Porcine reproductive and respiratory syndrome virus recombinant nanoparticle vaccine as well as preparation method and application thereof

Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus recombinant nanoparticle vaccine and a preparation method and application thereof, the vaccine contains a recombinant nanoparticle protein, and the protein comprises: (1) a recombinant protein designed based on a PRRSV structural protein epitope; (2) nano-particle protein designed on the basis of ferritin derived from bacteria; and (3) realizing the self-assembly of the recombinant nano particles by virtue of a SpyCatcher-SpyTag system. The vaccine can prevent and control NADC30PRRSV-like strains which are popular in China in recent years, and is used for suppressing propagation of PRRSV variants.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a porcine reproductive and respiratory syndrome virus recombinant nanoparticle vaccine and a preparation method and application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRSV infection in pigs. It is the disease that causes the most economic losses to the pig industry worldwide, except for African swine fever and porcine epidemic diarrhea. Although a variety of antiviral drugs have been reported to have the potential to control PRRSV infection, there is still no low-cost and convincing drug that is widely used in clinical practice. Although the gene-edited pigs that can resist PRRSV infection reported in recent years have brought new hope for the prevention and control of PRRSV, the potential risk verification and legal approval of such gene-edited pigs still require a lot of time to complete. Therefore, vaccines are still an important means to control the spread of the virus. Currently, commercial vaccines are divided into two categories: inactivated vaccines and live attenuated vaccines. However, although vaccine developers have made great efforts to optimize existing vaccines, inactivated vaccines are still questioned because they cannot effectively induce cellular immune responses and neutralizing antibody responses, and live attenuated vaccines are worrying because of their potential recombination and virulence risk. Disappointingly, these commercial vaccines have not shown sufficient immune protection or are even ineffective against the NADC30-like strains that have been prevalent in China in recent years. Therefore, it is urgent to develop a new generation of PRRSV vaccines.

[0003] As an enveloped single-stranded positive-strand RNA virus, PRRSV is prone to mutation and recombination during genome replication, resulting in changes in viral pathogenicity and antigenicity. This characteristic has brought great obstacles to the prevention and control of the virus through vaccines. Unlike the spike glycoprotein of coronaviruses such as SARS-CoV-2 or PEDV, which is a clear immune protection-related protein, there are multiple structural proteins with single or multiple transmembrane on the surface of the PRRSV envelope, forming heterodimers or trimers, which participate in the process related to viral infection. This special viral structure poses great difficulties in analyzing the target protein of neutralizing antibodies, causing confusion in the selection of protective antigens, and seriously hindering the development of subunit vaccines. Usually, after viral infection, humoral immunity and cellular immune responses work together to inhibit the spread of the virus in the body and gradually eliminate the virus. However, in different viral infections, there are certain differences in the role played by cellular immune responses and humoral immune responses. In viruses represented by monkeypox virus, new coronavirus and PCV2, the production of humoral immune responses plays a more important role in preventing and eliminating such viral infections. On the contrary, in viruses that can cause long-term infections, such as HIV, humoral immune responses can only play a limited role, and cellular immune responses are crucial in controlling HIV infection. For PRRSV, it is generally believed that cellular immune responses are more important in preventing and controlling the virus infection. Therefore, developing a vaccine that can effectively induce PRRSV-specific cellular immune responses is the focus of next-generation vaccine research.

[0004] The basis of specific cellular immune response is T cell epitopes. Therefore, analyzing the T cell epitopes related to the target pathogen and designing recombinant proteins based on them is an effective method to induce specific cellular immune response. This has been confirmed in SARS-CoV-2, but it is rarely involved in the study of PRRSV vaccinology. At the same time, in order to further enhance the immunogenicity of this recombinant protein designed based on T cell epitopes, the recombinant protein is displayed on the surface of nanoparticles, because displaying antigens on the surface of nanoparticles (NPs) has been proven to effectively enhance immunogenicity and can strongly activate humoral and cellular immune responses after immunization. By comparing the characteristics of a variety of reported nanoparticle proteins and pre-testing the difficulty of developing these nanoparticle proteins, ferritin nanoparticles derived from Pyrococcus furiosus were finally selected as a platform for the development of a new generation of PRRSV vaccines. This bacterial-derived nanoparticle is natural, non-toxic and safe. At the same time, its expression and purification are simple, rapid and inexpensive, and it has the potential for large-scale production. Through the SpyCatcher-SpyTag system, the target antigen can be easily and accurately loaded on the surface of the nanoparticle, and recombinant nanoparticles can be produced simply and quickly. Summary of the invention

[0005] In view of the technical problem that the existing commercial vaccines have insufficient protective effects against NADC30-like PRRSV strains, the present invention provides a porcine reproductive and respiratory syndrome virus recombinant nanoparticle vaccine and its preparation method and application. The vaccine developed based on the sequence of the NADC30-like PRRSV strain can effectively and strongly induce specific cellular immune response after immunizing piglets, and endow the immunized animals with immune protection against the NADC30-like PRRSV strain.

