Construction method of Brucella bovis mutant and application of outer membrane vesicles of Brucella bovis mutant

Abstract

The invention discloses a construction method of a Brucella abortus mutant and application of an outer membrane vesicle of the Brucella abortus mutant, Brucella abortus RB51 is used as a parent strain, and a triple deletion mutant is obtained by performing multi-gene editing homologous recombination on an eipB gene, a per gene and a wadC gene through step-by-step knockout. The mutants are constructed by knocking out genes such as eipB, the OMVs secretion amount of the mutants is remarkably increased, the particle size is smaller, the immune protection capacity is superior to that of a parent strain, the mutants are safe and free of replication capacity, and a foundation is laid for efficient and safe brucellosis vaccine development.

Description

Technical Field

[0001] The present invention relates to the technical field of Brucella bovis mutants, in particular to a method for constructing a Brucella bovis mutant and application of outer membrane vesicles thereof. Background Art

[0002] Brucellosis, also known as brucellosis, is a major bacterial zoonosis with global distribution, characterized by miscarriage and reproductive failure in livestock, severely impacting the development of animal husbandry and international trade. It is estimated that over 500,000 cases of brucellosis occur in humans each year, posing a serious threat to human public health. Live attenuated brucellosis vaccines are the primary means of controlling animal brucellosis, but their high virulence and difficulty in differential diagnosis limit their use in human prevention and control. To date, no brucellosis vaccine has been approved worldwide for human prevention and control.

[0003] Outer membrane vesicles, or OMVs, are nanoscale lipid bilayer structures released by bacteria during their natural growth. OMVs are rich in pathogen-associated molecular patterns, such as lipopolysaccharides, peptidoglycans, proteins, nucleic acids, and bacterial toxins, among other immunogens. They can interact with corresponding pattern recognition receptors on antigen-presenting cells to activate them and activate Toll-like receptors to elicit potent inflammatory responses. OMVs are one of the immunostimulants that have emerged in recent decades as a novel candidate for vaccines and drug delivery vehicles. Given that OMVs lack the ability to replicate like bacteria and their immunogenicity can be enhanced through genetic engineering, their application in the development of new brucellosis vaccines could offer improved safety and protective efficacy compared to attenuated or subunit vaccines. However, the outer membrane vesicles secreted by the parent Brucella bovis strain suffer from insufficient immunogenicity, necessitating methods for constructing mutant Brucella bovis strains and the application of these outer membrane vesicles. Summary of the Invention

[0004] The purpose of the present invention is to solve the problems of the prior art and to provide a method for constructing a Brucella bovis mutant and application of its outer membrane vesicles.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] On the one hand, a method for constructing a Brucella bovis mutant is provided, using Brucella bovis RB51 as the parent strain, and obtaining a triple deletion mutant by performing multi-gene editing homologous recombination on the step-by-step knockout of the eipB gene, the per gene, and the wadC gene.

[0007] Furthermore, using Brucella bovis RB51 as the parent strain, suicide plasmids were constructed for the eipB gene, per gene, and wadC gene, respectively, and then electroporated, and the eipB gene of the parent strain was knocked out and then sucrose screening was performed to obtain deletion mutants;

[0008] Performing a second electroporation transformation on the deletion mutant, knocking out the per gene of the deletion mutant and then performing sucrose screening to obtain a double deletion mutant;

[0009] The double deletion mutant was transformed by electroporation three times, and the wadC gene of the deletion mutant was knocked out and then screened with sucrose to obtain a triple deletion mutant.

[0010] Furthermore, the construction of the suicide plasmid specifically includes amplifying the upstream homology arm sequence and the downstream homology arm sequence of the target genes per, wadC, and eipB by PCR, fusing the upstream homology arm sequence and the downstream homology arm sequence respectively to obtain a fusion fragment, and connecting the fusion fragment to the suicide plasmid by ligase, wherein the suicide plasmid contains the sacB gene for sucrose negative selection.

[0011] Furthermore, the upstream forward primer of the eipB gene is shown as SEQ ID NO: 1; the upstream reverse primer of the eipB gene is shown as SEQ ID NO: 2; the downstream forward primer of the eipB gene is shown as SEQ ID NO: 3; and the downstream reverse primer of the eipB gene is shown as SEQ ID NO: 4.

