A recombinant Staphylococcus aureus enterotoxin B nanoparticle protein vaccine mSEB-mi3, its preparation method and application

Abstract

This invention provides a recombinant Staphylococcus aureus enterotoxin B nanoparticle protein vaccine, mSEB-mi3, its preparation method, and its applications. This invention utilizes a SpyTag / SpyCather protein linker system to connect the recombinant protein to the nanoparticle protein, allowing mSEB to be displayed on the surface of self-contained nanoparticles, thus obtaining the recombinant Staphylococcus aureus enterotoxin B nanoparticle protein mSEB-mi3. Both mSEB-SpyTag and SpyCather-mi3 proteins in this invention are induced to be expressed in soluble form in Escherichia coli. The mSEB-SpyTag and SpyCather-mi3 of this invention can be covalently bound via isopeptide bonds to form the mSEB-mi3 nanoparticle protein vaccine. The binding conditions are simple, and when combined with the adjuvant MF59, it can induce rapid antibody production in mice, generating a high-level immune response and exerting a protective effect. Furthermore, its antibody response rate and protective efficiency are superior to those of the monomeric protein mSEB.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a recombinant Staphylococcus aureus enterotoxin B nanoparticle protein mSEB-mi3, its preparation method, and its application. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen causing hospital-acquired and community-acquired infections, which can lead to severe skin and soft tissue infections, osteomyelitis, bacteremia, and even death. In 2019, approximately 1.07 million deaths worldwide were related to SA infection, making it the leading bacterial cause of death in humans after Mycobacterium tuberculosis. Methicillin-resistant Staphylococcus aureus The emergence and global spread of MRSA (metastatic leukemia) have significantly increased the complexity of clinical treatment and the disease burden, particularly in immunocompromised populations. The effectiveness of traditional antibiotic treatments remains limited, highlighting the urgent need to develop novel strategies to address SA infection. Vaccination is a highly promising approach: it can prevent infection at its source, reduce disease severity, lower mortality rates, and reduce antibiotic use, thereby helping to control SA infection and bacterial resistance.

[0003] Staphylococcal enterotoxin B produced by SA ( Staphylococcal Enterotoxin B SEB (Self-Suppressant Toxic Acid) is a key virulence factor driving disease development and bacterial immune evasion. As a superantigen, SEB can bind to MHC class II molecules and directly bind to co-stimulatory receptors CD28 and CD86, triggering massive T cell activation and a "cytokine storm," ultimately leading to severe toxic shock syndrome and accelerating patient death. SEB is detected at extremely high rates in isolates of predominantly community-associated SA strains. This toxin has a highly conserved structure and good stability, can be transmitted via aerosols, and is classified as a Category B potential biothalassemia. Therefore, SEB is a highly attractive antigenic target for SA vaccine development.

[0004] Detoxified SEB mutants have lost superantigen activity and have shown excellent protective effects in various animal models, including mice, rhesus monkeys, and piglets. Furthermore, mRNA vaccines encoding detoxified SEB can also induce a potent immune response in mice. Anti-SEB monoclonal antibodies (such as M0313 and MB102a) and SEB-targeting nanobodies (such as Nb6 and Nb8) also exhibit protective effects. Our research group has developed a pentagen SA vaccine (rFSAV) containing SEB carrying L45R, Y89A, and Y94A mutations (i.e., mSEB), which is currently in Phase III clinical trials (trial number: CTR2016004). However, similar to many protein subunit vaccines, mSEB alone exhibits poor immunogenicity, limiting its protective effect, especially in immunocompromised patients.

[0005] Nanoparticles can promote the uptake and cross-presentation of subunit antigens by antigen-presenting cells, thereby enhancing the immune response. mi3 nanoparticles, derived from 2-keto-3-deoxyphosphoglucose (KDPG) aldolase, can self-assemble into a cage-like scaffold structure composed of 60 subunits, making them a highly efficient antigen delivery carrier. Using the SpyTag-SpyCatcher system, researchers have successfully conjugated influenza virus neuraminidase (NA), classical swine fever virus E2 glycoprotein, and monkeypox virus M1R / A35R antigens to the surface of mi3 nanoparticles, significantly improving the protective efficacy of vaccines.

[0006] To enhance the immunogenicity and protective effect of mSEB, this study constructed a mi3 nanoparticle with mSEB on its surface (mSEB–mi3), systematically optimized the adjuvant pairing scheme of this nanoparticle platform, and evaluated its protective efficacy and potential mechanism of action in mouse toxin challenge and bacterial infection models. Summary of the Invention

[0007] This invention addresses the serious harm caused by Staphylococcus aureus enterotoxin B. Although the recombinant subunit protein mSEB greatly reduces toxicity and has some immunogenicity, its immunogenicity is weak and it is difficult to provide effective protection. This invention uses the SpyTag / SpyCather protein linker system to display the antigen mSEB on the surface of self-assembled mi3 nanoparticles, providing a Staphylococcus enterotoxin B self-assembled nanoparticle protein mSEB-mi3.

[0008] The present invention first provides a Staphylococcus aureus enterotoxin B mutant, the amino acid sequence of which is SEQ ID NO:5.

[0009] The present invention further provides a recombinant Staphylococcus aureus enterotoxin B nanoparticle protein, comprising covalently bound mSEB-SpyTag recombinant protein and SpyCatcher-mi3 recombinant protein, wherein the molar ratio of SpyCatcher-mi3 to mSEB-SpyTag is 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6, preferably 1:2; The SpyCatcher-mi3 is composed of SpyCatcher peptide and nanoparticle protein mi3 peptide, and the mSEB-SpyTag is composed of SpyTag peptide and Staphylococcus aureus enterotoxin B mutant as described in claim 1.

[0010] In one embodiment of the present invention, the above-mentioned recombinant Staphylococcus aureus enterotoxin B nanoparticle protein, wherein the amino acid sequence of the recombinant protein mSEB-SpyTag is SEQ ID NO:2; and the amino acid sequence of the recombinant protein SpyCatcher-mi3 is SEQ ID NO:4.

[0011] In another aspect, the present invention provides nucleotides for encoding the aforementioned recombinant Staphylococcus aureus enterotoxin B nanoparticle protein, comprising: a nucleotide encoding the recombinant protein mSEB-SpyTag, and / or a nucleotide encoding SpyCatcher-mi3; wherein the nucleotide sequence of the nucleotide encoding the recombinant protein mSEB-SpyTag is SEQ ID NO:1; and the nucleotide sequence of the nucleotide encoding SpyCatcher-mi3 is SEQ ID NO:3.

