Use of mycobacterium tuberculosis rv3921c recombinant protein in preparation of tuberculosis vaccine

Abstract

The application relates to application of a Mycobacterium tuberculosis Rv3921c recombinant protein in preparation of a tuberculosis vaccine and belongs to the field of immunology. The application provides a Mycobacterium tuberculosis Rv3921c recombinant protein, and the amino acid sequence is shown as SEQ ID NO. 1 or SEQ ID NO. 2. The Rv3921c recombinant protein can improve the total IgG antibody level in mouse serum and the IFN-gamma level in the spleen cell supernatant, indicating that the Rv3921c recombinant protein can have a certain effect on strengthening the immunoprotective force of BCG in the latent period, and can be used for preparing a tuberculosis vaccine.

Description

Technical Field

[0001] This invention relates to the field of immunology, and more particularly to the application of Mycobacterium tuberculosis Rv3921c recombinant protein in the preparation of tuberculosis vaccines. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease primarily caused by Mycobacterium tuberculosis (MTB) and transmitted through the respiratory tract. It is also one of the three major infectious diseases prioritized for prevention and control. With the diminishing protective effect of the Bacillus Calmette-Guérin Vaccine (BCG) and the current lack of a new, highly protective TB vaccine, the TB epidemic is showing a tendency to spread. Furthermore, the increasing number of multidrug-resistant TB patients and HIV patients presents an even more severe challenge to TB epidemic control.

[0003] BCG, prepared from a live attenuated suspension of Mycobacterium bovis, is the only licensed TB vaccine to date. It provides moderate immunization against severe types of tuberculosis such as tuberculous meningitis and hematogenous disseminated pulmonary tuberculosis in infants and young children. However, its effectiveness in preventing infectious pulmonary tuberculosis, which is the predominant type in adolescents and adults, varies significantly. Studies have shown that BCG vaccination or revaccination has no significant effect on tuberculosis control. Currently, TB vaccines under research can be categorized into live attenuated vaccines, inactivated mycobacterial or extract vaccines, recombinant subunit vaccines, and viral vector vaccines. Among these, live attenuated and inactivated vaccines have mature production processes, retain a complete MTB antigen library, can elicit a broader immune response, and do not require adjuvants. However, their immunity period is relatively short, usually requiring multiple doses to boost immunity. Vaccination in immunocompromised patients (such as HIV patients) may increase their risk of TB infection. Recombinant subunit vaccines are safe, highly effective, and can be mass-produced, but their immunogenicity is weak, requiring adjuvants. Their antigenicity is closely related to the selected expression system, making it difficult to find a good and suitable expression system. Viral vector vaccines have a simple vaccination method and require fewer doses, but can elicit a lower immune response. The selection of viral vectors is limited, and they also have potential safety issues.

[0004] Currently, the prevention and control mechanism of tuberculosis is still mainly based on cellular immunity. Although specific antibodies induced by antigens and their functional characteristics in anti-tuberculosis immunity have begun to receive attention, the exact mechanism and contribution of specific antibodies in the prevention and control of tuberculosis have not yet been determined by existing technologies. Summary of the Invention

[0005] The purpose of this invention is to address the deficiencies in the prior art by providing the application of Mycobacterium tuberculosis Rv3921c recombinant protein in the preparation of tuberculosis vaccines.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides a recombinant protein of Mycobacterium tuberculosis Rv3921c, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] The present invention also provides the application of the aforementioned Mycobacterium tuberculosis Rv3921c recombinant protein in the preparation of anti-tuberculosis formulations.

[0009] The present invention also provides a nucleotide sequence encoding the recombinant protein of Mycobacterium tuberculosis Rv3921c, the nucleotide sequence encoding SEQ ID NO.1 being shown in SEQ ID NO.3;

[0010] The nucleotide sequence encoding SEQ ID NO.2 is shown in SEQ ID NO.4.

[0011] The present invention also provides the application of the nucleotide sequence in the preparation of anti-tuberculosis formulations.

[0012] The present invention also provides a recombinant plasmid comprising the aforementioned nucleotide sequence and an empty vector;

[0013] The empty carrier is a pET-28a(+) carrier.

[0014] The present invention also provides the application of the recombinant plasmid in the preparation of anti-tuberculosis formulations.

