A bacterial outer membrane vesicle-based antigen display system, and a preparation method and application thereof

Abstract

This invention discloses an antigen display system based on bacterial outer membrane vesicles, its preparation method, and its application, belonging to the field of biomedicine. This system utilizes SpyCatcher / SpyTag covalent linkage technology, using the E. coli surface protein ClyA as an anchoring scaffold, and fuses the SpyCatcher003 protein to its C-terminus. Simultaneously, the target antigen is fused with the SpyTag003 peptide for expression. Through the spontaneously formed irreversible covalent bond between SpyCatcher003 and SpyTag003, efficient, targeted, and stable display of the antigen on the surface of OMVs is achieved. Using the hemagglutinin HA1 domain of canine influenza virus H3N2 subtype as the antigen target, OMVs displaying HA1 were prepared. In vitro experiments confirmed that it has significant immunostimulatory activity and good biosafety, possessing the characteristics of high efficiency, controllability, and strong versatility, providing a new strategy for vaccine development.

Description

Technical Field

[0001] This invention belongs to the field of nanozyme preparation and catalytic detection technology, specifically relating to an antigen display system based on bacterial outer membrane vesicles, its preparation method, and its application. Background Technology

[0002] Canine influenza virus (CIV) is a major pathogen causing respiratory diseases in dogs, with the H3N2 subtype being the predominantly prevalent subtype in Chinese canine populations. Studies have confirmed that this virus can infect other animals such as cats, demonstrating its potential for cross-species transmission. Vaccination is a crucial measure for controlling canine influenza epidemics and reducing virus transmission in animal populations. Currently, there are no approved canine influenza vaccines in my country. While commercially available inactivated vaccines exist abroad, they suffer from limitations such as short periods of immune protection and complex immunization procedures. Therefore, developing safe and highly effective novel canine influenza vaccines is of significant practical importance.

[0003] Mucosal immunity is the first line of defense against respiratory viral infections. Besides antibodies (humoral immunity) and sensitized T cells (cellular immunity) produced by systemic immunity, it can also induce the production of immune effector substances locally in the mucosa. Therefore, developing novel vaccines that can effectively activate mucosal immunity is crucial for CIV control. However, the development of mucosal vaccines faces many challenges, one of the key being the lack of a safe, efficient, and stable antigen delivery system. Bacterial outer membrane vesicles (OMVs) are natural nanoparticles secreted by Gram-negative bacteria. They contain various bacterial components such as lipopolysaccharide (LPS), lipoproteins, peptidoglycans, phospholipids, and nucleic acids, possessing natural adjuvant effects and highly efficient antigen delivery capabilities, making them a highly promising vaccine carrier platform. Through genetic engineering, exogenous antigens can be directionally displayed on the surface of OMVs, thereby preparing novel vaccines with both strong immunogenicity and good safety.

[0004] Despite the promising prospects of OMVs as antigen delivery carriers, achieving efficient, controllable, and targeted display of exogenous antigens on OMV surfaces remains a core technological bottleneck in this field. Existing methods largely rely on direct fusion expression of antigens with OMV membrane proteins. This method is often limited by the expression efficiency, correct folding, and membrane anchoring stability of the fusion protein, resulting in insufficient process flexibility and universality. To address these issues, this invention presents an OMV antigen display system based on the "plug-and-display" principle. This system uses the highly abundant proteolysin A (ClyA) on the surface of *E. coli* OMVs as an anchoring scaffold, fusing and displaying the SpyCatcher003 protein at its C-terminus; simultaneously, the target antigen is fused and expressed with the SpyTag003 peptide. Utilizing the high-strength, irreversible heteropeptide bonds that spontaneously form between SpyCatcher003 and SpyTag003, efficient, covalent, and targeted coupling of exogenous antigens on the OMV surface is achieved. Regarding antigen selection, this invention targets the H3N2 subtype of CIV, which is prevalent worldwide, and selects the head domain HA1 of its hemagglutinin (HA) protein as the antigen target. Although the HA1 sequence has low conservation among different HA subtypes, the HA1 protein contains an important immunodominant epitope that can effectively induce the production of neutralizing antibodies, making it an ideal target for vaccine preparation against currently prevalent strains. Summary of the Invention

[0005] Technical problem to be solved: The present invention aims to overcome the shortcomings of the prior art and solve the problems of poor universality, low display efficiency and complicated process in the existing OMVs antigen display technology.

[0006] Objective of this invention: The objective of this invention is to provide a method for preparing an antigen display system based on bacterial outer membrane vesicles. Using green fluorescent protein (EGFP) as a model antigen, it is directly fused and expressed at the C-terminus of the ClyA protein, demonstrating that ClyA can serve as an effective scaffold protein for displaying exogenous antigens on the surface of OMVs. Based on this, a SpyCatcher / SpyTag system is introduced, fusing SpyCatcher with ClyA and expressing it on the surface of OMVs, while simultaneously linking SpyTag to the target antigen. Utilizing the spontaneous formation of irreversible heteropeptide bonds between SpyTag and SpyCatcher, the extracted OMVs can be efficiently coupled with any exogenous protein carrying the SpyTag, constructing a "plug-and-display" antigen display platform. This platform was applied to the development of a canine influenza subunit vaccine, successfully displaying the HA1 antigen on the surface of OMVs. This strategy not only enables rapid and flexible display of multiple antigens but also possesses the potential for rapid response in the development of vaccines for various infectious diseases.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing an antigen display system based on bacterial outer membrane vesicles includes the following steps: S1. The fusion gene ClyA-EGFP, a combination of green fluorescent protein EGFP and ClyA, was obtained using fusion PCR. This gene was then cloned into the pET28a vector, and the recombinant plasmid was transformed into *E. coli* DH5α competent cells. The cells were plated, and positive colonies were selected by PCR screening and sequenced. The plasmid from the correctly identified bacterial cultures was extracted and transformed into *E. coli* BL21(DE3) competent cells for expression. OMVs were extracted, and Western blot was used to verify the successful expression of the ClyA-EGFP fusion protein in OMVs, thus confirming that ClyA can serve as an effective surface display scaffold for OMVs. S2. The ClyA and SpyCatcher003 genes were amplified separately, and the ClyA-SpyCatcher (C-SC) fusion gene was obtained by fusion pCR method. This gene was then ligated into the pETDuet-1 vector. The recombinant plasmid was then transformed into E. coli DH5α competent cells, plated, and positive colonies were screened by pCR and sequenced. The plasmid was extracted from the correctly identified bacterial cultures. The amino acid sequence of SpyCatcher003 is shown in SEQ ID NO.1: AMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGS; S3. The extracted C-SC plasmid was transformed into E. coli BL21 (DE3) competent cells for expression. After Western blot analysis, the bacterial culture was cultured in large quantities. After IPTG induction, OMVs (C-SC OMVs) were extracted. The expression of C-SC fusion protein was verified by Western blot, and its morphology was observed by transmission electron microscopy (TEM). The particle size distribution was determined by nanoparticle tracking analysis (NTA) to confirm the integrity and uniformity of C-SC OMVs. S4. The hemagglutinin head domain HA1 of the CIV canine influenza virus H3N2 subtype was selected as the target antigen. Its encoding gene was fused with SpyTag003 using a flexible linker to construct the SpyTag-HA1 fusion gene, which was then cloned into the pCold-1 vector. The recombinant plasmid was then transformed into E. coli DH5α competent cells, and positive colonies were screened. The plasmid was extracted and transformed into E. coli BL21(DE3) competent cells for expression and Western blot detection. The bacterial culture was further cultured, and the SpyTag-HA1 protein in the inclusion body form was purified by Ni-NTA affinity chromatography. The purified protein was mixed with the C-SC OMVs prepared in S3 and ligated overnight on a rotary mixer at 4°C to obtain OMVs displaying HA1, namely OMVs-HA1. The amino acid sequence of SpyTag003 is shown in SEQ ID NO.2: RGVPHIVMVDAYKRYK. S5. Systematically identify C-SC-ST-HA1 OMVs, including: verifying the successful display and immunogenicity of the HA1 antigen by Western blot; analyzing the integrity of its nanostructure by TEM and NTA; assessing its ability to be taken up by macrophages and its immunostimulatory activity inducing cytokine expression by cell experiments; and detecting its in vitro cytotoxicity by lactate dehydrogenase (LDH) release assay, and comprehensively evaluating its physicochemical properties, immunogenicity and biosafety.

