Brucella-tubercle bacillus bigeminy recombinant vaccine based on outer membrane protein OMP31 and application of brucella-tubercle bacillus bigeminy recombinant vaccine
By constructing the recombinant BCG-OMP31, the safety and effectiveness of the bi-linked vaccine of Brucella and Tuberculosis in the prior art was solved, effective protection of Brucella, and significantly improved the immune response and protective effect of mice.
Patent Information
- Application Number
- CN202510424562.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-07
- Publication Date
- 2025-07-04
AI Technical Summary
The prior art is difficult to provide a safe and effective double vaccine of Brucella and Tuberculosis, and cannot protect both pathogens at the same time. The existing BCG vaccine carrier vaccine has the problem of insufficient immune effect in preventing Brucella infection diseases.
The recombinant BCG-OMP31 was constructed using BCG-OMP31, and the Brucella OMP31 protein was expressed through gene recombinant technology to form a recombinant BCG, which was used to simultaneously protect Brucella and tuberculosis bacteria, and to verify its immune effect in immunized mice.
The successful construction of the recombinant BCG-OMP31 significantly improved the immune protection effect of mice, promoted T lymphocyte proliferation, formed an immune state dominated by Th1 immune response, and improved the immune protection against Brucella.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and particularly to a Brucella - Mycobacterium tuberculosis bivalent recombinant vaccine based on outer membrane protein OMP31 and its application. Background Art
[0002] Bacillus Calmette - Guérin (BCG) is an attenuated strain of Mycobacterium bovis and is mainly used for the prevention of tuberculosis (TB). BCG is one of the most widely used vaccines globally. It has been reported that BCG has a non - specific protective effect against pathogens other than tuberculosis and can significantly reduce child mortality. In the past few decades, recombinant BCG (rBCG) has been used as a vector to express heterologous antigens, thereby stimulating immune responses against bacterial, viral, and parasitic infections. BCG has the advantages of low toxicity, safety, effectiveness, thermal stability, easy large - scale production, low cost, and being unaffected by maternal antibodies. The neonatal BCG vaccination not only induces immunity but also cross - protection against many other infectious pathogens, including Candida albicans, Staphylococcus aureus, respiratory syncytial virus (RSV), influenza A virus, and herpes simplex virus type 2. This is consistent with its ability to reduce other respiratory infections in children. It provides a new idea for the development of Brucella vaccines based on the BCG vector. Summary of the Invention
[0003] The object of the present invention is to provide a Brucella - Mycobacterium tuberculosis bivalent recombinant vaccine based on outer membrane protein OMP31 and its application to solve the problems existing in the above - mentioned prior art. The present invention uses BCG as a vector to construct a safe and effective recombinant Brucella BCG vaccine, which serves as a bivalent vaccine that can protect against both Brucella and Mycobacterium tuberculosis simultaneously. After immunizing mice, experimental studies such as detecting antibody levels, in vitro splenocyte proliferation, IFN - γ release from splenocytes, and challenge protection are carried out to verify the immune effect of the novel Brucella - Mycobacterium tuberculosis bivalent vaccine based on the BCG vector.
[0004] To achieve the above object, the present invention provides the following solutions:
[0005] One of the technical solutions of the present invention is a recombinant BCG rBCG - OMP31, and the recombinant BCG rBCG - OMP31 comprises BCG and the OMP31 gene.
[0006] Another technical solution of the present invention is the application of the recombinant BCG rBCG - OMP31 in the preparation of a vaccine for preventing and / or treating Brucella - infected diseases.
[0007] A further technical solution of the present invention is a vaccine for preventing and / or treating Brucella - infected diseases, which comprises the recombinant BCG rBCG - OMP31.
[0008] Fourth technical solution of the present invention, an application of the recombinant BCG rBCG-OMP31, wherein the application is to prepare a kit for treating and / or preventing Brucella.
