Bovine-derived murine anti-brucella antibodies, methods for their production and use

Abstract

The application belongs to the technical field of biological pharmacy, and particularly relates to a bovine-derived mouse anti-Brucella antibody and a preparation method and application thereof. A bovine-derived mouse anti-Brucella antibody is prepared by synthesizing a tandem epitope protein of Brucella OMP25 proteins of bovine species, sheep testis species and canine species, immunizing mice, preparing a monoclonal antibody, cloning CDR fragments in Hv and Lv regions of the antibody to corresponding positions of bovine-derived IgG, and then performing overall mutation modification on the recombinant IgG to perform bovine-derived modification. The bovine-derived mouse anti-Brucella antibody preparation has good immunogenicity and protection effect, and can block and inhibit the propagation and spread of Brucella of bovine species, canine species and sheep testis species in an animal body.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biopharmaceuticals, and particularly relates to a bovine-derived murine anti-Brucella antibody and a preparation method and application thereof. BACKGROUND

[0002] Brucellosis is a major zoonosis, which is extremely harmful to both humans and animals, and belongs to the same class of infectious diseases as rabies, SARS, and highly pathogenic avian influenza. At present, all Brucella vaccines are live vaccines, which not only have a certain pathogenic effect on humans and animals, but also interfere with serological detection and cause abortion in pregnant animals after immunization of animals. Therefore, the current prevention and control of animal brucellosis mainly relies on purification, and the use of Brucella vaccine is limited to certain areas, and cannot be used on breeding animals and dairy animals, which leads to the fact that most areas in the country can only achieve disease purification through monitoring and killing. Therefore, an effective means of antagonizing and hindering the spread of Brucella in the animal body is urgently needed.

[0003] Monoclonal antibody preparations are an effective means of antagonizing pathogens in the body, but the progress of monoclonal antibody technology in clinical treatment applications has been relatively slow, and the main reason is that most monoclonal antibodies are of murine origin, and the use of murine monoclonal antibodies in other animals or human treatment has many problems: first, it cannot effectively activate the body's complement and Fc receptor-related effector system; second, it is recognized by the body's immune system, produces an antigenic response, and is quickly cleared from the body's circulatory system. Therefore, in order to maintain the activity of monoclonal antibodies and reduce or eliminate antigen activity, the use of Fab fragments of murine antibodies and Fc fragments of target animal antibodies to chimerize into complete and active chimeric antibodies has been developed. The main idea of chimeric antibodies or target animalization of antibodies is to use the IgG of the target animal as a template, retain the FR fragments on the Hv and Lv regions, and replace the CDR fragments with the corresponding murine fragments, which not only retains the activity of the antibody, but also ensures the maximum target animal similarity of the antibody molecule, and can play an ideal role in the body.

[0004] In recent years, Brucella outer membrane protein OMP25 has become a research hotspot. Studies have found that OMP25 protein plays an important role in Brucella infection and pathogenic process, and is involved in maintaining the stability of the outer membrane functional structure of the bacteria, related to the growth and reproduction of pathogenic bacteria in the host endosome, and has important immunogenicity and antigen protection. Therefore, by means of bioinformatics analysis and other means, the antigen epitope of Brucella outer membrane protein OMP25 with clinical significance is found, the monoclonal antibody is prepared and the target animal is modified, so that the monoclonal antibody with good molecular activity and target animal consistency can be obtained. The preparation of the antibody into an in vivo injection preparation will effectively improve the prevention and treatment of brucellosis, and has important significance for the prevention and control of brucellosis, public health safety and the like.

[0005] The patent for invention with publication number CN113354742A discloses a Brucella genetic engineering subunit vaccine and a preparation method and application thereof, specifically a prokaryotic expressed Brucella outer membrane protein tandem epitope protein; the amino acid sequence of the Brucella outer membrane protein tandem epitope protein is composed of the amino acid sequences of 16 kinds of proteins such as bovine Omp25 protein, canine Omp25 protein, porcine Omp25 protein, sheep testicular Omp25 protein and sheep Omp31 protein; compared with the patent, the amino acid sequence composition of the tandem epitope protein is more complex. SUMMARY

[0006] One of the purposes of the present application is to provide a Brucella outer membrane protein OMP25 tandem epitope protein, and the amino acid sequence of the Brucella outer membrane protein OMP25 tandem epitope protein is composed of the amino acid sequences of bovine OMP25 protein, canine OMP25 protein and sheep testicular OMP25 protein.