[0008] An antigen display system based on bacterial outer membrane vesicles prepared by the above-mentioned method is provided. This system uses a SpyCatcher and / or SpyTag system to couple exogenous antigens to the surface of bacterial outer membrane vesicles (OMVs), forming a plug-and-display platform. The SpyCatcher and / or SpyTag system comprises: a) OMVs with a first reaction chaperone SpyCatcher peptide or a functional variant thereof displayed on their surface, the first reaction chaperone being anchored to the outer membrane of the OMVs in the form of a fusion protein; b) a target antigen fused with a second reaction chaperone SpyTag peptide or a functional variant thereof, wherein the first reaction chaperone and the second reaction chaperone specifically recognize each other and form a covalent bond, thereby coupling the exogenous antigen to the surface of the bacterial outer membrane vesicles (OMVs).

[0009] Furthermore, the ClyA-SpyCatcher fusion protein was expressed in Escherichia coli prokaryotes, and OMVs were extracted from the supernatant after IPTG induction.

[0010] Furthermore, the exogenous antigen is the HA1 antigen of canine influenza virus H3N2 subtype, which is renatured after expression and purification via inclusion bodies.

[0011] Furthermore, the first reaction companion is SpyCatcher003, and the second reaction companion is SpyTag003.

[0012] Furthermore, the fusion protein is the ClyA-SpyCatcher003 fusion protein.

[0013] Furthermore, OMVs showed no significant cytotoxic effect on MDCK cells.

[0014] Furthermore, OMVs can be effectively taken up by macrophages, stimulating macrophages to produce cytokines, namely IFN-α and / or IL-1β.

[0015] A vaccine composition comprising the antigen display system based on bacterial outer membrane vesicles as described in any one of the preceding claims and a pharmaceutically acceptable carrier or excipient, the vaccine composition being administered by injection or orally.

[0016] This application also discloses the application of the antigen display system based on bacterial outer membrane vesicles as described in any of the above claims in the preparation of canine influenza virus subunit vaccines.

[0017] A nanoparticle vaccine displaying the HA1 antigen in OMVs, which, after intranasal immunization, can induce a significant increase in the level of secretory IgA (sIgA) in bronchoalveolar lavage fluid (BALF) of mice.

[0018] Furthermore, after intranasal immunization, the weight loss of challenged mice was significantly reduced. After intranasal immunization, the viral load in the feces and lungs of challenged mice was significantly reduced. After intranasal immunization, the viral load in the lungs of challenged mice was significantly reduced and the viral shedding time was significantly shortened. After intranasal immunization, the pathological damage score of lung tissue in challenged mice was significantly lower than that in the PBS challenge group and the whole virus inactivated vaccine group.

[0019] Technical Principle: This invention creatively combines the natural adjuvant effect and efficient antigen delivery capability of OMVs with the efficient, specific, and irreversible covalent linkage characteristics of SpyCatcher and / or SpyTag systems. Through genetic engineering, OMVs are modified into a universal antigen capture device with SpyCatcher grippers densely distributed on their surface. For any selected target antigen, it can be fused with a short SpyTag tag for expression or chemical synthesis. In vitro, the two are mixed, and heteropeptide bonds are spontaneously formed between SpyCatcher and SpyTag, clicking the antigen onto the surface of OMVs. This modular strategy of first preparing a universal vector and then coupling it with a specific antigen perfectly solves the shortcomings of existing technologies and achieves true plug-and-play functionality.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The OMV-based plug-and-display system prepared in this invention not only significantly improves the efficiency and controllability of antigen display, but also has good versatility and flexibility, and can quickly adapt to different pathogen antigens, providing an efficient and stable new strategy for developing novel mucosal vaccines against canine influenza and other respiratory diseases. 2. This invention utilizes the irreversible covalent bonds of the SpyCatcher and / or SpyTag systems to achieve a near 100% directional coupling efficiency of antigens on the surface of OMVs, and the antigen display is stable and not easily detached. 3. The OMVs-SpyCatcher vector can be prepared on a large scale in one go. As a universal platform, for different target antigens, only the expression of the SpyTag-antigen fusion protein needs to be changed to quickly assemble different vaccine candidates, which greatly shortens the research and development cycle and reduces costs. 4. Experiments have shown that the OMVs-antigen system constructed in this invention can be effectively taken up by antigen-presenting cells and significantly stimulate the expression of immune-related cytokines, indicating that it has good immunostimulatory activity. In vitro cytotoxicity tests have confirmed that it has good biosafety within the effective concentration range. 5. The conjugation of antigens to OMVs can be completed under mild in vitro conditions without the need for complex intracellular operations. The process is simple, controllable, and easy to scale up for production. 6. The OMVs-HA1 nanoparticle vaccine prepared in this invention can significantly induce a mucosal immune response in a mouse model after intranasal immunization, as evidenced by a significant increase in sIgA levels in BALF, indicating that the vaccine can establish an effective immune barrier in the respiratory tract. 7. Mice immunized with the OMVs-HA1 vaccine prepared in this invention showed a significantly reduced rate of weight loss after challenge, a significantly reduced viral load in feces and lungs, a significantly shortened viral shedding time, and a significantly lower lung tissue pathological damage score than the PBS challenge group and the whole virus inactivated vaccine group. Compared with the whole virus inactivated vaccine, the vaccine of this invention showed better protective effects in inhibiting viral replication, alleviating clinical symptoms, and reducing tissue damage. Attached Figure Description