[0009] Based on the above technical solutions, the present invention has the following technical effects:
[0010] The present invention aims to develop a Brucella - Mycobacterium tuberculosis combined vaccine. Based on the Brucella OMP31 gene and the tuberculosis vaccine strain BCG (rBCG) vector, gene recombination is carried out to construct a recombinant vector, and the immune protection effect of mice is verified. Methods: First, construct a recombinant shuttle vector; culture and identify BCG to obtain BCG competent cells; electrotransfer the shuttle vector into the competent cells, and verify by PCR, Western blot and growth curve; immunize mice with the successfully constructed recombinant BCG, and detect the spleen index and spleen CFU after challenge with Brucella to evaluate the vaccine protection. Results: The recombinant BCG rBCG-OMP31 was successfully constructed, and the recombinant BCG strain grew well; after immunizing mice, it promoted the proliferation of T lymphocytes, formed an immune state dominated by Th1-type immune response, and significantly improved the immune protection of mice. This provides data support for the development of a safe and effective BCG vector candidate vaccine against Brucella. Description of the Drawings
[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.
[0012] Figure 1 PCR verification of the recombinant shuttle plasmid. Among them, M, DNA marker; 1, negative control; 2, pMV361-OMP31.
[0013] Figure 2 Double digestion identification of the recombinant expression plasmid. Among them, 1, recombinant plasmid; 2, recombinant plasmid pMV361-OMP31 digested with BamH I - EcoR I; M, DNA marker.
[0014] Figure 3 Resistance screening of the recombinant BCG.
[0015] Figure 4 PCR identification of the genomic DNA of the recombinant BCG. Among them, M, DNA marker; 1, rBCG-pMV-361; 2, rBCG--OMP31.
[0016] Figure 5 To identify recombinant BCG by Western blot.
[0017] Figure 6 For the growth curve of recombinant BCG. Among them, A: BCG; B: rBCG-pMV361-OMP31.
[0018] Figure 7 To detect the IFN-γ secretion level of splenocytes from mice immunized with recombinant BCG by ELISpot. Among them, A is the IFN-γ dot plot and B is the statistical chart.
[0019] Figure 8 For the levels of specific antibodies IgG, IgG1 and IgG2a in the sera of mice immunized with recombinant BCG. Among them, A: changes in IgG antibody level; B: changes in IgG2a antibody level; C: changes in IgG1 antibody level; D: changes in IgG2a / IgG1 ratio.
[0020] Figure 9 For the virulent challenge protection effect of mice immunized with recombinant BCG. Among them, A: spleen index of mice; B: bacterial load in the spleens of mice. Specific implementation manners
[0021] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.
[0022] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.
[0024] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of this application are merely exemplary.
[0025] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.
[0026] The technical solutions of the present invention, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or are publicly available.
[0027] An embodiment of the present invention provides a recombinant BCG rBCG-OMP31, and the recombinant BCG rBCG-OMP31 includes BCG and the OMP31 gene.
[0028] In some specific embodiments, its nucleotide sequence is as shown in SEQ ID NO.1.
[0029] An embodiment of the present invention also provides the application of the recombinant BCG rBCG-OMP31 in the preparation of a vaccine for preventing and / or treating Brucella infection diseases.
[0030] An embodiment of the present invention also provides a vaccine for preventing and / or treating Brucella infection diseases, including the recombinant BCG rBCG-OMP31.
[0031] An embodiment of the present invention also provides an application of the recombinant BCG rBCG-OMP31, and the application is to prepare a kit for treating and / or preventing Brucella.
[0032] In some specific embodiments, the application is the application of the recombinant BCG rBCG-OMP31 in the preparation of a vaccine for inducing a Th1-type immune response.
[0033] Example 1
[0034] 1.1 Materials
[0035] 1.1.1 Strains and Vectors
[0036] The BCG strain and the pMV361 shuttle vector are preserved by Shihezi University; the Brucella M28 standard strain is preserved by Tiankang Biopharmaceutical Co., Ltd.
[0037] 1.1.2 Main Reagents and Main Instruments
[0038] Restriction endonucleases (BamH I, EcoR I), T4 DNA ligase were all purchased from Takara; the bacterial genomic DNA extraction kit was purchased from Shanghai Jingnuo Biotechnology Co., Ltd.; magnesium citrate, sodium glutamate, magnesium sulfate, potassium dihydrogen phosphate were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.; 7H9 Broth and 7H10 Ager were purchased from BD; Middlebrook ADC enrichment broth, Middlebrook OADC enrichment broth; the acid-fast staining kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. The McFarland turbidimeter (RSM-10051B) was purchased from Shanghai Ruiqi Biotechnology Co., Ltd.; the electroporator and electroporation cuvettes were purchased from Eppendorf.