[0012] The upstream forward primer of the per gene is shown in SEQ ID NO: 5; the upstream reverse primer of the per gene is shown in SEQ ID NO: 6; the downstream forward primer of the per gene is shown in SEQ ID NO: 7; and the downstream reverse primer of the per gene is shown in SEQ ID NO: 8;

[0013] The upstream forward primer of the wadC gene is shown in SEQ ID NO: 9; the upstream reverse primer of the wadC gene is shown in SEQ ID NO: 10; the downstream forward primer of the wadC gene is shown in SEQ ID NO: 11; and the downstream reverse primer of the wadC gene is shown in SEQ ID NO: 12.

[0014] On the other hand, a mutant of Brucella bovis, the mutant protein was deposited by the General Microbiology Center of China Culture Collection Administration on May 27, 2025, and was classified and named Brucella bovis remodeling body BAΔ3, with the deposit number CGMCC No.34791.

[0015] On the other hand, a mutant of Brucella bovis is used in the following aspects:

[0016] (1) Application of outer membrane vesicles of Brucella bovis mutants in enhancing immunogenicity;

[0017] (2) The use of outer membrane vesicles of mutant Brucella bovis to prepare brucellosis vaccines, wherein the brucellosis vaccine is used via a mucosal immunization route.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention constructs mutants by knocking out genes such as eipB. The secretion of OMVs is significantly increased, the particle size is smaller, the immune protection is better than that of the parent strain, and it is safe and has no replication ability, laying the foundation for the development of an efficient and safe vaccine for brucellosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a method for constructing a Brucella bovis mutant proposed in the present invention; and a diagram analyzing the secretion amount of OMVs of Brucella bovis remodeled bodies;

[0021] Figure 2 This is a method for constructing a Brucella bovis mutant proposed in the present invention, and a morphological analysis diagram of Brucella bovis remodeled bodies and their OMVs;

[0022] in, Figure 2 (A) Morphological observation of the parental strain Brucella bovis BA and its secreted OMVs, and the remodeled Brucella bovis BAΔ3 and its secreted OMVs under electron microscopy; Figure 2 (B) Diameter statistics of the parent strain Brucella bovis BA and the remodeled Brucella bovis BAΔ3 under electron microscopy; Figure 2 (C) Diameter size statistics of OMVBA and OMV BAΔ3 under electron microscopy.

[0023] Figure 3 This is a safety analysis diagram of a method for constructing a Brucella bovis mutant and remodeled Brucella bovis OMVs proposed in the present invention;

[0024] in, Figure 3 (A) Diagram of the animal model for safety evaluation of OMVs; Figure 3 (B) Percentage change in body weight of immunized mice.

[0025] Figure 4 This is a diagram showing the immune efficacy evaluation of Brucella bovis remodeled OMVs and a method for constructing a Brucella bovis mutant proposed in the present invention;

[0026] in, Figure 4 (A) Animal model for evaluating the immune efficacy of OMVs; Figure 4 (B) Analysis of bacterial load in the spleen of immunized mice after challenge.

[0027] Figure 5 Research on the humoral immune response induced by remodeled Brucella bovis OMVs of a Brucella bovis mutant construction method proposed in the present invention

[0028] in, Figure 5 (A) Schematic diagram of the animal model for evaluating humoral immune responses to OMVs; Figure 5 (B) Analysis of IgG antibody titers in the serum of immunized mice at week 2 and week 5; Figure 5 (C) Analysis of IgG2a / IgG1 antibody ratios in the serum of immunized mice at week 2 and week 5.

[0029] Figure 6 Research on the T cell immune response induced by Brucella bovis remodeled OMVs based on the method for constructing Brucella bovis mutants proposed in the present invention

[0030] in, Figure 6 (A) Animal model for evaluation of T cell immune responses to OMVs; Figure 6 (B) Analysis of the levels of CD4+ T cells secreting IFN-γ and TNF-α in the spleen of immunized mice at week 4 after infection. DETAILED DESCRIPTION

[0031] The present invention is described in detail below with reference to the embodiments and accompanying drawings. However, it should be understood that the embodiments and drawings are only used to illustrate the present invention and do not constitute any limitation on the scope of protection of the present invention.