[0015] The present invention also provides a recombinant bacterium, the recombinant bacterium comprising the recombinant plasmid and an empty vector bacterium;

[0016] The empty carrier bacteria is Escherichia coli.

[0017] The present invention also provides the application of the recombinant bacteria in the preparation of anti-tuberculosis preparations.

[0018] The present invention also provides a tuberculosis vaccine, the tuberculosis vaccine comprising the Mycobacterium tuberculosis Rv3921c recombinant protein, the nucleotide sequence, the recombinant plasmid, or the recombinant bacteria.

[0019] The present invention has the following technical effects and advantages:

[0020] The Rv3921c recombinant protein of the present invention can increase the level of total IgG antibody in mouse serum and the level of IFN-γ in mouse spleen cell supernatant, indicating that the Rv3921c recombinant protein has a certain effect on enhancing the immune protection of BCG and can be used to prepare tuberculosis vaccine. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the pET-28a / TB gene recombinant plasmid.

[0022] Figure 2 The results of bacterial culture PCR identification of the pET-28a / TB gene recombinant positive plasmid;

[0023] Figure 3 The image shows the SDS-PAGE results of candidate recombinant proteins in tuberculosis serum. Lane M is the marker. Lanes 1-8 contain recombinant proteins Rv1815, Rv3354, Rv3849, Rv1411c, Rv1566c, Rv1825, Rv3807c, and Rv1579, which are soluble proteins. Lanes 9-16 contain recombinant proteins Rv0436c, Rv0835, Rv0229c, Rv1146, Rv1977, Rv3206c, Rv3738c, and Rv3921c, which are inclusion body proteins.

[0024] Figure 4 The image shows the SDS-PAGE electrophoresis results of the purified candidate recombinant protein solution from tuberculosis serum. Lane M is the marker, and lanes 1-16 are recombinant proteins Rv1815, Rv3354, Rv3849, Rv1411c, Rv1566c, Rv1825, Rv3807c, Rv1579, Rv0436c, Rv0835, Rv0229c, Rv1146, Rv1977, Rv3206c, Rv3738c, and Rv3921c, respectively.

[0025] Figure 5The following are the SDS-PAGE electrophoresis results of the Rv3921c recombinant protein. Lane M is the marker, lane 1 is the uninduced Rv3921c recombinant protein, lane 2 is the Rv3921c recombinant protein after 1 h of induction, lane 3 is the Rv3921c recombinant protein after 2 h of induction, lane 4 is the Rv3921c recombinant protein after 3 h of induction, lane 5 is the Rv3921c recombinant protein after 4 h of induction, lane 6 is the Rv3921c recombinant protein after 5 h of induction, lane 7 is the soluble expression of BL21 cells after sonication, lane 8 is the inclusion body expression of BL21 cells after sonication, lane 9 is the recombinant protein solution after Ni-Sepharose column adsorption, lane 10 is the post-column solution sample, lane 11 is elution sample I, lane 12 is elution sample II, lane 13 is elution sample III, and lane 14 is elution sample IV.

[0026] Figure 6 Serum antibody levels were analyzed in the experimental and positive control groups of mice.

[0027] Figure 7 Analysis of spleen cytokine levels in mice in the experimental group, positive control group, and negative control group. Detailed Implementation

[0028] The present invention provides a recombinant protein of Mycobacterium tuberculosis Rv3921c, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0029] In this invention, SEQ ID NO.2 is located at positions 258 to 366 of SEQ ID NO.1.

[0030] In this invention, SEQ ID NO.2 is the amino acid sequence of the recombinant Mycobacterium tuberculosis Rv3921c protein optimized from SEQ ID NO.1.

[0031] The present invention also provides the application of the aforementioned Mycobacterium tuberculosis Rv3921c recombinant protein in the preparation of anti-tuberculosis formulations.

[0032] The present invention also provides a nucleotide sequence encoding the recombinant protein of Mycobacterium tuberculosis Rv3921c, the nucleotide sequence encoding SEQ ID NO.1 being shown in SEQ ID NO.3;

[0033] The nucleotide sequence encoding SEQ ID NO.2 is shown in SEQ ID NO.4.

[0034] In this invention, SEQ ID NO.4 is located at positions 772 to 1098 of SEQ ID NO.3.

[0035] The present invention also provides the application of the nucleotide sequence in the preparation of anti-tuberculosis formulations.

