CN116023505A - Mycobacterium tuberculosis fusion protein BfrB-GrpE, preparation method and application - Google Patents

Abstract

The invention discloses a mycobacterium tuberculosis fusion protein BfrB-GrpE, a preparation method and application thereof, wherein the mycobacterium tuberculosis fusion protein BfrB-GrpE is formed by combining a mycobacterium tuberculosis replication and dormancy immunodominant antigen BfrB and a dormancy immunodominant antigen GrpE; the invention successfully constructs, expresses and purifies the mycobacterium tuberculosis fusion protein BG without any label by utilizing the genetic engineering technology, solves the subsequent problem that the label carried by the fusion protein possibly affects animal experiments and later clinical experiments, and effectively purifies the unlabeled fusion protein by a method of combining salting-out and hydrophobic chromatography; the method has the advantages of high expression yield, stable expression, insensitivity to temperature and salt ion concentration, supernatant expression and the like, and can be used for industrial fermentation; the combined construction of the novel fusion protein BG and the adjuvant in the invention induces the biased Th1 type cell immune response.

Description

Mycobacterium tuberculosis fusion protein BfrB-GrpE and its preparation method and application

technical field

The invention belongs to the technical field of biomedical engineering, in particular to the construction of a novel tuberculosis subunit vaccine using a genetic engineering method.

Background technique

Tuberculosis is caused by Mycobacterium tuberculosis (Mycobacterium Tuberculosis, M.Tuberculosis) infection, and is one of the infectious diseases with the largest number of deaths caused by infection with a single pathogen. Currently, BCG (Bacille Calmette-Guérin, BCG) is the only vaccine used clinically to prevent tuberculosis, but its protective effect on adult tuberculosis is not good. A large number of studies have shown that tuberculosis subunit vaccine can provide long-term immune protection effect and good safety, so it has a good application and development prospect. After Mycobacterium tuberculosis infects the body, there are different metabolic states in the host body, such as multiple stages of resuscitation, replication and dormancy, and different metabolic states can be converted to each other under certain conditions . Therefore, the new tuberculosis subunit vaccine should contain immunodominant antigens of different metabolic stages to form a multi-stage vaccine, which can effectively improve the protective effect against tuberculosis.

Tuberculosis subunit vaccines are some immunologically active substances (such as proteins, polypeptides, glycolipids, etc.) in Mycobacterium tuberculosis. Compared with live vaccines, they have clear ingredients, low cost, high yield, good safety, and easier preparation , good quality control, and the immune memory of T cells can be enhanced through vaccination. Compared with whole-cell vaccines, subunit vaccines target a small number of selected antigens, usually six or even fewer. So far, only a few antigens have been identified as promising TB subunit vaccine candidates. In various animal models, the selection of suitable antigens is mainly based on antigenicity, immunogenicity and protection against infection. Due to the complex pathogenesis of tuberculosis, the above approach may have limitations. Therefore, the main challenge in developing protein subunit vaccines is to determine the type and quantity of antigens contained in such vaccines. Some researchers have screened immunodominant antigens at different growth stages for inclusion in candidate vaccines according to the metabolic state of Mycobacterium tuberculosis-infected organisms. In addition, compared with BCG, TB subunit vaccines face some unavoidable challenges, including poor immunogenicity and shorter duration of immune memory, so they need to be used in combination with adjuvants to produce better immune protection.

The MH(Mtb10.4-HspX), LT69(ESAT-6-Ag85B -MPT64190-198-Mtb8.4-HspX) and LT70 (ESAT-6-Ag85B-MPT64190–198-Mtb8.4-Rv2626c) multi-stage vaccines showed better anti-TB clade than single-stage antigen-constructed vaccines Bacillus strain H37Rv infection protective effect. The inventor screened out immunodominant antigens—BfrB and GrpE—through a large number of literature reviews and computer prediction of antigenic epitopes. Mycobacterium tuberculosis BfrB (Rv3841) is an iron storage protein with strong immunogenicity, which can induce an immune response equivalent to the classic antigen Ag85B of Mycobacterium tuberculosis in mice; GrpE (Rv3051) is a group of HSP70 complex proteins One part of it has strong immunogenicity, and the immune memory response induced in mice is equivalent to that of ESAT6, and the ability of a single antigen against Mycobacterium tuberculosis strains is equivalent to that of traditional tuberculosis vaccine BCG. Therefore, the tag-free fusion protein BfrB-GrpE (BG for short) was constructed by combining BfrB and GrpE antigens, aiming at developing a more effective multi-stage subunit vaccine.

Contents of the invention

The present invention provides a novel fusion protein BG that combines the replication and dormant immunodominant antigen BfrB of Mycobacterium tuberculosis and the dormant immunodominant antigen GrpE, and proposes a method for constructing, expressing and purifying the fusion protein.