[0032] The present invention is further illustrated below with reference to specific embodiments:

[0033] Example 1 Remodeling of Brucella bovis strains

[0034] The mutant protein was deposited by the General Microbiology Center of the China National Committee for the Collection of Microorganisms on May 27, 2025, and was classified as Brucella bovis remodeled body BAΔ3, with a deposit number of CGMCC No. 34791. The reference biological material is RB51ΔeipBΔperΔwadC. The OMVs produced by the natural strain of Brucella bovis have the defect of insufficient Brucella immune protection. The strain can be reshaped by genetic engineering to improve the immune protection of Brucella bovis OMVs. The present invention selects the key LPS genes per, wadC and the key outer membrane gene eipB of Brucella bovis to reshape Brucella bovis.

[0035] 1.1 Construction of target gene suicide plasmid.

[0036] Homology primers were designed for the upstream and downstream target gene sequences (see Table 1 for sequence information). PCR was used to amplify the upstream and downstream homology arm sequences of the target genes per, wadC, and eipB. Overlap-PCR was then used to fuse the upstream and downstream homology arm sequences to generate fusion fragments. The fusion fragments were then ligated with the suicide plasmid pUCML using T4 ligase to construct the suicide plasmids pUCML-ΔeipB, pUCML-Δper, and pUCML-ΔwadC. The suicide plasmid pUCML contains the sacB gene.

[0037] Table 1 Sequence information

[0038]

[0039]

[0040] 1.2 Construction of target gene deletion strains.

[0041] First, prepare competent cells of Brucella bovis: spread Brucella bovis on TSA plates, culture at 37°C for 3 days, pick a single colony and shake culture in 100 mL TSB medium until OD 600 After incubating the bacteria on ice for 30 minutes, centrifuge at 5000 rpm for 10 minutes, collect the pellet, wash three times with ultrapure water containing 10% glycerol, resuspend in ultrapure water with 10% glycerol, and aliquot 100 μL per tube for freezing. Subsequently, the resulting suicide plasmid containing the fusion fragment was added to competent Brucella bovis cells and mixed thoroughly. After incubating on ice for 30 minutes, the cells were transformed by electroporation. After electroporation, 800 μL of SOC resuscitation solution was added and the cells were incubated at 37°C with shaking at 180 rpm for 12 hours. The transformation solution was then plated on TSA plates containing 50 μg / mL kanamycin and incubated at 37°C for 3 days. Finally, single colonies were picked from the TSA plates containing 50 μg / mL kanamycin and plated in an appropriate amount of TSB solution. The culture was shaken for 24 hours. The bacterial solution was then appropriately diluted and plated on TSA plates containing 5% sucrose and incubated at 37°C for 4 days. Colonies growing on the plates were identified by PCR, and those positive by PCR were identified as the remodeled Brucella bovis strain. Using this method, using Brucella bovis RB51 (BA) as the parent strain, the target genes eipB, per, and wadC were sequentially deleted, ultimately yielding the remodeled Brucella bovis RB51ΔeipBΔperΔwadC (BAΔ3).

[0042] Example 2 Extraction and morphological analysis of OMVs

[0043] In order to explore the effect of remodeling Brucella bovis on the secretion of OMVs, the present invention extracted OMVs of Brucella bovis and remodeled Brucella bovis and performed morphological analysis.

[0044] The specific method is as follows:

[0045] 1.1 Extraction of Brucella bovis OMVs.

[0046] Culture 1L of Brucella bovis to OD 600 Reaching about 2.0, a bacterial culture fluid was obtained. EDTA with a pH of 8.0 and a concentration of 100mM was added to the bacterial culture fluid, ice-bathed for 1 hour, and then centrifuged at 10,000×g for 10 minutes at 4°C, and the bacteria were discarded to obtain the supernatant. The culture supernatant was filtered with a 0.45μm pore size filter to remove residual bacteria, and concentrated using a Vivaflow200 system equipped with a 100kDa filter membrane, followed by ultracentrifugation at 120,000×g, 4°C for 2 hours. After the end, the supernatant after ultracentrifugation was discarded, and the precipitate was resuspended in sterile 0.1×PBS with a pH of 7.4, and centrifuged again at 12,000r / min, 4°C for 10 minutes. The supernatant was filtered with a 0.22μm pore size membrane and stored at -80°C for use. According to this method, OMVs secreted by Brucella bovis BA and Brucella bovis remodeling body BAΔ3 were extracted respectively, and finally OMVs were obtained. BA and OMV BAΔ3 .