[0007] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0008] The Brucella outer membrane protein OMP25 tandem epitope protein, and the amino acid sequence of the Brucella OMP25 protein tandem epitope protein is shown as SEQ ID NO. 1.

[0009] Further, the nucleotide sequence of the Brucella OMP25 protein tandem epitope protein is shown as SEQ ID NO. 2.

[0010] Further, the amino acid sequence of the Brucella outer membrane protein OMP25 tandem epitope protein is composed of the amino acid sequences of bovine OMP25 protein, canine OMP25 protein and sheep testicular OMP25 protein.

[0011] Further, the amino acid sequence of the Brucella outer membrane protein tandem epitope protein is composed of the amino acid sequences at positions 4-46, 143-187 of the bovine OMP25 protein, positions 79-130 of the canine OMP25 protein and positions 20-55 of the sheep testis OMP25 protein.

[0012] The second object of the present application is to provide a recombinant plasmid for expressing a Brucella outer membrane protein OMP25 tandem epitope protein.

[0013] To achieve the above object, the present application adopts the following technical scheme:

[0014] The recombinant plasmid for expressing the Brucella outer membrane protein OMP25 tandem epitope protein comprises the nucleotide sequence of the Brucella outer membrane protein OMP25 tandem epitope protein.

[0015] The third object of the present application is to provide a genetically engineered strain.

[0016] To achieve the above object, the present application adopts the following technical scheme:

[0017] The genetically engineered strain contains the recombinant plasmid for expressing the Brucella outer membrane protein OMP25 tandem epitope protein.

[0018] Further, the genetically engineered strain is constructed by transforming a host bacterium with the recombinant plasmid, and the genetically engineered strain is prepared into a seed strain of the Brucella outer membrane protein tandem epitope protein genetically engineered strain after identification, and the host bacterium is an Escherichia coli BL21 strain.

[0019] Further, the identification method of the genetically engineered strain is as follows: the genetically engineered expression strain is cultured in an LB medium at 37℃ under shaking until the logarithmic phase, then 1mM of IPTG is added to the LB medium to continue culturing for 2-3 hours. The bacterial body is collected by centrifugation, resuspended with ice-cold PBS buffer at a ratio of 1:10 (W / V), then the bacterial body is collected by centrifugation again, and the resuspension and centrifugation are repeated for 2-3 times to remove the medium components. The bacterial body is detected by SDS-PAGE electrophoresis, and compared with the untransformed Escherichia coli BL21 host bacterium, there is an obvious band at 20kD.

[0020] The fourth object of the present application is to provide a hybridoma cell.

[0021] To achieve the above object, the present application adopts the following technical scheme:

[0022] The hybridoma cell can secrete a mouse anti-Brucella outer membrane protein tandem epitope protein OMP25 antibody.

[0023] The fifth object of the present application is to provide a bovineized murine anti-OMP25 tandem epitope protein antibody.

[0024] To achieve the above object, the present application adopts the following technical solutions.

[0025] A bovineized murine anti-OMP25 tandem epitope protein antibody, the amino acid sequence of the antibody comprising a murine anti-OMP25 tandem epitope protein IgG VH region CDR region amino acid fragment and a VL region CDR region amino acid fragment.

[0026] Further, the antibody comprises bovine IgG immunoglobulin H and L chains.

[0027] Further, the antibody comprises bovine IgG immunoglobulin H and L chains.

[0028] Further, the amino acid sequence of the Hv chain of the antibody is shown in SEQ ID NO. 3; the amino acid sequence of the Lv chain of the antibody is shown in SEQ ID NO. 5.

[0029] Further, the nucleotide sequence of the Hv chain of the antibody is shown in SEQ ID NO. 4; the nucleotide sequence of the Lv chain of the antibody is shown in SEQ ID NO. 6.

[0030] Further, the amino acid sequence of the CDR1 fragment H chain of the antibody is shown in SEQ ID NO. 8, and the amino acid sequence of the L chain is shown in SEQ ID NO. 9; the amino acid sequence of the CDR2 fragment H chain is shown in SEQ ID NO. 10, and the amino acid sequence of the L chain is shown in SEQ ID NO. 11; the amino acid sequence of the CDR3 fragment H chain is shown in SEQ ID NO. 12, and the amino acid sequence of the L chain is shown in SEQ ID NO. 13.

[0031] The sixth object of the present application is to provide a recombinant eukaryotic expression vector for expressing the bovineized murine anti-OMP25 tandem epitope protein antibody.

