution and placed at room temperature for 1 minute. 100 μL of DDA solution was added dropwise to the mixed solution, and then fully emulsified to make the vaccine uniformly emulsified to obtain the subunit vaccine LT57-DDA / Poly I:C (LT57-DP). When the vaccine is used for immunization, the dosage is 200 μl / mouse.

[0070] Example 3 Evaluation of Immunology and Protection Efficacy of Fusion Protein LT57

[0071] 1. Materials: subunit vaccine LT57-DDA / Poly I:C; Bacillus Calmette-Guérin (BCG); phosphate buffered saline (PBS);

[0072] 2. Experimental animals: C57BL / 6 mice;

[0073] 3. Experimental animal grouping (six groups in total):

[0074] (1) PBS; (2) BCG; (3) DDA / Poly I:C; (4) Rv0518-DDA / Poly I:C; (5) PE25-PPE41-DDA / Poly I:C; (6) LT57-DDA / Poly I:C;

[0075] 4. Animal immunization plan:

[0076] In the 0th week, mice in the BCG group were injected subcutaneously in the groin with 200 μl (5×10 6 CFU), immunized once. The other vaccine groups were immunized subcutaneously in the groin of mice at 0, 3, and 6 weeks, for a total of 3 immunizations (200 μl / mouse). Immunogenicity detection and protection evaluation against attenuated strain H37Ra were performed 6 and 12 weeks after the last immunization.

[0077] 5. Immune index determination method

[0078] (1) Detection of CD4 in mouse spleen by intracellular factor staining + T cell IFN-γ, IL-2, and IL-17A secretion levels.

[0079] Six weeks after the last immunization of mice, spleen lymphocytes were isolated and stimulated in vitro with the corresponding single antigen (5ug / ml) for 4h. 1ul of protein transport inhibitor was added and incubated for 8h before detecting CD4 + The secretion levels of IFN-γ, IL-2 and IL-17A by T cells ( Figure 3 , 4 ).

[0080] Figure 3 The results show that 6 weeks after the last immunization with LT57, flow cytometry was used to detect the secretion of cytokines by mouse lymphocytes stimulated by Rv0518 antigen. The BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT57 / DP group immunized C57BL / 6 mice 3 times at weeks 0, 3, and 6. Six weeks after the last immunization, the spleen lymphocytes of mice were isolated and incubated with the antigen Rv0518 (5 μg / mL) for 12 hours. The secretion of IFN-γ, IL-2, and IL-17A by CD4 + The proportion of T cells secreting granzymes was detected by ELISA +The proportion of T cells. (A) CD4 + Statistical analysis of the proportion of T cells; (B) CD4 + Statistical analysis of the proportion of T cells; (C) CD4 + Statistical analysis of the proportion of T cells; (D) CD4 + Statistical analysis of T cell numbers; (E) IL-2 secreting CD4 + Statistical analysis of T cell numbers; (F) IL-17A secreting CD4 + Statistical analysis of T cell numbers; (G) CD8 + Statistical analysis of T cell granzyme B secretion levels. n=5, *p<0.05, p<0.01, *p<0.001.

[0081] Figure 4 The results show that 6 weeks after the last immunization with LT57, flow cytometry was used to detect the levels of cytokine secretion by mouse lymphocytes stimulated with PE25-PPE41 antigens; the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT57 / DP group immunized C57BL / 6 mice 3 times at weeks 0, 3, and 6. 6 weeks after the last immunization, the mouse spleen lymphocytes were isolated and incubated with the antigen PE25-PPE41 (5 μg / mL) for 12 hours, and the CD4 secretion of IFN-γ, IL-2, and IL-17A was detected by flow cytometry. + The proportion of T cells secreting granzymes was detected by ELISA + The proportion of T cells. (A) CD4 + Statistical analysis of the proportion of T cells; (B) CD4 + Statistical analysis of the proportion of T cells; (C) CD4 + Statistical analysis of the proportion of T cells; (D) CD4 + Statistical analysis of T cell numbers; (E) IL-2 secreting CD4 + Statistical analysis of T cell numbers; (F) IL-17A secreting CD4 + Statistical analysis of T cell numbers; (G) CD8 + Statistical analysis of T cell granzyme B secretion levels. n=5, *p<0.05, p<0.01, *p<0.001.

[0082] Twelve weeks after the last immunization of mice, splenic lymphocytes were isolated from mice immunized with LT57 (10 μg / mouse) antigen 3 days after the mice were immunized. They were then stimulated in vitro with a single antigen (5 μg / mL) for 4 h, and 1 μl of protein transport inhibitor was added. After incubation for 8 h, CD4+ The secretion levels of IFN-γ, IL-2 and IL-17A by T cells ( Figure 5 , 6).