[0006] The present invention can be implemented by the following technical solutions:

[0007] In a first aspect, the present invention claims a method for preparing a recombinant nanoparticle protein, the method comprising the following steps:

[0008] (1) Designing recombinant antigenic proteins based on PRRSV structural protein epitopes;

[0009] (2) The designed recombinant antigen protein was fused with the SpyCatcher003 protein through a flexible linker to form the recombinant antigen protein-SpyCatcher;

[0010] (3) selecting the ferritin amino acid sequence from Pyrococcus furiosus, fusing the SpyTag003 tag to the N-terminus of the ferritin amino acid sequence through a flexible linker to obtain the SpyTag-ferritin recombinant protein, expressing the SpyTag-ferritin recombinant protein in large quantities and purifying it through ion exchange and size exclusion chromatography to obtain ferritin nanoparticles SpyTag-Ferritin;

[0011] (4) Based on the SpyCatcher-SpyTag system, the recombinant antigen protein-SpyCatcher is loaded on the surface of ferritin nanoparticles SpyTag-Ferritin to obtain recombinant nanoparticle protein.

[0012] In a specific embodiment of the present invention, technicians identified short peptides that can induce IFN-γ production in the structural proteins of NADC30-like PRRSV strains, and based on this and publicly published epitope information, they recombined the regions rich in T cell epitopes and B cell epitopes in GP3, GP4 and GP5 proteins to design three recombinant antigen proteins GP3m, GP4m and GP5m.

[0013] In order to achieve the purpose of in vitro self-assembly based on the SpyCatcher-SpyTag system, the three recombinant antigen proteins GP3m, GP4m and GP5m were constructed and fused with the SpyCatcher003 protein through a flexible linker to form new recombinant proteins. The secretory expression of the recombinant proteins was achieved in the baculovirus-insect cell expression system, and the vaccine core antigen components were obtained after affinity chromatography purification: recombinant antigen protein-SpyCatcher (GP3m-spycatcher, GP4m-spycatcher and GP5m-spycatcher).

[0014] In order to display the vaccine core antigen on the surface of nanoparticle protein, the present invention selected the ferritin amino acid sequence derived from Pyrococcus furiosus, fused the SpyTag003 tag to the N-terminus of ferritin through a flexible linker, and achieved large-scale expression of SpyTag-ferritin recombinant protein in the Escherichia coli system. After purification by ion exchange and size exclusion chromatography, the vaccine nanoparticle carrier component (ferritin nanoparticle SpyTag-Ferritin) was obtained.

[0015] By mixing the vaccine core antigen component and the vaccine nanoparticle carrier component in vitro at a preferred molar volume ratio, the recombinant antigen protein can be displayed on the surface of ferritin nanoparticles with the help of the SpyCatcher-SpyTag system.

[0016] The recombinant antigen protein-SpyCatcher is at least one of GP3m-spycatcher, GP4m-spycatcher and GP5m-spycatcher;

[0017] The GP3m-spycatcher has an amino acid sequence as shown in SEQ ID NO.1;

[0018] The GP4m-spycatcher has an amino acid sequence as shown in SEQ ID NO.3;

[0019] The GP5m-spycatcher has an amino acid sequence as shown in SEQ ID NO.5;

[0020] The ferritin nanoparticle SpyTag-Ferritin has an amino acid sequence as shown in SEQ ID NO.7;

[0021] The obtained recombinant nanoparticle protein is at least one of GP3m-Fe, GP4m-Fe and GP5m-Fe.

[0022] Furthermore, in step (4), the ferritin nanoparticles SpyTag-Ferritin and the recombinant antigen protein-SpyCatcher are mixed in a molar ratio of 1:0.5-1:3 and then self-assembled in vitro to obtain the recombinant nanoparticle protein. Preferably, the ferritin nanoparticles SpyTag-Ferritin and the recombinant antigen protein-SpyCatcher are mixed in a molar ratio of 1:2-1:3.

[0023] In a second aspect, the present invention seeks to protect the recombinant nanoparticle protein prepared by the above method.

[0024] In a third aspect, the present invention claims protection for a recombinant nanoparticle protein, comprising: (I) a recombinant antigen protein-SpyCatcher, and (II) a ferritin nanoparticle SpyTag-Ferritin; the recombinant antigen protein-SpyCatcher is loaded on the surface of the ferritin nanoparticle SpyTag-Ferritin through a SpyCatcher-SpyTag system;

[0025] The recombinant antigen protein-SpyCatcher is at least one of GP3m-spycatcher, GP4m-spycatcher and GP5m-spycatcher;

[0026] The GP3m-spycatcher has an amino acid sequence as shown in SEQ ID NO.1;

[0027] The GP4m-spycatcher has an amino acid sequence as shown in SEQ ID NO.3;

[0028] The GP5m-spycatcher has an amino acid sequence as shown in SEQ ID NO.5;

[0029] The ferritin nanoparticles have an amino acid sequence as shown in SEQ ID NO.7;

[0030] The obtained recombinant nanoparticle protein is at least one of GP3m-Fe, GP4m-Fe and GP5m-Fe.