[0032] Further, by means of artificial synthesis, a DNA fragment with a nucleotide sequence as shown in SEQ ID NO. 4 and SEQ ID NO. 6 is added with a DNA Linker and cloned into a multiple cloning site of a pCDNA3.1 vector, i.e. a pCDNA3.1 eukaryotic expression vector of a bovine-derived murine anti-OMP25 tandem epitope protein antibody of the present application.

[0033] Further, a nucleotide sequence of the DNA Lingker is shown in SEQ ID NO. 7.

[0034] A seventh object of the present application is to provide a cell transfected with the recombinant eukaryotic expression vector.

[0035] Further, the recombinant eukaryotic expression vector is transformed into an expression cell and cultured, and the culture is purified to obtain the bovine-derived murine anti-Brucella antibody.

[0036] Further, the recombinant eukaryotic expression vector is a pCDNA3.1 vector; and the expression cell is a VERO cell.

[0037] A VERO cell is selected as a target cell, a synthetic recombinant eukaryotic expression vector is transfected into the VERO cell by means of a liposome method, and then a cell line with stable high expression is screened. The specific steps are as follows: 1) in a 35mm hole, 1.5µg of the recombinant eukaryotic expression vector is added; 2) 5µL of liposomes prepared with OptiMEM are added, and incubated at room temperature for 45min. Then added to the host cell which is rinsed with OptiMEM, and incubated for 5h; the vector-liposome mixture is removed by centrifugation, and DMEM is added to the cell for culture; 3) the cell strain expressing the bMABRU monoclonal antibody is selected by pressure screening.

[0038] An eighth object of the present application is to provide an application of the bovine-derived murine anti-OMP25 tandem epitope protein antibody and / or the cell of the seventh object in preparing a medicine for preventing and / or treating brucellosis.

[0039] A ninth object of the present application is to provide an application of the bovine-derived murine anti-OMP25 tandem epitope protein antibody in preparing a medicine for blocking and inhibiting the propagation and spread of Brucella in bovine, canine and sheep testes.

[0040] A tenth object of the present application is to provide a bovine-derived murine anti-OMP25 tandem epitope protein antibody preparation.

[0041] To achieve the above objects, the present application adopts the following technical solutions:

[0042] A bovine-derived murine anti-OMP25 tandem epitope protein antibody preparation containing the bovine-derived murine anti-OMP25 tandem epitope protein antibody.

[0043] Further, the bovine-derived murine anti-OMP25 tandem epitope protein antibody preparation can block and inhibit the multiplication and spread of Brucella abortus, Brucella canis and Brucella ovis in animals.

[0044] Further, the bovine-derived murine anti-OMP25 tandem epitope protein antibody preparation is in the form of an injection, and the preparation method is as follows: filtering and sterilizing the purified bMABRU monoclonal antibody stock solution, adjusting the concentration to 50 µg / mL with sterile normal saline, and adding glucose to a final concentration of 15 µg / mL, and then loading the solution into a sterile vial, sealing and storing at 2-8°C.

[0045] The purification method of the bMABRU monoclonal antibody is as follows: repeatedly freezing and thawing the cell culture, and collecting all the cell culture liquid, and centrifuging at 4°C and 12000g for 30 min to remove impurities. The supernatant is subjected to chromatography purification with Protein G affinity chromatography filler which has been equilibrated with PBS at pH 6.5-7.5, and the eluent is PBS containing 0.2-0.5M sodium chloride at pH 6.5-7.5, and the elution peak is the bMABRU stock solution, i.e. the antibody stock solution.

[0046] The present application has the following advantages:

[0047] 1. The bovine-derived murine anti-OMP25 tandem epitope protein antibody of the present application is in the form of an injection, and has a uniform appearance, no odor and no bacteria, and no pyrogen, abnormal toxicity and acute toxicity reaction, and is safe; the detection result is negative by ELISA method using goat anti-mouse IgG (GAM-IgG), and the detection titer can reach 1:12800 or more by using goat anti-bovine IgG (GAH-IgG), and the antibody has good immunogenicity.

[0048] 2. The bovine-derived murine anti-OMP25 tandem epitope protein antibody of the present application can block and inhibit the multiplication and spread of Brucella abortus, Brucella canis and Brucella ovis in animals.

[0049] 3. The bovine-derived murine anti-OMP25 tandem epitope protein antibody of the present application can produce good protection effect, and the protection rate is more than 90%. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 For SDS-PAGE detection of tandem epitope protein expression, 1-4 are detection samples taken at different induction times; 5 is detection after protein purification; and 6 is a blank BL21.