The novel fusion protein BG and the adjuvant provided by the present invention are combined to construct a tuberculosis subunit vaccine, which induces a Th1-biased cellular immune response, and is expected to become a more effective new candidate tuberculosis vaccine in the later stage.

The present invention adopts the following technical scheme: Mycobacterium tuberculosis fusion protein BfrB-GrpE, which is jointly constructed by Mycobacterium tuberculosis replication and dormant immunodominant antigen BfrB and dormant immunodominant antigen GrpE, the amino acid sequence is:

MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLDRDLRVEIPGVDTVRNQFDRPREALALDQERTVTDQVGRLTAVARDEGDFLGEQFMQWFLQEQIEEVALMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRLGGGGS GGGGSGGGGSVTDGNQKPDGNSGEQVTVTDKRRIDPETGEVRHVPPGDMPGGTAAADAAHTEDKVAELTADLQRVQADFANYRKRALRDQQAAADRAKASVVSQLLGVLDDLERARKHGDLESGPLKSVADKLDSALTGLGLVAFGAEGEDFDPVLHEAVQHEGDGGQGSKPVIGTVMRQGYQLGEQVLR HALVGVVDTVVVDAAELESVDDGTAVADTAENDQADQGNSADTSGEQAESEPSGS.

The method for preparing the fusion protein BfrB-GrpE of Mycobacterium tuberculosis comprises the following steps: 1. Construction of the fusion protein BG; 2. Small batch expression of the fusion protein BG; 3. Fermentative expression of the fusion protein BG; 5. Purification of fusion protein BG.

Optionally, the step 1 is specifically: designing a BfrB-GrpE fusion gene, adding a linker between the two genes, and translating the amino acid sequence into:

MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLDRDLRVEIPGVDTVRNQFDRPRREALALLDQERTVTDQVGRLTAVARDEGDFLGEQFMQWFLQEQIEEVALMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRL GGG GSGGGGSGGGGS VTDGNQKPDGNSGEQVTVTDKRRIDPETGEVRHVPPGDMPGGTAAADAAHTEDKVAELTADLQRVQADFANYRKRALRDQQAAADRAKASVVSQLLGVLDDLERARKHGDLESGPLKSVADKLDSALTGLGLVAFGAEGEDFDPVLHEAVQHEGDGGQGSKPVIGTVMRQGYQLGEQVL RHALVGVVDTVVVDAAELESVDDGTAVADTAENDQADQGNSADTSGEQAESEPSGS*

Underline is the linker

The pET30a(+)-BfrB-GrpE gene was optimized and synthesized to obtain the pET30a(+)-BfrB-GrpE vector.

Optionally, the second step is specifically: transforming the successfully constructed pET30a(+)-BfrB-GrpE vector into Escherichia coli E.coliBL21(DE3)-pLySs competent cells for later purpose protein expression; After activation, inoculate at 37°C for shaking culture overnight, take 2ml of activated cells and add them to 200ml LB medium for shaking culture at 37°C for 6 hours; add protein inducer IPTG to a final concentration of 0.5mmol/L, induce shaking at 37°C for 6 hours; 4°C, Centrifuge at 8000rpm for 20min to harvest the wet bacteria; resuspend the bacteria in PB buffer at 10ml/g of wet bacteria, and sonicate the bacteria three times in an ice bath; collect the supernatant and the precipitate after centrifuging at 8000rpm for 20min at 4°C, and separate the supernatant and the precipitate Acrylamide gel electrophoresis.

Optionally, the step three is specifically:

BG was fermented and expressed in the E.coliBL21(DE3)-pLySs strain, and a single colony was obtained after three-section line culture, and a single colony was picked and inoculated in LB liquid medium containing chloramphenicol and kana antibiotics at 37°C, 180rpm Cultivate for 12-16 hours as first-class seeds; transfer the first-class seeds according to the inoculation ratio of 1:1000, shake and culture at 37°C and 180rpm for 12-16 hours, and use them as second-class seeds; inoculate 70mL of second-class seed bacterial liquid into Add 2mL of kanamycin solution and chloramphenicol solution to the fermenter containing 2L medium, adjust the pH to 6.8-7.4, set the dissolved oxygen to about 20% in the enrichment stage, and the associated speed; 6h At the beginning, feed according to the dissolved oxygen; add 1mL IPTG inducer at the 7th hour to a final concentration of 1.0mmol/L, and stop the fermentation after 5 hours of cultivation; after 12 hours of fermentation, the obtained bacteria were washed, resuspended, and homogenized under high pressure in sequence Lysis and centrifugation; BG whole bacteria, BG supernatant and BG precipitate resuspension were taken for 15% SDS-PAGE gel electrophoresis analysis.

