A method for constructing a mutant of Brucella bovis and its application in outer membrane vesicles

Abstract

This invention discloses a method for constructing a Brucella bovis mutant and its application in outer membrane vesicles. Using Brucella bovis RB51 as the parent strain, a triple-deletion mutant was obtained through multi-gene editing and homologous recombination by stepwise knockout of the eipB, per, and wadC genes. This invention, by knocking out genes such as eipB, constructs a mutant with significantly increased OMV secretion, smaller particle size, and superior immunoprotective efficacy compared to the parent strain. Furthermore, it is safe and lacks replication ability, laying the foundation for the development of a highly effective and safe brucellosis vaccine.

Description

Technical Field

[0001] This invention relates to the field of Brucella bovis mutant technology, and more particularly to a method for constructing Brucella bovis mutants and the application of their outer membrane vesicles. Background Technology

[0002] Brucellosis, commonly known as brucellosis, is a major zoonotic bacterial disease characterized by abortion and reproductive disorders in livestock, distributed globally. It severely impacts animal husbandry and international trade. It is estimated that over 500,000 cases of brucellosis occur in humans annually, posing a serious threat to public health. Live attenuated brucellosis vaccines are the primary means of controlling brucellosis in animals; however, these vaccines have limitations such as high virulence and difficulty in diagnosis, which prevents their use in controlling human brucellosis. To date, no brucellosis vaccine has been approved worldwide for the control of human brucellosis.

[0003] Outer membrane vesicles, abbreviated as OMVs, are nanoscale lipid bilayer vesicle structures released by bacteria during natural growth. OMVs are rich in pathogen-associated molecular patterns, such as lipopolysaccharides, peptidoglycans, proteins, nucleic acids, and bacterial toxins, which are abundant immunogens. They can interact with corresponding pattern recognition receptors on antigen-presenting cells to activate them; they can also activate Toll-like receptors to trigger a potent inflammatory response. OMVs are one of the emerging immunostimulants for novel vaccine and drug delivery vectors that have gained popularity in recent decades. Given that OMVs lack bacterial replication capabilities and their immunogenicity can be enhanced through genetic engineering, their application in the development of novel brucellosis vaccines is expected to demonstrate superior safety and protective efficacy compared to attenuated live vaccines or subunit vaccines. However, the outer membrane vesicles secreted by the parental *Brucella bovis* have insufficient immunogenicity, thus necessitating a method for constructing *Brucella bovis* mutants and the application of their outer membrane vesicles. Summary of the Invention

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

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] On one hand, a method for constructing a Brucella bovis mutant involves using Brucella bovis RB51 as the parent strain and obtaining a triple deletion mutant through multi-gene editing and homologous recombination by stepwise knockout of the eipB gene, per gene, and 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 transformed by electroporation. The parent strain was then subjected to eipB gene knockout and sucrose screening to obtain deletion mutants.

[0008] The deletion mutant was subjected to a second electroporation transformation, and the per gene was knocked out of the deletion mutant, followed by sucrose screening to obtain a double deletion mutant.

[0009] The double deletion mutant was subjected to three electroporation transformations, and the wadC gene was knocked out of the deletion mutant, followed by sucrose screening to obtain the triple deletion mutant.

[0010] Furthermore, the construction of the suicide plasmid specifically includes amplifying the upstream and downstream homologous arm sequences of the target genes per, wadC, and eipB by PCR, fusing the upstream and downstream homologous arm sequences to obtain a fusion fragment, and ligating the fusion fragment to the suicide plasmid using a ligase. The suicide plasmid contains the sacB gene for sucrose negative selection.

[0011] Furthermore, 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; and the downstream reverse primer of the eipB gene is shown in 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 Brucella bovis mutant, whose mutant protein was deposited by the China General Microbiological Culture Collection Center on May 27, 2025, was classified and named Brucella bovis remodeled BAΔ3, with accession number CGMCC No. 34791.

[0015] On the other hand, a bovine Brucella mutant has the following applications:

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

[0017] (2) Application of brucellosis vaccine prepared from outer membrane vesicles of bovine Brucella mutant, wherein the brucellosis vaccine is used via mucosal immunization.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention constructs mutants by knocking out genes such as eipB, which significantly increase the secretion of OMVs, have smaller particle size, and provide better immune protection than the parent strain. Moreover, these mutants are safe and have no replication ability, laying the foundation for the development of highly effective and safe brucellosis vaccines. Attached Figure Description

[0020] Figure 1 This is a diagram showing the secretion volume analysis of OMVs of bovine Brucella remodeling, based on a method for constructing bovine Brucella mutants proposed in this invention.