[0051] Figure 2 For the RT-PCR results of Example 4, 1 is heavy chain DNA; 2 is light chain DNA;

[0052] Figure 3 For the WB hybridization results, 1 is the purified tandem epitope protein, and the detection antibody is the prepared bovine-derived antibody;

[0053] Figures 4-6 For the flow cytometry graph of Example 10. DETAILED DESCRIPTION

[0054] The technical solutions of the present application will be described further in detail below in combination with specific examples. Obviously, the described examples are only some of the embodiments of the present application, but not all the embodiments. Therefore, all other embodiments obtained by those skilled in the art based on the examples in the present application without creative labor fall within the protection scope of the present application.

[0055] Example 1. Construction of recombinant expression vector

[0056] Bioinformatics analysis was performed on the amino acid sequences of the outer membrane proteins OMP25 of Brucella bovine, Brucella ovis and Brucella canis from Genebank. The amino acid sequences of the tandem epitope protein of Brucella outer membrane protein OMP25 were obtained from the 4-46th, 187-197th and 143-154th positions of the OMP25 protein of bovine (GenBank: AFJ79953.1), the 87-94th position of the OMP25 protein of canine (GenBank: CDL76104.1) and the 10-27th position of the OMP25 protein of ovine testis (GenBank: ABU93464.1), as shown in SEQ ID NO. 1. The amino acid sequences were translated and codon-optimized by bioinformatics software to obtain the nucleotide sequence of the Brucella OMP25 tandem epitope protein, as shown in SEQ ID NO. 2. The DNA fragment of the protein was artificially synthesized, and a BamH I enzyme cutting site was introduced at the 5' end of the DNA fragment, and a Xho I enzyme cutting site was introduced at the 3' end of the DNA fragment. The DNA fragment was connected to the multiple cloning site of the pET28a prokaryotic expression vector to construct a recombinant expression vector.

[0057] Example 2. Preparation of OMP25 recombinant epitope protein engineering bacteria

[0058] The E. coli BL21 cell was selected as the host bacteria, and the recombinant expression vector constructed in Example 1 was transformed into the competent cells of E. coli BL21 by heat shock method, and then the genetically engineered strain stably expressing the target protein was screened. The specific steps are as follows: thaw the prepared competent E. coli BL21 cells 100 µL on ice, then add 1.5 µg of the prepared recombinant expression vector. Mix well, then place on ice for 30 min, 45°C water bath for 90 s, then place on ice again for 3 min. Add 700 µL of LB medium, activate at 37°C for 1 h, then spread on LB solid plate with kanamycin concentration of 100 µg / mL, and incubate at 37°C overnight. Randomly select a full single colony, inoculate in 5 mL of LB liquid medium with kanamycin concentration of 100 µg / mL, and incubate at 37°C with shaking. After 6 h, add IPTG to a final concentration of 1 mM to induce expression for 2-3 h. Centrifuge to collect the bacterial pellet, and detect by SDS-PAGE electrophoresis. The detection result is shown in Figure 1 Compared with the untransformed E. coli BL21 host bacteria, there is an obvious band at 20 kD.

[0059] Example 3. Preparation of OMP25 recombinant epitope protein

[0060] The engineered bacteria prepared in Example 2 were inoculated in an appropriate amount of LB liquid medium with kanamycin concentration of 100 µg / mL, and incubated at 37°C with shaking. After 6 h, IPTG was added to a final concentration of 1 mM to induce expression for 2-3 h. The bacterial pellet after fermentation was collected by centrifugation, resuspended with ice-cold PBS buffer at a ratio of 1:10 (W / V), and then the bacterial pellet was collected again by centrifugation, repeated for 2-3 times to remove the culture medium components. The bacterial pellet was resuspended with ice-cold PBS buffer at a ratio of 1:10 (W / V), and the bacterial pellet was broken by a high-pressure homogenizer. The supernatant after breaking was collected by centrifugation. The supernatant was ultrafiltered by a hollow fiber column with a pore size of 0.45 µm to remove impurities. The filtrate was collected, and concentrated by ultrafiltration with a hollow fiber column with a molecular weight cutoff of 10 kD to 20-30% of the original volume. The backflow liquid after concentration was detected by BSA method for protein quantification, and the total protein content should be not less than 30 mg / mL. The detection result is shown in Figure 1 The target protein band should account for not less than 80%. The concentrated liquid was subjected to affinity chromatography with nickel ion purification filler. The equilibrium liquid was PBS with pH 6.5-7.5 and containing 50 mM imidazole, and the eluent was PBS with pH 6.5-7.5 and containing 500 mM imidazole. The eluent was dialyzed by a dialysis bag with a molecular weight cutoff of 10 kD to remove imidazole, which was the purified OMP25 recombinant epitope protein for preparing monoclonal antibody hybridoma cells by immunizing mice.