[0039] 1.2 Methods
[0040] 1.2.1 Primer design and synthesis
[0041] The primers designed in this experiment were specific amplification primers and recombinant plasmid identification primers targeting the pMV361 plasmid and the OMP31 (NC_003318.1) gene sequence. They were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. After centrifuging the synthesized primers at 12000 rpm for 10 min, they were dissolved in sterile water to a storage solution with a concentration of 100 pmol / μL and stored at -20°C for later use. The specific information of the primers is shown in Table 1.
[0042] Table 1 Primer design
[0043]
[0044] 1.2.2 Synthesis of the target gene
[0045] The codons of the target gene sequence were optimized using online software. BamH I - EcoR I restriction sites were introduced, and a stop codon (TAA) was introduced downstream. It was synthesized by Anhui General Biotechnology Co., Ltd. Subsequently, the target gene was inserted into the pMD-19T vector to construct the recombinant plasmid pMD-19T-OMP31, which was stored at -20°C for later use.
[0046] 1.2.3 Identification of the recombinant plasmid by colony PCR
[0047] The above-synthesized recombinant positive plasmid pMD-19T-OMP31 was streaked on LB solid medium, and a single colony was picked and inoculated into LB liquid medium (containing Amp resistance). It was cultured with shaking at 37°C for 2 h. 2 μL of the bacterial liquid was taken as a template for colony PCR amplification. The bacterial liquid identified as positive was transferred to 20 mL of LB medium and cultured with shaking overnight at 37°C.
[0048] Table 2 PCR reaction system
[0049]
[0050] The successfully constructed recombinant plasmid was identified by colony PCR. The results showed that pMV361-OMP31( Figure 1 ) amplified a band of 597 bp in size, which was consistent with the size of the target gene fragment, indicating that the recombinant plasmid was successfully constructed.
[0051] 1.2.4 Construction of recombinant shuttle vector
[0052] The shuttle vector pMV361 was digested with BamHI / EcoR I restriction endonucleases, and then the gel was recovered and the pMD-19T-OMP31 obtained by enzyme digestion and recovery was ligated to the shuttle vector pMV361 with T4 ligase to construct a recombinant shuttle vector.
[0053] 1.2.5 Identification of recombinant shuttle vector
[0054] The positive colonies selected from the LB culture plate of the recombinant shuttle vector were shaken and activated, and then identified by colony PCR. The bacterial solution with correct PCR amplification was transferred to a 20 mL conical flask for shaking culture, and the plasmid was extracted. Double digestion identification was carried out with BamHI / EcoR I. The recombinant plasmid with correct enzyme digestion was sent for sequencing. The recombinant shuttle plasmid with correct sequencing was named pMV361-OMP31 and stored in a glycerol tube at -20 °C for standby.
[0055] The recombinant shuttle plasmid pMV361-OMP31 was digested with BamH I-EcoR I. The results of 1.5% agarose gel electrophoresis showed that a vector fragment band of 4684 bp and three target bands of 597 bp were obtained, which were consistent with the size of the inserted target gene sequence. After subsequent sequencing identification, the recombinant shuttle plasmid was successfully constructed( Figure 2 ).
[0056] 1.2.6 Preparation of BCG competent cells
[0057] BCG was inoculated into 7H9 liquid medium and cultured for about 15 - 20 days. The bacterial solution was placed in an ice box for ice bath for 50 min. The pre-cooled BCG was collected and centrifuged at 8000 rpm for 15 min in a 4 °C centrifuge, and the supernatant was discarded. BCG was resuspended with pre-cooled sterile 10% glycerol water, centrifuged at 8000 rpm for 5 min, washed repeatedly 3 times, and the supernatant was discarded. Finally, BCG was resuspended with pre-cooled sterile 10% glycerol and aliquoted for use.