[0021] Figure 2 This invention presents a method for constructing Brucella bovis mutants, and morphological analysis of Brucella bovis remodels and their OMVs.

[0022] in, Figure 2 (A) Morphological observation of the parent strain Brucella bovis BA and its secreted OMVs and Brucella bovis remodeling BAΔ3 and its secreted OMVs under electron microscopy. Figure 2 (B) Statistical analysis of the diameter of the parental Brucella bovis strain BA and the Brucella bovis remodeled body BAΔ3 under electron microscopy; Figure 2 (C)Statistical analysis of the diameters of OMVBA and OMV BAΔ3 under electron microscopy.

[0023] Figure 3 This is a safety analysis diagram of the method for constructing Brucella bovis mutants proposed in this invention, and Brucella bovis remodeled OMVs.

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

[0025] Figure 4 This is a diagram evaluating the immunogenicity of bovine Brucella remodeling OMVs, based on a method for constructing bovine Brucella mutants proposed in this invention.

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

[0027] Figure 5 This invention relates to a method for constructing bovine Brucella mutants and the study of how OMVs, remodeled bovine Brucella, induce humoral immune responses.

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

[0029] Figure 6 This invention relates to a method for constructing a Brucella bovis mutant and the study of T cell immune responses induced by OMVs of the Brucella bovis remodeled strain.

[0030] in, Figure 6 (A) Animal model diagram for evaluating OMVsT cell immune response; Figure 6 (B) Analysis of CD4+ T cell levels secreting IFN-γ and TNF-α in the spleen of immunized mice at week 4 after challenge. Detailed Implementation

[0031] The present invention will be described in detail below with reference to the embodiments and accompanying drawings. However, it should be understood that the embodiments and accompanying 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 will be further illustrated below with reference to specific embodiments:

[0033] Example 1: Reconstructed Brucella bovis strain

[0034] The mutant protein was deposited by the China General Microbiological Culture Collection Center (CGMCC) on May 27, 2025, and classified as Brucella bovis remodeled BAΔ3, with accession number CGMCC No. 34791. The reference biological material was RB51ΔeipBΔperΔwadC. The OMVs produced by natural Brucella bovis strains exhibit insufficient Brucella immunoprotection. Genetic engineering can be used to remodel the strains to improve the immunoprotective effect of Brucella bovis OMVs. This invention selects the key LPS genes per and wadC and the key outer membrane gene eipB of Brucella bovis to remodel Brucella bovis.

[0035] 1.1 Construction of suicide plasmid for target gene.

[0036] Homologous arm primers were designed for the upstream and downstream of the target gene sequences (as shown in Sequence Information Table 1). The upstream and downstream homologous arm sequences of the target genes per, wadC, and eipB were amplified by PCR. The upstream and downstream homologous arm sequences were then fused using overlap-PCR to obtain fusion fragments. These fusion fragments were ligated to 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 Table

[0038]

[0039]

[0040] 1.2 Construction of strains with target gene deletion.

[0041] First, competent bovine Brucella cells were prepared: Bovine Brucella bacteria were spread onto TSA plates and incubated at 37°C for 3 days. Single colonies were then picked and cultured in 100 mL of TSB medium with shaking until OD (Oxygen Demand). 600 The bacterial culture was incubated on ice for 30 min, then centrifuged at 5000 rpm for 10 min to collect the bacterial pellet. The pellet was washed three times with ultrapure water containing 10% glycerol, resuspended in ultrapure water containing 10% glycerol, and aliquoted into 100 μL tubes for cryopreservation. Subsequently, the suicide plasmid containing the fusion fragment was added to competent Brucella bovis cells and mixed thoroughly. After incubation on ice for 30 min, electroporation transformation was performed. Following electroporation, 800 μL of SOC resuscitation medium was added and the cells were incubated at 37°C with shaking at 180 rpm for 12 hours. The transformation solution was then plated onto 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 placed in an appropriate amount of TSB solution, incubated with shaking for 24 hours, and then the bacterial culture was appropriately diluted and placed on TSA plates containing 5% sucrose and incubated at 37°C for 4 days. Colonies grown on the plate were identified by PCR. Positive colonies identified by PCR were identified as reconstituted Brucella bovis strains. Using this method, Brucella bovis RB51 (BA) was used as the parent strain, and the target genes eipB, per, and wadC were knocked out sequentially to obtain the Brucella bovis reconstituted strain RB51ΔeipBΔperΔwadC (BAΔ3).