[0036] The present invention also provides a recombinant plasmid comprising the aforementioned nucleotide sequence and an empty vector;

[0037] The empty carrier is a pET-28a(+) carrier.

[0038] The present invention also provides the application of the recombinant plasmid in the preparation of anti-tuberculosis formulations.

[0039] The present invention also provides a recombinant bacterium, the recombinant bacterium comprising the recombinant plasmid and an empty vector bacterium;

[0040] The empty carrier bacteria is Escherichia coli;

[0041] The preferred strain of Escherichia coli is Escherichia coli BL21.

[0042] The present invention also provides the application of the recombinant bacteria in the preparation of anti-tuberculosis preparations.

[0043] The present invention also provides a tuberculosis vaccine, the tuberculosis vaccine comprising the Mycobacterium tuberculosis Rv3921c recombinant protein, the nucleotide sequence, the recombinant plasmid, or the recombinant bacteria.

[0044] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] The biological materials used in this application were as follows: Escherichia coli DH5α competent cells and Escherichia coli BL21 expressing bacteria were purchased from Tiangen Biotech (Beijing) Co., Ltd., BALB / c mice were purchased from the Animal Center of Naval Medical University, LEAF-CD3ε antibody was purchased from Biolegend, purified protein derivative (PPD) of Mycobacterium tuberculosis was purchased from Chengdu Institute of Biological Products, and bovine serum albumin (BSA) was purchased from Gibco.

[0046] The reagents used in this application included: pET-28a(+) vector purchased from Guangzhou Baihui Biotechnology Co., Ltd., Ni-Sepharose column purchased from GE, incomplete Freund's adjuvant purchased from Abcam, TMB chromogenic solution purchased from eBioscience, erythrocyte lysis buffer (ACK) purchased from Shenzhen Dakwei Biotechnology Co., Ltd., RPMI-1640 medium purchased from Shanghai Yisheng Biotechnology Co., Ltd., and BCA kit purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0047] In the test solution of this application:

[0048] Preparation of cell lysis buffer: Add 1.4 mmol NaCl, 0.027 mmol KCl, 0.1 mmol MgCl2, 0.1 mmol Na2HPO4 and 0.018 mmol KH2PO4 to 10 mL of water. Before use, add 2 g 100× lysozyme, 250 mg 100× Dnase I, 250 mg 100× RNase I, 10 mmol 100× PMSF and 100 mmol 100× DTT, and add Triton-X100 to a final concentration of 0.5%.

[0049] Preparation of supernatant equilibrium solution: Add 20 mmol Na3PO4 and 0.5 mol NaCl to 1 L of water and adjust the pH to 7.4-8.1;

[0050] Preparation of supernatant washing solution: Add 20 mmol Na3PO4, 0.5 mol NaCl and 60 mmol imidazole to 1 L of water and adjust the pH to 7.4-8.1;

[0051] Preparation of the washing and descaling solution: Add 20 mmol Na3PO4, 0.5 mol NaCl and 500 mmol imidazole to 1 L of water and adjust the pH to 7.4-8.1;

[0052] Preparation of inclusion body equilibrium solution: Add 20 mmol Na3PO4, 8 mol urea and 0.5 mol NaCl to 1 L of water and adjust the pH to 7.4-8.1;

[0053] Preparation of inclusion body washing solution: Add 20 mmol Na3PO4, 8 mol urea, 0.5 mol NaCl and 10 mmol imidazole to 1 L of water and adjust the pH to 7.4-8.1;

[0054] Preparation of inclusion body eluent: Add 20 mmol Na3PO4, 8 mol urea, 0.5 mol NaCl and 500 mmol imidazole to 1 L of water and adjust the pH to 7.4-8.1;

[0055] Preparation of the improved C7 refolding solution: Add 50 mmol Tris-HCl, 1 mmol TCEP, 250 mmol NaCl and 0.2 mol L-arginine to 1 L of water and adjust the pH to 7.5;

[0056] Preparation of coating solution: Add 0.05 mol Na2CO3-NaHCO3 to 1 L of water and adjust the pH to 9.6;

[0057] Preparation of blocking solution: Add 137 mmol NaCl, 2.7 mmol KCl, 10 mmol Na2HPO4, and 2 mmol KH2PO4 to 1 L of water and add Tween-20 to a final concentration of 0.05%.