[0012] The present invention also provides a recombinant expression vector, characterized in that it comprises the above-mentioned nucleotides and an expression plasmid; the expression plasmid is selected from any one of the pGEX series vectors, pET series vectors or pQE series vectors; preferably, the recombinant expression vector for expressing mSEB-SpyTag is constructed based on the expression vector pET-32a(+), and / or, the recombinant expression vector for expressing SpyCther-mi3 is constructed based on the expression vector pGEX-6P-1.

[0013] In another aspect, the present invention provides a recombinant strain comprising the above-mentioned recombinant expression vector and host bacteria; preferably, the host bacteria is selected from any one of Escherichia coli XL1-blue strain, BL21 series strain and HMS174 series strain, and preferably Escherichia coli BL21 strain.

[0014] This invention further provides a method for preparing the above-mentioned recombinant Staphylococcus aureus enterotoxin B nanoparticle protein, characterized by comprising the following steps: 1) The mSEB gene is fused with SpyTag (ST) to construct a first recombinant gene, and SpyCatcher (SC) is fused with mi3 to construct a second recombinant gene; preferably, the nucleotide sequence of the first recombinant gene is SEQ ID NO:1; and / or, the nucleotide sequence of the second recombinant gene is SEQ ID NO:3; 2) The first recombinant gene is ligated into a vector plasmid to obtain a first expression vector; the second recombinant gene is ligated into a vector plasmid to obtain a second expression vector; 3) The first expression vector is transformed into different host bacteria to obtain the first recombinant bacteria, and the second expression vector is transformed into the second host bacteria to obtain the second recombinant bacteria; 4) After inducing expression in the first host bacterium, the recombinant protein mSEB-SpyTag is extracted and purified; after inducing expression in the second host bacterium, the recombinant protein SpyCatcher-mi3 is extracted and purified; preferably, the amino acid sequence of the recombinant protein mSEB-SpyTag is SEQ ID NO:2; and / or, the amino acid sequence of the recombinant protein SpyCatcher-mi3 is SEQ ID NO:4; 5) Recombinant proteins mSEB-SpyTag and SpyCatcher-mi3 were co-dissolved in a buffer solution of 25 mM Tris-HCl + 150 mM NaCl at a molar ratio of 1-5:1, and bound at 25°C; preferably, the recombinant proteins SpyCatcher-mi3 and mSEB-SpyTag were purified by GST and Ni2+ affinity chromatography, respectively, before the binding operation. 6) The mSEB-mi3 recombinant protein is obtained after purification; preferably, the recombinant protein SpyCatcher-mi3 and the recombinant protein mSEB-SpyTag are combined and then purified by size exclusion chromatography. The present invention also provides the application of the above-mentioned Staphylococcus aureus enterotoxin B mutant, recombinant Staphylococcus aureus enterotoxin B nanoparticle protein mSEB-mi3, encoding gene, recombinant expression vector, or recombinant strain in the preparation of subunit vaccines against Staphylococcus aureus enterotoxin B.

[0015] Another aspect of the present invention provides a vaccine for the prevention or treatment of Staphylococcus aureus enterotoxin B infection, which contains the above-mentioned recombinant Staphylococcus aureus enterotoxin B nanoparticle protein.

[0016] In one embodiment of the invention, it further comprises a pharmaceutically acceptable adjuvant; preferably, the adjuvant is selected from any one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant, and mycobacterial BCG adjuvant.

[0017] The beneficial effects of the above-described technical solution of the present invention are as follows: 1) This invention involves analyzing the charge, spatial structure, and energy kinetic parameters of staphylococcal enterotoxin mSEB protein and mi3 nanoparticle protein. SpyCatcher is then fused with mi3 for expression, and mSEB is further fused with SpyTag for expression. The recombinant proteins SpyCatcher-mi3 and mSEB-SpyTag of this invention are expressed using the SpyTag / Spycatcher conjugation system. This is a highly efficient site-specific protein conjugation method. Although this method still relies on gene manipulation and protein purification, it eliminates the need for starting from scratch; it only requires the production of modular protein components, which are then assembled as needed. This method also minimizes the disruption of protein spatial conformation and biological activity.

[0018] 2) The recombinant proteins SpyCatcher-mi3 and mSEB-SpyTag of this invention are both induced to be expressed in soluble form in E. coli. The histidine tag at the N-terminus of the expression vector is retained, and the protein is processed via GST and Ni... 2+ Purification techniques such as packing affinity chromatography and size exclusion chromatography were used to obtain recombinant proteins that are stable in buffer systems, have uniform particle size, and are of high purity.

[0019] 3) The SpyCatcher-mi3 and mSEB-SpyTag recombinant protein of the present invention can be covalently linked by isopeptide bonds to form mSEB-mi3 nanovaccine molecules. The binding conditions are simple, the coupling efficiency is high, and they appear as nanoparticles with uniform particle size under transmission electron microscopy.

[0020] 4) The mSEB-mi3 nanovaccine of this invention, when combined with adjuvant MF59 and administered via intramuscular injection, can induce the production of mSEB-specific IgGs antibodies in mouse serum, thereby exerting a highly efficient immune protection effect. The speed of antibody response induction and the protective efficiency are significantly better than those of monomeric mSEB protein. Attached Figure Description

[0021] Figure 1 SDS-PAGE electrophoresis images of mSEB-SpyTag, SpyCather-mi3, and mSEB-mi3 are shown, where M is the standard molecule, 1 is mSEB-SpyTag, 2 is SpyCather-mi3, and 3 is mSEB-mi3.

[0022] Figure 2 To detect the molecular weight profiles of proteins mSEB-SpyTag, SpyCather-mi3, and mSEB-mi3 using molecular sieve chromatography.

[0023] Figure 3 The image shows the dynamic light scattering nanoparticle size detection of SpyCatcher-mi3 and mSEB-mi3.

[0024] Figure 4 Transmission electron microscopy (TEM) images of the nanoparticle sizes of mSEB, mi3, and mSEB-mi3.

[0025] Figure 5 Antibody titer detection in the short term after single-injection intramuscular immunization with different adjuvants for mSEB-mi3.

[0026] Figure 6 The image shows the results of mSEB-specific IgG antibody subtype detection induced 10 days after a single intramuscular immunization with MF59 in combination with mSEB-mi3.