[0042] Example 2: Extraction and Morphological Analysis of OMVs

[0043] To investigate the effect of remodeling Brucella bovis on secreted OMVs, this invention extracted Brucella bovis and the OMVs of Brucella bovis remodeled bodies and performed morphological analysis.

[0044] The specific method is as follows:

[0045] 1.1 Extraction of bovine Brucella OMVs.

[0046] 1L of bovine Brucella was cultured to OD. 600 The bacterial culture medium was obtained when the pH reached approximately 2.0. EDTA at a concentration of 100 mM and a pH of 8.0 was added to the bacterial culture medium, and the mixture was incubated on ice for 1 hour. Then, it was centrifuged at 10,000 × g for 10 minutes at 4°C, and the bacteria were discarded, collecting the supernatant. The culture supernatant was filtered through a 0.45 μm filter to remove residual bacteria, concentrated using a Vivaflow 200 system equipped with a 100 kDa filter membrane, and then ultracentrifuged at 120,000 × g at 4°C for 2 hours. After ultracentrifugation, the supernatant was discarded, and the precipitate was resuspended in sterile 0.1 × PBS at pH 7.4. The mixture was then centrifuged again at 12,000 r / min at 4°C for 10 minutes, and the supernatant was filtered through a 0.22 μm membrane and stored at -80°C for later use. Based on this method, OMVs secreted by *Brucella bovis* BA and *Brucella bovis* remodeled BAΔ3 were extracted, ultimately obtaining the OMVs. BA and OMV BAΔ3 .

[0047] 1.2 Analysis of the secretion of bovine Brucella remnants OMVs

[0048] The OMV extracted in 1.1 was detected using a BCA protein concentration assay kit. BA and OMV BAΔ3 Total volume. The specific method is as follows: Add 25 μL of BCA protein standard and OMVs sample to a 96-well plate; add 200 μL of reaction solution to each well and shake on a shaker for 30 s to ensure thorough mixing. Incubate at 37℃ for 30 min; after cooling to room temperature, measure the absorbance at 562 nm using a spectrophotometer. Plot a standard curve and calculate the OMVs sample concentration.

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

[0050] 1.3 Morphological analysis of bovine Brucella remnants and their OMVs:

[0051] The parental strain of Brucella bovis BA and the remodeled Brucella bovis BAΔ3 were cultured separately to OD.600 The bacterial culture medium was obtained when the pH reached approximately 1.0. 1 mL of the bacterial culture medium was centrifuged at 8,000 × g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS for washing. This washing process was repeated twice with PBS. After three washes, the bacterial cells were resuspended in 800 μL of 2.5% glutaraldehyde solution and incubated at 4°C for 24 h. The treated bacteria were stained and observed using a transmission electron microscope (TEM). The morphology of the bacteria was analyzed based on the imaging results. Similarly, the isolated and purified OMV was... BA and OMV BAΔ3 After staining according to the above steps, the OMVs were observed using a transmission electron microscope, and their morphology was analyzed based on the imaging results.

[0052] The results are as follows Figure 2 As shown, the morphology of the Brucella bovis remodel BAΔ3 is similar to that of its parent strain, Brucella bovis BA, appearing as a short rod-shaped organism with a size of approximately 0.78–1.48 μm. Furthermore, the OMV secreted by the Brucella bovis remodel is also observed. BAΔ3 OMV secreted by the parent strain of Brucella bovis BA Similar in shape, but OMV BAΔ3 The average diameter is 54.15 nm, which is significantly smaller than that of OMV, which has an average diameter of 91.73 nm. BA The results showed that remodeling of *Brucella bovis* did not affect the bacterial morphology, but it did affect the size of its secreted OMVs.

[0053] Example 3: Safety Study of OMVs

[0054] To investigate the separation and purification of OMV BA and OMV BAΔ3 The safety of the OMVs in mice was determined by immunizing mice via nasal drop (in) immunization.

[0055] The specific operating procedure is as follows: Six-week-old BALB / c mice were divided into PBS group and OMV group. BA Group and OMV BAΔ3 Group 3, with the PBS group consisting of mice immunized with PBS solution via the in immunotherapy route, serving as a negative control group; OMV BA Mice in this group were immunized with 15 μg of OMV via the in route. BA ;OMV BAΔ3 Mice in this group were immunized with 15 μg of OMV via the in route. BAΔ3 Three weeks after the initial immunization, the immunized mice received a booster immunization. During the immunization period, the immune OMV was evaluated by monitoring changes in body weight in each group of mice. BA and OMV BAΔ3Safety in mice. Among them, OMV BA Secreted by the parent strain of Brucella bovis BA, OMV BAΔ3 It is secreted by the bovine Brucella remodeling BAΔ3.