[0058] Example 1: Screening of candidate recombinant proteins from tuberculosis serum

[0059] Specific antibody levels for tuberculosis serum antigens were obtained by comparing serological screening of a GTS-TB fusion expression screening library composed of 408 tuberculosis antigens (see PubMed PMID: 26481294). Combined with literature review and bioinformatics prediction, 16 strongly positive (OD) antibodies for tuberculosis serum antigens were ultimately obtained. 450nm Candidate recombinant proteins for tuberculosis serum with values ​​> 0.5 and for which no immunogenicity studies have been reported are shown in Table 1.

[0060] Table 1. Candidate recombinant proteins obtained from tuberculosis serum screening.

[0061]

[0062]

[0063] Example 2: Construction of pET-28a / TB gene recombinant plasmid

[0064] (1) Primer design: Using the H37Rv Mycobacterium tuberculosis standard strain genome (provided by the Department of Tuberculosis, Shanghai Pulmonary Hospital, see NCBI ID: 1773) as a template, primer sequences for synthesizing candidate recombinant protein genes of various tuberculosis serum were designed and sent to BGI Genomics for synthesis. The nucleotide sequences of each primer are shown in Table 2.

[0065] Table 2 Primer sequences used for synthesizing candidate recombinant protein genes from tuberculosis serum.

[0066]

[0067]

[0068] (2) PCR amplification: The PCR amplification system, in 50 μL volume, includes 25 μL of 2×GC Buffer, 1 μL of H37Rv Mycobacterium tuberculosis genome template, 0.5 μL each of the forward primer (F) and reverse primer (R) with an independent concentration of 10 μmol / L as described in step (1), 5 μL of dNTPs, 0.2 μL of Takara PCR enzyme, and the remainder ddH2O. After mixing, PCR amplification is performed according to the following program: denaturation at 94℃ for 5 min → (denaturation at 94℃ for 30 s → annealing at 58℃ for 30 s → extension at 72℃ for 1 min) × 35 cycles → extension at 72℃ for 5 min → storage at 10℃ to obtain the candidate recombinant protein gene of tuberculosis serum.

[0069] (3) Construction of recombinant plasmids: The candidate recombinant protein genes from tuberculosis serum obtained in step (2) were double-digested with the restriction endonucleases listed in Table 2 to obtain the digested recombinant protein genes. The pET-28a(+) vector was then digested with the corresponding restriction endonucleases to obtain the digested vectors. Following the instructions for using the pET-28a(+) vector, each digested recombinant protein gene was ligated into the digested vector to obtain the pET-28a / TB gene recombinant plasmid, as shown below. Figure 1 As shown.

[0070] (4) Transformation: Following the instructions for E. coli DH5α competent cells, the pET-28a / TB gene recombinant plasmids obtained in step (3) were transformed into DH5α competent cells. After being plated on LB medium (100 μg / mL Kan+) and cultured overnight at 37°C, monoclonal cells were obtained. Monoclonal cells were picked and mixed with 1 μL ddH2O to obtain bacterial suspensions for PCR positive identification.

[0071] (5) Bacterial culture PCR positive identification: The bacterial culture PCR positive identification system, in 15 μL volume, includes 7.5 μL of 2×GC Buffer, 1 μL of the bacterial culture described in step (4), 0.5 μL each of the forward primer (F) and reverse primer (R) with an independent concentration of 10 μmol / L described in step (1), 2.5 μL of dNTPs, 0.1 μL of Takara PCR enzyme, and the remainder ddH2O. After mixing, the bacterial culture PCR positive identification and DNA sequencing were performed according to the PCR amplification program described in step (2), and the pET-28a / TB gene recombinant positive plasmid was obtained. The results are as follows. Figure 2 As shown.

[0072] The results showed that the recombinant positive plasmids of each pET-28a / TB gene were successfully constructed.

[0073] Example 3: Expression and detection of candidate recombinant proteins in tuberculosis serum

[0074] (1) Transformation of recombinant positive plasmids: Following the instructions for Escherichia coli BL21 expression, the recombinant positive plasmids of pET-28a / TB gene obtained in Example 2 were transformed into BL21 expression bacteria. After being plated on LB medium (100 μg / mL Kan+) and cultured overnight at 37°C, positive monoclonal cells were obtained. Each positive monoclonal cell was then picked and seeded into 5 mL of LB medium (100 μg / mL Kan+) and cultured overnight at 37°C until OD. 600 =0.6, thus obtaining BL21 seed solution.