[0047] 1.2 Analysis of secretion of remodeled OMVs of Brucella bovis

[0048] Use BCA protein concentration assay kit to detect OMV extracted in 1.1 BA and OMV BAΔ3 The specific method is as follows: Add 25 μL of BCA protein standard and OMV sample to a 96-well plate; add 200 μL of the reaction solution to each well and shake on a plate shaker for 30 seconds to mix thoroughly. Incubate at 37°C for 30 minutes. After cooling to room temperature, measure the absorbance at 562 nm on a spectrophotometer. Plot a standard curve and calculate the OMV sample concentration.

[0049] The results are as follows Figure 1 As shown, compared with the parent strain Brucella bovis BA, the OMVs secretion of the remodeled Brucella bovis BAΔ3 was significantly increased, indicating that the remodeling of Brucella bovis affected the secretion of its OMVs.

[0050] 1.3 Morphological analysis of Brucella bovis remodeling bodies and their OMVs:

[0051] The parent strain Brucella bovis BA and the remodeled Brucella bovis BAΔ3 were cultured to OD600 Reaching about 1.0, bacterial culture fluid was obtained. Take 1mL of bacterial culture fluid and centrifuge at 8,000×g for 5 minutes, discard the supernatant, resuspend the precipitate with PBS to wash the bacteria, repeat the centrifugation and wash the bacteria twice with PBS, resuspend the bacteria after three washes with 800μL of 2.5% glutaraldehyde solution, and then incubate at 4℃ for 24h. After staining the treated bacteria, observe them with a transmission electron microscope, and analyze the morphology of the bacteria based on the imaging results. Similarly, the isolated and purified OMV BA and OMV BAΔ3 After staining according to the above steps, the cells were observed using a transmission electron microscope, and the morphology of OMVs was analyzed based on the imaging results.

[0052] The results are as follows Figure 2 As shown, the morphology of the remodeled Brucella bovis BAΔ3 is similar to that of the parent strain Brucella bovis BA, which is short rod-shaped and has a size of about 0.78 to 1.48 μm. In addition, the OMVs secreted by the remodeled Brucella bovis BAΔ3 OMVs secreted by the parent strain Brucella bovis BA Similar, spherical morphology, but OMV BAΔ3 The average diameter of OMVs was 54.15 nm, which was significantly smaller than the average diameter of OMVs of 91.73 nm. BA The results showed that remodeling of Brucella bovis did not affect the bacterial morphology, but affected the size of the OMVs secreted by it.

[0053] Example 3 Safety Study of OMVs

[0054] To explore the separation and purification of OMV BA and OMV BAΔ3 The safety of the OMVs to mice was determined by immunizing mice through intranasal (abbreviated as in) immunization.

[0055] The specific operation method is as follows: Six-week-old BALB / c mice were divided into PBS group, OMV group BA Group and OMV BAΔ3 There were three groups, of which the mice in the PBS group were inoculated with PBS solution via the in immunization route and served as the negative control group; BA Mice in the immunization group were inoculated with 15 μg of OMV. BA ;OMV BAΔ3 Mice in the immunization group were inoculated with 15 μg of OMV. BAΔ3 Three weeks after the initial immunization, the mice were given a booster immunization. During the immunization period, the weight changes of each group of mice were monitored to evaluate the immune OMV. BA and OMV BAΔ3Safety in mice. BA Secreted by the parent strain Brucella bovis BA, OMV BAΔ3 Secreted by the Brucella bovis remodeler BAΔ3.

[0056] The results are as follows Figure 3 As shown, OMV BA and OMV BAΔ3 After inoculation, mice were in good spirits and no deaths occurred. BA The body weight of mice in the immunized group decreased by about 4% three days after immunization. BAΔ3 The body weight of mice in the immunized group decreased by about 10% three days after immunization, and then gradually returned to normal. BA and OMV BAΔ3 The safety of the vaccine in mice was good after inoculation.

[0057] Example 4 Study on the immune efficacy of OMVs

[0058] To explore the separation and purification of OMV BA and OMV BAΔ3 To determine the immune protection of mice, the OMVs were administered via intranasal immunization (abbreviated as in) and then subjected to a challenge test to determine the immune protection efficacy of the OMVs on mice.