[0021] Figure 1This diagram shows the expression and identification of ClyA-EGFP in OMVs according to the present invention. A represents the expression of EGFP in bacterial cells, B represents the expression of EGFP in OMVs, where 1 is an empty vector plasmid, 2 is EGFP, and 3 is ClyA-EGFP; C represents the Western blot detection results using ClyA as the primary antibody, D represents the Western blot detection results using GFP as the primary antibody, and E represents the Western blot detection results using His tag antibody as the primary antibody, where 1 represents ClyA OMVs, 2 represents EGFP OMVs, and 3 represents ClyA-EGFP OMVs. Figure 2 The diagram shows the expression of the C-SC protein in bacterial cells and OMVs according to this invention. A represents the expression of the C-SC protein in OMVs, where 1 is the supernatant and 2 is the bacterial cells; B represents the expression of the C-SC protein in OMVs; and C represents the optimized expression conditions of the C-SC protein in OMVs, where 1 is induced by 1 mM IPTG, 2 is induced by 0.5 mM IPTG, 3 is induced by 0.1 mM IPTG, and 4 is induced by 0.02 mM IPTG. Figure 3 This is a verification diagram showing the positioning of the C-SC of the present invention on the surface of OMVs; Figure 4 This is a TEM observation and NTA analysis of C-SC OMVs of the present invention, wherein A is a TEM observation image of C-SC OMVs with a scale bar of 200 nm, and B is an NTA analysis image of C-SC OMVs. Figure 5 The diagrams show the expression, purification, and identification of recombinant OMVs of CIV HA1 protein in this invention. A is an SDS-PAGE analysis of ST-HA1 protein expression, with 1-3 and 4-6 representing the supernatant and precipitate induced by 1mM, 0.5mM, and 0.2mM IPTG, respectively. B is an SDS-PAGE analysis of purified ST-HA1 protein, with 1 representing unpurified inclusion bodies and 2 representing purified protein. C is a Western blot result of OMVs-HA1 using anti-ClyA polyclonal antibody as the primary antibody. D is a Western blot result of OMVs-HA1 using His-tagged antibody as the primary antibody. E is a Western blot result of OMVs-HA1 using anti-CIV whole virus antiserum as the primary antibody. F is a Western blot result of OMVs-HA1 using anti-HA polyclonal antibody as the primary antibody, with 1 representing C-SC OMVs, 2 representing ST-HA1 protein, and 3 representing OMVs-HA1. Figure 6This is a quantitative analysis diagram of HA1 protein carried by OMVs in this invention. A is the gradient Western blot analysis of HA1 protein samples, where 1-5 represent HA1 protein samples with loading amounts of 2.5, 2, 1.5, 1, and 0.5 μg, respectively. B is the quantitative standard curve of HA1 protein Western blot band gray values. Figure 7 The images show TEM observation and NTA analysis of the C-SC-ST-HA1 OMVs of this invention, where A is the TEM observation image with a scale bar of 200 nm and B is the NTA analysis image. Figure 8 This is a fluorescence microscopy image showing the uptake of OMVs by macrophages according to the present invention. The scale bar in the image is 100 μm. Figure 9 The images show the RT-qPCR results of key cytokine mRNA levels in macrophages after OMVs treatment according to this invention. The left image shows the TNF-α mRNA expression level, the middle image shows the IL-1β mRNA level, and the right image shows the IL-6 mRNA level. Figure 10 This is a graph showing the cytotoxic effect of OMVs-HA1 on MDCK according to the present invention. Figure 11 This is a schematic diagram illustrating the principle of antigen-OMV coupling based on the SpyCatcher / SpyTag system of this invention. Figure 12 This invention relates to the detection of serum IgG antibody levels in mice after immunization. Figure 13 This invention relates to the detection of mouse BALF sIgA antibody levels after immunization, where A is the linear regression curve of the standard and B is the detection result of mouse BALF sIgA antibody levels. Figure 14 This invention relates to the determination of neutralizing antibody titers in mouse serum after immunization; Figure 15 This invention relates to the detection of changes in mouse body weight after immunization and challenge with the virus. Figure 16 The JS / 10 M gene standard curve constructed for this invention; Figure 17 This invention describes the detection of viral load in the feces and lungs of mice after immunization and challenge with the virus. Figure A shows the results of viral load detection in the feces, and figure B shows the results of viral load detection in the lungs. Figure 18 For the pathological observation and analysis of mouse lung tissue after immunization and challenge according to the present invention, A is a pathological section of lung tissue in the OMVs-HA1 group, B is a pathological section of lung tissue in the inactivated vaccine group, C is a pathological section of lung tissue in the PBS-M group, and D is a pathological section of lung tissue in the PBS-Ch group. Figure 19 The pathological damage score of mouse lung tissue after immunization and challenge with the virus is given by this invention. Detailed Implementation

[0022] To better illustrate the purpose, technical solution, and advantages of this invention, the following will provide further explanation of this application in conjunction with specific embodiments.

[0023] Experimental materials used in the embodiments of this invention: Strains and plasmids: Escherichia coli DH5α and BL21 (DE3 competent cells) were purchased from Tolo Biotechnology Co., Ltd.; RAW264.7 cells, canine kidney epithelial cells (Madin-Darby Canine kidney, MDCK) and plasmids such as pET28a, pCold-1, and pETDuet-1 were preserved in our laboratory; H3N2 subtype CIV strain A / Canine / Jiangsu / 06 / 2010 (JS / 10), GenBank accession numbers JN247616~JN247623, were isolated and preserved in our laboratory.

[0024] Main reagents: 2×phanta Max MasteRMix (Dyeplus) high-fidelity DNA polymerase, Green TaqMix DNA polymerase, recombinant cloning kit, and reverse transcription real-time quantitative PCR (RT-qPCR) detection kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; gel extraction kit and plasmid extraction kit were purchased from Omega Bio-Tek; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG, ECL high-sensitivity chemiluminescence solution, 4% paraformaldehyde fixative, and DAPI-containing anti-fluorescence quenching mounting solution were purchased from Beijing Lanjieke Technology Co., Ltd.; FITC-labeled goat anti-mouse IgG was purchased from Wuhan Sanying Biotechnology Co., Ltd.; proteinase K (PK) solution was purchased from Biosharp Biotechnology Co., Ltd.; ethylenediaminetetraacetic acid (EDTA) was purchased from Maclean's Reagents Co., Ltd.; glycine was purchased from BioFroxx; BCA protein assay kit was purchased from Beyotime Biotechnology Co., Ltd.; and various specifications of DNA... MarkeR and protein gel preparation kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.; protein markers were purchased from Shanghai Yamei Biomedical Technology Co., Ltd.; kanamycin and ampicillin were purchased from Invitrogen. GoldView TM Nucleic acid dyes were purchased from Beijing Dingguo Biotechnology Co., Ltd.; mouse IgA ELISA kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.; tissue fixative: 4% formaldehyde, prepared fresh for immediate use.

[0025] The primer sequences required for this invention are shown in Tables 1-3 below and were synthesized by Qingke Biotechnology Co., Ltd. or Genscript Biotech Co., Ltd.

[0026] Table 1 ; Table 2 ; Table 3 .