[0075] (2) Expression of candidate recombinant proteins in tuberculosis serum: 1 mL of each BL21 seed culture from step (1) was inoculated into 100 mL of 2×YT culture medium (100 μg / mL Kan+) and cultured at 37℃ and 250 rpm for 4 h until OD. 600 =0.7, add IPTG to a final concentration of 1 mmol / L to induce the expression of candidate recombinant proteins from tuberculosis serum, and continue culturing for 5 h to obtain BL21 expression solution. Centrifuge each BL21 expression solution at 8000 rpm for 10 min at 4℃ to obtain BL21 cells.

[0076] (3) Detection of candidate recombinant proteins in tuberculosis serum: The BL21 cells obtained in step (2) were repeatedly freeze-thawed twice at -80℃ and room temperature. 10 mL of cell lysis buffer was added to each cell for ultrasonic disruption. The ultrasonic disruption power was set to 500 W, and the disruption was performed for 10 seconds per disruption, with a 10-second interval between each disruption, for a total of 10 minutes, to obtain disrupted BL21 cells. SDS-PAGE was used to detect the presence of candidate recombinant proteins in the disrupted BL21 cells. The results are as follows: Figure 3 As shown.

[0077] The results showed that among the candidate recombinant proteins in tuberculosis serum, Rv1815, Rv3354, Rv3849, Rv1411c, Rv1566c, Rv1825, Rv3807c, and Rv1579 were expressed in soluble form, while Rv0436c, Rv0835, Rv0229c, Rv1146, Rv1977, Rv3206c, Rv3738c, and Rv3921c were expressed as inclusion bodies.

[0078] Example 4: Purification of candidate recombinant proteins from tuberculosis serum

[0079] (1) Purification of soluble tuberculosis serum candidate recombinant proteins: The Ni-Sepharose column was pre-equilibrated with 25 mL of supernatant equilibration buffer, and the post-column liquid sample was collected. The soluble tuberculosis serum candidate recombinant proteins obtained in Example 3 were filtered through a 0.45 μm filter to remove impurities and then loaded onto a Ni-Sepharose column. Then, 15 mL of supernatant equilibration buffer, 15 mL of supernatant washing buffer, and 15 mL of supernatant equilibration buffer were added sequentially to wash the Ni-Sepharose column. Finally, 15 mL of supernatant washing buffer was added to elute the soluble tuberculosis serum candidate recombinant proteins from the Ni-Sepharose column, and the eluent was collected into centrifuge tubes to obtain the purified soluble tuberculosis serum candidate recombinant protein solution. The purified soluble tuberculosis serum candidate recombinant protein solution was subjected to SDS-PAGE electrophoresis. The specific steps were as follows: 4 μL of supernatant was added to each sample. 5×SDS-PAGE protein loading buffer (containing mercaptoethanol) was used to thoroughly denature candidate recombinant proteins from tuberculosis serum by boiling in a water bath for 5 min. After each sample cooled to room temperature, it was added to the wells of the SDS-PAGE gel. The upper gel was electrophoresed at a constant voltage of 80V for 20 min, and the lower gel was electrophoresed at a constant voltage of 120V until bromophenol blue ran out of the gel. After SDS-PAGE electrophoresis, the gel plate was removed, stained with Coomassie Brilliant Blue for 10 min, and destained repeatedly until the background was clear. The results are as follows. Figure 4 Lanes 1 to 8 are shown in the diagram.

[0080] (2) Purification of tuberculosis serum candidate recombinant proteins expressed by inclusion bodies: The tuberculosis serum candidate recombinant proteins expressed by inclusion bodies obtained in Example 3 were purified according to the method described in step (1) to obtain a purified solution of tuberculosis serum candidate recombinant proteins expressed by inclusion bodies. The difference is that in this step, the buffers used are inclusion body equilibration buffer, inclusion body washing buffer, and inclusion body elution buffer, respectively. The results are as follows: Figure 4 Lanes 9-16 are shown in the figure. The feasibility of the purification method for candidate recombinant proteins from tuberculosis serum was verified using Rv3921c recombinant protein as an example. The results are as follows: Figure 5 As shown.