[0083] Figure 5 The results show that 12 weeks after the last immunization with LT57, flow cytometry was used to detect the secretion of cytokines by mouse lymphocytes stimulated by Rv0518 antigen. The BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT57 / DP group was immunized three times at weeks 0, 3, and 6 in C57BL / 6 mice. 12 weeks after the last immunization, 3 days after the mice were immunized with LT57 (10 μg / mouse), the mouse spleen lymphocytes were isolated and incubated with the antigen Rv0518 (5 μg / mL) for 12 hours, and the secretion of IFN-γ, IL-2, and IL-17A by CD4 + The proportion of T cells secreting granzymes was detected by ELISA + The proportion of T cells. (A) CD4 + Statistical analysis of the proportion of T cells; (B) IL-2 secreting CD4 + Statistical analysis of the proportion of T cells; (C) CD4 + Statistical analysis of the proportion of T cells; (D) CD4 + Statistical analysis of T cell numbers; (E) IL-2 secreting CD4 + Statistical analysis of T cell numbers; (F) IL-17A secreting CD4 + Statistical analysis of T cell numbers; (G) CD8 + Statistical analysis of T cell granzyme B secretion levels. n=5, *p<0.05, p<0.01, *p<0.001.

[0084] Figure 6 The results show that 12 weeks after the last immunization with LT57, the levels of cytokine secretion by lymphocytes stimulated by PE25-PPE41 antigen were detected by flow cytometry; the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT57 / DP group was immunized three times at weeks 0, 3, and 6 in C57BL / 6 mice. 12 weeks after the last immunization, 3 days after the mice were immunized with LT57 (10 μg / mouse) antigen, the spleen lymphocytes of the mice were isolated and incubated with the antigen PE25-PPE41 (5 μg / mL) for 12 hours, and the CD4 secretion of IFN-γ, IL-2, and IL-17A was detected by flow cytometry. + The proportion of T cells secreting granzymes was detected by ELISA + The proportion of T cells. (A) CD4 + Statistical analysis of the proportion of T cells; (B) IL-2 secreting CD4+ Statistical analysis of the proportion of T cells; (C) CD4 + Statistical analysis of the proportion of T cells; (D) CD4 + Statistical analysis of T cell numbers; (E) IL-2 secreting CD4 + Statistical analysis of T cell numbers; (F) IL-17A secreting CD4 + Statistical analysis of T cell numbers; (G) CD8 + Statistical analysis of T cell granzyme B secretion levels. n=5, *p<0.05, p<0.01, *p<0.001.

[0085] (2) ELISA to detect mouse spleen CD8 + T cell granzyme B secretion levels.

[0086] Six weeks after the last immunization of mice, spleen lymphocytes were isolated and stimulated in vitro with the corresponding single antigen (5ug / ml) for 72h. The supernatant was taken and CD8 + The level of granzyme secreted by T cells ( Figure 3 G. Figure 4 G).

[0087] Twelve weeks after the last immunization of mice, splenic lymphocytes were isolated from mice immunized with LT57 (10 μg / mouse) antigen 3 days after the mice were immunized. Then, after in vitro stimulation with a single antigen (5 μg / mL) for 72 h, the supernatant was collected and CD8 + The level of granzyme secreted by T cells ( Figure 5 G. Figure 6 G).

[0088] (3) Flow cytometry was used to detect the proliferation level of mouse antigen-specific splenic lymphocytes.

[0089] Twelve weeks after the last immunization of mice, splenic lymphocytes were isolated from mice immunized with LT57 (10 μg / mouse) antigen 3 days later, and splenic lymphocytes were stimulated in vitro with a single antigen (5 μg / mL) for 3 days. EdU was added at a final concentration of 30 μM, and CD4 + T and CD8 + The proliferation capacity of T cells Figure 7 , 8 ).

[0090] Figure 7The flow cytometry was used to detect the proliferation of mouse lymphocytes stimulated by Rv0518 antigen 12 weeks after the last immunization with LT57. The BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT57 / DP group was immunized three times at weeks 0, 3, and 6 in C57BL / 6 mice. 12 weeks after the last immunization, the spleen lymphocytes of mice were isolated 3 days after the mice were immunized with LT57 (10 μg / mouse) antigen, and the spleen lymphocytes were stimulated in vitro with Rv0518 antigen for 3 days. EdU was added at a final concentration of 30 μM, and CD4 was detected by flow cytometry. + T and CD8 + T cell proliferation ability. (A) Proliferation of CD4 + T cell statistical analysis; (B) Proliferation of CD8 + T cell statistical analysis. n=5, *p<0.05, p<0.01, *p<0.001.