[0027] Example 1: This example provides a method for preparing an antigen display system based on bacterial outer membrane vesicles, including the following steps: S1. The fusion gene ClyA-EGFP, a combination of green fluorescent protein EGFP and ClyA, was obtained using fusion PCR. This gene was then cloned into the pET28a vector, and the recombinant plasmid was transformed into *E. coli* DH5α competent cells. The cells were plated, and positive colonies were selected by PCR screening and sequenced. The plasmid from the correctly identified bacterial cultures was extracted and transformed into *E. coli* BL21(DE3) competent cells for expression. OMVs were extracted, and Western blot was used to verify the successful expression of the ClyA-EGFP fusion protein in OMVs, thus confirming that ClyA can serve as an effective surface display scaffold for OMVs. S2. The ClyA and SpyCatcher003 genes were amplified separately, and the ClyA-SpyCatcher (C-SC) fusion gene was obtained by fusion pCR method. This gene was then ligated into the pETDuet-1 vector. The recombinant plasmid was then transformed into E. coli DH5α competent cells, plated, and positive colonies were screened by pCR and sequenced. The plasmid was extracted from the correctly identified bacterial cultures. The amino acid sequence of SpyCatcher003 is shown in SEQ ID NO.1: AMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGS; S3. The extracted C-SC plasmid was transformed into E. coli BL21 (DE3) competent cells for expression. After Western blot analysis, the bacterial culture was cultured in large quantities. After IPTG induction, OMVs (C-SC OMVs) were extracted. The expression of C-SC fusion protein was verified by Western blot, and its morphology was observed by transmission electron microscopy (TEM). The particle size distribution was determined by nanoparticle tracking analysis (NTA) to confirm the integrity and uniformity of C-SC OMVs. S4. The hemagglutinin head domain HA1 of the CIV canine influenza virus H3N2 subtype was selected as the target antigen. Its encoding gene was fused with SpyTag003 using a flexible linker to construct the SpyTag-HA1 (ST-HA1) fusion gene, which was then cloned into the pCold-1 vector. The recombinant plasmid was then transformed into E. coli DH5α competent cells, and positive colonies were screened. The plasmid was extracted and transformed into E. coli BL21(DE3) competent cells for expression and Western blot detection. The bacterial culture was further cultured, and the SpyTag-HA1 protein in inclusion body form was purified by Ni-NTA affinity chromatography. The purified protein was mixed with the C-SC OMVs prepared in S3 and ligated overnight on a rotary mixer at 4°C to obtain OMVs displaying HA1, namely C-SC-ST-HA1 OMVs. The amino acid sequence of SpyTag003 is shown in SEQ ID NO.2: RGVPHIVMVDAYKRYK. S5. Systematically identify C-SC-ST-HA1 OMVs, including: verifying the successful display and immunogenicity of the HA1 antigen by Western blot; analyzing the integrity of its nanostructure by TEM and NTA; evaluating its ability to be taken up by macrophages and its immunostimulatory activity in inducing cytokine expression by cell experiments; and detecting its in vitro cytotoxicity by lactate dehydrogenase (LDH) release assay, and comprehensively evaluating its physicochemical properties, immunogenicity, and biosafety.

[0028] The antigen display system based on bacterial outer membrane vesicles prepared by the method described herein couples exogenous antigens to the surface of bacterial outer membrane vesicles (OMVs) via a SpyCatcher and / or SpyTag system, forming a plug-and-display antigen display platform. The SpyCatcher and / or SpyTag system comprises: a) OMVs with a first reaction chaperone SpyCatcher peptide or a functional variant thereof displayed on their surface, the first reaction chaperone being anchored to the outer membrane of the OMVs in the form of a fusion protein; b) a target antigen fused with a second reaction chaperone SpyTag peptide or a functional variant thereof, wherein the first reaction chaperone and the second reaction chaperone specifically recognize each other and form a covalent bond, thereby coupling the exogenous antigen to the surface of bacterial outer membrane vesicles (OMVs).

[0029] The ClyA-SpyCatcher fusion protein was expressed in Escherichia coli prokaryotic cells. After IPTG induction, OMVs were extracted from the supernatant. The exogenous antigen was canine influenza virus H3N2 subtype HA1 antigen, which was purified by inclusion body expression and then refolded. The first reaction chaperone was SpyCatcher003, the second reaction chaperone was SpyTag003, and the fusion protein was the ClyA-SpyCatcher003 fusion protein. OMVs had no significant cytotoxic effect on MDCK cells. OMVs could be effectively taken up by macrophages and stimulate macrophages to produce cytokines, namely IFN-α, IL-6, and / or IL-1β.

[0030] A vaccine composition comprising the antigen display system based on bacterial outer membrane vesicles as described in any one of the preceding claims and a pharmaceutically acceptable carrier or excipient, the vaccine composition being administered by injection or orally.

[0031] The application of any of the above-described antigen display systems based on bacterial outer membrane vesicles in the preparation of canine influenza virus subunit vaccines.

[0032] ClyA serves as a validation of the effectiveness of OMVs surface display brackets: First, using enhanced green fluorescent protein (EGFP) as a model antigen, its encoding gene was ligated to the C-terminus of the ClyA protein via fusion PCR to prepare a fusion gene fragment. This fragment was cloned into the pET28a expression vector, successfully preparing the recombinant plasmid pET28a-ClyA-EGFP. This plasmid was transformed into *E. coli* BL21 (DE3) competent cells, and after IPTG induction, the cells exhibited obvious green fluorescence under excitation light. Figure 1 As shown in Figure A. The bacterial supernatant was collected and purified by ultracentrifugation to obtain OMVs. The purified OMVs also showed a detectable green fluorescence signal under the same conditions, as shown in Figure A. Figure 1 As shown in Figure B. To confirm the expression of the fusion protein at the molecular level, Western blot analysis was performed on the purified OMVs samples using ClyA antibody, GFP antibody, and His-tagged antibody as primary antibodies. The results showed that a single specific band was detected at approximately 65 kDa in all OMVs samples, as shown in Figure B. Figure 1 C~ Figure 1 As shown in Figure E, this molecular weight is consistent with the theoretically calculated size of the ClyA-EGFP fusion protein. These results fully demonstrate that ClyA can serve as an effective scaffold protein, enabling the display of exogenous proteins on the surface of OMVs.

[0033] Example 2, Preparation and optimization of the ClyA-SpyCatcher (C-SC) fusion protein expression system: Preparation of the C-SC fusion protein expression system: To prepare a vector system capable of directionally displaying the SpyCatcher003 protein on the surface of OMVs, the gene sequences encoding the E. coli ClyA protein and the SpyCatcher003 protein were first amplified by PCR. Then, the SpyCatcher003 gene was precisely ligated to the C-terminus of the ClyA gene using a flexible linker peptide (GGGGS)3, obtaining the ClyA-SpyCatcher003 (hereinafter referred to as C-SC OMVs) fusion gene. This fusion gene fragment was then processed... BamHI and HindIII After double enzyme digestion, the plasmid was cloned into the pETDuet-1 vector to prepare a recombinant plasmid. The plasmid was transformed into E. coli DH5α competent cells, and positive clones were screened by ampicillin resistance plates. The fusion gene sequence was confirmed to be correct and the reading frame was correct by colony PCR and DNA sequencing. The verified recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium and cultured at 37°C until OD500. 600 When the pH was 0.6–0.8, IPTG was added, and expression was induced at 16°C for 16 h. After induction, the expression of C-SC protein in bacterial cells was detected by Western blot. The results showed that C-SC protein was successfully expressed, with a size of 55 kDa. Figure 2 As shown in Figure A.

