Brucella vaccine of recombinant adenovirus based on BP26 and IFNG and application

Abstract

The invention discloses a Brucella vaccine of recombinant adenovirus based on BP26 and IFNG and application, and belongs to the technical field of biological medicine. In order to develop a safe and effective brucella recombinant adenovirus vector vaccine, a brucella immunodominance antigen is designed through a genetic engineering technology, a recombinant adenovirus of an antigen-coupled molecular adjuvant IFN-gamma is constructed, and the constructed recombinant adenovirus is identified through PCR, WB, an electron microscope and the like. And the immune stimulation capability and the protective efficacy of the brucella recombinant adenovirus vector vaccine are evaluated by verifying the cellular immune response level and the humoral immune response level induced by the vaccine at a mouse level and the challenge protection efficiency, so that a foundation is laid for research and development of a brucella recombinant adenovirus candidate vaccine.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a Brucella vaccine based on recombinant adenovirus of BP26 and IFNG and its application. Background Art

[0002] Brucella is a Gram-negative facultative intracellular parasite, which can cause fetal abortion in animals, and in humans, it shows common diseases such as undulant fever and osteoarthritis. Brucellosis is prevalent in China, especially in Xinjiang, Inner Mongolia and other places with relatively high incidence rates. Implementing the prevention, control and purification of brucellosis is an important task for ensuring the development of animal husbandry and public health safety in China at present. Vaccination is the key strategy for clearing and purifying brucellosis. In recent decades, attenuated live Brucella vaccines have been considered the most effective vaccines for controlling the spread of brucellosis globally, but problems such as poor safety and serological interference have still not been solved, which has hindered the purification and clearance of brucellosis. Therefore, developing a safer and more effective brucellosis vaccine is the main research direction. With the increasing update of emerging biotechnology such as microbiology, genetic modification, molecular cloning, vector construction, etc., inactivated vaccines, attenuated live vaccines, vector vaccines, subunit vaccines, nucleic acid DNA vaccines, and nanoparticle vaccines have become the focus of research by scientists all over the world.

[0003] Adenovirus is an enveloped double-stranded DNA virus with a molecular mass of about 36 Kb. The virus particle has an icosahedral symmetric structure with a diameter of 70-100 nm. It has a wide host range, a simple structure, strong thermal stability, and is easy to achieve genetic modification. Adenovirus vector vaccines can induce innate and acquired immune responses. Adenovirus can induce strong humoral and cellular immunity and is an important engineered live vector for gene transfer and expression. The immune response induced by adenovirus vectors is closely related to the maturity of dendritic cells. Some studies have shown that adenovirus vectors can activate dendritic cells, promote the maturation of dendritic cells, improve antigen presentation ability, and thus enhance T cell responses. Currently, all adenoviruses used in vaccine research are of the E1 or E3 gene deletion type. After the virus enters the body, it can enter the cell by adsorbing to CAR on the host cell, and then the viral DNA enters the nucleus, and finally the purpose of viral transcription is achieved. Adenovirus vectors generally do not integrate into the host genome, and the viral backbone remains outside the membrane, rarely causing host genome mutations, avoiding the mutation risk caused by random integration of foreign gene insertion.

[0004] However, there are few reports on the research of novel Brucella vaccines based on adenovirus vectors. Summary of the Invention

[0005] The purpose of the present invention is to provide a Brucella vaccine based on recombinant adenovirus of BP26 and IFNG and its application to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a shuttle plasmid, which carries the BP26-IFNG gene.

[0008] Another technical solution of the present invention is a recombinant adenovirus rAd-BP26-IFNG. The preparation method of the recombinant adenovirus rAd-BP26-IFNG is as follows: co-transfect the shuttle plasmid and the adenovirus backbone plasmid pBHG into 293A cells through Lipofectamine2000 to perform homologous recombination in the cells.

[0009] Another technical solution of the present invention is the application of the shuttle plasmid or the recombinant adenovirus rAd-BP26-IFNG in the preparation of a vaccine for preventing and / or treating Brucella infection diseases.