Optionally, the fourth step is specifically:

The Mycobacterium tuberculosis fusion protein BfrB-GrpE is fermented and expressed in a 30L fermenter, and the fermentation parameters are shown in Table 1.

Table 1 BG-20200909 fermentation parameters

Optionally, the fifth step is specifically:

Resuspend the fermented Escherichia coli in a 10% ratio in 20mM PB (pH=7.4) buffer, lyse by high-pressure homogenization, centrifuge at 12000rpm for 20min, and collect the supernatant containing the target protein; first, use the volume fraction of the supernatant 35% saturated ammonium sulfate solution was used for salting out, and the salted out protein was resuspended in PB buffer; then, the supernatant was collected by centrifugation for hydrophobic chromatography, and 5mL of Butyl Sepharose High Performance was used for purification, and finally the loading buffer system was determined 20mM PB (pH=8.0)+2M NaCl; set 100-0% B pump [20mM PB(pH=8.0)+2M NaCl] after loading the sample, 40min for linear elution; UV value is greater than 40 to collect, less than 100 to stop ; The final purified product was analyzed by SDS-PAGE electrophoresis, and the electrophoretic gel image was scanned by image Lab software to analyze the protein purity. The detection sensitivity was set to 50%, and the purity of the fusion protein BG was greater than 95%.

The preparation method of mycobacterium tuberculosis fusion protein BfrB-GrpE subunit vaccine comprises the steps of: preparing 1mg/mL PolyI:C with PBS; preparing cationic liposome dimethyl hexadecyl ammonium and cholesterol mixed solution with PBS , the concentration of DDA in this mixture is 5mg/mL, the concentration of cholesterol is 0.5mg/mL, dissolve in a water bath at 80°C for 10min, cool to room temperature for later use; dilute the BG fusion protein with PBS buffer to the required concentration; dilute the protein and Poly The I:C solution was thoroughly mixed at a volume ratio of 1:1, and left at room temperature for 1 min; then an equal volume of the DDA solution was added dropwise to the mixture, emulsified, and the vaccine was uniformly creamy, and a subunit vaccine was obtained.

The beneficial effect of the present invention is that: the present invention utilizes genetic engineering technology to successfully construct, express and purify the Mycobacterium tuberculosis fusion protein BG without any label, and solve the problem that the label carried by the fusion protein may affect animal experiments As well as follow-up questions for late-stage clinical trials, and the tag-free fusion protein was effectively purified by a combination of salting-out and hydrophobic chromatography. Moreover, it has the advantages of high expression yield, stable expression, insensitivity to temperature and salt ion concentration, and expression in supernatant, and can carry out industrial fermentation; the novel fusion protein BG and adjuvant in the present invention are jointly constructed tuberculosis subunit vaccine, the vaccine Induces a Th1-biased cellular immune response. The early fusion protein MH of the vaccine immunized mice, and the protective effect against Mycobacterium tuberculosis was better than that of the single fusion vaccine group. The vaccine has no obvious toxic and side effects in the mouse immunization process; the invention is expected to become a candidate vaccine for clinical tuberculosis prevention and treatment.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, and are used together with the embodiments of the present invention to explain the present invention, and do not constitute a limitation to the present invention. In the attached picture:

Fig. 1 is a graph showing the expression and purification results of the Mycobacterium tuberculosis fusion protein BfrB-GrpE in Escherichia coli E.coliBL21(DE3)-pLySs.

Figure 2 is a graph showing the industrial fermentation results of the 20200909 fusion protein BG.

Figure 3 is a graph showing the crude extraction results of the fusion protein BG ammonium sulfate.

Fig. 4 is a graph showing the results of hydrophobic chromatography of the fusion protein BG.

Figure 5 is the detection of the levels of IFN-γ and IL-2 secreted by spleen cells by ELISA.

Figure 6 is a graph showing the results of serum antibody detection after immunizing mice in different vaccine groups.

Figure 7 is a diagram for evaluating the protection effect.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1: Construction and expression of fusion protein BG

1. Construction of fusion protein BG

Design the BfrB-GrpE fusion gene, add a linker between the two genes, and translate the amino acid sequence into: MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLDRDLRVEIPGVDTVRNQFDRPRREALALLDQERTVTDQVGRLTAVARDEGDFLGEQFMQWFLQEQIEEVALMA TLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRL GGGGSGGGGSGGGGS VTDGNQKPDGNSGEQVTVTDKRRIDPETGEVRHVPPGDMPGGTAAADAAHTEDKVAELTADLQRVQADFANYRKRALRDQQAAADRAKASVVSQLLGVLDDLERARKHGDLESGPLKSVADKLDSALTGLGLVAFGAEGEDFDPVLHEAVQHEGDGGQGSKPVIGTVMRQGYQLGEQVLRHALVGVVDTV VVDAAELESVDDGTAVADTAENDQADQGNSADTSGEQAESEPSGS* (Note: The linker is underlined)

The pET30a(+)-BfrB-GrpE gene was optimized and synthesized to obtain the pET30a(+)-BfrB-GrpE vector.