[0058] 1.2.7 Electroporation
[0059] Thaw the recombinant shuttle plasmid and BCG competent cells on ice. Sterilely aspirate 10 μL of the recombinant shuttle plasmid in a laminar flow hood and add it to 100 μL of BCG competent cells. Gently pipette to mix well, avoiding the generation of bubbles, and incubate on ice for 30 min. Slowly add the plasmid-competent cell mixture to a pre-chilled electroporation cuvette. Place it in an electroporator, set the voltage to 1800 v, the capacitance to 25 μF, the resistance to 400 Ω, and the transformation time to 5 ms. Electroporate once, twice, and three times respectively, with a 90 s interval each time. Quickly take out the electroporation cuvette and add 1 mL of antibiotic-free 7H9 liquid medium to it. Pipette to mix well, transfer it to an empty 2 mL centrifuge tube, and place it in a 37 °C constant temperature shaker for 48 h. After centrifuging at 8000 rpm for 5 min, discard the culture medium and retain 100 μL of the culture medium to resuspend the strain. Use a spreading rod to evenly spread it on a 7H10 solid medium (containing Kan resistance). Invert and place it in a 37 °C incubator for culture. Add 2 mL of sterile water to the medium every 3 - 5 days to ensure the humidity of the culture environment and prevent the medium from drying out. After culturing for about 20 days, when single colonies grow on the plate, randomly pick 10 single colonies and inoculate them into a 7H9 liquid medium with Kan resistance for culture. Collect the bacterial liquid for identification of recombinants.
[0060] 1.2.8 PCR Verification of Recombinant BCG
[0061] Collect the recombinant BCG bacterial liquid, grind the bacteria with a grinder, and extract the genomic DNA of recombinant BCG using a Mycobacterium genomic DNA extraction kit. Using the extracted genomic DNA as a template, amplify the target gene by PCR, then send the recombinant bacteria with correct PCR identification for sequencing, and store the recombinant strains with correct sequencing in a glycerol bacterial preservation tube and place it at -20 °C for storage.
[0062] Electrotransform the recombinant shuttle plasmid pMV361-OMP31 into BCG competent cells, spread it on a 7H10 solid culture plate containing Kan resistance, and incubate it inverted for about 20 days. When single colonies grow on the culture plate, perform PCR identification. As Figure 3 shown. Amplify a target band of about 597 bp in size, which is consistent with the expected size ( Figure 4 ), indicating that the recombinant shuttle plasmid has been successfully transferred into BCG competent cells. The sequences are all correct after sequencing, indicating that the recombinant BCG is successfully constructed and named rBCG-OMP31. The gene sequence of rBCG-OMP31 is shown in SEQ ID NO.1.
[0063]
[0064] 1.2.9 Western blot Verification of Recombinant BCG
[0065] Total bacterial proteins of the recombinant strain were extracted using a total bacterial protein extraction kit, mixed with protein loading buffer, and boiled for 10 min, followed by Western blot verification. Rabbit polyclonal antibody was used as the primary antibody, and goat anti-rabbit IgG was used as the secondary antibody for incubation.
[0066] The recombinant strains identified as positive by PCR were subcultured in 100 mL of 7H9 liquid medium. After 20 days, the bacterial cells were collected, and total bacterial proteins were extracted for SDS-PAGE and Western blot analysis. The results showed that specific bands could be detected in all three recombinant BCG strains when incubated with the polyclonal antibody. The size of the bands was approximately 18 kDa, which was consistent with the theoretical molecular weight of the inserted protein ( Figure 5 ), indicating the successful construction of recombinant BCG rBCG-OMP31.
[0067] 1.2.10 Growth Curve Plotting of Recombinant BCG
[0068] The correctly identified recombinant BCG was inoculated into 100 mL of 7H9 liquid medium at a ratio of 1:1000 and cultured in a shaker at 37 °C. Every three days, 1 mL of the homogenate was taken, ground, and the absorbance at OD 600 was measured to plot the growth curve and determine the logarithmic growth phase of the recombinant BCG. The growth activities of the parental strain and the recombinant strain were compared. The recombinant BCG in the logarithmic growth phase was recultured, and the bacterial solution was ground and serially diluted 10-fold. Then, 100 μL of the serially diluted BCG was evenly spread on a 7H10 solid culture plate and cultured at 37 °C for about 10 - 15 days. The colonies in the counting plate were counted and the average value was calculated to obtain the colony CFU in the logarithmic growth phase.