[0059] The specific operation method is as follows: Six-week-old BALB / c mice were divided into PBS group, OMV group BA Group and OMV BAΔ3 There were three groups, of which the mice in the PBS group were inoculated with PBS solution via the in immunization route and served as the negative control group; BA Mice in the immunization group were inoculated with 15 μg of OMV. BA ;OMV BAΔ3 Mice in the immunization group were inoculated with 15 μg of OMV. BAΔ3 Three weeks after the initial immunization, the immunized mice were given a booster immunization. Three weeks after the booster immunization, a challenge protection test was conducted. The immunized mice were challenged with 5×10 5 CFU of bovine Brucella, compared with PBS control group and OMV control group 4 weeks after infection BA Group and OMV BAΔ3 The bacterial load in the spleen of mice in the two groups was evaluated by OMV BA and OMV BAΔ3 Immunoprotection of mice. BA Secreted by the parent strain Brucella bovis BA, OMV BAΔ3 Secreted by the Brucella bovis remodeler BAΔ3.

[0060] The results are as follows Figure 4 As shown in the figure, compared with the PBS group, the bacterial load in the spleen of the OMVBA group of mice immunized by the in route was significantly reduced after infection with Brucella bovis; BA Compared with the mice in the group, OMVs immunized by in route BAΔ3 After the mice in the group were infected with Brucella bovis, the bacterial load in the spleen was also significantly reduced. The results showed that mice were immunized with mouse OMV through the in route. BA After that, it can induce a certain immune protection, but mice are immunized with mouse OMV through the in route BAΔ3 OMV BA Therefore, remodeling of Brucella bovis allows the OMVs it secretes to have higher immune protection.

[0061] Example 5 Study on OMVs-induced humoral immune response

[0062] To explore the separation and purification of OMV BA and OMV BAΔ3 The humoral immune response was induced in mice, and the mice were immunized via intranasal (abbreviated as in) immunization route. The serum of the mice was isolated and the levels of IgG, IgG1, and IgG2a antibodies in the serum were determined by ELASA.

[0063] The specific operation method is as follows: Six-week-old BALB / c mice were divided into PBS group, OMV group BA Group and OMV BAΔ3 There were three groups, of which the mice in the PBS group were inoculated with PBS solution via the in immunization route and served as the negative control group; BA Mice in the immunization group were inoculated with 15 μg of OMV. BA ;OMV BAΔ3 Mice in the immunization group were inoculated with 15 μg of OMV. BAΔ3 Two weeks after the initial immunization, whole blood was collected from the immunized mice and serum was separated. Three weeks after the initial immunization, the immunized mice were given a booster immunization. Five weeks after the initial immunization, whole blood was collected from the immunized mice and serum was separated. ELASA assay was used to determine the levels of IgG, IgG1, and IgG2a antibodies in the serum and to evaluate the OMV BA and OMV BAΔ3 The humoral immune response induced in mice. BA Secreted by the parent strain Brucella bovis BA, OMV BAΔ3 Secreted by the Brucella bovis remodeler BAΔ3.

[0064] The results are as follows Figure 5As shown, the PBS group did not produce anti-Brucella IgG antibodies; OMV BA Group and OMV BAΔ3 The mice in the two groups could induce strong anti-Brucella IgG titers; BA Compared with the mice in the same group, OMV BAΔ3 The mice in the group produced higher levels of anti-Brucella IgG titers. The results showed that mice were immunized with mouse OMVs through the in route. BA After that, it can induce a certain level of humoral immune response in mice, but mice are immunized with mouse OMVs through the in route. BAΔ3 OMV BA A stronger humoral immune response results in higher immune protection of the OMVs secreted by remodeled Brucella bovis.

[0065] The PBS group did not produce anti-Brucella IgG1, IgG2a antibodies; OMV BA Group and OMV BAΔ3 The mice in the two groups were able to induce strong anti-Brucella IgG1 and IgG2a titers. By calculating the IgG2a / IgG1 ratio, it was found that OMV BA Group and OMV BAΔ3 The IgG2a / IgG1 ratios of mice in the group were all less than 1. The results showed that mice were immunized with mouse OMVs via the in route. BA The humoral immune response induced by the vaccine in mice plays a key role in combating Brucella.

[0066] Example 6 Study on OMVs-induced T cell immune response

[0067] To explore the separation and purification of OMV BA and OMV BAΔ3 The T cell immune response was induced in mice, and mice were immunized by intranasal immunization (abbreviated as in) and then challenged with the virus. The spleens of the mice were taken out in the fourth week after the challenge, and the lymphocytes in the spleens were isolated. The CD4+ cells secreting IFN-γ and TNF-α in the spleens were determined by flow cytometry. + T cell levels.