[0034] Extraction of C-SC OMVs: A small amount of the above bacterial culture was inoculated into 1 L of LB medium and cultured at 37°C until OD500 reached. 600 The bacterial culture was cooled to 16°C to a concentration of 0.6–0.8, and then 0.1 mM IPTG and 1% glycine were added. After induction at 16°C for 16 h, the culture was centrifuged at 5000 g for 10 min at 4°C, and the bacterial cells were discarded. The supernatant was filtered through 0.45 μm filter paper and then concentrated to below 100 mL using an ultrafiltration membrane or a 100-kDa ultrafiltration tube. The concentrate was then centrifuged at 120,000 g for 2 h at 4°C. The collected OMVs were washed with PBS and then resuspended in PBS. Western blot analysis of the extracted OMVs was performed using a His-tagged antibody as the primary antibody. A single, clear band was detected at approximately 53 kDa. Figure 2 As shown in Figure B, the molecular weight of the C-SC fusion protein is consistent with the theoretically calculated value, confirming that the C-SC fusion protein has been successfully expressed and localized in OMVs.

[0035] Optimization of C-SC expression conditions in OMVs: To further optimize the expression efficiency of C-SC in OMVs, expression was induced at 16℃ using IPTG concentration gradients (1, 0.5, 0.1, 0.02 mM), and OMVs were extracted for Western blot analysis. The results showed... Figure 2 As shown in Figure C, the C-SC fusion protein exhibited the highest expression level in OMVs at an IPTG concentration of 0.1 mM, with a single protein band and no significant degradation. Therefore, 0.1 mM IPTG was determined to be the optimal induction concentration for this system, which can be used for subsequent large-scale preparation of OMVs displaying SpyCatcher003.

[0036] Example 3, Verification of the localization of C-SC protein on the surface of OMVs: To verify whether the C-SC fusion protein is correctly located on the surface of OMVs, thus ensuring its function as an antigen capture scaffold, this study performed a proteinase K (PK) digestion experiment. The principle is that PK cannot penetrate the intact OMV membrane structure and can only degrade proteins exposed on the outer surface of the vesicles; while ethylenediaminetetraacetic acid (EDTA) can disrupt membrane integrity, exposing internal proteins. Therefore, by comparing the protein degradation in different treatment groups, protein localization can be clearly defined.

[0037] The experiment was conducted in three groups: Group A, with PK added (final concentration 100 μg / mL); Group B, with EDTA added (final concentration 0.1 mol / L); and Group C, with both PK and EDTA added (final concentrations as above). After incubating the OMVs samples at 37℃ for 3 hours, the C-SC protein bands were detected by Western blotting (using a His-tagged antibody as the primary antibody). The results are as follows: Figure 3 As shown, after treatment with PK alone (Group A), the C-SC protein band completely disappeared; while after treatment with EDTA alone (Group B), the protein band remained intact. This result indicates that the C-SC fusion protein is exposed on the surface of OMVs. This key localization characteristic confirms that the fusion protein can serve as an effective surface display scaffold, laying a reliable foundation for the subsequent efficient and targeted conjugation of target antigens on OMVs using the SpyCatcher / SpyTag system.

[0038] Example 4, TEM observation and NTA analysis of 4C-SC OMVs, including the following steps: C-SC OMVs were characterized by TEM and NTA, such as Figure 4As shown. The purified OMVs sample was diluted with PBS, and 10 μL was dropped onto a copper grid supporting a carbon membrane and allowed to stand for 1 min. Excess liquid was absorbed from the edge of the droplet with filter paper. Then, 10 μL of phosphotungstic acid negative staining solution was added for staining for 1 min, and the staining solution was again absorbed with filter paper. After the copper grid was completely dried, images were observed and acquired under a TEM. The OMVs were diluted to a certain proportion, and their concentration and diameter distribution were analyzed using NTA. The results showed that C-SC OMVs exhibited a classic spherical vesicle structure, and their phospholipid bilayer membrane was clearly visible. Quantitative analysis using NTA revealed that the concentration of OMVs was 1.27 × 10⁻⁶. 11 particles·mL -1 It is abundant in content, with an average particle size of (68±3) nm and a concentrated particle size distribution, which further confirms its size uniformity.

[0039] Example 5: CIV HA1 antigen display and validation based on OMVs: Preparation and purification of the SpyTag-HA1 (ST-HA1) protein expression system: To prepare canine influenza virus (CIV) antigen that can be directionally displayed on the surface of OMVs, the gene encoding the CIV H3N2 hemagglutinin head domain (HA1) was linked to the SpyTag003 gene via a flexible linker and cloned into the pCold-1 vector to prepare a recombinant plasmid. This plasmid was transformed into *E. coli* BL21 (DE3) and expression was induced under different concentrations of IPTG (1, 0.5, 0.2 mM). SDS-PAGE analysis showed that the ST-HA1 fusion protein was mainly expressed in inclusion body form, and the expression level was highest at an IPTG concentration of 1.0 mM. Figure 5 As shown in Figure A. Then, the inclusion body form of ST-HA1 was purified by Ni-NTA affinity chromatography, as shown... Figure 5 As shown in Figure B, the recombinant ST-HA1 protein was purified by refolding via gradient dialysis.

[0040] Display and validation of HA1 protein on OMVs: The refolded ST-HA1 protein was mixed with OMVs prepared in Example 2, with SpyCatcher003 displayed on the surface, and ligated overnight at 4°C using a rotary mixer. Covalent ligation was achieved using spontaneously formed isopeptide bonds between SpyCatcher003 and SpyTag003. The ligation products were analyzed by Western blot, using ClyA antibody and His-tagged antibody for detection. Specific bands were observed at approximately 95 kDa in both samples. Figure 5 C and such Figure 5 As shown in Figure D, the molecular weight of C-SC-ST-HA1 is consistent with the theoretically calculated value, confirming that the HA1 antigen has been successfully attached to the surface of OMVs.

[0041] Quantitative analysis of HA1 protein carried by OMVs: To accurately determine the content of HA1 protein displayed on the surface of OMVs, Western blot combined with grayscale analysis was used for quantification. First, purified recombinant ST-HA1 protein of known concentrations was prepared into gradient concentration samples (0.5, 1.0, 1.5, 2.0, 2.5 μg) as standards, which were then subjected to SDS-PAGE electrophoresis together with the OMVs-HA1 samples to be tested. Western blot detection was performed using an anti-His tag antibody as the primary antibody. The results are shown below. Figure 6 As shown in Figure A. ImageJ software was used to analyze the grayscale values ​​of each concentration band of the standard. A standard curve was plotted with the protein loading amount as the x-axis and the corresponding band grayscale values ​​as the y-axis, as shown in Figure A. Figure 6 As shown in Figure B, the standard curve exhibits good linearity (R²). 2 >0.98), meeting the quantitative requirements. Substituting the gray value of the OMVs-HA1 sample band into the standard curve, the concentration of C-SC-ST-HA1 fusion protein in OMVs was calculated to be approximately 240 μg·mL⁻¹. -1 .

[0042] TEM observation and NTA analysis of SC-ST-HA1 OMVs: To verify the impact of the antigen display process on the basic structure of OMVs vectors, C-SC OMVs were characterized by TEM and NTA, such as... Figure 7 As shown. The specific operation method is as described in Example 4. TEM observation results show that the OMVs displaying the CIV HA1 antigen exhibit typical, well-defined spherical or near-spherical vesicle structures, with their phospholipid bilayer structure fully visible, indicating that the SpyTag / SpyCatcher display system of this invention does not disrupt the basic nanovesicle morphology of the OMVs. Quantitative analysis of the hydrodynamic particle size of C-SC-ST-HA1 OMVs was performed using NTA, as shown... Figure 7 As shown in Figure B, its average particle size is (70±3) nm, and the particle size distribution is relatively uniform. Compared with the C-SCOMVs characterized in the previous examples, the average particle size is as follows... Figure 4 Compared to Figure B, there were no statistically significant differences in average particle size and distribution range between the two. These results indicate that the successful display and conjugation of the exogenous HA1 antigen did not significantly alter the structural integrity, particle size, or distribution characteristics of the OMVs vector, further confirming the mildness and controllability of this display strategy.