[0010] Another technical solution of the present invention is a vaccine for preventing and / or treating Brucella infection diseases, which includes the shuttle plasmid or the recombinant adenovirus rAd-BP26-IFNG.

[0011] Another technical solution of the present invention is the application of the recombinant adenovirus rAd-BP26-IFNG, and the application is to prepare a kit for treating and / or preventing Brucella.

[0012] Based on the above technical solutions, the present invention has the following technical effects:

[0013] The present invention aims to develop a safe and effective recombinant adenovirus vector vaccine against Brucella. Through genetic engineering technology, the immune dominant antigen of Brucella is designed and a recombinant adenovirus conjugated with the antigen-coupled molecular adjuvant IFN-γ is constructed. The constructed recombinant adenovirus is identified by means such as PCR, WB, and electron microscopy, and the cellular immune response level, humoral immune response level induced by the vaccine, and the challenge protection efficiency are verified at the mouse level to evaluate the immune stimulation ability and protective efficacy of the recombinant adenovirus vector vaccine against Brucella, laying a foundation for the research and development of a candidate recombinant adenovirus vaccine against Brucella. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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 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, other drawings can be obtained based on these drawings without creative efforts.

[0015] Figure 1PCR identification of the positive clone of the recombinant adenovirus shuttle vector. Among them, M, DNA Marker; 1-2, PCR products of the recombinant plasmid.

[0016] Figure 2 Double digestion identification of the recombinant adenovirus shuttle vector. Among them, M, 1Kb DNA Marker; 1, plasmid; 2, digestion product of EcoR I - BamH I.

[0017] Figure 3 Packaging of the recombinant adenovirus (100×).

[0018] Figure 4 PCR identification of the recombinant adenovirus.

[0019] Figure 5 Western blot identification of the recombinant adenovirus. Among them, 1: total protein of 293A cells; 2: recombinant adenovirus.

[0020] Figure 6 Transmission electron microscopy identification of the recombinant adenovirus (50000×).

[0021] Figure 7 ELISpot detection of the IFN-γ secretion level of splenocytes of mice immunized with the recombinant adenovirus.

[0022] Figure 8 Levels of specific antibodies IgG, IgG1 and IgG2 in the sera of mice immunized with the recombinant adenovirus. Among them, A: change in IgG antibody level; B: change in IgG2a antibody level; C: change in IgG1 antibody level; D: change in IgG2a / IgG1 ratio.

[0023] Figure 9 Virus challenge protection effect of mice immunized with the recombinant adenovirus. Among them, A: spleen index of mice; B: bacterial load in the spleen of mice. Specific implementation manners

[0024] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered 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.

[0025] 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 can be independently included or excluded from the range.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this 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 this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0027] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this application are merely exemplary.

[0028] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0029] The technical solutions described in this 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 have been made public.

[0030] An embodiment of this invention provides a shuttle plasmid, and the shuttle plasmid carries the BP26-IFNG gene.

[0031] In some specific embodiments, the nucleotide sequence of the BP26-IFNG gene is as shown in SEQ ID NO.1.

[0032] An embodiment of this invention also provides a recombinant adenovirus rAd-BP26-IFNG, and the preparation method of the recombinant adenovirus rAd-BP26-IFNG is: co-transfecting the shuttle plasmid and the adenovirus backbone plasmid pBHG into 293A cells through Lipofectamine2000 to perform homologous recombination in the cells.

[0033] An embodiment of this invention also provides the application of the shuttle plasmid or the recombinant adenovirus rAd-BP26-IFNG in the preparation of a vaccine for preventing and / or treating Brucella infection diseases.

[0034] An embodiment of this invention also provides a vaccine for preventing and / or treating Brucella infection diseases, including the shuttle plasmid or the recombinant adenovirus rAd-BP26-IFNG.

[0035] An embodiment of the present invention also provides an application of the recombinant adenovirus rAd-BP26-IFNG, and the application is to prepare a kit for treating and / or preventing Brucella.