[0091] Figure 8 The flow cytometry was used to detect the proliferation of mouse lymphocytes stimulated by PE25-PPE41 antigen 12 weeks after the last immunization with LT57. The BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT57 / DP group was immunized three times at weeks 0, 3, and 6 in C57BL / 6 mice. 12 weeks after the last immunization, the spleen lymphocytes of mice were isolated 3 days after the mice were immunized with LT57 (10 μg / mouse) antigen, and the spleen lymphocytes were stimulated in vitro with PE25-PPE41 antigen for 3 days. EdU was added at a final concentration of 30 μM, and CD4 was detected by flow cytometry. + T and CD8 + T cell proliferation ability. (A) Proliferation of CD4 + T cell statistical analysis; (B) Proliferation of CD8 + T cell statistical analysis. n=5, *p<0.05, p<0.01, *p<0.001.

[0092] (4) The ELISA method was used to detect the levels of mouse serum specific antibodies IgG, IgG1 and IgG2c.

[0093] Serum was collected 6 and 12 weeks after the last immunization of mice. 96-well plates were coated with the corresponding single antigens (2.5ug / 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 by the same method ( Fig. 9 , 10 , 11, 12).

[0094] Fig. 9 The ELISA test of Rv0518-specific antibody levels in mouse sera was shown 6 weeks after the last immunization with LT57; the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT57 / DP group was immunized 3 times at weeks 0, 3, and 6 in C57BL / 6 mice. Six weeks after the last immunization, mouse sera were collected to detect the titers of Rv0518 antigen-specific IgG, IgG1, and IgG2c antibodies. (AC) Rv0518 antigen coating was used to detect IgG, IgG1, and IgG2c antibody levels; (D) Antibody titers of different groups. n=5, *p<0.05, p<0.01, *p<0.001.

[0095] Fig.10 The ELISA test of PE25-PPE41-specific antibody levels in mouse serum 6 weeks after the last immunization with LT57 was shown; the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT57 / DP group was immunized 3 times at weeks 0, 3, and 6 in C57BL / 6 mice. Six weeks after the last immunization, mouse serum was collected to detect the titers of IgG, IgG1, and IgG2c antibodies specific to the PE25-PPE41 antigen. (AC) PE25-PPE41 antigen was coated to detect IgG, IgG1, and IgG2c antibody levels; (D) Antibody titers of different groups. 0n=5, *p<0.05, p<0.01, *p<0.001.

[0096] Fig.11The ELISA test of Rv0518-specific antibody levels in mouse serum 12 weeks after the last immunization with LT57 was shown; the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT57 / DP group was immunized three times at weeks 0, 3, and 6 in C57BL / 6 mice. 12 weeks after the last immunization, mouse serum was collected to detect the titers of Rv0518 antigen-specific IgG, IgG1, and IgG2c antibodies. (AC) Rv0518 antigen coating was used to detect IgG, IgG1, and IgG2c antibody levels; (D) Antibody titers of different groups. n=5, *p<0.05, p<0.01, *p<0.001.

[0097] Fig.12 The ELISA test of PE25PPE41-specific antibody levels in mouse serum 12 weeks after the last immunization with LT57 was shown; the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine LT57 / DP group was immunized three times at weeks 0, 3, and 6 in C57BL / 6 mice. 12 weeks after the last immunization, mouse serum was collected to detect the titers of IgG, IgG1, and IgG2c antibodies specific to the PE25-PPE41 antigen. (AC) PE25-PPE41 antigen coating was used to detect IgG, IgG1, and IgG2c antibody levels, respectively; (D) Antibody titers of different groups. n=5, *p<0.05, p<0.01, *p<0.001.

[0098] (3) Bacterial load index determination method

[0099] Twelve weeks after the last immunization, all experimental mice were infected with the H37Ra strain by aerosol infection at a dose of 5×10 6 CFU / mouse. 42 days after infection, mice were killed, lungs were collected, ground and smeared on plates to count CFU for tuberculosis load detection.

[0100] Fig.13 The immune protection effect after challenge with the attenuated strain H37Ra was demonstrated; the BCG group was immunized once at week 0, and the tuberculosis subunit vaccine group was immunized three times at weeks 0, 3, and 6 in C57BL / 6 mice. Twelve weeks after the last immunization, the mice were infected with the H37Ra strain by intranasal drops, and the bacterial load in the lungs of the mice in each experimental group was detected 3 weeks after infection. N = 5 mice per group, the results are expressed as log10 CFU ± SD, *p < 0.05, p < 0.01 and *p < 0.001.