[0068] The specific operation method is as follows: Six-week-old BALB / c mice were divided into PBS group, OMV group BA Group and OMV BAΔ3 There were three groups, of which the mice in the PBS group were inoculated with PBS solution via the in immunization route and served as the negative control group; BA Mice in the immunization group were inoculated with 15 μg of OMV. BA ;OMV BAΔ3 Mice in the immunization group were inoculated with 15 μg of OMV.BAΔ3 Three weeks after the initial immunization, the immunized mice were given a booster immunization. Three weeks after the booster immunization, a challenge protection test was conducted. The immunized mice were challenged with 5×10 5 CFU of bovine Brucella, lymphocytes from the spleen of immunized mice were isolated 4 weeks after the challenge, and the PBS control group, OMV BA Group and OMV BAΔ3 CD4 T cells secreting IFN-γ and TNF-α in the spleen of group 1 mice + T cell levels, evaluation of OMV BA and OMV BAΔ3 Induced T cell immune response in mice. BA Secreted by the parent strain Brucella bovis BA, OMV BAΔ3 Secreted by the Brucella bovis remodeler BAΔ3.

[0069] The results are as follows Figure 6 As shown, with OMV BA Compared with the mice in the group, OMVs immunized by in route Bru-M3 After the mice in the group were infected with Brucella bovis, the CD4 + The number of T cells increased significantly. The results showed that mice were immunized with mouse OMV via the in route BAΔ3 OMV BA A stronger T cell immune response is also an important reason why the OMVs secreted by remodeled bovine Brucella have higher immune protection.

[0070] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for constructing a Brucella bovis mutant, characterized in that: Using Brucella bovis RB51 as the parental strain, a triple deletion mutant was obtained by stepwise knockout of the eipB gene, per gene, and wadC gene through multi-gene editing and homologous recombination.

2. The method for constructing a Brucella bovis mutant according to claim 1, wherein Using Brucella bovis RB51 as the parent strain, suicide plasmids were constructed for the eipB gene, per gene, and wadC gene, and then electroporated, and the eipB gene of the parent strain was knocked out and then sucrose screening was performed to obtain deletion mutants; Performing a second electroporation transformation on the deletion mutant, knocking out the per gene of the deletion mutant and then performing sucrose screening to obtain a double deletion mutant; The double deletion mutant was transformed by electroporation three times, and the wadC gene of the deletion mutant was knocked out and then screened with sucrose to obtain a triple deletion mutant.

3. The method for constructing a Brucella bovis mutant according to claim 2, wherein: The construction of the suicide plasmid specifically includes amplifying the upstream homology arm sequence and the downstream homology arm sequence of the target genes per, wadC, and eipB by PCR, fusing the upstream homology arm sequence and the downstream homology arm sequence respectively to obtain a fusion fragment, and connecting the fusion fragment to the suicide plasmid by T4 ligase, wherein the suicide plasmid contains the sacB gene for sucrose negative selection.

4. The method for constructing a Brucella bovis mutant according to claim 1 or 2, wherein: The upstream forward primer of the eipB gene is shown in SEQ ID NO: 1; the upstream reverse primer of the eipB gene is shown in SEQ ID NO: 2; the downstream forward primer of the eipB gene is shown in SEQ ID NO: 3; the downstream reverse primer of the eipB gene is shown in SEQ ID NO: 4; The upstream forward primer of the per gene is shown in SEQ ID NO: 5; the upstream reverse primer of the per gene is shown in SEQ ID NO: 6; the downstream forward primer of the per gene is shown in SEQ ID NO: 7; the downstream reverse primer of the per gene is shown in SEQ ID NO: 8; The upstream forward primer of the wadC gene is shown in SEQ ID NO: 9; the upstream reverse primer of the wadC gene is shown in SEQ ID NO: 10; the downstream forward primer of the wadC gene is shown in SEQ ID NO: 11; and the downstream reverse primer of the wadC gene is shown in SEQ ID NO:

12.

5. A Brucella bovis mutant, characterized in that The mutant protein was deposited by the General Microbiology Center of the China Microorganism Culture Collection Administration on May 27, 2025, and was classified and named Brucella bovis remodeling body BAΔ3, with the deposit number CGMCC No.34791.

6. Use of the Brucella bovis mutant according to claim 5 in the following aspects: (1) Application of outer membrane vesicles of Brucella bovis mutants in enhancing immunogenicity; (2) The use of outer membrane vesicles of mutant Brucella bovis to prepare brucellosis vaccines, wherein the brucellosis vaccine is used via a mucosal immunization route.

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