[0027] Figure 7 Figure 1 shows the results of detecting mSEB-specific IgG antibodies induced in the short and long term after a single intramuscular immunization with MF59 in combination with mSEB-mi3.

[0028] Figure 8 Survival curves of mice 10 days after a single intramuscular immunization with mSEB-mi3 and MF59 and subsequent systemic infection with Staphylococcus aureus ST59.

[0029] Figure 9 Survival curves of mice 10 days after systemic infection with Staphylococcus aureus enterotoxin B following a single intramuscular immunization with mSEB-mi3 combined with MF59.

[0030] Figure 10 Survival curves of mice immunized intramuscularly with a single injection of MF59 in combination with mSEB-mi3 after systemic infection with Staphylococcus aureus enterotoxin B 200 days later. Detailed Implementation

[0031] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0032] As used throughout the specification and claims, the terms "comprising" or "including" are open-ended and should be interpreted as "comprising but not limited to". The subsequent descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0033] Example 1: Construction of pET-32a(+)-mSEB-SpyTag recombinant plasmid Test method: (1) Based on previous research results, using the SAR1916 gene of Staphylococcus aureus MRSA252 genome (BX571856.1 GI:49240382) as a template, the mature peptide encoding gene was selected for recombinant plasmid construction. Through analysis of the SEB crystal structure, it was found that amino acids at positions 45, 89, and 94 are key amino acids for SEB to function. After point mutation, they can be made non-toxic, resulting in mSEB (L45R, Y89A, Y94A), whose amino acid sequence is SEQ ID NO:5. The codons were then optimized to obtain the final protein nucleotide sequence of mSEB, whose sequence is SEQ ID NO:6. The SpyTag nucleotide sequence obtained by searching the NCBI database was then ligated to its C-terminus and synthesized by Nanjing Genscript Biotech Co., Ltd. to obtain mSEB-SpyTag recombinant DNA, whose nucleotide sequence is SEQ ID NO:1, which was cloned into the pET-32a vector.

[0034] (2) Transformation: Dissolve the plasmid powder in 40 μL ddH2O. In a clean bench, add 1 μL of mSEB-SpyTag plasmid to 50 μL of BL21(DE3) competent cells (Tiangen Biotech Co., Ltd.). Gently mix and place in an ice-water bath for 30 min. Then heat the competent cells in a metal bath for 90 s and immediately place on ice for 1 min. Add 1 mL of antibiotic-free liquid LB medium to the competent cells in a clean bench and incubate at 37℃, 220 rpm for 40-60 min. After removing the centrifuge tube, centrifuge at 5000 rpm for 5 min, collect 800 μL of the supernatant and discard it. Wash and mix again. Spread 50 μL of the liquid onto LB solid medium containing ampicillin sodium resistance. Incubate upright for 30 min, then invert and place in a 37℃ incubator overnight. The next day, store at 4℃.

[0035] (3) Cultivating engineered bacteria: Using a 10 μL pipette tip, pick a single colony from a petri dish in a clean workbench and add it to 10 mL of LB liquid medium. Add 5 μL of ampicillin sodium (Sangon Biotech) solution and incubate overnight at 37°C and 220 rpm on a shaker.

[0036] (4) Plasmid extraction: Plasmids were extracted using the plasmid mini-prep kit DP103 (Tiangen Biotech Co., Ltd.).

[0037] 2. Experimental Results The plasmid was sent to Hanjin Kairui Co., Ltd. for sequencing, and the sequencing results were identical to the target sequence.

[0038] Example 2: Construction of pGEX-6P-1-SpyCatcher-mi3 recombinant plasmid 1. Test Methods (1) The coding gene of SpyCatcher-mi3 was obtained by searching the NCBI database for the nucleotide sequences of mi3 and SpyCatcher. Its nucleotide sequence is SEQ ID NO:3. It was cloned into the pGEX-6P-1 vector and synthesized by Nanjing Genscript Biotech Co., Ltd.

[0039] (2) Conversion: Take a centrifuge tube containing 4 μg of lyophilized plasmid powder and centrifuge at 4000 rpm for 2 min to centrifuge the plasmid powder to the bottom. Slowly add 40 μL of autoclaved water to dissolve it and transfer it to a clean bench. E. coli 50 μL of BL21(DE3) competent cells were slowly thawed on ice, transferred to a clean bench, and then 1 μL of plasmid solution was added to the thawed cells. E. coli Gently mix BL21(DE3) competent cells, place on ice for 30 min, heat shock at 42℃ for 90 s, and place on ice again for 3 min; add 1 mL of liquid LB medium, and incubate at 37℃ and 180 rpm for 40 min; centrifuge the culture medium at 8000 rpm for 2 min, discard 800 μL of the supernatant, resuspend the cells, transfer 50 μL of the cell suspension to solid LB medium (Amp), spread evenly, and incubate at 37℃ overnight.

[0040] (3) Cultivating engineered bacteria: Observe whether single colonies grow on the overnight culture plates, and invert them to store them in a 4℃ refrigerator for later use. Take a 10 μL pipette tip to pick up a single colony and put it into 10 ml LB medium, add 5 μL ampicillin sodium solution, and place the conical flask in a shaker for overnight cultivation at 37℃ and 180 rpm.

[0041] (4) Plasmid extraction: Plasmids were extracted using the plasmid mini-prep kit DP103 (Tiangen Biotech Co., Ltd.).

[0042] 2. Experimental Results The plasmid was sent to Nanjing GenScript Biotech Co., Ltd. for sequencing, and the sequencing results were identical to the target sequence.

[0043] Example 3: Preparation and biochemical characterization of mSEB-SpyTag, SpyCatcher-mi3 recombinant protein and mSEB-mi3 binding protein 1. Test Methods 1.1 Preparation of mSEB-SpyTag and SpyCatcher-mi3 recombinant proteins 1.1.1 Expression identification (1) Take 20 ml of liquid LB solution and add it to the conical flask. Then add 10 μL of ampicillin sodium (to make the final concentration of antibiotic 100 μg / mL). Then take 200 μL of the cultured bacterial solution and add it to the conical flask.

[0044] (2) Second stage: Place the conical flask in a shaker, 37℃, 220rpm, and incubate for 2~4h to ensure OD 600 It is around 0.8 to 1.2.