[0043] Example 6: Validation of uptake of the CIV HA1 antigen display system based on OMVs in macrophages: To evaluate the immunodelivery potential of the CIV HA1 antigen display system, OMVs-HA1, prepared in this invention, its ability to be internalized by antigen-presenting cells was verified by a macrophage uptake experiment. The specific steps are as follows: RAW 264.7 mouse macrophage cells were revived and passaged, and the cells were seeded into 24-well plates containing cell spreaders. When the cell density reached 70%–80%, purified OMVs-HA1 was added and co-incubated for 3 h. After incubation, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde at 4°C for 15 min, permeabilized with 0.1% Triton X-100 at 4°C for 10 min, and then blocked with 3% bovine serum albumin (BSA) at room temperature for 30 min. Immunofluorescence staining was then performed: first, anti-ClyA mouse polyclonal antibody was used as the primary antibody and incubated overnight at 4°C; after washing, FITC-labeled goat anti-mouse IgG was used as the secondary antibody and incubated at room temperature in the dark for 1 h. Finally, the cell nuclei were stained with DAPI, and the slides were mounted and observed under a fluorescence microscope.

[0044] The results are as follows Figure 8 As shown, a clear fluorescent signal was observed in the FITC channel (green fluorescence) concentrated in the cytoplasm, particularly around the nucleus. This signal was adjacent to, but did not overlap with, the DAPI channel (blue, nucleus) signal. This indicates that OMVs-HA1 can be effectively internalized by macrophages and primarily localized in the cytoplasm and perinuclear region. This result provides direct cellular evidence that this OMVs vaccine vector can be effectively taken up by immune cells and may initiate subsequent antigen presentation processes.

[0045] Example 7, RT-qPCR detection of the stimulatory effect of OMVs on cytokines: To investigate the stimulatory effect of OMVs on immune cells, the mRNA expression levels of some cytokines in macrophages were measured using RT-qPCR. RAW 264.7 cells were passaged after resuscitation and seeded in 24-well plates. Once the cells reached 70%–80% confluence, they were injected with 2 μg / mL OMV. -1 Cells were treated with C-SC OMVs, ST-HA1 protein, and OMVs-HA1 for 6 h, with PBS and LPS serving as negative and positive controls, respectively. Total RNA was then extracted for reverse transcription, using β-actin as an internal reference gene. −ΔΔCT The relative expression levels of target genes were calculated. Results showed that OMVs-HA1 treatment significantly upregulated the mRNA expression levels of TNF-α, IL-1β, and IL-6, and this induction effect was significantly stronger than that of single C-SC OMVs or ST-HA1 protein treatment groups; however, compared with the LPS treatment group, the cytokine expression levels induced by OMVs-HA1 were still significantly lower, such as... Figure 9As shown, this indicates that it potentially reduces the risk of triggering an excessive inflammatory response while retaining sufficient adjuvant activity.

[0046] Example 8 demonstrates the in vitro evaluation of the immunostimulatory effect of CIV HA1 OMVs on macrophages: This invention evaluates the immunoactivation properties of a prepared CIV HA1-OMVs vaccine candidate by detecting the expression levels of key pro-inflammatory cytokines in a macrophage model. The specific method is as follows: RAW 264.7 cells were seeded in 24-well plates. When the cell confluence reached 70%–80%, the cells were treated for 6 hours with OMVs-HA1 (experimental group), C-SC OMVs (vector control group), soluble ST-HA1 protein (antigen control group), PBS (negative control group), and LPS (positive control group), respectively. All OMVs and proteins were treated at a concentration of 2 μg / mL. -1 After treatment, total RNA was extracted from cells and reverse transcribed into cDNA. Using β-actin as an internal reference gene, the mRNA expression levels of TNF-α, IL-1β, and IL-6 were detected by real-time quantitative PCR. -ΔΔCt The relative expression level was calculated using this method. The results showed that, as... Figure 10 As shown, compared with the PBS group, the OMVs-HA1 treatment group showed the most significant induction of TNF-α, IL-1β, and IL-6 mRNA expression, with a significantly stronger effect than the C-SC OMVs or ST-HA1 protein treatment groups alone. However, this induction level was still significantly lower than that of the LPS positive control group. These results demonstrate that the OMVs vaccine system of the present invention can effectively activate the immune response while potentially reducing the risk of triggering an excessive inflammatory response.

[0047] Example 9 demonstrates the cytotoxicity assessment of CIV HA1's OMVs: The potential cytotoxicity of OMVs-HA1 on canine kidney epithelial cells (MDCK) was assessed using the lactate dehydrogenase (LDH) release assay, following the instructions of the commercially available assay kit. MDCK cells were seeded in 96-well plates, and after cell attachment, different final concentrations (5, 10, 20, 40, and 100 μg / mL) were administered. -1 Cells were treated with OMVs-HA1 for 24 h, and a solution of 20 μg·mL⁻¹ was prepared. -1 Positive control group treated with lipopolysaccharide (LPS) and negative control group treated with PBS. After treatment, cell culture supernatant was collected, LDH detection working solution was added, and the absorbance value at 490 nm was measured using an ELISA reader. The percentage of cytotoxicity was calculated using the following formula.

[0048] Cytotoxicity (%) = × 100 The results showed that at ≤40 μg·mL -1 Within the concentration range, the OMVs-HA1 treatment group did not show a statistically significant increase in cytotoxicity, and its LDH release level was comparable to that of the negative control group. Even at the highest tested concentration (100 μg·mL⁻¹), -1 Even at this level, its cytotoxicity remained below 10%, significantly lower than 20 μg·mL⁻¹. -1 The LPS-positive control group showed greater than 40% cytotoxicity. This result confirms that the OMVs-HA1 prepared in this invention exhibits cytotoxicity within ≤100 μg·mL⁻¹. -1 The concentration range did not produce significant membrane damage to MDCK cells, indicating that the vaccine candidate has good in vitro cell safety.

[0049] Example 10, Mouse Immunization and Challenge: 120 five-week-old female SPF BALB / c mice were randomly divided into four groups of 30 mice each. A three-dose immunization strategy was adopted, with each immunization two weeks apart. The OMVs-HA1 group was immunized via intranasal administration; a whole-virus inactivated vaccine group (using 15% Montanide Gel 02 as an adjuvant) was set up as the immunization control group, the PBS challenge group (PBS-Ch) as the negative control to evaluate the challenge model and protective effect, and the PBS non-challenge group (PBS-M) as the blank control to define the baseline levels of various indicators. Two weeks after the last immunization, 50 μL of 10⁷ EID₅₀ JS / 10 chicken embryo cultured virus was administered intranasally.