[0101] LT57 gene sequence

[0102] The italic part is the restriction site, and the part marked with “” is the nucleic acid sequence of PE25; is the nucleic acid sequence of PPE41, is the nucleic acid sequence of Rv0518; the nucleic acid sequence of linker (GGGGS) 3 is in bold;

[0103]

[0104]

[0105] Amino acid sequence of LT57

[0106] The italic part is the restriction site, and the part marked with “_” is the amino acid sequence of PE25; is the amino acid sequence of PPE41, is the amino acid sequence of Rv0518; the bold is the amino acid sequence of linker (GGGGS) 3;

[0107]

[0108] 1. Construction of recombinant gene vector pET30a(+)-LT57(PE25-linker-PPE41-Rv0518)

[0109] The single antigens PE25, PPE41 and Rv0518 were connected to the vector pET30a by full gene synthesis, with EcoRⅠ (CATATG) at the 5' end and HindⅢ (AAGCTT) at the 3' end. At the same time, a linker sequence (GGGGS) was added between the single antigens PE25 and PPE41 to promote protein folding and increase stability, completing the construction of the recombinant gene vector pET30a(+)-LT57 (PE25-linker-PPE41-Rv0518).

[0110] The tuberculosis subunit vaccine is composed of MTB proteins, polypeptides, glycolipids and other immunologically active components and adjuvants. It has the advantages of clear ingredients, low cost, high yield, good safety, easy preparation and good quality control.

[0111] At present, BCG is the only vaccine used for clinical prevention of tuberculosis, but its protective effect against adult tuberculosis is poor. The Chinese population is generally vaccinated with BCG, and the booster immunization of subunit vaccines has huge market potential.

[0112] The LT57 fusion protein constructed by the present invention was combined with DP adjuvant and the attenuated strain H37Ra (5×10 6 CFU / mouse) infected experimental mice, the LT57 / DP group was able to induce a protective efficiency similar to that of BCG (compared with BCG, the LT57 / DP group reduced 0.004log10 CFU).

[0113] The following are two specific embodiments based on the above technical solution:

[0114] Example 1: Construction and expression of fusion protein LT57

[0115] Background: MTB is the pathogen that causes tuberculosis. The development of new vaccine components is of great significance in the fight against tuberculosis. The fusion protein LT57 is composed of three antigens: PE25, PPE41 and Rv0518, and has good immunogenicity.

[0116] 1. Gene construction:

[0117] Using genetic engineering technology, gene fragments encoding PE25, PPE41 and Rv0518 were obtained by PCR amplification, and specific linker sequences were added between the gene fragments.

[0118] The amplified gene fragments were sequentially inserted into the prokaryotic expression vector pET30a(+) to construct a recombinant expression plasmid pET30a(+)-LT57.

[0119] After the plasmid construction was completed, the correctness of the plasmid was verified by restriction digestion and sequencing.

[0120] The plasmid pET30a(+)-LT57 was constructed by complete gene synthesis.

[0121] 2. Protein expression:

[0122] The recombinant expression plasmid pET30a(+)-LT57 was transformed into Escherichia coli BL21(DE3) host cells.

[0123] The expression of fusion protein LT57 was induced by IPTG. After culturing at 37°C for 4 hours, 100ul of protein inducer IPTG (1.0mmol / L) was added, and then induced shaking culture was performed at 25°C for 12 hours to induce protein expression.

[0124] The expressed protein was detected by SDS-PAGE, and it was found that the fusion protein LT57 mainly existed in the form of inclusion bodies.

[0125] Technical effects:

[0126] The expression system of fusion protein LT57 was successfully constructed, and the fusion protein was efficiently expressed in Escherichia coli.

[0127] The protein mainly exists in the form of inclusion bodies, which provides a basis for subsequent purification steps.

[0128] Example 2: Purification and vaccine application of fusion protein LT57

[0129] In order to obtain a high-purity LT57 fusion protein with biological activity and evaluate its application effect in a recombinant tuberculosis vaccine, the fusion protein LT57 was purified in this example and applied to vaccine preparation.

[0130] 1. Protein purification:

[0131] The expressed fusion protein LT57 inclusion bodies were denatured and dissolved with 8M urea to obtain a preliminary dissolved protein solution.

[0132] Denatured proteins were renatured using molecular sieve columns and some impurities were removed by preliminary purification.