[0045] (3) SpyCatcher-mi3 induction expression: Adjust the shaker temperature to 16℃, 150 rpm, and after cooling, add 4 μL of 1M IPTG (isopropyl-β-D-thiogalactoside) (Sangon Biotech) (to a final concentration of 0.2 mM / L). Then place the conical flask in the shaker and incubate at 16℃ for 16 h at 150 rpm; another flask without IPTG was prepared. mSEB-SpyTag induction expression: Adjust the shaker temperature to 16℃, 200 rpm, and after cooling, add 4 μL of 1M IPTG (to a final concentration of 0.2 mM / L). Then place the conical flask in the shaker and incubate at 16℃ for 16 h at 200 rpm; another flask without IPTG was prepared.

[0046] (4) Sterilization: Transfer the cultured bacterial solution to a 10 mL centrifuge tube and centrifuge at 8000 rpm for 5 min at room temperature. Discard the supernatant and resuspend the solution in 2 mL Tris-HCl buffer and take a sample. After resuspending, sonicate the bacterial solution in an ice-water bath using a sonic cell disruptor (SONICS) for 5-8 min. After sonication, centrifuge the bacterial solution at 12000 rpm and 4℃ for 30 min. Transfer the lysis supernatant to a new 2 mL centrifuge tube. Resuspend the lysis pellet in 2 mL Tris-HCl buffer. Both the lysis supernatant and the pellet should be sampled. (6) SDS-PAGE detection of protein expression: Prepare electrophoresis gel, then add 10 μL of 6× protein loading buffer to the sample, mix well, and heat at 100℃ for 7 min; take 5 μL of protein marker and 10 μL of each protein sample prepared above, and gently inject them into the sample wells along the top of the front glass plate. Connect the electrophoresis cap and electrophoresis apparatus according to the positive and negative electrodes, set the voltage parameter to 80V, and adjust the voltage parameter to 140V to 200V when the bromophenol blue indicator enters the separating gel. Observe the position of the indicator and stop electrophoresis when it reaches the bottom of the gel. Open the electrophoresis tank and take out the glass plate. Pry open the front glass plate and take out the gel and transfer it to the staining box. Wash with primary water 3 times, take an appropriate amount of instant blue staining solution into the staining box, close the cap, transfer the staining box to a horizontal shaker, set the shaker parameter to 40 rpm, stain at room temperature for 1 h, decolorize with primary water overnight, take pictures using a gel imaging system, and analyze the results using Image Lab software.

[0047] 1.1.2 mSEB-SpyTag protein purification (1) Add 10 mL of nickel ion affinity chromatography packing to the affinity chromatography column, elute the preservation solution, add 40 mL of Tris-HCl protein buffer solution containing 1 M imidazole to the affinity chromatography column, mix thoroughly and wash, discard the washing solution and repeat the washing 3 times; add Tris-HCl buffer to the affinity chromatography column, mix thoroughly and wash, discard the washing solution and repeat the washing 3 times; Add the collected supernatant to the washed affinity chromatography column, mix thoroughly, and place on a vertical mixer at 25 °C for 1 h. Discard the supernatant, wash three times with Tris-HCl buffer, and elute sequentially with Tris-HCl buffer (25 mM Tris, 1 M NaCl, pH=7.8) containing 20 mM, 50 mM, 100 mM, 200 mM, 300 mM, 500 mM, and 1000 mM, collecting the eluent. Wash the nickel ion affinity chromatography packing material three times with Tris-HCl protein buffer containing 1 M imidazole, wash three times with Tris-HCl buffer, and finally wash three times with 20% ethanol solution. Add 20 mL of 20% ethanol solution and store at 4 °C.

[0048] (2) Replacement buffer: SDS-PAGE was used to detect the above samples. Samples with the correct target protein position and high protein purity were selected. The high-salt buffer was replaced with Tris-HCl buffer by gel filtration chromatography on the AKTA pure purifier to obtain the nanoparticles SpyCatcher-mi3 and stored at -80 ℃ for later use.

[0049] (3) Cation exchange chromatography Thaw the above-mentioned mSEB-SpyTag initial purified solution at room temperature. Add PreScission Protease enzyme (Chongqing Yuanlun Biotechnology Co., Ltd.) at a ratio of 1:0.1 (mSEB-SpyTag initial purified solution volume: PreScission Protease enzyme volume) to cleave the solubilizing tag enzyme on the mSEB-SpyTag. Filter the phosphate-protein buffer and high-salt phosphate buffer through a 0.22 μm filter membrane. Install the cation exchange chromatography column (HiTrap™ SP HP) onto the AKTA pure purifier. Set the purifier parameters to a flow rate of 5 mL / min, column inlet pressure of 0.5 MPa, and column pressure of 0.3 MPa. After using a first-stage water-balanced cation exchange chromatography column, place pump A in the phosphate-protein buffer and pump B in the high-salt phosphate buffer. Run the program to add 100% solution B and use the high-salt buffer to clean the pathway and cation exchange chromatography column. After the absorbance at 280 nm stabilizes, adjust the program to add 100% solution A. After rinsing to remove high-salt buffer and waiting for the conductivity to decrease and stabilize, the mSEB-SpyTag pre-purified solution was introduced into the cation exchange chromatography column via pump A. After sample loading, pump A was re-introduced with phosphate-protein buffer for equilibration. After equilibration, the program was adjusted to 5 min, with a gradient increase until 100% solution B was introduced. Samples were collected at different peak positions based on the absorbance curve at 280 nm. After collection, pumps A and B were rinsed in 20% ethanol solution to clean the pathway and anion exchange chromatography column, and then stored.

[0050] (4) Replacement buffer: Same as above.

[0051] 1.1.3 Purification of SpyCatcher-mi3 (1) GST affinity chromatography: Add 20 mL of GST affinity chromatography packing material to the affinity chromatography column, elute the preservation solution, add 40 mL of 0.1 M NaOH solution to the affinity chromatography column and mix thoroughly to wash, discard the washing solution and repeat the washing 3 times; add Tris-HCl buffer to the affinity chromatography column and mix thoroughly to wash, discard the washing solution and repeat the washing 3 times; add the supernatant collected above to the washed affinity chromatography column, place it on a vertical mixer and mix thoroughly, bind at 25℃ for 4 h; discard the supernatant after binding, wash 3 times with Tris-HCl buffer, wash 3 times with Tris-HCl high salt buffer (25 mM Tris, 1 M NaCl, pH=8.0), wash 3 times with Tris-HCl buffer, wash 3 times with Tris-HCl buffer and add 20 mL of Tris-HCl buffer; add 2 mL of PreScission Protease to the affinity chromatography column according to the ratio of buffer volume: packing material volume: PreScission Protease enzyme volume 1:1:0.1. The enzyme was digested at 4°C for 16 h on a vertical mixer. The supernatant after digestion, i.e., SpyCatcher-mi3 pre-purified solution, was collected and stored at -80°C for later use. The GST packing material was then washed three times with 0.1 M NaOH solution, three times with Tris-HCl buffer, and finally three times with 20% ethanol solution. 20 mL of 20% ethanol solution was added, and the GST packing material was stored at 4°C.