[0069] The correctly identified recombinant BCG was transferred to 100 mL of 7H9 liquid medium for culture. Every 3 days, 1 mL of the strain was taken to measure the OD600 value and plot the growth curve of the recombinant BCG, as Figure 6 shown. It can be seen from the curve that the growth rate of the recombinant BCG was similar to that of the parental strain BCG. The proliferation rate reached its peak at 15 days. The logarithmic growth phase was at an OD600 of about 0.9, and the bacterial solution concentration was 5×10 9 CFU / mL.
[0070] 1.2.11 Immunization of Mice with Recombinant BCG
[0071] Female BALB / c mice aged 4 - 6 weeks were randomly divided into 3 groups of 12 mice each. After colony counting of the recombinant BCG rBCG-OMP31 and the BCG strain, they were adjusted to 1×10 7CFU / mouse, subcutaneous injection, 100 μL per mouse. Set the injection of PBS as the blank control. Booster immunization was carried out 14 days after the first immunization. All vaccine groups were bled from the tail vein every 7 days after the first immunization to collect serum, which was stored at -20 °C for standby.
[0072] 1.2.12 ELISpot assay for detecting the secretion of IFN-γ by splenocytes of mice
[0073] Isolation of splenocytes from mice
[0074] (1) Three mice in each group at 28 days and 42 days after the first immunization were randomly sacrificed by cervical dislocation and soaked in 75% alcohol for 10 minutes.
[0075] (2) The spleens of mice were aseptically removed and placed in a 2 mL EP tube to weigh the spleen.
[0076] (3) Add 1 mL of homogenate to cut the spleen into pieces, grind all the tissues, and rinse with the homogenate while grinding. Filter through a 70 μm sterile cell sieve and finally collect into a 15 mL centrifuge tube, and centrifuge at 450 × g for 10 minutes.
[0077] (4) Discard the supernatant, and resuspend the splenocytes by adding 1 - 4 mL of sample diluent.
[0078] (5) Take another 15 mL centrifuge tube and add 4 mL of separation solution. Slowly add the cell suspension from the previous step to the upper layer of the separation solution and centrifuge at 490 × g for 25 minutes.
[0079] (6) Slowly aspirate the middle milky white lymphocyte layer into a new 15 mL centrifuge tube. Add 10 mL of washing solution and centrifuge at 400 × g for 10 minutes.
[0080] (7) Discard the supernatant, resuspend the cells by adding 5 mL of washing solution, and centrifuge at 250 × g for 10 minutes.
[0081] (8) Discard the supernatant and repeat step 7.
[0082] (9) Discard the supernatant, add 1 mL of 1640 culture medium (containing 10% fetal bovine serum) to the tube to resuspend the splenocytes of mice, and take 10 μL of the cells for cell counting.
[0083] ELISpot assay for detecting the secretion of IFN-γ by splenocytes of mice
[0084] (1) Wash the ELISPOT plate 4 times with sterile PBS (200 μL / well);
[0085] (2) Add 200 μL of 1640 cell culture medium containing 10% fetal bovine serum and incubate at room temperature for 30 minutes;
[0086] (3) Pour out the culture medium and spread 1×10 6 spleen cells;
[0087] (4) Add 10 μL of ConA (100 μg / mL) as the positive control well, 10 μL of PBS as the negative control well; 10 μL of protein stimulator (100 μg / mL) as the experimental group. Seal the plate with a sealing film and incubate it in a 37°C, 5% CO₂ incubator for 12 - 48 h;
[0088] (5) Discard the cell culture in the plate and wash it 5 times with PBS;
[0089] (6) Dilute the detection antibody to 1 μg / mL with PBS containing 0.5% fetal bovine serum, filter it through a 0.22 μm filter membrane and add 100 μL, then incubate at room temperature for 2 h;
[0090] (7) Discard the detection antibody in the plate and wash it 5 times with PBS;
[0091] (8) Dilute streptavidin - ALP with PBS (containing 0.5% fetal bovine serum) at a ratio of 1:1000, add 100 μL to each well and incubate at room temperature for 1 h;
[0092] (9) Discard the streptavidin - ALP in the plate and wash it 5 times with PBS;
[0093] (10) Filter the substrate solution (BCIP / NBT - plus) through a 0.45 μm filter membrane and add 100 μL for color development;
[0094] (11) Rinse with tap water to terminate the reaction
[0095] (12) Use an ELISpot reader to count the spots, take pictures for preservation and perform statistical analysis.