[0061] Example 4. Obtaining of mouse anti-OMP25 recombinant epitope protein antibody sequence

[0062] The OMP25 recombinant epitope protein was sent to a biotechnology company to prepare mouse monoclonal antibody hybridoma cells, total RNA of the hybridoma cells was extracted, and DNA fragments of IgG protein VH region and VL region were cloned by RT-PCR, and the RT-PCR results are shown in Figure 2 The nucleotide sequences of the DNA fragments were sequenced and analyzed, and the nucleotide sequences of each CDR fragment and FR fragment on the VH region and VL region were determined by bioinformatics alignment and analysis.

[0063] Example 5. Obtaining bovine-derived murine anti-OMP recombinant epitope protein antibody sequence

[0064] The nucleotide sequences of the murine anti-Brucella OMP25 tandem epitope protein antibody and the H chain and L chain Hv region and Lv region nucleotide sequences of bovine-derived IgG were divided into FR1, FR2, FR3, FR4, CDR1, CDR2, and CDR3 by bioinformatics methods. The CDR fragments on the bovine-derived IgG were replaced with the corresponding regions of the murine-derived IgG by computer-aided technology. The recombinant IgG was subjected to amino acid prediction and point mutation by bioinformatics means, and the mutation sites were as follows: 3 K-Q and 19 R-K in the FR1 fragment of the Hv region; 9 A-G, 13 M-V, and 14 A-G in the FR2 fragment of the Hv region; 13 N-D, 17 I-T, 18 L-A, and 32 T-A in the FR3 fragment of the Hv region; 22 S-T in the FR1 fragment of the Lv region; 8 G-K and 10 V-P in the FR2 fragment of the Lv region; 17 L-F, 24 Q-P, and 25 G-E in the FR3 fragment of the Lv region. The amino acid and nucleotide sequences of the bovine-derived murine anti-Brucella OMP25 tandem epitope protein IgG H chain and L chain were finally obtained. The amino acid sequence of the Hv chain of the bMABRU monoclonal antibody is shown in SEQ ID NO. 3, and the nucleotide sequence is shown in SEQ ID NO. 4; the amino acid sequence of the Lv chain of the bMABRU monoclonal antibody is shown in SEQ ID NO. 5, and the nucleotide sequence is shown in SEQ ID NO. 6.

[0065] The amino acid sequence of the CDR1 fragment of the H chain of the bMABRU monoclonal antibody is shown in SEQ ID NO. 8, and the amino acid sequence of the L chain is shown in SEQ ID NO. 9; the amino acid sequence of the CDR2 fragment of the H chain is shown in SEQ ID NO. 10, and the amino acid sequence of the L chain is shown in SEQ ID NO. 11; the amino acid sequence of the CDR3 fragment of the H chain is shown in SEQ ID NO. 12, and the amino acid sequence of the L chain is shown in SEQ ID NO. 13.

[0066] Example 6. Construction of a eukaryotic expression cell strain of the bMABRU monoclonal antibody

[0067] The DNA fragment with the nucleotide sequence of Example 5 is added with a DNA Linker by an artificial synthesis method, and cloned into a multiple cloning site of a pCDNA3.1 vector, i.e. a pCDNA3.1 eukaryotic expression vector of the bMABRU monoclonal antibody of the application, and the nucleotide sequence of the DNA Linker is shown in SEQ ID NO. 7. VERO cells are selected as target cells, and the artificial synthesized recombinant eukaryotic expression vector is transfected into the VERO cells by a liposome method, and then a cell line with stable high expression is screened. The specific steps are as follows: 1) in a 35mm hole, 1.5μg of the recombinant eukaryotic expression vector is added; 2) 5μL of the liposome prepared with OptiMEM is added, and incubated at room temperature for 45min. Then added into the host cells which are rinsed with OptiMEM, and incubated for 5h; the vector-liposome mixture is removed by centrifugation, and DMEM is added into the cells for culture; 3) the cell strain expressing the bMABRU monoclonal antibody is screened by pressure.