[0056] The results are as follows Figure 3 As shown, OMV BA and OMV BAΔ3 Following immunization with in, the mice were in good spirits, with no deaths, and OMV was [not specified]. BA The immunized mice experienced a weight loss of approximately 4% three days post-immunization, OMV BAΔ3 The immunized mice experienced a weight loss of approximately 10% three days post-immunization, after which their weight gradually returned to normal. Therefore, OMV... BA and OMV BAΔ3 The in-immunization method showed good safety in mice.

[0057] Example 4: Immunopotency Study of OMVs

[0058] To investigate the separation and purification of OMV BA and OMV BAΔ3 To assess the immunoprotective efficacy of OMVs in mice, mice were immunized via nasal drop (in) followed by a challenge test to determine the immunoprotective efficacy of the OMVs in mice.

[0059] The specific operating procedure is as follows: Six-week-old BALB / c mice were divided into PBS group and OMV group. BA Group and OMV BAΔ3 Group 3, with the PBS group consisting of mice immunized with PBS solution via the in immunotherapy route, serving as a negative control group; OMV BA Mice in this group were immunized with 15 μg of OMV via the in route. BA ;OMV BAΔ3 Mice in this group were immunized with 15 μg of OMV via the in route. BAΔ3 Three weeks after the initial immunization, the mice received a booster immunization. Three weeks after the booster immunization, a challenge protection test was conducted, with the immunized mice challenged by intraperitoneal injection (ip) of 5 x 10^6 mmol / L virus. 5 CFU-treated Brucella bovis was compared with PBS control group and OMV 4 weeks after challenge. BA Group and OMV BAΔ3 The bacterial load in the spleen of mice was used to evaluate OMV. BA and OMV BAΔ3 Immunoprotective efficacy in mice. Among them, OMV BA Secreted by the parent strain of Brucella bovis BA, OMV BAΔ3 It is secreted by the bovine Brucella remodeling BAΔ3.

[0060] The results are as follows Figure 4 As shown, compared with the PBS group mice, the OMVBA group mice immunized via the in route had significantly lower bacterial load in their spleens after infection with Brucella bovis; compared with OMV... BA Compared with the control group of mice, OMV immunized via the in route... BAΔ3 In mice infected with Brucella bovis, the bacterial load in the spleen was significantly reduced. This indicates that immunization of mice with OMV via the intransitive route... BA It can induce a certain degree of immune protection, but mice immunized with OMV via the in route can also be affected. BAΔ3 It can then cause more than OMV BA Better immune protection. Therefore, remodeling bovine Brucella results in OMVs that have higher immune protection.

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

[0062] To investigate the separation and purification of OMV BA and OMV BAΔ3 The humoral immune response in mice was induced by immunizing the mice via nasal drops (in), and the serum of the mice was separated. The levels of IgG, IgG1, and IgG2a antibodies in the serum were determined by ELISA.

[0063] The specific operating procedure is as follows: Six-week-old BALB / c mice were divided into PBS group and OMV group. BA Group and OMV BAΔ3 Group 3, with the PBS group consisting of mice immunized with PBS solution via the in immunotherapy route, serving as a negative control group; OMV BA Mice in this group were immunized with 15 μg of OMV via the in route. BA ;OMV BAΔ3 Mice in this group were immunized with 15 μg of OMV via the in route. BAΔ3 Two weeks after the initial immunization, whole blood was collected from immunized mice and serum was separated. Three weeks after the initial immunization, immunized mice received a booster immunization. Five weeks after the initial immunization, whole blood was collected from immunized mice and serum was separated. The levels of IgG, IgG1, and IgG2a antibodies in the serum were determined using an ELISA assay to evaluate OMV. BA and OMV BAΔ3 The induced humoral immune response in mice. Among them, OMV BA Secreted by the parent strain of Brucella bovis BA, OMV BAΔ3 It is secreted by the bovine Brucella remodeling BAΔ3.

[0064] The results are as follows Figure 5As shown, the PBS group did not produce anti-brucellosis IgG antibodies; OMV BA Group and OMV BAΔ3 All groups of mice were able to induce strong anti-brucellosis IgG titers; compared with OMV BA Compared with the control group of mice, OMV BAΔ3 The mice in this group produced higher levels of anti-brucellosis IgG titers. The results indicate that immunization of mice with OMV via the in route [unclear - likely a specific route or method]... BA It can induce a certain level of humoral immune response in mice, but mice are immunized with OMV via the in route. BAΔ3 It can then cause more than OMV BA A stronger humoral immune response results in greater immune protection from OMVs secreted by remodeled bovine Brucella.