2. Small batch expression of fusion protein BG

The successfully constructed pET30a(+)-BfrB-GrpE vector was transformed into Escherichia coli E.coliBL21(DE3)-pLySs competent cells for later expression of the target protein. Inoculate the expression strain with shaking at 37°C overnight after activation, take 2ml of activated bacteria and add it to 200ml LB medium for shaking at 37°C for 6 hours; add protein inducer IPTG (1.0mmol/L) 100ul to a final concentration of 0.5mmol/L, Induce and shake at 37°C for 6 hours; 4°C, 8000rpm, centrifuge for 20 minutes to harvest the wet bacteria; resuspend the bacteria in PB buffer (Na2HPO4·12H2O 20mMol/L, Na2HPO4·12H2O 20mMol/L, pH7.4) 10ml/g wet bacteria, Sonicate the bacteria 3 times in an ice bath (600-800W, 3s and 8s, 99 cycles); 4°C, 8000rpm, centrifuge for 20min, collect the supernatant and precipitate respectively, and perform acrylamide gel electrophoresis on the supernatant and precipitate respectively; SDS-PAGE electrophoresis analysis showed that compared with E.coliBL21(DE3)-pLySs empty bacteria, there were obvious specific protein expression bands at a molecular weight of about 45kDa, mainly expressed in the supernatant form, and the protein in the precipitate was relatively small. As shown in Figure 1.

3. Fermentation expression of fusion protein BG

Fermentative expression of BG in E.coliBL21(DE3)-pLySs strain. A single colony was obtained after three-section line culture, and a single colony was picked and inoculated in LB liquid medium containing chloramphenicol and kana antibiotics at 37°C and 180rpm for 12-16h as the first-class seed; Transplant at an inoculation ratio of 1:1000, and shake culture at 37°C and 180rpm for 12-16h as secondary seeds; inoculate 70mL of secondary seed bacterial liquid into a fermenter containing 2L of medium, and then add 2mL of kana For mymycin solution and chloramphenicol solution, adjust the pH to 6.8-7.4, set the dissolved oxygen to about 20% in the enrichment stage, and the associated speed (180-800rpm); start from the 6th hour, feed according to the dissolved oxygen, and induce The dissolved oxygen is controlled at about 5% in the stage; 1mL IPTG inducer is added at the 7th hour to a final concentration of 1.0mmol/L, and the fermentation is stopped after 5 hours of cultivation; after 12 hours of fermentation, about 83g of wet bacteria can be obtained, and the obtained bacteria are washed in turn , resuspension (10mL of 1g wet bacteria resuspended in 20mM PB), high-pressure homogenate lysis and centrifugation (4°C, 10000rpm, 20min.) processing; take BG whole bacteria, BG supernatant and BG precipitate resuspension, carry out 15 %SDS-PAGE gel electrophoresis analysis; it can be seen that the fusion protein BG (at 45kDa) is expressed in the supernatant, as shown in FIG. 1 .

4. Industrial fermentation of fusion protein BG

After a small amount of expression and fermentation, the fusion protein BG has the advantages of high expression yield, stable expression, insensitivity to temperature and salt ion concentration, and expression in the supernatant. Therefore, the fusion protein BG was fermented to express the target protein in a 30L fermenter in Lanzhou Institute of Biological Products. The fermentation parameters are shown in Table 1, and the results are shown in Figure 2.

Table 1 BG-20200909 fermentation parameters

5. Purification of Fusion Protein BG

Resuspend the fermented Escherichia coli in 20mM PB (pH=7.4) buffer at a ratio of 10%, lyse with high-pressure homogenate (break once at 600bar, repeat three times at 900bar), centrifuge at 12000rpm for 20min and collect the supernatant containing the target protein clear. First, the supernatant was salted out with a saturated ammonium sulfate solution with a volume fraction of 35%, and the salted out protein was resuspended with PB buffer. Then, the supernatant was collected by centrifugation for hydrophobic chromatography, and 5 mL of Butyl Sepharose High Performance (GE Healthcare) was selected for purification, and the loading buffer system was finally determined to be 20 mM PB (pH=8.0)+2M NaCl. After loading the sample, set the 100-0% B pump [20mM PB (pH=8.0) + 2M NaCl] for 40min for linear elution. UV value greater than 40 to collect, less than 100 to stop. The final purified product was analyzed by SDS-PAGE electrophoresis, and the electrophoretic gel image was scanned by image Lab software to analyze the protein purity. The detection sensitivity was set to 50%, and the purity of the fusion protein BG was greater than 95%. As shown in Figure 3 and 4.