[0068] Example 7. Purification of the bMABRU monoclonal antibody

[0069] The bMABRU monoclonal antibody cell strain is cultured in a large amount by using a cell fermentation tank or a suitable device, the culture medium is DMEM medium, and 100U / mL of penicillin and 100μg / mL of streptomycin are added, and then 2-10% of fetal bovine serum by volume fraction is added. The cell culture solution is collected, centrifuged at 12000g at 4℃ for 30min, and the cells and impurities are removed. The supernatant is added into an ultrafiltration concentration device with a molecular weight cutoff of 10kD for ultrafiltration concentration. The concentrated solution is collected, and subjected to antibody affinity chromatography by using Protein G purification filler. The equilibrium solution is PBS containing 0.05-0.15M sodium chloride at pH 6.5-7.5, and the eluent is PBS containing 0.2-0.5M sodium chloride at pH 6.5-7.5. The eluent is collected, and is the purified bMABRU antibody stock solution.

[0070] Example 8. Formulation of the injection type of the bMABRU monoclonal antibody

[0071] The purified bMABRU monoclonal antibody stock solution is filtered and sterilized, diluted with sterile normal saline to 50μg / mL, and 15μg / mL of glucose is added as the final concentration. The solution is added into a vial and sealed, and stored at 2-8℃ for standby.

[0072] Example 9: Determination experiment of the bMABRU monoclonal antibody of the application

[0073] The injection form of the prepared bMABRU monoclonal antibody of the application has uniform appearance, no peculiar smell and no bacterial contamination; no pyrogen, abnormal toxicity and acute toxicity reaction is found through guinea pig and rabbit tests; the protein content is in the dose range through BSA method; the detection result is negative through ELISA method using goat anti-mouse IgG (GAM-IgG) and the detection titer is above 1:12800 using goat anti-cow IgG (GAH-IgG); the OMP25 protein is detected using the bovine-derived antibody prepared in Example 8, and the WB hybridization result is shown in Figure 3 .

[0074] Experimental Example 10: Therapeutic effect experiment of the bMABRU monoclonal antibody of the application in mice

[0075] The 5-6 week old mice (BALB / c mice) are tail vein injected with the virulent Brucella strain 2308 (5x104CFU), and 24 hours later, the prepared bMABRU monoclonal antibody of the application is used as the experimental group, and PBS is used as the control group, and the experimental mice are administered; 14 days after the administration, the mortality of the mice in each group is counted, and the eyeballs are taken out to collect blood, separate serum, and collect lung grinding liquid; the results show that there is no death phenomenon in each experimental group, and the mortality of the control group is 83%; the serum antibody titer is above 1:6400 through ELISA method, and the important cytokines, inflammatory molecules, chemotactic factors, eosinophils and neutrophils in the serum are measured by flow cytometry, and it is found that the cytokine over-release is not induced, which is shown in Figures 4-6 ; the lung anatomy observation shows that the appearance is normal, and no inflammatory reaction is induced due to excessive cytokines.

[0076] Experimental Example 11: Protective effect experiment of the bMABRU monoclonal antibody of the application in mice

[0077] The bMABRU monoclonal antibody prepared in Example 5 of the application is used as the test group, and PBS is used as the control group, and the 5-6 week old mice are administered; 24 hours after the administration, the mice in each test group and the control group are attacked with the virulent strain 2308 (5x104CFU), and the protective effect is observed. 14 days after the attack, the results show that the mortality of the experimental group is less than 1%, and the mortality of the control group is 75%; it is shown that the bMABRU monoclonal antibody of the application can produce good protective effect, and the protection rate is above 90%.

Claims

1. A bovine-derived mouse antibody against OMP25 tandem epitope protein, characterized in that, The amino acid sequence of the OMP25 tandem epitope protein is shown in SEQ ID NO.1; the H chain amino acid sequence of the CDR1 fragment of the antibody is shown in SEQ ID NO.8, and the L chain amino acid sequence is shown in SEQ ID NO.9; the H chain amino acid sequence of the CDR2 fragment of the antibody is shown in SEQ ID NO.10, and the L chain amino acid sequence is shown in SEQ ID NO.11; the H chain amino acid sequence of the CDR3 fragment of the antibody is shown in SEQ ID NO.12, and the L chain amino acid sequence is shown in SEQ ID NO.

13.

2. The use of the bovine-derived mouse anti-OMP25 tandem epitope protein antibody of claim 1 in the preparation of a medicament for the prevention and / or treatment of brucellosis.

3. A bovine-derived mouse anti-OMP25 tandem epitope protein antibody formulation, characterized in that, The formulation contains the bovine-derived mouse anti-OMP25 tandem epitope protein antibody as described in claim 1.

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