[0052] (3) Anion exchange chromatography: Thaw the SpyCatcher-mi3 initial purified solution at room temperature. Filter the Tris-HCl buffer and Tris-HCl high-salt buffer using a 0.22 μm filter membrane. Install the anion exchange chromatography column (HiTrap™ QHP) onto the AKTA pure purifier. Set the purifier parameters to a flow rate of 5 mL / min, column inlet pressure of 0.5 MPa, and column pressure of 0.3 MPa. After using a first-stage water-balanced anion exchange chromatography column, place pump A in the Tris-HCl buffer and pump B in the Tris-HCl high-salt buffer. Run the program to add 100% B solution to clean the pathway and anion exchange chromatography column with the high-salt buffer. After the absorbance at 280 nm stabilizes, adjust the program to add 100% A solution to rinse and remove the high-salt buffer. After the conductivity decreases and stabilizes, add the SpyCatcher-mi3 initial purified solution to A. Pump A is inserted into the anion exchange chromatography column. After sample loading, pump A is reconnected to Tris-HCl buffer for equilibration. After equilibration, the program is adjusted to 5 min, with a gradient increase until 100% solution B is added. Samples are collected at different peak positions based on the absorbance curve at 280 nm. After collection, pumps A and B are rinsed in 20% ethanol solution to clean the passageway and anion exchange chromatography column, and then stored.

[0053] (4) Replacement buffer: SDS-PAGE was used to detect the above samples. Samples with the correct target protein position and high protein purity were selected. The high-salt buffer was replaced with Tris-HCl buffer by gel filtration chromatography on the AKTA pure purifier to obtain the nanoparticles SpyCatcher-mi3 and stored at -80 ℃ for later use.

[0054] 1.2 Preparation and biochemical characterization of the binding protein mSEB-mi3 (1) Preparation of binding protein mSEB-mi3: SpyCatcher-mi3 is a 60-mer with a single molecule molecular weight of about 34 kDa. The theoretical molecular weight of mSEB-SpyTag is about 29 kDa. SpyCatcher-mi3: mSEB-SpyTag were set to bind at a molar ratio of 1:1, 1:2, 1:3, 1:4 and 1:5 in an incubator at 25 ℃ for 16 h. The optimal binding ratio was detected by SDS-PAGE.

[0055] (2) Molecular sieve chromatography for molecular weight determination: mSEB-SpyTag and SpyCatcher-mi3 were incubated at 25 ℃ for 16 h according to the optimal binding ratio; Tris-HCl buffer was filtered through a 0.22 μm filter membrane, and the molecular sieve chromatography column was installed on an AKTA pure purifier; the purifier parameters were set as follows: flow rate 1 mL / min, column inlet pressure 0.5 MPa, column pressure 0.3 MPa, and pump A was placed in Tris-HCl buffer; the program was run to connect 100% A solution to the equilibration pathway and the molecular sieve chromatography column. After the conductivity and absorbance at 280 nm wavelength stabilized, the self-assembled nanoparticles mSEB-mi3 obtained after binding were connected to pump A and fed into the molecular sieve chromatography column. After the sample loading was completed, pump A was reconnected to Tris-HCl buffer; samples with different peak positions were collected according to the absorbance curve at 280 nm wavelength, and the collected samples were analyzed by SDS-PAGE. Preliminary identification was performed, and samples that matched the theoretical band positions were selected to obtain the mSEB-mi3 protein.

[0056] (2) Dynamic light scattering (DLS) data of mSEB-mi3 nanoparticles were collected using a ZS90 nanoparticle size analyzer. Each protein was diluted to 0.1 mg / ml, and 1.2 ml of each protein was taken into a cuvette. The instrument parameters were set as follows: angle 90°, temperature 25 ℃, and number of times 3.

[0057] (3) Transmission electron microscopy: The protein was diluted to a final concentration of 0.01 mg / ml. After glow discharge treatment of the carbon-coated copper mesh, 8 μL of sample was added until it was adsorbed on the copper mesh for 2 minutes. After removing the excess liquid with filter paper, the copper mesh was placed in a 2% formic acid and uranium acetate solution for negative staining for 2 minutes. After the copper mesh dried, the sample was observed and the image was recorded using a transmission electron microscope at an accelerating voltage of 120 kV.

[0058] 2. Test Results The recombinant plasmid pET-32a(+)-mSEB-SpyTag was transformed into *E. coli* to prepare the pET28a-mSEB-SpyTag / BL21(DE3) engineered strain. The strain was induced with IPTG at 16 °C, followed by ultrasonic lysis, and the supernatant and precipitate were separated by centrifugation. A band of approximately 29 kDa was found in the lysed supernatant, indicating that this protein can be expressed in a soluble form in *E. coli*. To obtain the purified mSEB-SpyTag protein, the pET28a-mSEB-SpyTag / BL21(DE3) engineered strain was induced with IPTG, and the supernatant after ultrasonic lysis and centrifugation was mixed with Ni... 2+After binding with the packing material and washing away contaminating proteins, the mSEB-SpyTag protein was eluted with a 200 mM, 1M imidazole buffer. The obtained mSEB-SpyTag protein was then desalted using a HiPrep™ 26 / 10 Desalting column. The recombinant plasmid pGEX-6P-1-SpyCatcher-mi3 was transformed into *E. coli* to prepare the pGEX-6P-1-SpyCatcher-mi3 / BL21(DE3) engineered strain. The strain was induced with IPTG at 16°C, followed by sonication to lyse the bacteria, and the supernatant and precipitate were separated by centrifugation. A band of approximately 34 kDa was observed in the lysed supernatant, indicating that it could be expressed in a soluble form in *E. coli*. After IPTG induction, the sonicated supernatant of the pGEX-SpyCatcher-mi3 / BL21(DE3) engineered strain was bound to GST packing material, collected, and flow-throughd for SDS-PAGE. The protein was bound to the nanoparticles at molar ratios (SpyCatcher-mi3:mSEB-SpyTag) of 1:1, 1:2, 1:3, 1:4, and 1:5. The results showed that 1:2 was the optimal binding ratio. Excess mSEB-SpyTag monomeric protein was then removed by size exclusion chromatography to obtain the final mSEB-mi3 nanoparticle protein. The results are as follows... Figure 1 .