[0036] Example 1

[0037] 1.1 Materials

[0038] 1.1.1 Cells, Strains

[0039] 293A cells were purchased from Beijing Beina Chuanglian Biotechnology Research Institute; Escherichia coli Top10 competent cells were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Escherichia coli BJ5183 was preserved in this laboratory.

[0040] 1.1.2 Main Reagents and Instruments

[0041] EcoR I, BamH I, and Cla restriction endonucleases were purchased from TaKaRa; PmeI enzyme, PacI enzyme, and Lipofectamine2000 were purchased from Thermo scientific; pAdTrack-CMV and pAdeasy-1 were purchased from Miaoling Biotechnology Co., Ltd.; Endotoxin-free plasmid large extraction kit, DNA gel recovery kit, and plasmid small extraction kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0042] 1.1.3 Main Instruments

[0043] The fluorescence inverted microscope was purchased from Nikon. The emulsifier and high-speed refrigerated centrifuge were purchased from Eppendorf; the microplate reader was purchased from TECAN; the ultrasonic crusher was purchased from Sonics. The electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory; the constant temperature incubator, PCR instrument, fully automatic microplate reader, Nanodrop 2000 ultra-micro spectrophotometer, and autoclave were purchased from ZEALWAY, USA; the gel imaging system was purchased from BioRad.

[0044] 1.2 Methods

[0045] 1.2.1 Design and Synthesis of Target Genes

[0046] Download the gene sequences from the NCBI database according to the Brucella melitensis gene sequence numbers released by GenBank: BP26 (NC_003317.1) and ovine IFNG gene sequence number (NM_001009803.1). Use DNAstar software to optimize the codons of the target gene sequences. After introducing the ovine IFNG gene fragment into the Brucella immunodominant antigen, additionally introduce the EcoRI (GAATTC)-BamHI (GGATCC) restriction enzyme sites, and carry a His tag at the N-terminus. The gene synthesis was carried out by Anhui General Biotechnology Co., Ltd. It was named BP26-IFNG, and the primer sequences are shown in Table 1.

[0047] Table 1 Primer synthesis

[0048]

[0049] 1.2.2 Construction of recombinant adenovirus shuttle plasmid

[0050] Digest the BP26-IFNG gene and the shuttle vector VD051-max-cmv-mcs-EF1-zsgreen with the restriction enzymes EcoR I and BamH I at the same time, and recover the target fragment and the shuttle vector. Ligate the target gene and the shuttle vector overnight in a 16°C water bath. The ligation system is shown in Table 2. The next day, transform the ligation product into Escherichia coli Top10 competent cells by heat shock method, and spread them on LB solid medium (containing Amp+) respectively, and culture them inverted at 37°C for 12h - 16h.

[0051] Table 2 Ligation reaction system of target gene and vector

[0052]

[0053] 1.2.3 Identification of recombinant adenovirus shuttle plasmid

[0054] 1.2.3.1 Verification by bacterial liquid PCR

[0055] Pick a single colony into LB liquid medium (containing Amp+) for activation, culture it in a shaker at 37°C and 200 rpm for 3 - 4 h, and use this bacterial liquid as a template for PCR detection. The PCR reaction system is shown in Table 3.

[0056] Table 3 PCR reaction system

[0057]

[0058]

[0059] PCR reaction conditions: pre-denaturation at 95°C for 5 min, 30 cycles of (denaturation at 94°C for 40 s, annealing at 55 - 60°C for 30 s, extension at 72°C for 1 min), and final extension at 72°C for 10 min.

[0060] The recombinant shuttle vector was identified by PCR amplification. The results are as Figure 1 shown. The positive clone of VD051 - BP26 - IFNG was approximately 1254 bp, which was consistent with the size of the inserted target fragment.

[0061] 1.2.3.2 Restriction enzyme verification

[0062] The correct single bacterial solution identified by PCR was transferred and cultured for expansion, the bacteria were harvested, and the plasmid was extracted on a small scale. The extracted plasmid was identified by restriction enzymes EcoR I and BamH I. The correctly identified vector was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared with the target sequence using SnapGene software. After confirmation, all vectors and strains were aliquoted and stored at -80°C in the refrigerator for future use.