[0133] The renatured protein was further purified using an ion exchange chromatography QHP column. 0-40Ms / cm high-salt PB buffer was used to elute the impurities, and 40-48Ms / cm high-salt PB buffer was used to collect the high-purity target protein LT57.

[0134] By SDS-PAGE and Gel Doc TM XR+ analysis verified the protein purity, and the results showed that the purity reached more than 90%.

[0135] 2. Vaccine application:

[0136] The purified fusion protein LT57 was mixed with DP adjuvant to prepare the recombinant tuberculosis subunit vaccine.

[0137] The immune effect of the vaccine was evaluated using a mouse model. The experiment showed that mice vaccinated with the recombinant protein vaccine produced an effective anti-tuberculosis immune response and showed a good protective effect.

[0138] Technical effects:

[0139] Through an effective purification method, high-purity LT57 fusion protein was successfully obtained.

[0140] The recombinant tuberculosis subunit vaccine prepared using this protein showed good immune effects in mouse experiments, indicating that it has potential clinical application value.

[0141] These two examples demonstrate the entire process of LT57 fusion protein from construction, expression to purification and application in tuberculosis vaccine development, verifying its feasibility and effectiveness as a component of anti-tuberculosis candidate vaccine.

[0142] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for constructing a Mycobacterium tuberculosis fusion protein LT57, characterized in that: The following steps are involved: a) Genes PE25, PPE41 and Rv0518 were recombined into the prokaryotic expression vector pET30a(+) by genetic engineering technology to construct a recombinant expression plasmid pET30a(+)-LT57 (PE25-linker-PPE41-Rv0518); b) Transforming the recombinant expression plasmid into host cells to induce the expression of fusion protein LT57.

2. The method for expressing and purifying the Mycobacterium tuberculosis fusion protein LT57 according to claim 1, characterized in that: The expression and purification method comprises the following steps: a) The fusion protein LT57 was expressed in both the supernatant and the inclusion bodies, with the inclusion body being the main expression. First, the fusion protein expressed in the inclusion body was denatured and dissolved with urea, and then preliminarily purified using a molecular sieve column renaturation method; b) After initial purification by molecular sieve, the sample containing the target protein was further purified using an ion exchange chromatography QHP column, the impurities were eluted with 0-40Ms / cm high salt PB buffer, and the target protein was collected with 40-48Ms / cm high salt PB buffer.

3. The method for purifying the Mycobacterium tuberculosis fusion protein LT57 according to claim 2, characterized in that: The purification process includes expressing the supernatant and inclusion bodies of the fusion protein LT57, with the inclusion body expression being the main method, and purifying by combining molecular sieve column renaturation with ion exchange chromatography QHP column to obtain a high-purity target protein.

4. The application method of the Mycobacterium tuberculosis fusion protein LT57 according to claim 3, characterized in that: The fusion protein LT57 and the DP adjuvant together constitute a recombinant tuberculosis subunit vaccine, and the recombinant protein vaccine is used for the prevention of anti-tuberculosis.

5. A recombinant tuberculosis subunit vaccine for anti-tuberculosis prevention, characterized in that: The vaccine comprises Mycobacterium tuberculosis fusion protein LT57 and DP adjuvant. The fusion protein LT57 is obtained by genetic engineering technology and is expressed and purified before being mixed with the adjuvant for preventing tuberculosis infection.

6. The method for using the recombinant tuberculosis subunit vaccine according to claim 1, characterized in that: The vaccine is used for anti-tuberculosis prevention through injection. The fusion protein LT57 in the vaccine can induce an immune response against Mycobacterium tuberculosis, thereby enhancing the body's resistance to tuberculosis infection.

7. A method for obtaining Mycobacterium tuberculosis fusion protein LT57, characterized in that: The method comprises: recombining genes PE25, PPE41 and Rv0518 into a prokaryotic expression vector pET30a(+) by genetic engineering technology, constructing a recombinant expression plasmid pET30a(+)-LT57 (PE25-linker-PPE41-Rv0518), and expressing it in a host cell to obtain a fusion protein LT57.

8. A recombinant gene vector pET30a(+)-LT57(PE25-linker-PPE41-Rv0518), characterized in that: The single antigens PE25, PPE41 and Rv0518 were connected to the vector pET30a by full gene synthesis, with EcoRⅠ (CATATG) at the 5' end and HindⅢ (AAGCTT) at the 3' end. At the same time, a linker sequence (GGGGS) was added between the single antigens PE25 and PPE41 to promote protein folding and increase stability, completing the construction of the recombinant gene vector pET30a(+)-LT57 (PE25-linker-PPE41-Rv0518).

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