[0059] Purified mSEB-Spytag, mi3-SpyCatcher, and mSEB-mi3 were analyzed by size exclusion chromatography using a Superose 6 column. All three proteins exhibited single, symmetrical peaks, indicating good homogeneity. Their elution volumes were 19.6 mL, 13.6 mL, and 10.2 mL, respectively. Compared to mi3 nanoparticles, the peak volume of mSEB-mi3 protein decreased by approximately 3.4 mL, indicating a significant increase in molecular weight. This suggests that mSEB-mi3 protein formed uniform nanoparticles. The results are as follows... Figure 2 .

[0060] Dynamic light scattering (DLS) experiments showed that the average particle sizes of SpyCatcher-mi3 and mSEB-mi3 nanoparticles were 43.8 nm and 105.7 nm, respectively. Compared with mi3 nanoparticles, the nanoparticle size of mSEB-mi3 protein increased by 61.9 nm, suggesting that mSEB-mi3 protein forms uniform nanoparticles. Figure 3 .

[0061] Transmission electron microscopy (TME) experiments showed that mSEB-mi3 exhibited spherical nanoparticles with good dispersibility, as indicated by the results. Figure 4 .

[0062] Example 4: Evaluation of short-term immune response to mSEB-mi3 in combination with different adjuvants 1. Experimental Methods Humoral immune response detection Mouse Immunization: Purified mSEB-mi3 protein was combined with different adjuvants to prepare mSEB-mi3 vaccines with different adjuvants, including MF59, ALPO4, CpG ODN 1018, and AS01. A control group without adjuvants was used. The appropriate dosage of each adjuvant was selected according to the instructions. The mSEB-mi3 immunization dose per mouse was 43 µg, with immunization doses of MF59 50 μL, ALPO4 50 μL, CpG ODN 1018 50 μL, and AS01 50 μL, respectively. On day 0, 6-8 week old female BALB / c mice were randomly divided into groups of 5 mice each. Each group received an intramuscular injection in the thigh of MF59 + mSEB-mi3, ALPO4 + mSEB-mi3, CpG ODN 1018 + mSEB-mi3, or AS01 + mSEB-mi3, respectively, for immunization.

[0063] Serum collection: Tail vein blood was collected from mice in each immunization group on days 4, 6, 8 and 10 after immunization and placed in clean EP tubes. After being placed in a 37°C incubator for 1 hour, the upper serum was centrifuged at 12,000 rpm for 5 minutes and collected into clean EP tubes. The serum was stored at -80°C for later use.

[0064] Indirect ELISA detection of mouse serum mSEB-specific IgG antibody levels 1) Coating: Coat mSEB to 96-well plates. Dilute mSEB protein to 6 μg / mL with coating buffer and coat 100 μL per well in 96-well plates. Coat overnight at 4°C or 2 h at 37°C. 2) Blocking: After washing the coated microplate with a plate washer, add 250 μL of blocking solution to each well and block at 37℃ for 2 h; 3) Primary antibody: After washing and blocking the ELISA plate with a plate washer, the serum was diluted with PBST at an appropriate ratio and added to the plate. The serum was also serially diluted 2-fold in 96-well plates. A negative control was set up in each plate. The plate was incubated at 37°C for 1 h. 4) Secondary antibody: After washing the plate with a plate washer and incubating the primary antibody, dilute the secondary antibody at a ratio of 1:7500 and add 100 μL to each well of the plate. Incubate at 37°C for 45 min. 5) Color development: After washing the plate with the secondary antibody, add 100 μL of TMB color development solution to each well and incubate at 37°C for 10 min. 6) Termination: Remove the ELISA plate after color development and add 50 μL of stop solution to each well; 7) Plate reading: Preheat the microplate reader 30 minutes in advance. After stopping the color development, use the microplate reader to detect the absorbance of each well of the microplate at 450 nm and 690 nm and export the data. 8) Analysis: Calculate OD 450 -OD 690 The cut-off value was used to analyze the antibody titer of each group of mice.

[0065] 2. Experimental Results When mSEB-mi3 protein was combined with MF59, the titer of anti-mSEB-specific IgG antibodies showed a rapid increase, and was significantly higher than other groups on days 4, 6, 8, and 10. Therefore, MF59 was selected as an adjuvant for the vaccine in the subsequent four experiments. The results are as follows: Figure 5 .

[0066] Example 5: Antibody subtype detection after single intramuscular immunization of mice with mSEB-mi3 and MF59 1. Experimental Methods The mSEB-specific antibody subtypes induced in mice in the mSEB-mi3+MF59 group were detected by the same indirect ELISA method as described above on day 10 after immunization, except that the secondary antibody was changed from IgG to IgG1, IgG2a, and IgG2b, while the antibody dilution factor remained unchanged.

[0067] Experimental results On day 10, the serum of mice was analyzed for anti-mSEB antibody subtypes. Both the mSEB-mi3 group and the mSEB group showed equally strong IgG1 and IgG2 antibodies. Among them, the IgG1, IgG2a, and IgG2b levels in the mSEB-mi3 group were significantly higher than those in the mSEB group. P <0.0001), indicating that mSEB-mi3 can simultaneously induce strong Th1 and Th2 immune responses, as shown in the results. Figure 6 .

[0068] Example 6: Short-term and long-term antibody titer detection after single intramuscular immunization of mice with mSEB-mi3 and MF59 1. Experimental Methods The MF59 adjuvant was combined with purified mSEB-mi3 and mSEB proteins to prepare a vaccine. The dosage was as shown in 1.1 of Experiment 4. Five 6-8 week old female BALB / c mice were selected for each group and injected into the thigh intramuscularly on day 0. The antibody titers of each group of mice were then continuously detected. The antibody titer detection method was as shown in 1.1 of Experiment 4.