[0050] Example 11, Detection of mouse serum IgG antibody levels: Two weeks after each immunization, four mice from each group were randomly selected, and blood was collected via orbital sampling to obtain serum. Serum IgG antibody levels were detected using ELISA, and the results are as follows: Figure 12 As shown, the IgG antibody level in the OMVs-HA1 group was slightly lower than that in the inactivated vaccine group, but there was no statistically significant difference.

[0051] Example 12, Detection of sIgA antibody levels in mouse bronchoalveolar lavage fluid (BALF): Two weeks after the second booster immunization, four mice were randomly selected from each group, and BALF (basal body fluid) was collected. The specific method was as follows: after euthanizing and restraining the mice, the skin at the neck was cut open to expose the trachea. An indwelling needle was carefully inserted parallel to the trachea, the needle tip was removed, leaving only the tubing inside. A 1 mL syringe was then inserted into the tubing, and the trachea and needle were tied tightly with thread. Pre-cooled PBS was slowly injected, and the mouse's chest bulged. The chest returned to its original position when the syringe was slowly withdrawn. This process was repeated twice. The collected BALF was placed on ice. The IgA level in the BALF was detected using a one-step sandwich ELISA kit with double antibodies. The sample, standard, and HRP-labeled detection antibody were added sequentially to the coated microwells containing IgA antibody, incubated, and then washed. The substrate TMB was used for color development. TMB is converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The absorbance (OD value) was measured at 450 nm using a microplate reader. A linear regression curve of the standard concentration was plotted on the x-axis and the corresponding OD value on the y-axis to calculate the sample concentration. The results are as follows Figure 13 As shown, the BALF sIgA level in the OMVs-HA1 nasal immunization group was significantly higher than that in other groups (P<0.0001), indicating that nasal immunization successfully induced a strong humoral immune response in the respiratory mucosa.

[0052] Example 13, Detection of antibody neutralization levels in mouse serum: Serum from two weeks after the first booster immunization was used for the neutralization experiment. The experiment included an experimental group, a cell control group (cells + maintenance medium), a virus control group (virus + maintenance medium), and positive and negative serum control groups. The specific procedures are as follows: (1) Cell plating: Add 100 μL of cell suspension to each well of a 96-well plate and incubate overnight at 37°C in a CO2 incubator until the cells reach 80%.

[0053] (2) Dilute the virus to 200 TCID50 with serum-free DMEM; treat the serum antibody in a 56℃ water bath for 30 min to remove serum complement, adjust to the initial concentration of 1 mg / mL, and perform serial dilution. Then mix 50 μL of virus solution and 50 μL of serum, and incubate in a 96-well plate at 37℃ for 1 h.

[0054] (3) Discard the liquid in the cell plate and wash once with PBS. Add 100 μL of the prepared mixture from (2) to the corresponding well. Incubate at 37°C for 1-2 h.

[0055] (4) After adsorption is complete, discard the liquid in the well, add 100 μL of cell maintenance medium containing TPCK-trypsin to each well, and put it back into the incubator to continue culturing.

[0056] (5) Culture the cells at 37℃ for 48-72 h. When 100% cytopathic effect occurs in the MDCK cells of the virus-positive control, observe the cytopathic effect in the test wells. The neutralizing titer is expressed as the highest dilution that neutralizes the virus.

[0057] The results showed that, Figure 14 As shown, when the positive control MDCK cells exhibited significant cytopathic effects, the OMVs-HA1 group showed no significant cytopathic effects at a 32-fold dilution of the antiserum, but some cells showed cytopathic effects at a 64-fold dilution, with subsequent dilutions showing more severe cytopathic effects. In the inactivated vaccine group, no significant cytopathic effects were observed at a 64-fold dilution; however, some cells showed cytopathic effects at a 128-fold dilution, with subsequent dilutions showing more severe cytopathic effects. The experiment was repeated three times, with consistent results, indicating that the neutralizing titer of the OMVs-HA1 group was 1:32, and the neutralizing titer of the inactivated vaccine group was 1:64.

[0058] Example 14, Clinical symptoms and weight changes in mice: Mice were observed daily after challenge, and their weight was recorded on days 2, 4, 6, 10, and 14 post-challenge. Two to four days post-challenge, mice in the PBS-challenge group exhibited lethargy, huddling, and decreased appetite, while the vaccine-treated group showed milder symptoms. Post-challenge weight change is an important indicator for evaluating vaccine efficacy; therefore, mice were weighed every two days (n=4) after challenge. Results are as follows... Figure 15 As shown, the PBS challenge group experienced a continuous decrease in body weight over 6 days post-challenge, reaching its lowest point on day 6 with a decrease of 15%. In contrast, all vaccine immunization groups showed significant protective effects (P<0.001), with the OMVs-HA1 immunization group exhibiting the least weight fluctuation, showing only a slight decrease 2–4 days post-challenge followed by rapid recovery.

[0059] Example 15, Determination of viral load in mouse feces and lungs: Four mice were randomly sacrificed on days 2, 4, 6, 10, and 14 after viral challenge. The mice were immobilized, and their lungs were aseptically harvested while feces were collected. The diseased tissue was photographed and recorded. The lungs were divided into three portions: one for tissue sectioning; the other two portions were weighed and recorded separately, and placed in an RNA Keeper at -80°C for analysis of viral load and related cytokine detection. RNA was extracted from all samples collected at all time points using the Trizol method. Samples were first thawed at room temperature, and the surface moisture was blotted dry with filter paper before being placed in a homogenization tube. 0.1 g of tissue was added to 1 mL of lysis buffer and a grinding bead, and the mixture was homogenized thoroughly. The homogenate was transferred to a 1.5 mL EP tube and centrifuged at 10,000 rpm for 5 min. The supernatant was then collected for further analysis. The same procedure was performed on mouse feces. Using cDNA obtained by reverse transcription of viral RNA extracted from lungs and feces as a template, primer pairs targeting a highly conserved sequence of the JS / 10 Matrix gene (M gene) were designed. Absolute quantification was used to determine the viral load in lungs and feces. First, a standard plasmid was constructed by PCR amplification of the M gene, followed by ligation into the pMD-19T vector. Positive bacteria were identified and screened, and the plasmid was extracted and used as a standard at a ratio of 10-10. -3 -10 -9 Dilute the sample and test it together with the test sample. Plot a standard curve by comparing the Ct value of the standard with the copy number. Figure 16 As shown, the copy number of the sample to be tested can be calculated. The formula for calculating the viral copy number is as follows: Copy number / gram of tissue (copies / g) = copies / mL = 6.02 × 10²³ × (plasmid concentration × 10⁻⁶) -6 (ng / μL) / (total bases × 660).

[0060] From 0 to 14 days post-challenge, the viral load in the feces and lungs of mice in the PBS-challenge group was significantly higher than that in other groups at all time points (P<0.001), reaching its peak on day 6. The viral load in the IV group was the second highest, indicating that the inactivated vaccine had some antiviral effect but failed to completely eliminate the virus. The viral load in the OMVs-HA1 group was the lowest at all time points, decreasing to extremely low levels from days 10 to 14, with the shortest viral shedding time, demonstrating the strongest inhibitory ability against viral replication. Its effect was significantly better than that of traditional inactivated vaccines. Figure 17 As shown.