Embodiment 2: the preparation of fusion protein BG subunit vaccine

Use PBS to prepare 1mg/mL PolyI:C; use PBS to prepare cationic liposome dimethyl hexadecyl ammonium (DDA) and cholesterol mixture, the concentration of DDA in this mixture is 5mg/mL, and the concentration of cholesterol is 0.5mg/mL mL, dissolved in a water bath at 80°C for 10 minutes, cooled to room temperature for later use; dilute the BG fusion protein with PBS buffer to the required concentration. Fully mix the protein and Poly I:C solution at a volume ratio of 1:1, and place at room temperature for 1 min; then add an equal volume of DDA solution to the mixture drop by drop, and emulsify the vaccine so that the vaccine is in the form of a uniform cream, and the sub unit vaccine. When the vaccine is used for immunization, the dosage is 200 μL/mouse.

Experimental example: Detection of immunological activity of fusion protein BG and its subunit vaccine combined with fusion protein MH

1. Experimental animals: C57BL/6 mice

2. Grouping of experimental animals (a total of seven groups):

BfrB/DPC group (6 rats), GrpE/DPC group (6 rats), BG/DPC group (12 rats), MH/DPC group (12 rats), BG+MH/DPC group (12 rats), PBS group ( 18), BCG group (18)

3. Immunization of animals:

The mice were immunized by subcutaneous injection in the groin at 0, 4, and 12 weeks respectively, and immunized three times in total. The dose of immunization: PBS group was injected with 200 μL PBS, BCG group was immunized with 200 μL (5×106 CFU), MH/DPC group was immunized with 200 μL (5 μg), BG /DPC group was immunized with 200 μL (5 μg), and MH+BG/DPC group was immunized with 200 μL (5 μg each for MH and BG, the total dose was 10 μg). Twelve weeks after the last immunization, the immunogenicity was tested and the protective effect of the attenuated strain H37Ra challenged was evaluated.

4. Immune index determination method:

(1) ELISA method to detect the secretion levels of IFN-γ and IL-2 in mouse spleen lymphocytes

①Method: 12 weeks after the last immunization, the mouse spleen was aseptically removed, ground and filtered through a 200-mesh nylon mesh, and lymphocytes were separated with lymphocyte separation medium, and the lymphocyte density was adjusted to 1×107 cells/mL. Take 200 μL of cells and add them to a 24-well plate, then add 300 μL of RPMI 1640 complete medium containing BfrB or GrpE antigen (the total volume of each well solution is 500 μL, the final concentration of antigen is 5 μg/mL), and incubate at 37 °C and 5% CO2 Cultured in medium for 72h. Add the cell culture supernatant to a 96-well ELISA plate, 100ul/well, add detection antibodies and other reagents in sequence according to the ELISA instructions, wash the plate, develop color, terminate the reaction, read the plate with a microplate reader, and calculate the IFN-γ value according to the standard curve Value (pg/ml).

②Results: BfrB stimulated spleen cells of immunized mice in BfrB/DPC and BG/DPC vaccine groups respectively, and the levels of cytokines IFN-γ and IL-2 secreted by them were higher than those in BCG group (p<0.05). GrpE stimulated the splenocytes of mice immunized with GrpE/DPC in a single antigen group, and there were individual differences in the ability to secrete the cytokine IFN-γ, and the level of secreted IL-2 was significantly higher than that of the BCG group (p<0.0001); GrpE stimulated The level of cytokine IFN-γ secreted by the splenocytes of mice immunized with BG/DPC in fusion antigen group was higher than that of BCG group (p<0.05), but the secreted level of IL-2 was not significantly different from that of BCG group. As shown in Figure 5.

Twelve weeks after the last immunization, mouse spleen cells were isolated, and the spleen cells were stimulated in vitro with BfrB and GrpE single antigens respectively. After 72 hours of culture, the supernatant was collected to detect the secretion levels of IFN-γ and IL-2. Figure 5A, Figure 5B: BfrB respectively stimulates the levels of IFN-γ and IL-2 secreted by lymphocytes of mice in the single antigen BfrB and fusion antigen BG vaccine groups; Figure 5C, Figure 5D: GrpE stimulates the single antigen GrpE and fusion antigen BG vaccine respectively The levels of IFN-γ and IL-2 secreted by lymphocytes of mice in the same group. There are at least three mice in each group, Mean±SD, *p<0.05, ***p<0.001, ****p<0.0001. (2) Antigen-specific antibody level detection in serum