[0081] (3) Analysis of expression level and purity of candidate recombinant proteins in tuberculosis serum

[0082] Take 10 mL of each tuberculosis serum candidate recombinant protein purification solution obtained in steps (1) to (2) and put them into dialysis bags with a molecular weight cutoff of 7 kDa. Dialyze each soluble tuberculosis serum candidate recombinant protein purification solution with 200 mL of PBS buffer and each inclusion body tuberculosis serum candidate recombinant protein purification solution with 200 mL of modified C7 refolding solution. The dialysis temperature is 4℃. Change the PBS buffer or modified C7 refolding solution every 6 hours and repeat the dialysis 3 times to obtain tuberculosis serum candidate recombinant protein dialysate. Use an ultrafiltration concentrator to concentrate each tuberculosis serum candidate recombinant protein dialysate to 1 mL. Refer to the operation instructions of the BCA kit to determine the concentration of each tuberculosis serum candidate recombinant protein and calculate the expression level. Perform SDS-PAGE electrophoresis on each tuberculosis serum candidate recombinant protein dialysate according to the SDS-PAGE electrophoresis method described in step (1). Use Quality One software to perform grayscale analysis and determine the purity of each tuberculosis serum candidate recombinant protein. The results are shown in Table 3.

[0083] Table 3 Expression levels and purity of candidate recombinant proteins in tuberculosis serum

[0084]

[0085]

[0086] Example 5: Serum antibody analysis of mice immunized with candidate recombinant proteins from tuberculosis serum

[0087] (1) Primary immunization of mice with BCG-enhanced recombinant protein candidate for tuberculosis serum: 90 BALB / c mice were selected and injected subcutaneously with 100 μL of 0.5 mg / mL BCG in the groin. After 8 weeks of culture, mice were obtained by primary immunization with BCG. 20 μg of each recombinant protein candidate for tuberculosis serum obtained in Example 4 was taken and diluted to 50 μL with PBS buffer. Then, 50 μL of incomplete Freund's adjuvant was added and emulsified at 8000 rpm for 10 min using a high-speed homogenizer to obtain the recombinant protein candidate for tuberculosis serum. The BCG-immunized mice were randomly divided into 18 groups, of which 16 groups were experimental groups. Each experimental group received 100 μL of the candidate recombinant protein solution for tuberculosis serum via a three-point subcutaneous injection in the neck and back. Simultaneously, BCG-immunized mice injected with 100 μL of Ag85B protein solution (containing 20 μg Ag85B protein) served as a positive control group, and BCG-immunized mice injected with 100 μL of BSA protein solution (containing 20 μg BSA protein) served as a negative control group. After culturing the experimental, positive, and negative control mice for 2 weeks, they were boosted with the candidate recombinant protein solution for tuberculosis serum according to the immunization method described above. After culturing for another 2 weeks, the experimental, positive, and negative control mice were sacrificed by cervical dislocation.

[0088] (2) Serum antibody analysis of immunized mice: Starting one week before the BCG priming, blood samples were collected weekly from each mouse via tail vein sampling. Serum samples were obtained by centrifugation at 3800 rpm for 20 min at 4°C. Candidate recombinant proteins, Ag85B protein, and BSA protein from each tuberculosis serum were diluted to a final concentration of 2 μg / mL using coating buffer. 100 μL of each protein was then coated onto a 96-well plate, incubated overnight at 4°C, and the liquid was discarded. The plate was washed three times with 300 μL of PBST solution. 200 μL of blocking buffer was added to each well, and the plate was blocked at room temperature for 2 h. The liquid was then discarded, and the plate was washed three times with 300 μL of PBST solution. The serum samples were diluted 200-fold with PBST solution containing 3% skim milk powder and added to a 96-well plate at 100 μL per well. The plate was incubated at room temperature for 2 h, and the liquid was discarded. The plate was then washed three times with 300 μL of PBST solution. Wash the plate three times with PBST solution; dilute the horseradish peroxidase (HRP)-conjugated anti-mouse IgG, IgG1, and IgG2a antibodies 5000-fold with PBST solution containing 3% skim milk powder, and add 100 μL to each well of a 96-well plate. Incubate at room temperature for 1 hour, then discard the liquid and wash the plate three times with 300 μL of PBST solution; add 100 μL of TMB chromogenic solution to each well of a 96-well plate according to the instructions for TMB chromogenic solution, incubate in the dark for 10 minutes, and then add 50 μL of 2 mol / L H2SO4 to terminate the reaction; use an ELISA reader with A... 630nm Read OD for reference wavelength 450nm Value, result as Figure 6 As shown.