[0096] The cellular immune level of recombinant BCG was detected by ELISpot. 42 days after immunization of mice, spleen lymphocytes were isolated. The results showed ( Figure 7 ): After immunization of mice with recombinant BCG, after specific antigen restimulation of spleen lymphocytes, the secretion of IFN - γ could be significantly increased. Among them, the recombinant BCG rBCG - OMP31 group was significantly higher than the PBS control group and the BCG control group (p < 0.01). The results indicate that recombinant Brucella BCG rBCG - OMP31 can significantly induce the improvement of cellular immune level in mice.
[0097] 1.2.13 Detection of specific antibody IgG, IgG1, IgG2a levels by indirect ELISA
[0098] Take the above - collected immune mouse serum as the serum to be tested, and use indirect ELISA to detect the expression levels of specific antibodies IgG, IgG1, and IgG2a of a single antigen in the sera to be tested in each group. First, use the checkerboard titration method to screen the optimal dilution concentrations of the ELISA - coated antigen, the sera to be tested, and the secondary antibody.
[0099] (1) Dilute the purified recombinant protein with ELISA coating buffer, i.e., 50 mM sodium bicarbonate solution (pH = 9.6), to 4 concentration gradients: 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL as the coating antigen. Add 100 μL / well to a 96 - well ELISA plate, seal it with a sealing film, and incubate it overnight at 4°C for coating.
[0100] (2) The next day, discard the coating buffer, wash the plate 5 times with PBST, 300 μL / well. After blotting the liquid dry with blotting paper, add 5% BSA blocking solution, 200 μL / well, and block it at 37°C for 2 h.
[0101] (3) Discard the blocking solution, wash it 5 times with PBST. Use the serum of mice immunized with Brucella melitensis M5 as the primary antibody, designated as the positive serum. Use the serum of normal mice as the primary antibody, designated as the negative serum, and dilute them with PBST to 1:50, 1:100, 1:200, and 1:400 respectively. Add them to the 96 - well ELISA plate, 100 μL / well, and incubate at 37°C for 1 h.
[0102] (4) Discard the primary antibody, wash it 5 times with PBST. Use HRP - labeled goat anti - mouse IgG, goat anti - mouse IgG1, and goat anti - mouse IgG2a as the secondary antibodies respectively, and dilute them at 1:10000, 1:50000, and 1:100000 respectively. Add 100 μL / well and incubate at 37°C for 1 h.
[0103] (5) Discard the secondary antibody, wash it 5 times with PBST. Add 100 μL of single - component TMB chromogenic solution to each well, incubate at room temperature in the dark for 15 min. Add 50 μL / well of the termination solution to terminate the reaction. Immediately place it in an enzyme - linked immunosorbent assay reader and read the OD value at a wavelength of 450 nm. Result determination: Set the OD value of the positive serum as the P value and the OD value of the negative serum as the N value. Take P / N value ≥ 2.1 as the valid value, and select the antigen - coating concentration, the dilution degree of the sera to be tested, and the dilution concentration of the secondary antibody corresponding to the maximum P / N value as the optimal conditions.
[0104] (6) Establish an indirect ELISA detection method according to the explored optimal conditions to detect the levels of specific antibodies IgG, IgG1, and IgG2a in the sera to be tested of mice immunized with each group of vaccines at 7 d, 14 d, 21 d, 28 d, 35 d, and 42 d. And calculate the ratio of IgG2a to IgG1.