[0069] 2. Experimental Results Mice were immunized with MF59 in combination with mSEB-mi3 and mSEB, respectively. The results showed that significant differences occurred between the mSEB-mi3 and mSEB groups starting from day 4. P <0.05), and subsequently the antibody titer continued to increase. Continuous monitoring revealed that, within the mSEB-mi3 group, there was no significant difference in antibody titer after day 10 compared to the previous test. This indicates that mice in the mSEB-mi3 group reached a high level of antibody titer on day 10 and maintained it for nearly 188 days, while the mSEB monomer group only reached a high level of titer on day 28. The results are as follows. Figure 7 .

[0070] Example 7: Evaluation of immunoprotective effect of a mouse model of systemic Staphylococcus aureus infection 10 days after a single intramuscular immunization with mSEB-mi3 combined with MF59. 1. Experimental Methods (1) Mouse immunization mSEB-mi3 group: mSEB-mi3 43μg, MF59 adjuvant 10μg; mSEB group: mSEB 20μg, MF59 adjuvant 10μg; mi3 group: mi3 23μg (when the mSEB-mi3 vaccine contains 20μg mSEB, the mass of mi3 is calculated to be 23μg), MF59 adjuvant 10μg; control group: equal volume of PBS buffer; 6-8 week old SPF-grade female BABL / c mice were randomly selected and grouped, and different groups of vaccines were injected intramuscularly into the thigh of the mice.

[0071] (2) Resuscitation, culture and conditioning of Staphylococcus aureus ST59 1) Take the ST59 strain out of the -80℃ freezer and thaw it. Use an inoculation loop to take the bacterial solution and streak it on a non-resistant TSA solid plate using the three-zone streak method. Incubate it overnight in a 37℃ incubator.

[0072] 2) Pick a single colony from the plate and inoculate it into 10 mL of TSB liquid medium. Incubate overnight at 37°C and 180 rpm for 14-16 h.

[0073] 3) Take out the culture flask, take 200 μL from the live bacteria solution and add it to 20 mL of TSB liquid culture medium, and incubate at 37℃ and 220 rpm for 3 h.

[0074] 4) Centrifuge the two live bacterial cultures at 8500 rpm for 5 min, discard the supernatant, wash the bacterial cells twice with physiological saline, resuspend and mix well, and measure the OD in a UV spectrophotometer. 600 Adjust the bacterial concentration to OD using physiological saline. 600= 1.0, which is 1 × 10 9 CFU / mL.

[0075] 5) Dilute the bacterial culture with physiological saline to adjust the concentration to 1×10⁻⁶. 8 CFU / mL.

[0076] (3) Systemic infection model of lethal dose ST59 mice challenged with the virus Mice from different immunization groups were challenged with the virus on day 10 after a single injection. The tail veins of the mice were dilated by irradiation under an infrared lamp. The mice were then placed on a mouse restraint device, and the prepared bacterial solution was injected into the tail veins of the mice using an insulin needle. The survival of the mice after the challenge was observed and recorded, and the observation continued for 1 week (168 hours).

[0077] 2. Experimental Results To verify the protective effect of the mSEB-mi3 nanoparticle vaccine on mice, mice were challenged with a lethal dose of ST59 Staphylococcus aureus 10 days after immunization. Survival was observed for 7 days after challenge. The results showed that the survival rate was 80% in the mSEB-mi3 immunization group, 40% in the mSEB group, 20% in the mi3 group, and 0% in the PBS group. The comparison revealed a significantly prolonged survival time in the mSEB-mi3 immunization group, with statistically significant differences. P <0.05), the result is as follows Figure 8 .

[0078] Example 8: Immunoprotective effect of a mouse model of systemic infection with Staphylococcus aureus enterotoxin B following a single intramuscular immunization with mSEB-mi3 combined with MF59. 1. Test Methods (1) Mouse immunization mSEB-mi3 group: mSEB-mi3 43μg, MF59 adjuvant 10μg; mSEB group: mSEB 20μg, MF59 adjuvant 10μg; mi3 group: mi3 23μg (when the mSEB-mi3 vaccine contains 20μg mSEB, the mass of mi3 is calculated to be 23μg), MF59 adjuvant 10μg; control group: equal volume of PBS buffer; 6-8 week old SPF-grade female BABL / c mice were randomly selected and grouped, and different groups of vaccines were injected intramuscularly into the thigh of the mice.

[0079] (2) Lethal dose SEB mouse infection model Six- to eight-week-old SPF-grade female BABL / c mice were randomly selected and grouped. The purified SEB protein was diluted to different concentration gradients (2.5 μg / mouse, 5 μg / mouse, 10 μg / mouse, 20 μg / mouse) and injected via the tail vein. The lethal dose of the mice was then observed and selected.

[0080] (3) Systemic infection model of mice with lethal dose of SEB virus challenge Mice from different immunization groups were challenged with a lethal dose of the virus on day 10 after a single injection. The tail veins of the mice were dilated by irradiation under an infrared lamp. The mice were then placed on a mouse restraint device, and the prepared bacterial solution was injected into the tail veins of the mice using an insulin needle. The survival of the mice after the challenge was observed and recorded.

[0081] Test results To verify the protective effect of the mSEB-mi3 nanoparticle vaccine in mice, mice were challenged with a lethal dose of SEB via tail vein 10 days after immunization. Since SEB infection is an acute inflammatory condition, the lethal dose for mice was found to be 5 μg / mouse in previous dose-finding studies. Furthermore, it was observed that mice were most likely to die within 12-24 hours after toxication; therefore, the survival rate of mice within 24 hours was chosen to verify the protective effect. The results showed that the survival rate of mice in the mSEB-mi3 group was 80%, while the survival rates in the mSEB, mi3, and PBS groups were 40%, 20%, and 0%, respectively. The survival time in the mSEB-mi3 immunization group was significantly prolonged, and the difference was statistically significant. P <0.05), the result is as follows Figure 9 .

[0082] Example 9: Evaluation of the immunoprotective effect of a single-injection intramuscular immunization with mSEB-mi3 combined with MF59 for 200 days in a systemic Staphylococcus aureus enterotoxin B infection model. 1. Test Methods (1) Mouse immunization mSEB-mi3 group: mSEB-mi3 20 μg (based on loaded mSEB), MF59 adjuvant 50 μL; mSEB group: mSEB 20 μg, MF59 adjuvant 50 μL; mi3 group: mi3 23 μg (when the mSEB-mi3 vaccine contains 20 μg mSEB, the mass of mi3 is calculated to be 23 μg), MF59 adjuvant 50 μL; control group: equal volume of PBS buffer; 6-8 week old SPF-grade female BABL / c mice were randomly selected and grouped, and different groups of vaccines were injected intramuscularly into the thigh of the mice.