[0061] Example 16, Pathological observation and pathological damage scoring of mouse lung tissue: Tissue blocks fixed in 4% formaldehyde were dehydrated, cleared, paraffin-embedded, embedded, and sectioned to obtain sections. After HE staining, pathological changes in each tissue were observed. A double-blind method was used to assess the severity of lesions from 0 to 3 points based on histopathological characteristics, and the average score was taken as the final pathological score. The scoring criteria comprehensively considered indicators such as alveolar structural integrity, degree of inflammatory cell infiltration, alveolar septal widening, and alveolar exudate. Pathological section observation results showed that, compared with the PBS-M group, such as... Figure 18 As shown in Figure A, the lung tissue of mice in the PBS-challenged group showed obvious pathological damage, such as... Figure 18 As shown in Figure D, a small number of exfoliated cells were visible in the bronchioles, with lymphocyte infiltration around the bronchioles. The alveolar walls were moderately thickened, the alveolar septa were widened, and numerous granulocytes were infiltrated. Macrophages were visible in some alveolar cavities. In contrast, the lung tissue structure of the OMVs-HA1 group mice was basically normal, with intact alveolar walls, clear alveolar cavities, slight alveolar dilation in some areas, and no obvious inflammatory cell infiltration. Figure 18 As shown in Figure A, the lung tissue lesions in the inactivated vaccine immunization group were mild, showing alveolar fusion, slight thickening of the alveolar walls, and a small amount of inflammatory cell infiltration, such as... Figure 18 As shown in Figure B. A double-blind method was used to score lung tissue pathological damage. The results showed that the PBS-challenged mice had the most severe lung damage; the inactivated vaccine immunized group had the second most severe damage; the OMVs-HA1 group had the least damage, with basically normal lung tissue structure and only minor pathological changes, such as... Figure 19 As shown.

[0062] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0063] It should also be noted that the various specific technical features and steps described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0064] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing an antigen display system based on bacterial outer membrane vesicles, characterized in that, Includes the following steps: S1. The fusion gene ClyA-EGFP, a combination of green fluorescent protein EGFP and ClyA, was obtained using fusion PCR. This gene was then cloned into the pET28a vector, and the recombinant plasmid was transformed into *E. coli* DH5α competent cells. The cells were plated, and positive colonies were selected by PCR screening and sequenced. The plasmid from the correctly identified bacterial cultures was extracted and transformed into *E. coli* BL21(DE3) competent cells for expression. OMVs were extracted, and Western blot was used to verify the successful expression of the ClyA-EGFP fusion protein in OMVs, thus confirming that ClyA can serve as an effective surface display scaffold for OMVs. S2. The ClyA and SpyCatcher003 genes were amplified separately, and the ClyA-SpyCatcher (C-SC) fusion gene was obtained by fusion PCR. This gene was then ligated into the pETDuet-1 vector, and the recombinant plasmid was transformed into E. coli DH5α competent cells. The cells were plated, and positive colonies were screened by bacterial culture PCR and sequenced. The plasmid was extracted from the correctly identified bacterial cultures. The amino acid sequence of SpyCatcher003 is shown in SEQ ID NO.1: AMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGS; S3. The extracted C-SC plasmid was transformed into E. coli BL21 (DE3) competent cells for expression. After Western blot analysis, the bacterial culture was cultured in large quantities. After IPTG induction, OMVs (C-SC OMVs) were extracted. The expression of C-SC fusion protein was verified by Western blot, and its morphology was observed by transmission electron microscopy (TEM). The particle size distribution was determined by nanoparticle tracking analysis (NTA) to confirm the integrity and uniformity of C-SC OMVs. S4. The hemagglutinin head domain HA1 of the CIV canine influenza virus H3N2 subtype was selected as the target antigen. Its encoding gene was fused with SpyTag003 using a flexible linker to construct the SpyTag-HA1 (ST-HA1) fusion gene, which was then cloned into the pCold-1 vector. The recombinant plasmid was then transformed into E. coli DH5α competent cells, and positive colonies were screened. The plasmid was extracted and transformed into E. coli BL21 (DE3) competent cells for expression, and Western blot detection was performed. The bacterial culture was further cultured, and the SpyTag-HA1 protein in inclusion body form was purified by Ni-NTA affinity chromatography. The purified protein was mixed with the C-SC OMVs prepared in S3 and ligated overnight on a rotary mixer at 4°C to obtain OMVs displaying HA1, namely C-SC-ST-HA1OMVs. The amino acid sequence of SpyTag003 is shown in SEQ ID NO.2: RGVPHIVMVDAYKRYK. S5. Systematically identify C-SC-ST-HA1 OMVs, including: verifying the successful display and immunogenicity of the HA1 antigen by Western blot; The integrity of its nanostructure was analyzed by TEM and NTA; its ability to be taken up by macrophages and its immunostimulatory activity in inducing cytokine expression were evaluated by cell experiments; and its in vitro cytotoxicity was detected by lactate dehydrogenase (LDH) release assay. These methods were used to comprehensively evaluate its physicochemical properties, immunogenicity, and biosafety.

2. An antigen display system based on bacterial outer membrane vesicles prepared by the method of claim 1, characterized in that, Exogenous antigens are coupled to the surface of bacterial outer membrane vesicles (OMVs) using the SpyCatcher and / or SpyTag system, forming a plug-and-display antigen display platform. The SpyCatcher and / or SpyTag system comprises: a) OMVs with a first reaction chaperone SpyCatcher peptide or a functional variant thereof displayed on their surface, the first reaction chaperone being anchored to the outer membrane of the OMVs in the form of a fusion protein; b) a target antigen fused with a second reaction chaperone SpyTag peptide or a functional variant thereof, the first reaction chaperone and the second reaction chaperone specifically recognizing each other and forming a covalent bond, thereby coupling the exogenous antigen to the surface of bacterial outer membrane vesicles (OMVs).

3. The antigen display system based on bacterial outer membrane vesicles according to claim 2, characterized in that, The ClyA-SpyCatcher fusion protein was expressed in Escherichia coli prokaryotes, and OMVs were extracted from the supernatant after IPTG induction.

4. The antigen display system based on bacterial outer membrane vesicles according to claim 2, characterized in that, The exogenous antigen is the HA1 antigen of canine influenza virus H3N2 subtype, which is renatured after expression and purification via inclusion bodies.

5. The antigen display system based on bacterial outer membrane vesicles according to claim 2, characterized in that, The first reaction companion is SpyCatcher003, and the second reaction companion is SpyTag003.

6. The antigen display system based on bacterial outer membrane vesicles according to claim 3, characterized in that, The fusion protein is the ClyA-SpyCatcher003 fusion protein.

7. The antigen display system based on bacterial outer membrane vesicles according to claim 3, characterized in that, OMVs had no significant cytotoxic effect on MDCK cells.

8. The antigen display system based on bacterial outer membrane vesicles according to claim 3, characterized in that, OMVs can be effectively taken up by macrophages, stimulating macrophages to produce cytokines, namely IFN-α and / or IL-1β.

9. A vaccine composition, characterized in that: The vaccine composition comprises the antigen display system based on bacterial outer membrane vesicles as described in any one of claims 2-8, and a pharmaceutically acceptable carrier or excipient, and is administered by injection or orally.

10. The use of the antigen display system based on bacterial outer membrane vesicles as described in any one of claims 2-8 in the preparation of a canine influenza virus subunit vaccine.

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