[0063] Table 4 Double restriction enzyme reaction system of the vector

[0064]

[0065] The recombinant plasmid VD051 - BP26 - IFNG was verified using two restriction enzymes, EcoR I - BamH I. The results are as Figure 2 shown. VD051 - BP26 - IFNG was digested into two bands of 5196 bp and 1254 bp. The sizes of the enzyme digestion bands of the recombinant plasmid were all consistent with the expected values, indicating that the recombinant adenovirus shuttle vector was successfully constructed.

[0066] 1.2.4 Packaging of recombinant adenovirus

[0067] Culture 293A cells until they reach a monolayer, and co - transfect the recombinant shuttle plasmid VD051 - MAX - cmv - mcs - EF1a - ZsGreen and the adenovirus backbone plasmid pBHG into 293A cells:

[0068] (1) One day before transfection, seed 293A cells into a 6 - well plate. When the cell density reaches 70 - 80%, perform transfection;

[0069] (2) Replace the cells with fresh medium (serum - free) before transfection;

[0070] (3) Preparation of the complex of transfection reagent Lipofectamine 2000 and plasmid: 4 μg of viral vector plasmid (backbone plasmid: shuttle plasmid = 1:1) was dissolved in serum-free DMEM medium and gently mixed; the transfection reagent was mixed with serum-free DMEM medium; added to the plasmid mixture and gently mixed, and placed at room temperature for 20 min to allow the plasmid DNA and the transfection reagent to fully combine to form a transfection complex;

[0071] (4) Take out a 6-well cell plate, add the prepared DNA-transfection reagent complex to the cell culture plate, and put it back into the incubator;

[0072] (5) After culturing for 6 h, aspirate the medium, wash once with PBS, add 2 mL of fresh complete medium, and observe the fluorescence and cytopathic effect every day;

[0073] (6) Comet-shaped CPE appeared after culturing for about 7 - 10 days. When the cells gradually detached and floated, collect the cells and supernatant. Place them in liquid nitrogen and freeze-thaw 3 times repeatedly, then centrifuge at 3000 rpm for 10 min, collect the supernatant, filter with a 0.22 μm filter membrane, store in a -80 °C refrigerator, and record this virus as the P1 generation recombinant adenovirus rAd-BP26-IFNG.

[0074] The linearized adenovirus shuttle plasmid VD051-BP26-IFNG and the adenovirus backbone plasmid pBHG were co-transfected into 293A cells to perform homologous recombination in the cells. After culturing the cells for 7 - 10 d, when obvious CPE such as bright green fluorescence, pyknosis, and vacuoles appeared in the cells, harvest the cells and supernatant for identification. Record the correctly identified recombinant adenovirus as the P1 generation, named rAd-BP26-IFNG as Figure 3 shown.

[0075] 1.2.5 PCR identification of recombinant adenovirus

[0076] Take 5 μL of the primary virus of the recombinant adenovirus rAd-BP26-IFNG, add 10 μL of proteinase K, incubate in a 55 °C water bath for 1 h, and then boil in a 100 °C metal bath for 5 min. This is used as the genomic DNA of the recombinant adenovirus. Take 2 μL as the template for PCR amplification. Perform 1.5% agarose gel electrophoresis on the PCR product for identification. At the same time, cut the correct target band of the PCR amplification, recover the gel, and send it for sequencing.

[0077] Perform PCR amplification on the propagated recombinant adenovirus. The results are as Figure 4 shown. rAd-BP26-IFNG has a bright target band at 1254 bp. The size of the target band is consistent with the inserted target gene and can be stably passaged.

[0078] The BP26-IFNG gene sequence is shown in SEQ ID NO.1.