[0083] (2) Systemic infection model of mice with lethal dose of SEB virus challenge Mice from different immunization groups were challenged with the virus 200 days after a single injection. The tail veins of the mice were dilated by irradiation under an infrared lamp. The mice were then placed on a mouse restraint device, and the prepared bacterial solution was injected into the tail veins of the mice using an insulin needle. The survival of the mice after the challenge was observed and recorded.

[0084] 2. Test Results Mice 200 days after a single immunization were challenged via tail vein with a lethal dose of the drug, and their survival within 24 hours was observed. The results showed that the survival rate of mice in the mSEB-mi3 group was 60%, while that in the mSEB, mi3, and PBS groups was 20%, 0%, and 0%, respectively. The survival time in the mSEB-mi3 immunization group was significantly prolonged, and the difference was statistically significant. P <0.05), which indicates that the vaccine still has a certain protective effect even long after immunization, as the result is... Figure 10 .

[0085] The above experimental results demonstrate that the mSEB-mi3 nanoparticle vaccine has a good protective effect against Staphylococcus aureus ST59 or SEB toxin.

[0086] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Staphylococcus aureus enterotoxin B mutant, the amino acid sequence of which is SEQ ID NO:

5.

2. A recombinant Staphylococcus aureus enterotoxin B nanoparticle protein, comprising, covalently bound mSEB-SpyTag recombinant protein and SpyCatcher-mi3 recombinant protein, wherein, The molar ratio of SpyCatcher-mi3 to mSEB-SpyTag is 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6, preferably 1:2; The SpyCatcher-mi3 is composed of SpyCatcher peptide and nanoparticle protein mi3 peptide, and the mSEB-SpyTag is composed of SpyTag peptide and Staphylococcus aureus enterotoxin B mutant as described in claim 1.

3. The recombinant Staphylococcus aureus enterotoxin B nanoparticle protein as described in claim 2, wherein, The amino acid sequence of the recombinant protein mSEB-SpyTag is SEQ ID NO:2; the amino acid sequence of the recombinant protein SpyCatcher-mi3 is SEQ ID NO:

4.

4. A nucleotide encoding the recombinant Staphylococcus aureus enterotoxin B nanoparticle protein as described in claim 2 or 3, comprising: a nucleotide encoding the recombinant protein mSEB-SpyTag, and / or, a nucleotide encoding SpyCatcher-mi3; wherein, The nucleotide sequence of the nucleotide encoding the recombinant protein mSEB-SpyTag is SEQ ID NO:1; the nucleotide sequence of the nucleotide encoding SpyCatcher-mi3 is SEQ ID NO:

3.

5. A recombinant expression vector, characterized in that, It comprises the nucleotide as described in claim 4, and an expression plasmid; the expression plasmid is selected from any one of the pGEX series vectors, pET series vectors, or pQE series vectors; preferably, the recombinant expression vector for expressing mSEB-SpyTag is constructed based on the expression vector pET-32a(+), and / or, the recombinant expression vector for expressing SpyCther-mi3 is constructed based on the expression vector pGEX-6P-1.

6. A recombinant bacterial strain, characterized in that, It comprises the recombinant expression vector as described in claim 5 and the host bacterium; preferably, the host bacterium is selected from any one of Escherichia coli XL1-blue strain, BL21 series strain and HMS174 series strain, preferably Escherichia coli BL21 strain.

7. A method for preparing recombinant Staphylococcus aureus enterotoxin B nanoparticle protein as described in claim 2 or 3, characterized in that, Includes the following steps: 1) The mSEB gene is fused with SpyTag (ST) to construct a first recombinant gene, and SpyCatcher (SC) is fused with mi3 to construct a second recombinant gene; preferably, the nucleotide sequence of the first recombinant gene is SEQ ID NO:1; and / or, the nucleotide sequence of the second recombinant gene is SEQ ID NO:3; 2) The first recombinant gene is ligated into a vector plasmid to obtain a first expression vector; the second recombinant gene is ligated into a vector plasmid to obtain a second expression vector; 3) The first expression vector is transformed into different host bacteria to obtain the first recombinant bacteria, and the second expression vector is transformed into the second host bacteria to obtain the second recombinant bacteria; 4) After inducing expression in the first host bacterium, the recombinant protein mSEB-SpyTag is extracted and purified; after inducing expression in the second host bacterium, the recombinant protein SpyCatcher-mi3 is extracted and purified; preferably, the amino acid sequence of the recombinant protein mSEB-SpyTag is SEQ ID NO:2; and / or, the amino acid sequence of the recombinant protein SpyCatcher-mi3 is SEQ ID NO:4; 5) Recombinant protein mSEB-SpyTag and recombinant protein SpyCatcher-mi3 were co-dissolved in a buffer solution of 25mM Tris-HCl + 150mM NaCl at pH 8.0 at a molar ratio of 1-5:1, and bound at 25°C; preferably, the recombinant protein SpyCatcher-mi3 and recombinant protein mSEB-SpyTag were subjected to GST and NiO2 respectively. 2+ After affinity chromatography purification, the binding operation is then performed; 6) The mSEB-mi3 recombinant protein is obtained after purification; preferably, the recombinant protein SpyCatcher-mi3 and the recombinant protein mSEB-SpyTag are combined and then purified by size exclusion chromatography.

8. The use of the Staphylococcus aureus enterotoxin B mutant of claim 1, the recombinant Staphylococcus aureus enterotoxin B nanoparticle protein mSEB-mi3 of claim 2 or 3, the encoding gene of claim 4, the recombinant expression vector of claim 5, or the recombinant strain of claim 6 in the preparation of a subunit vaccine against Staphylococcus aureus enterotoxin B.

9. A vaccine for the prevention or treatment of Staphylococcus aureus enterotoxin B infection, comprising recombinant Staphylococcus aureus enterotoxin B nanoparticle protein as described in claim 2 or 3.

10. The vaccine of claim 9, further comprising a pharmaceutically acceptable adjuvant; preferably, the adjuvant is selected from any one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant, and mycobacterial BCG adjuvant.

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