①Method: 12 weeks after the last immunization, the mouse serum was collected, and the antibody titers of BfrB and GrpE antigen-specific IgG, IgG1 and IgG2c in the mouse serum were detected by indirect ELISA. Individual antigens BfrB and GrpE (5 μg/mL) were respectively coated on 96-well plates (100 μL/well), and incubated overnight at 4°C; 100 μL/well of 5% calf serum/PBS solution was added, placed at 37°C, and incubated 1h; add washing solution PBST 280μL/well, wash 3 times, 1min each time; dilute serum from 1:200 to 1:51200, add to 96 empty plates respectively, place at 37°C, incubate for 1h (with 5% Serum diluted with BSA solution); wash the plate, add 1:10000 diluted rabbit anti-mouse IgG, IgG2c or IgG1 100 μL/well, place at 37°C for 1 hour; wash the plate, add TMB 100 μL/well, react in the dark at 37°C for 20 minutes, and then terminate the addition 50 μL/well; detect the absorbance OD value at a wavelength of 450 nm, and calculate the antibody titer.

②Results: BG/DPC and single antigen vaccine groups immunized mice to stimulate the body to produce single antigen BfrB and GrpE specific antibodies, but no specific antibodies were detected in BCG group and PBS group. The mouse serum antibody titer 1gG1/lgG2c ratio showed that the antigenic components of the vaccine group caused a Th1-biased immune response. As shown in Figure 6.

The BCG vaccine group was only immunized once at week 0, and the mice in the BfrB-DPC, GrpE-DPC and BG-DPC vaccine groups were immunized three times at 0, 4, and 12 weeks. Twelve weeks after the last immunization, mouse serum was collected. Individual antigens BfrB and GrpE were coated on 96-well plates to detect the levels of specific antibodies (IgG, IgG2c, IgG1). Figures 6A, 6C and 6E are the levels of IgG, IgG1 and IgG2c antibodies detected by BfrB antigen coating respectively; Figures 6B, 6D and 6F are the levels of IgG, IgG1 and IgG2c antibodies detected by GrpE antigen coating respectively.

Individual antigen-specific antibody titers in mouse sera

Note: Blood was collected from the eyeball 12 weeks after the last immunization, and the anti-BfrB and GrpE specific antibodies in the mouse serum were detected by indirect ELISA technology. At least three experimental mice per group. Anti-BfrB and GrpE specific antibodies were not detected in PBS and BCG controls.

(3) Evaluation of the protective effect (bacteria load) after H37Ra strain challenge

① Method: Inject the attenuated strain H37Ra strain 5×106 CFU/mouse through tail vein. Three weeks after the challenge, the lungs and spleens of the mice were collected, the organs were ground with a sterile mortar, and 1 mL of PBST buffer containing 0.05% Tween-80 was added to resuspend them as original. Then, the original sample was diluted 10 times in sequence to three gradients, and 100 μL of each sample and each gradient solution was drawn and spread on the 7H10 medium, and the duplicate plate was set at the same time. The plates were cultured in a 37°C incubator for 4 weeks and CFU counts were performed. The counting results of bacterial load were expressed in Log10 (CFU). Calculation formula: CFU/g = number of growing colonies × dilution factor × [suspension volume (mL) + tissue weight (g)]/tissue weight (g)/inoculation volume (mL).

②Results: Compared with the control PBS group, the lungs of the tuberculosis subunit vaccine BG/DPC group decreased by 0.35Log10 CFU (p<0.05), and there was no statistical difference in the spleen; while the combined fusion protein vaccine BG+MH/DPC group decreased the lung 1.49 Log10CFU (p<0.0001); the spleen decreased by 1.81 Log10CFU (p<0.0001), and its protective effect against H37Ra attenuated strain was equivalent to that of BCG.

Spleen and lung organs of mice were removed by aseptic operation, weighed, ground with a mortar, and resuspended by adding 1 mL of PBST buffer. This solution was then diluted in 3 gradients and plated on 7H10 solid culture plates. Incubate in a 37°C incubator for 4 weeks, count the colonies, and evaluate the protective effect of the vaccine on mice. Figure 7A and Figure 7B are the bacterial loads in the spleen and lung of the mice challenged with the tail vein of the attenuated H37Ra strain. There are at least three mice in each experimental group, Mean±SD, **p<0.01, ***p<0.001, ****p<0.0001.