[0079]

[0080] 1.2.6 Expansion Culture, Purification and Concentration of Recombinant Adenovirus

[0081] Seed 293A cells into 30 - 40 10-cm cell culture plates. When the cell density reaches 80%, discard the culture medium, wash the cells 3 times with PBS. Add 8 mL of cell culture medium containing 2% fetal bovine serum to each plate of cells, and inoculate 100 μL of the P1 generation recombinant adenovirus rAd-BP26-IFNG. Place the cells in an incubator and culture for 2 - 3 days. Observe the cytopathic effect (CPE) and green fluorescence expression under a microscope every day. After obvious CPE with green fluorescence appears, harvest the supernatant and cell precipitate, freeze-thaw them 3 times repeatedly in liquid nitrogen and 37 °C water, centrifuge at 12,000 rpm for 10 min, discard the cell debris, collect the supernatant virus solution. Add 50 mL of virus precipitation solution to every 100 mL of the supernatant virus solution, place it on ice for 1 h to precipitate the virus. Then centrifuge at 8,000 rpm for 5 min at 4 °C using CsCl solution, and collect the virus solution suspended in the upper layer. Finally, add 1.40 g / mL CsCl, 1.30 g / mL CsCl solution and the virus solution to the ultracentrifuge tube in sequence. Centrifuge at 22,800 rpm for 2.5 - 4 h at 4 °C in an ultracentrifuge. Collect the virus and dialyze it in a dialysis bag. Collect the virus solution, add it to a 100 kDa ultrafiltration tube in batches, and concentrate the virus at 5,000 rpm for 20 - 50 min. Collect the concentrated virus solution, aliquot it into 1.5 mL EP tubes and store it in a -80 °C refrigerator for later use.

[0082] 1.2.7 Determination of Recombinant Adenovirus Titer and Western Blot Identification

[0083] Seed 293A cells into a 96-well plate, 1×10 4 cells / well. After overnight culture, discard the culture medium in the 96-well cell plate and wash the cells 3 times with PBS. Dilute the adenovirus in gradient: First, take 10 1.5 mL EP tubes, add 900 μL of serum-free DMEM culture medium to each tube. Add 100 μL of adenovirus to the first tube, mix well by shaking, then aspirate 100 μL and add it to the second tube, and perform 10-fold gradient dilution in this way. Add the 10 diluted virus solutions to the 96-well cell plate, add 8 wells in a column for each dilution as duplicates, 100 μL / well. Do not add virus to the last column as a blank control well. Place it in a 37 °C, 5% CO2 incubator for culture. Observe the fluorescence and CPE of the recombinant adenovirus every day. After 7 days, record the number of wells with complete CPE in each plate, and calculate the virus titer TCID50 according to the Reed-Muench method. After lysing the harvested virus solution with proteinase K, mix it with the protein loading buffer and boil it, perform SDS-PAGE gel electrophoresis, and do Western blot verification.

[0084] Using the recombinant adenovirus rAd-BP26-IFNG as an antigen, SDS-PAGE electrophoresis was performed, with the total protein of 293A cells as a negative control, and the His-tag antibody was used as the primary antibody for Western blot identification. The results were as follows Figure 5 shown. The recombinant adenovirus rAd-BP26-IFNG showed a band around 48 kDa, which was consistent with the size of the target fragment, while no band was shown in the negative control group; indicating that the recombinant adenovirus could be stably and correctly expressed in 293A cells.

[0085] 1.2.8 Electron microscopy identification of recombinant adenovirus

[0086] Take 10 μL of the recombinant adenovirus verified by Western blot, drop it onto the carbon support film of the copper mesh, dry it, and then add 10 μL of 2% phosphotungstic acid negative staining solution for negative staining, and observe the morphology of the recombinant adenovirus under a transmission electron microscope.

[0087] The purified recombinant adenovirus sample was observed using a transmission electron microscope. The results were as follows Figure 6 shown: The recombinant adenovirus presented typical morphological characteristics of adenovirus particles. The diameter of the virus particles was approximately 70 - 90 nm, and the surface of the capsid was arranged with neat non-enveloped particles, which were composed of capsomeres arranged in an icosahedron.

[0088] The correctly identified recombinant adenovirus was inoculated into 293A cells, propagated in large quantities, the virus solution was collected, and after concentration, TCID 50 detection was performed. The results are shown in Table 5: The TCID 50 of rAd-BP26-IFNG was 10 -8.5 / 0.1 mL.