[0065] The PBS group did not produce antibodies against Brucella IgG1 and IgG2a; OMV BA Group and OMV BAΔ3 All groups of mice were able to induce strong anti-brucellosis IgG1 and IgG2a titers; by calculating the IgG2a / IgG1 ratio, it was found that OMV BA Group and OMV BAΔ3 The IgG2a / IgG1 ratio in all mice was less than 1. This indicates that immunizing mice with OMV via the in route [unclear - likely a specific route or method]... BA It can induce a humoral immune response in mice, which plays a key role in combating Brucella.

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

[0067] To investigate the separation and purification of OMV BA and OMV BAΔ3 T-cell immune responses were induced in mice. Mice were immunized via intranasal (in) administration followed by a challenge experiment. At week four post-challenge, the spleens of the mice were harvested, and lymphocytes were isolated. Flow cytometry was used to identify CD4+ cells in the spleen that secrete IFN-γ and TNF-α. + T cell levels.

[0068] The specific operating procedure is as follows: Six-week-old BALB / c mice were divided into PBS group and OMV group. BA Group and OMV BAΔ3 Group 3, with the PBS group consisting of mice immunized with PBS solution via the in immunotherapy route, serving as a negative control group; OMV BA Mice in this group were immunized with 15 μg of OMV via the in route. BA ;OMV BAΔ3 Mice in this group were immunized with 15 μg of OMV via the in route.BAΔ3 Three weeks after the initial immunization, the mice received a booster immunization. Three weeks after the booster immunization, a challenge protection test was conducted, with the immunized mice challenged by intraperitoneal injection (ip) of 5 x 10^6 mmol / L virus. 5 CFU-containing Brucella bovis was used to isolate lymphocytes from the spleen of immunized mice 4 weeks after challenge. Flow cytometry was used to determine the PBS control group and OMV. BA Group and OMV BAΔ3 CD4+ cells secreting IFN-γ and TNF-α in the spleen of mice in this group + T cell levels, evaluating OMV BA and OMV BAΔ3 It induces a T-cell immune response in mice. Among them, OMV BA Secreted by the parent strain of Brucella bovis BA, OMV BAΔ3 It is secreted by the bovine Brucella remodeling BAΔ3.

[0069] The results are as follows Figure 6 As shown, with OMV BA Compared with the control group of mice, OMV immunized via the in route... Bru-M3 After mice were infected with Brucella bovis, CD4+ cells secreting IFN-γ and TNF-α were found in the spleen. + The number of T cells increased significantly. The results indicate that immunization of mice with OMV via the in route... BAΔ3 It can then cause more than OMV BA A stronger T-cell immune response is also an important reason why OMVs secreted by remodeled bovine Brucella have higher immunoprotective efficacy.

[0070] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

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

2. The method for constructing a bovine Brucella mutant as described in claim 1, characterized in that, Using Brucella bovis RB51 as the parent strain, suicide plasmids were constructed for the eipB, per, and wadC genes, and then transformed by electroporation. The eipB gene was knocked out of the parent strain, and sucrose screening was performed to obtain deletion mutants. The deletion mutant was subjected to a second electroporation transformation, and the per gene was knocked out of the deletion mutant, followed by sucrose screening to obtain a double deletion mutant; The double deletion mutant was subjected to three electroporation transformations, and the wadC gene was knocked out of the deletion mutant, followed by sucrose screening to obtain the triple deletion mutant.

3. A method for constructing a bovine Brucella mutant as described in claim 2, characterized in that, The construction of the suicide plasmid specifically involves amplifying the upstream and downstream homologous arm sequences of the target genes per, wadC, and eipB by PCR, fusing the upstream and downstream homologous arm sequences to obtain a fusion fragment, and ligating the fusion fragment to the suicide plasmid using T4 ligase. The suicide plasmid contains the sacB gene for sucrose negative selection.

4. A mutant of Brucella bovis, characterized in that, The Brucella bovis mutant was deposited by the China General Microbiological Culture Collection Center (CGMCC) on May 27, 2025, and classified as Brucella bovis remodeled BAΔ3, with accession number CGMCC No. 34791.

5. The application of the bovine Brucella mutant as described in claim 4 in the following aspects: (1) Application of outer membrane vesicles of bovine Brucella mutant in the preparation of drugs with enhanced immunogenicity; (2) Application of brucellosis vaccine prepared from outer membrane vesicles of bovine Brucella mutant, wherein the brucellosis vaccine is used via mucosal immunization.

Images