The invention establishes a method for purifying and preparing the fusion protein BG, obtains the target protein with a purity greater than 95%, and meets the conditions for industrial fermentation. Compared with BCG group, the levels of BfrB-specific IFN-γ and IL-2 secreted by splenocytes of mice in BfrB/DPC and BG/DPC vaccine groups were increased; splenocytes of mice in GrpE/DPC vaccine group secreted GrpE-specific IL The level of -2 was significantly increased, and the level of GrpE-specific IFN-γ secreted by splenocytes of mice in the BG/DPC vaccine group was increased. Both the single antigen and the fusion antigen vaccine groups could produce higher levels of IgG, IgG1, IgG2c antibodies than the BCG group. The IgG1/IgG2c ratio of the antibody titer in the BG/DPC group was less than 1, suggesting that the BG/DPC subunit vaccine induced a Th1-biased cellular immune response. Mice immunized with fusion protein BG combined with DPC adjuvant did not have a good protective effect against the attenuated strain of Mycobacterium tuberculosis H37Ra; however, mice immunized with early fusion protein MH provided better protection against Mycobacterium tuberculosis than the single fusion vaccine group .

In order to develop a novel tuberculosis vaccine with better protection effect and longer protection time, the invention constructs a novel subunit vaccine containing multi-stage antigens of Mycobacterium tuberculosis. In the present invention, the replication and dormant immunodominant antigen BfrB of Mycobacterium tuberculosis and the dormant immunodominant antigen GrpE are jointly constructed into an unlabeled fusion protein BfrB-GrpE (abbreviated as BG). .coliBL21(DE3)-pLySs was expressed and purified, combined with an adjuvant to construct a tuberculosis subunit vaccine, and the vaccine was evaluated immunologically in mouse models. The fusion protein BG is stably expressed in Escherichia coli BL21(DE3)-pLySs, and the expression yield is high, and the target protein with a purity greater than 95% can be obtained through ammonium sulfate precipitation and hydrophobic column exchange chromatography. Mice immunized with fusion protein BG combined with DP(C) adjuvant showed good immunogenicity, but the protective effect against the attenuated strain of Mycobacterium tuberculosis H37Ra was not good. The present invention successfully constructs, expresses and purifies the novel fusion protein BG without any label, and the protein fuses the replication stage and dormant stage antigens of Mycobacterium tuberculosis, and can induce antigen-specific immunity. The invention can be applied to prepare effective candidate vaccines for clinical tuberculosis prevention and treatment.

Finally, it should be noted that: the above 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 foregoing embodiments, for those skilled in the art, it still The technical solutions recorded in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (9)

1. Mycobacterium tuberculosis fusion protein BfrB-GrpE is characterized in that: it is jointly constructed by Mycobacterium tuberculosis replication and dormant immunodominant antigen BfrB and dormant immunodominant antigen GrpE, and the amino acid sequence is: MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLDRDLRVEIPGVDTVRNQFDRPREALALDQERTVTDQVGRLTAVARDEGDFLGEQFMQWFLQEQIEEVALMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRLGGGGS GGGGSGGGGSVTDGNQKPDGNSGEQVTVTDKRRIDPETGEVRHVPPGDMPGGTAAADAAHTEDKVAELTADLQRVQADFANYRKRALRDQQAAADRAKASVVSQLLGVLDDLERARKHGDLESGPLKSVADKLDSALTGLGLVAFGAEGEDFDPVLHEAVQHEGDGGQGSKPVIGTVMRQGYQLGEQVLR HALVGVVDTVVVDAAELESVDDGTAVADTAENDQADQGNSADTSGEQAESEPSGS. 2. the preparation method of mycobacterium tuberculosis fusion protein BfrB-GrpE, is characterized in that: comprise the following steps: 1. Construction of Mycobacterium tuberculosis fusion protein BfrB-GrpE 2. Small batch expression of Mycobacterium tuberculosis fusion protein BfrB-GrpE 3. Fermentation expression of Mycobacterium tuberculosis fusion protein BfrB-GrpE 4. Industrial fermentation of Mycobacterium tuberculosis fusion protein BfrB-GrpE 5. Purification of Mycobacterium tuberculosis fusion protein BfrB-GrpE. 3. the preparation method of mycobacterium tuberculosis fusion protein BfrB-GrpE according to claim 2, is characterized in that: described step one is specifically: design BfrB-GrpE fusion gene, add linker between two genes, translate into The amino acid sequence is: MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLDRDLRVEIPGVDTVRNQFDRPRREALALLDQERTVTDQVGRLTAVARDEGDFLGEQFMQWFLQEQIEEVALMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRL GGG GSGGGGSGGGGS VTDGNQKPDGNSGEQVTVTDKRRIDPETGEVRHVPPGDMPGGTAAADAAHTEDKVAELTADLQRVQADFANYRKRALRDQQAAADRAKASVVSQLLGVLDDLERARKHGDLESGPLKSVADKLDSALTGLGLVAFGAEGEDFDPVLHEAVQHEGDGGQGSKPVIGTVMRQGYQLGEQVL RHALVGVVDTVVVDAAELESVDDGTAVADTAENDQADQGNSADTSGEQAESEPSGS* Underline is the linker The pET30a(+)-BfrB-GrpE gene was optimized and synthesized to obtain the pET30a(+)-BfrB-GrpE vector. 4. the preparation method of mycobacterium tuberculosis fusion protein BfrB-GrpE according to claim 2, is characterized in that: described step 2 is specially: the pET30a (+)-BfrB-GrpE carrier that constructs success is transformed into escherichia coli E.coliBL21(DE3)-pLySs competent cells are used to express the target protein in the later stage; after the expression strain is activated, inoculate at 37°C for shaking culture overnight, take 2ml of the activated bacteria and add it to 200ml LB medium for shaking at 37°C for 6 hours; add protein Inducer IPTG to a final concentration of 0.5mmol/L, induced shaking culture at 37°C for 6h; 4°C, 8000rpm, centrifuged for 20min to harvest wet bacteria; bacteria resuspended in PB buffer 10ml/g wet bacteria, ultrasonically disrupted bacteria in ice bath 3 centrifuge at 4°C, 8000 rpm for 20 min, and collect the supernatant and the precipitate, respectively, and perform acrylamide gel electrophoresis on the supernatant and the precipitate respectively. 5. the preparation method of mycobacterium tuberculosis fusion protein BfrB-GrpE according to claim 2, is characterized in that: described step 3 is specifically: BG was fermented and expressed in E.coli BL21(DE3)-pLySs strain, and a single colony was obtained after three-section line culture, and a single colony was picked and inoculated in LB liquid medium containing chloramphenicol and kana antibiotics at 37°C, Cultivate at 180rpm for 12-16h, as the first-class seed; transfer the first-class seed according to the inoculation ratio of 1:1000, 37°C, 180rpm shaking culture for 12-16h, as the second-class seed; inoculate 70mL of the second-class seed bacterial liquid Add 2mL of kanamycin solution and chloramphenicol solution to the fermenter containing 2L medium, adjust the pH to 6.8-7.4, set the dissolved oxygen to about 20% in the enrichment stage, and correlate with the rotation speed; Start at 6h, feed according to dissolved oxygen; add 1mL IPTG inducer at 7h to a final concentration of 1.0mmol/L, stop fermentation after 5h of cultivation; after 12h of fermentation, the bacteria obtained are washed, resuspended, and homogenized under high pressure Slurry lysis and centrifugation; BG whole bacteria, BG supernatant and BG precipitate resuspension were taken for 15% SDS-PAGE gel electrophoresis analysis. 6. the preparation method of mycobacterium tuberculosis fusion protein BfrB-GrpE according to claim 2, is characterized in that: described step 4 is specifically: The fusion protein BG uses a 30L fermenter to ferment and express the target protein. The fermentation parameters are shown in Table 1. Table 1BG-20200909 Fermentation Culture Parameters