[0031] Furthermore, the molar ratio of the ferritin nanoparticles SpyTag-Ferritin and the recombinant antigen protein-SpyCatcher during in vitro self-assembly is as described above.

[0032] In a fourth aspect, the present invention seeks to protect the use of the aforementioned recombinant nanoparticle protein in the preparation of a recombinant nanoparticle vaccine for porcine reproductive and respiratory syndrome virus.

[0033] In a fifth aspect, the present invention claims a porcine reproductive and respiratory syndrome virus recombinant nanoparticle vaccine, comprising the aforementioned recombinant nanoparticle protein. Further, the recombinant nanoparticle protein is combined with an aluminum gel adjuvant. Furthermore, the recombinant nanoparticle protein is at least one of GP3m-Fe, GP4m-Fe and GP5m-Fe.

[0034] In a specific embodiment of the present invention, the recombinant nanoparticle protein is (1) GP3m-Fe, GP4m-Fe or GP5m-Fe; or (2) FeCocktail (cocktail vaccine) obtained by mixing GP3m-Fe, GP4m-Fe and GP5m-Fe; specifically, the GP3m-Fe, GP4m-Fe and GP5m-Fe are mixed in a mass ratio of 1:1:1, but not limited thereto.

[0035] The room temperature described in the present invention is 25±10°C.

[0036] Beneficial effects of the present invention:

[0037] The present invention prepares epitope-based PRRSV recombinant protein antigens, displays the recombinant protein on the surface of ferritin nanoparticles with the help of SpyCatcher-SpyTag system, and forms a new vaccine with aluminum gel adjuvant. The vaccine can effectively and strongly induce specific cellular immune response after immunizing piglets, and endow the immunized animals with immune protection against NADC30-like PRRSV strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Determination of cellular immune response levels in piglets at different time points after challenge.

[0039] Figure 2 :Results of PRRSV-specific T cell responses to synthetic peptides detected by IFN-γ ELISPOT.

[0040] Figure 3 : Results of T cell epitope conservation analysis.

[0041] Figure 4 : Diagram of recombinant protein design strategy.

[0042] Figure 5 : Results of Coomassie Brilliant Blue staining (left) and Western blot (right) of the purified protein.

[0043] Figure 6 :Schematic diagram of SpyTag-Ferritin molecular design (top) and Alphafold protein structure prediction results (bottom).

[0044] Figure 7:The identification results of SpyTag-Ferritin protein molecular screening and Coomassie Brilliant Blue staining.

[0045] Figure 8 : DLS detection results of SpyTag-Ferritin protein after purification.

[0046] Fig. 9 : The results of the purified SpyTag-Ferritin protein were photographed under a transmission electron microscope.

[0047] Fig.10 :Schematic diagram of in vitro self-assembly of SpyTag and SpyCatcher proteins.

[0048] Fig.11 : Results of SDS-PAGE protein electrophoresis after mixing SpyTag-Ferritin and recombinant protein-SpyCatcher in different molar ratios.

[0049] Fig.12 : Identification results of recombinant nanoparticles molecular sieve and Coomassie brilliant blue staining.

[0050] Fig.13 : DLS detection results of recombinant nanoparticles.

[0051] Fig.14 :The results of recombinant nanoparticles taken under a transmission electron microscope.

[0052] Fig.15 : Results of determination of humoral immune response antibody levels after mice were immunized with recombinant nanoparticles.

[0053] Fig.16 :Results of determination of specific cellular immune response level after mice were immunized with recombinant nanoparticles.

[0054] Fig.17 : Results of determination of humoral immune response antibody levels in piglets immunized with recombinant nanoparticles.

[0055] Fig.18 :The results of lymphocyte proliferation experiment after piglets were immunized with recombinant nanoparticles.

[0056] Fig.19 : IFN-γ ELISPOT test results after piglets were immunized with recombinant nanoparticles.

[0057] Fig. 20 :The results of flow cytometry detection of intracellular cytokines in piglets after immunization with recombinant nanoparticles.

[0058] Fig.21 : Body temperature monitoring results of piglets in each group after virus attack.

[0059] Fig. 22 : Statistical results of clinical symptom scores of piglets in each group after virus attack.

[0060] Fig.23 : Statistical results of the average daily weight gain of piglets in each group after virus attack.

[0061] Fig.24 : Results of serum viral load determination of each group of piglets after virus attack.