[0089] Table 5 Determination of TCID 50 of recombinant adenovirus rAd-BP26-IFNG

[0090]

[0091]

[0092] 1.2.9 Immunization of mice with recombinant adenovirus vaccine

[0093] Female BALB / c mice aged 4 - 6 weeks were randomly divided into 2 groups, with 12 mice in each group. Each mouse in the rAd-BP26-IFNG group was subcutaneously immunized with 100 μL of concentrated adenovirus (containing 10 -8 TCID50), and the PBS control group: immunized with 100 μL of PBS. Immunization was performed once at 0 day and 14 days respectively. After immunization, blood was collected from the tail vein once a week. After centrifugation at 3000 r / min for 15 min at 4°C, the serum was collected and stored at -20°C for later use.

[0094] 1.2.10 Detection of IFN-γ secretion from mouse spleen lymphocytes by ELISpot

[0095] First, isolate mouse spleen lymphocytes. Randomly decapitate and sacrifice 3 mice in each group, and soak them in 75% alcohol for 10 min. Sterilely remove the mouse spleens and place them in 2 mL EP tubes to weigh the spleen weights. Add 1 mL of homogenate to cut and grind the spleens, rinse and filter with the homogenate, and finally collect in a 15 mL centrifuge tube and centrifuge at 450×g for 10 min. Discard the supernatant, add 1 - 4 mL of sample diluent to resuspend the spleen cells. Slowly add 4 mL of the upper layer of the separation solution and centrifuge at 490×g for 25 min. Slowly collect the middle ring-shaped milky white lymphocyte layer. Add 10 mL of washing solution and cleaning solution to wash the cells, and take 10 μL of the cells for cell counting. Wash the ELISPOT plate (200 μL / well) 4 times with sterile PBS; add 200 μL of 10% fetal bovine serum 1640 cell culture medium and incubate at room temperature for 30 min; pour out the culture medium, seed 1×10 6 spleen cells; 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 stimulant (100 μg / mL) as the experimental group. Seal the plate with a sealing film and place it in an incubator at 37°C and 5% CO2 for 12 - 48 h; discard the cell culture in the plate and wash 5 times with PBS; dilute the detection antibody with PBS containing 0.5% fetal bovine serum to 1 μg / mL, filter through a 0.22 μm filter membrane and add 100 μL, and incubate at room temperature for 2 h; discard the detection antibody in the plate and wash 5 times with PBS; 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. Discard the streptavidin-ALP in the plate and wash 5 times with PBS; filter the substrate solution (BCIP / NBT-plus) through a 0.45 μm filter membrane, add 100 μL for color development. Rinse with tap water to terminate the reaction. Use an ELISpot reader to count the spots, take pictures and save them, and perform statistical analysis.

[0096] Use ELISpot to detect the level of specific IFN-γ released by recombinant adenovirus-stimulated mice, so as to evaluate the cellular immune level induced by the vaccine. The results are as Figure 7 shown: The recombinant adenovirus vaccine rAd-BP26-IFNG can increase the level of IFN-γ produced by specific T cells, which is significantly higher than that of the PBS control group (p < 0.01). This indicates that the Brucella recombinant adenovirus vector vaccine constructed in the present invention can induce a high level of cellular immune response.

[0097] 1.2.11 Detection of specific antibody IgG, IgG1, IgG2a levels by indirect ELISA

[0098] Use the above - collected immune mouse serum as the serum to be tested, and detect the expression levels of specific antibodies IgG, IgG1, and IgG2a against a single antigen in the sera to be tested in each group by indirect ELISA. First, use the checkerboard titration method to screen the optimal dilution concentrations of the ELISA - coated antigen, the serum to be tested, and the secondary antibody. Establish an indirect ELISA detection method according to the optimal conditions explored to detect the levels of specific antibodies IgG, IgG1, and IgG2a in the sera to be tested 7d, 14d, 21d, 28d, 35d, and 42d after vaccination of mice in each group with the vaccine. And calculate the ratio of IgG2a to IgG1.