[0089] The results showed that the total IgG antibody levels in the serum of mice immunized with various candidate recombinant proteins from tuberculosis serum were significantly increased. Specifically, the IgG2a levels in the serum of mice immunized with recombinant proteins Rv0436c, Rv0835, Rv1815, Rv3354, Rv3849, Rv1825, and Rv3921c were significantly higher than the IgG1 levels, exhibiting humoral immune characteristics similar to those shown in the positive control group. This indicates that the recombinant proteins Rv0436c, Rv0835, Rv1815, Rv3354, Rv3849, Rv1825, and Rv3921c have a significant effect on the immune response in mice. The immune response induced in vivo was biased towards the Th1 type; in the serum of mice immunized with recombinant proteins Rv0229c, Rv1566c, Rv3738c and Rv1579c, the level of IgG2a was significantly lower than that of IgG1, indicating that the immune response induced by recombinant proteins Rv0229c, Rv1566c, Rv3738c and Rv1579c in mice was biased towards the Th2 type; while in the serum of mice immunized with recombinant proteins Rv1411c, Rv1146, Rv1977, Rv3206c and Rv3807c, the levels of IgG1 and IgG2a were not significantly increased.

[0090] Example 6: Analysis of spleen cytokine levels in mice immunized with candidate recombinant proteins from tuberculosis serum

[0091] (1) Processing of mouse spleen: Mice in the experimental group, positive control group, and negative control group described in Example 5 were sacrificed by cervical dislocation and immersed in 75% ethanol for 3 min. The spleens were then removed and ground in 5 mL of RPMI-1640 medium until no obvious tissue fragments remained, yielding mouse spleen cell grinding fluid. The mouse spleen cell grinding fluid was centrifuged at 1500 rpm for 5 min at room temperature, the supernatant was discarded, 2 mL of ACK was added, mixed well, and allowed to stand for 5 min. The mixture was then resuspended in 8 mL of RPMI-1640 medium, centrifuged at 1500 rpm for 5 min at room temperature, the supernatant was discarded, and then 10 mL of RPMI-1640 medium was added again for resuspending. The mixture was then centrifuged at 1500 rpm for 5 min at room temperature, the supernatant was discarded, and then 10 mL of RPMI-1640 medium was added again for resuspending. Resuspend the cells in RPMI-1640 medium to obtain mouse spleen cell suspensions. Add 10 μL of each mouse spleen cell suspension to a cell counting chamber for cell counting. Centrifuge the remaining mouse spleen cell suspensions at 1500 rpm for 5 min at room temperature, discard the supernatant, and then dilute with complete RPMI-1640 medium to a final concentration of 1 × 10⁻⁶. 7 The cell count was reduced to 1 / mL to obtain a diluted solution of mouse spleen cells.

[0092] (2) Stimulation of mouse spleen cells with recombinant proteins: The mouse spleen cell dilutions described in step (1) were added to 96-well plates, with two replicate wells (A and B) for each group, 500 μL per well; 100 μL of a mixture of PPD and 400 μL of complete RPMI-1640 medium was added to well A of each mouse spleen cell dilution in the experimental group, and 20 μg of the corresponding tuberculosis serum candidate recombinant proteins obtained in Example 4 and 500 μL of complete RPMI-1640 medium was added to well B; 1 μL of each mouse spleen cell dilution was independently added to wells A and B of the positive control group. A mixture of LEAF-CD3ε antibody and 500 μL of complete RPMI-1640 medium was prepared. In wells A and B of the mouse spleen cell dilution in the negative control group, 20 μg of GST protein and 500 μL of complete RPMI-1640 medium were added independently. The cells were then cultured at 37 °C and 5% CO2 for 48 h, and the mouse spleen cell supernatant was obtained by centrifugation at 1500 rpm for 5 min at room temperature.