[0105] The increase and decrease patterns of specific antibodies IgG and its subtypes IgG1 and IgG2a in the sera of mice at 7d, 14d, 21d, 28d, 35d, and 42d were detected by indirect ELISA to evaluate the humoral immune level induced by the recombinant BCG vector vaccine. The increase and decrease pattern of specific antibody IgG: The expression of specific antibody IgG showed a gradually increasing trend from 7d to 42d after immunization, reached the peak at 21d and then remained at a high level without a decreasing trend. The rBCG-OMP31 group was significantly higher than the PBS control group (p < 0.01) ( Figure 8 in A). This indicates that the recombinant BCG can produce a high level of specific IgG antibodies in immunized mice. The increase and decrease pattern of specific antibody IgG2a: The expression of specific antibody IgG2a showed a gradually increasing trend from 7d to 42d after immunization, reached the peak at 21d and then remained at a high level, significantly higher than the PBS control group (p < 0.01) ( Figure 8 in B). This indicates that the recombinant BCG can produce a high level of specific IgG2a antibodies in immunized mice, mediating Th1-type humoral immune responses. The increase and decrease pattern of specific antibody IgG1: The level of specific antibody IgG1 began to increase and then decrease after 14d of immunization. The recombinant BCG can produce a low level of specific IgG1 antibodies in immunized mice, mediating Th2-type humoral immune responses. The statistical results of the IgG2a / IgG1 ratio showed that the IgG2a / IgG1 ratios in the rBCG-OMP31 group of the recombinant BCG vector vaccine group were all greater than 1, indicating that the immune responses it induced were all biased towards Th1-type immune responses. And the IgG2a / IgG1 ratio was the highest at 28d of immunization, indicating that the body was still in a high-level immune response stage at 28d of immunization( Figure 8 in D).
[0106] 1.2.14 Brucella challenge
[0107] After 42d of immunization of mice in each group, 100 μL (containing 1×10 5 CFU) of the virulent strain of Brucella melitensis (M28 strain) was intraperitoneally inoculated. After challenge, the feeding and mental status changes of the mice were observed daily. After 14d, the mice were sacrificed, their spleens were taken and weighed, and the spleens of each group were separated, ground and plated for colony counting.
[0108] 1.2.15 CFU counting of mouse spleens
[0109] Six mice were randomly selected from each group 14 days after Brucella challenge, dislocated and sacrificed, weighed, soaked in 75% alcohol for 10 min, the spleen was aseptically removed and weighed, the spleen was placed in a 2 mL centrifuge tube, 1 mL of 0.2% Triton X-100 and steel beads were added, and homogenized in a homogenizer until it became a meat paste. 100 μL of tissue homogenate was taken from each group for gradient dilution, 100 μL of each gradient was spread on Brucella solid medium (TSA), inverted and cultured in a 37 °C incubator for 3-5 days, and CFU counting was performed after colonies grew on the plate. And statistical analysis was done.
[0110] To evaluate the protective efficacy of the BCG vector vaccine in immunized mice, the challenge was carried out 42 days after immunization in this experiment, the mice were sacrificed 14 days later, the spleen was aseptically removed and weighed, and the vaccine protective effect was judged by evaluating the degree of spleen swelling and the amount of bacteria carried in the spleen. The results are as Figure 9 shown. After challenge, the degree of spleen enlargement in the vaccine rBCG-OMP31 group was significantly smaller than that in the PBS control group, with a significant difference (p < 0.01) ( Figure 9 in A). The amount of bacteria carried in the spleen was significantly smaller than that in the PBS control group (p < 0.05) ( Figure 9 in B). The results indicate that the BCG vector vaccine can reduce the degree of spleen enlargement and the amount of bacteria carried during reinfection in mice.
[0111] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.
Claims
1. A recombinant BCG rBCG-OMP31, characterized in that, The recombinant BCG rBCG-OMP31 comprises BCG and the OMP31 gene.
2. The recombinant BCG rBCG-OMP31 according to claim 1, wherein Its nucleotide sequence is as shown in SEQ ID NO.
1.
3. The use of the recombinant BCG rBCG-OMP31 according to claim 1 or 2 in the preparation of a vaccine for preventing and / or treating Brucella infection diseases.
4. A vaccine for preventing and / or treating Brucella infection diseases, characterized in that, Comprising the recombinant BCG rBCG-OMP31 according to claim 1 or 2.
5. Use of the recombinant BCG rBCG-OMP31 according to claim 1 or 2, characterized in that, The said use is for the preparation of a kit for treating and / or preventing Brucella.
6. The application according to claim 5, wherein The said use is the use of the recombinant BCG rBCG-OMP31 in the preparation of a vaccine for eliciting a Th1-type immune response.