7. the preparation method of mycobacterium tuberculosis fusion protein BfrB-GrpE according to claim 2, is characterized in that: described step five is specifically: Resuspend the fermented Escherichia coli in a 10% ratio in 20mM PB (pH=7.4) buffer, lyse by high-pressure homogenization, centrifuge at 12000rpm for 20min, and collect the supernatant containing the target protein; first, use the volume fraction of the supernatant 35% saturated ammonium sulfate solution was used for salting out, and the salted out protein was resuspended in PB buffer; then, the supernatant was collected by centrifugation for hydrophobic chromatography, and 5mL of Butyl Sepharose High Performance was used for purification, and finally the loading buffer system was determined 20mM PB (pH=8.0)+2M NaCl; set 100-0% B pump [20mM PB(pH=8.0)+2M NaCl] after loading the sample, 40min for linear elution; UV value is greater than 40 to collect, less than 100 to stop ; The final purified product was analyzed by SDS-PAGE electrophoresis, and the electrophoretic gel image was scanned by image Lab software to analyze the protein purity. The detection sensitivity was set to 50%, and the purity of the fusion protein BG was greater than 95%. 8. The application of the Mycobacterium tuberculosis fusion protein BfrB-GrpE in the preparation of clinical tuberculosis prevention and treatment vaccines. 9. the preparation method of mycobacterium tuberculosis fusion protein BfrB-GrpE subunit vaccine, is characterized in that: Prepare 1 mg/mL PolyI:C with PBS; prepare cationic liposome dimethyl hexadecyl ammonium and cholesterol mixed solution with PBS, the DDA concentration in this mixed solution is 5 mg/mL, the cholesterol concentration is 0.5 mg/mL, 80 ℃ water bath for 10 minutes, cooled to room temperature for later use; dilute the BG fusion protein with PBS buffer to the desired concentration; fully mix the protein and Poly I:C solution at a volume ratio of 1:1, and place at room temperature for 1 min; The same volume of DDA solution as the mixed solution was added dropwise and emulsified to make the vaccine uniform in the form of cream to obtain the subunit vaccine.

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