[0093] (3) Analysis of cytokine levels in mice: Three replicate wells (a, b, and c) were set up according to the supernatant of splenic cells from each mouse. The mouse primary antibody was diluted with coating buffer at a ratio of 1:500 to a final concentration of 1 μg / mL and added to 50 μL of each well in a 96-well plate. After incubation at 4°C overnight, the plate was washed three times with 300 μL of PBST solution. 200 μL of blocking buffer was added to each well, and the plate was blocked at room temperature for 1 h. After washing three times with 300 μL of PBST solution, 50 μL of the corresponding splenic cell supernatant from step (2) diluted with PBS buffer at a ratio of 1:20 was added to well a to detect IFN-γ levels. 50 μL of the corresponding splenic cell supernatant from step (2) diluted with PBS buffer at a ratio of 1:6 was added to well b to detect IL-4 levels. Add 50 μL of the supernatant of each mouse spleen cell as described in step (2) diluted 1:1 with PBS buffer to detect IL-10 levels. After incubation at 37°C for 1 h, wash the plate 5 times with 300 μL PBST solution. Dilute biotin-labeled mouse secondary antibody with PBST solution at a ratio of 1:500 and add 50 μL to each well of a 96-well plate. Incubate at room temperature for 2 h and wash the plate 5 times with 300 μL PBST solution. Dilute avidin-labeled HRP with PBST solution at a ratio of 1:1000 and add 50 μL to each well of a 96-well plate. Incubate at room temperature for 30 min and wash the plate 7 times with 300 μL PBST solution. Add 50 μL of TMB chromogenic solution to each well of a 96-well plate according to the instructions for using TMB chromogenic solution. Incubate in the dark for 10 min and then add 25 μL of 2 mol / L H2SO4 to terminate the reaction. Use an ELISA reader with A 630nm Read OD for reference wavelength 450nm The value is calculated based on the standard curve formula y = 2.3855x - 0.4132(R). 2 =0.991, x is OD 450nm The concentration of IFN-γ in the supernatant of spleen cells from each mouse was calculated using the IFN-γ value (y = IFN-γ concentration). The results are shown in Table 4 and 5. Figure 7 As shown.

[0094] Table 4. IFN-γ concentrations in the supernatant of spleen cells from various mice

[0095]

[0096]

[0097] The results showed that the IFN-γ level in the spleen cell supernatant of the positive control group was significantly higher than that of the negative control group (P<0.01). In the experimental group, the IFN-γ level in the spleen cell supernatant immunized with Rv3921c recombinant protein was significantly higher than that of the negative control group (P<0.01). The IFN-γ concentrations in the spleen cell supernatant immunized with the other 15 recombinant proteins ranged from 500 to 800 pg / mL, significantly lower than those in the positive control group, and showed no statistically significant difference compared to the negative control group. These results indicate that the Rv3921c recombinant protein can enhance the immunoprotective effect of BCG.

[0098] As can be seen from the above embodiments, the present invention provides the application of Mycobacterium tuberculosis Rv3921c recombinant protein in the preparation of tuberculosis vaccines. Immunization of mice with the Rv3921c recombinant protein of the present invention significantly increased the total IgG antibody level in mouse serum, and the IgG2a level was significantly higher than the IgG1 level, indicating that the immune response induced in mice was biased towards the Th1 type. The IFN-γ level in the supernatant of mouse spleen cells was significantly increased, indicating that the Rv3921c recombinant protein has a certain effect on enhancing the immune protection of BCG and can be used to prepare tuberculosis vaccines.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of Mycobacterium tuberculosis Rv3921c recombinant protein in the preparation of anti-tuberculosis agents, characterized in that, The amino acid sequence of the recombinant protein of Mycobacterium tuberculosis Rv3921c is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The nucleotide sequence encoding SEQ ID NO.2 is shown in SEQ ID NO.

4.

3. The use of the nucleotide sequence shown in SEQ ID NO.4 in the preparation of anti-tuberculosis formulations.

4. The application of a recombinant plasmid in the preparation of an anti-tuberculosis agent, characterized in that, The recombinant plasmid includes the nucleotide sequence described in claim 3 and an empty vector; The empty carrier is a pET-28a(+) carrier.

5. The application of a recombinant bacterium in the preparation of an anti-tuberculosis agent, characterized in that, The recombinant bacteria include the recombinant plasmid of claim 4 and the empty vector bacteria; the empty vector bacteria is Escherichia coli.

6. A tuberculosis vaccine, characterized in that, The tuberculosis vaccine comprises the Mycobacterium tuberculosis Rv3921c recombinant protein as described in claim 1, the nucleotide sequence as described in claim 2, the recombinant plasmid as described in claim 4, or the recombinant bacteria as described in claim 5.

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