[0099] Use indirect ELISA to detect and analyze the growth and decline patterns of specific antibody IgG and its subtypes IgG1 and IgG2a in mouse sera at different times, and evaluate the humoral immune level of the recombinant adenovirus vector vaccine. The results are as Figure 8 shown: The growth and decline patterns of specific antibodies IgG and IgG2a showed a gradually increasing trend during 7d - 42d of immunization and remained at a high level. The recombinant adenovirus vector vaccine rAd - BP26 - IFNG was significantly higher than the PBS control group (p < 0.01). There was no significant difference in the expression of IgG1 compared with the PBS control group.

[0100] The statistical results of the IgG2a / IgG1 ratio showed that they were all greater than 1, indicating that the immune responses induced by the recombinant adenovirus vector vaccine group were all biased towards Th1 - type immune responses.

[0101] 1.2.12 Brucella challenge

[0102] After immunizing mice in each group for 42d, intraperitoneally inoculate 100 μL (containing 1×10 5 CFU) of the virulent strain of Brucella melitensis (strain M28). After challenge, observe the changes in the feeding and mental status of the mice daily. After 14d, sacrifice the mice, take their spleens and weigh them. Separate the spleens of each group, grind and smear them on plates for colony counting.

[0103] 1.2.13 CFU counting of mouse spleens

[0104] 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 spleens were aseptically removed and weighed. The spleens were placed in 2 - mL centrifuge tubes, 1 mL of 0.2% Triton X - 100 and steel beads were added, and homogenized in a homogenizer until it became a meat - paste shape. 100 μL of the tissue homogenate was taken from each group for gradient dilution, and 100 μL of each gradient was spread on Brucella solid medium (TSA), inverted and placed in an incubator at 37°C for 3 - 5 days. After colonies grew on the plate, CFU counting was performed. And statistical analysis was carried out.

[0105] To evaluate the immunoprotective effect of the recombinant adenovirus vector vaccine on mice. In this study, the mice were challenged 42 days after immunization, and the spleens of the mice were removed to calculate the spleen index and the bacterial load in the spleen. The results are as Figure 9 shown. The degree of spleen enlargement of the recombinant adenovirus vector vaccine rAd-BP26-IFNG was significantly less than that of the PBS control group, and the difference was extremely significant (p < 0.0001). The bacterial load in the spleen was also significantly lower than that of the PBS control group (p < 0.01).

[0106] The results indicate that the recombinant adenovirus vector vaccine rAd-BP26-IFNG can provide a high level of immunoprotection for mice.

[0107] In summary, the present invention prepared an adenovirus vector vaccine. This adenovirus is a non-replicating live virus that can only carry the target gene into cells, but cannot replicate in the body, will not cause infection, and is not likely to cause serum interference. The target gene of the present invention is a single antigen, and it is easily detected by serological tests.

[0108] Obviously, the above embodiments of the present invention are only examples for clearly explaining the present invention, and are not intended to limit the implementation manner 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 list all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A shuttle plasmid, characterized in that, The shuttle plasmid carries the BP26-IFNG gene.

2. The shuttle plasmid according to claim 1, characterized in that, The nucleotide sequence of the BP26-IFNG gene is shown as SEQ ID NO.

1.

3. A recombinant adenovirus rAd-BP26-IFNG, characterized in that, The preparation method of the recombinant adenovirus rAd-BP26-IFNG is as follows: The shuttle plasmid described in claim 1 or 2 and the adenovirus backbone plasmid pBHG are co-transfected into 293A cells by Lipofectamine 2000, and homologous recombination is carried out in the cells.

4. The application of the shuttle plasmid described in claim 1 or 2 or the recombinant adenovirus rAd-BP26-IFNG described in claim 3 in the preparation of a vaccine for preventing and / or treating Brucella infection diseases.

5. A vaccine for preventing and / or treating Brucella infection diseases, characterized in that, It includes the shuttle plasmid described in claim 1 or 2 or the recombinant adenovirus rAd-BP26-IFNG described in claim 3.

6. Use of the recombinant adenovirus rAd-BP26-IFNG according to claim 3, characterized in that, The application is to prepare a kit for treating and / or preventing Brucella.

Images