[0062] Fig.25 : Observation results of lung pathological sections staining in the PBS immunization group after virus challenge.

[0063] Fig.26 : Observation results of lung pathological sections staining in the FeCocktail vaccine immunization group after challenge.

[0064] Fig. 27 : Statistical results of lung pathological lesion scores of piglets in each group after virus attack.

[0065] Fig.28 : The results of lung virus load determination in each group of piglets after virus attack. DETAILED DESCRIPTION

[0066] The present invention is described in detail below in conjunction with specific embodiments. According to the following description and implementation, those skilled in the art can determine the basic characteristics of the present invention, and can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention to make it suitable for various uses and conditions.

[0067] Unless otherwise specified, the reagents and materials used in the following examples can be obtained through commercial channels or prepared using methods disclosed in the prior art.

[0068] Example 1: Analysis of T cell epitopes of structural proteins of PRRSV NADC30 strain FJ1402

[0069] 1. Experimental materials:

[0070] 1. Virus: PRRSV NADC30 strain FJ1402 is a conventional strain isolated and preserved in our laboratory.

[0071] 2. Reagents: IFN-γ ELISPOT kit was purchased from MABTECH, Sweden; porcine lymphocyte separation medium was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.; fetal bovine serum and RPMI1640 were purchased from GIBCO; ELISPOT reader was purchased from AID ELISPOT READER, Germany; ConA was purchased from Sigma; other conventional reagents were of analytical grade.

[0072] 3. Experimental animals: 11 piglets negative for PRRSV, ASFV, CSFV and PCV2 nucleic acid and antibody were purchased from farmers in Rugao County, Jiangsu Province.

[0073] 4. Peptides: All peptides were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0074] (II) Experimental methods:

[0075] 1.1 Prediction and synthesis of T cell epitopes of structural proteins of PRRSV NADC30 strain FJ1402

[0076] Potential SLA class I binding peptides (8 to 10 amino acids) were predicted from the amino acid sequence of PRRSV FJ1402 structural proteins using the online prediction software NetMHCpan 4.1 (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ) and IEDB (http: / / tools.iedb.org / mhci / ). A total of 4536 SLA binding short peptides (8 to 10 amino acids) were predicted. Peptides with a predicted ranking of less than 0.5 were considered potential epitopes, and the 58th highest ranked peptide was selected for synthesis in the experimental evaluation (see Table 1 for specific sequences). All selected short peptides were synthesized by Nanjing GenScript Biotechnology Co., Ltd. with a purity of at least 95%, and peptide stock solutions were prepared and distributed according to the manufacturer's recommendations. Peptide working stock solutions were freshly prepared in RPMI 1640 complete medium and added to cells.

[0077] Table 1 Sequences of synthetic peptides and their positions in viral proteins

[0078]

[0079]

[0080]

[0081] 1.2 Piglet challenge and cellular immune response level determination

[0082] Eleven healthy piglets aged 4 weeks were injected intramuscularly with 2 mL and intranasally with 2 mL of FJ1402 F5 virus solution (10 5 TCID 50 / mL), and challenged again with the same dose and challenge method on day 28. Blood was collected on days 28 and 42 after challenge to isolate PBMC (peripheral blood mononuclear cells), and the level of specific cellular immune response of challenged piglets was determined by IFN-γ ELISPOT method.

[0083] The specific separation and preparation method of PBMC is as follows:

[0084] (1) Reagent preparation:

[0085] Anticoagulant: trisodium citrate (Na3C6H5O7·2H2O) 15g, glucose (C6H 12 O6) 12.5g, citric acid (C6H8O7) 5g, add 500mL deionized water, filter with 0.22μm filter, and store at 4℃.

[0086] Red blood cell lysis solution: ammonium chloride (NH4Cl) 3.375g, tris(hydroxymethylaminomethane) (Tris, C4H 11 NO3) 1.3 g, add 500 mL of deionized water and adjust the pH to 7.2-7.6, filter with a 0.22 μm filter and store at 4 °C.

[0087] (2) Blood collection: Use a 10 mL syringe to draw 2 mL of anticoagulant before blood collection (10 mL / tube). Perform subsequent cell separation work in a clean bench.

[0088] (3) Aliquot the lymphocyte separation solution into 10 mL glass centrifuge tubes (4 mL / tube). Tilt the glass tube, place a syringe (without the needle) against the tube wall, and slowly add anticoagulant blood to the 10 mL mark (RPMI1640 can be used to make up the volume if it is insufficient).

[0089] (4) Place the tube containing the anticoagulated blood in a horizontal centrifuge and centrifuge at 1,700 rpm for 20 minutes. Discard the plasma layer, slowly aspirate the lymphocyte layer into a new tube, add PBS buffer to the same volume, and centrifuge at 1,500 rpm for 10 minutes.

[0090] (5) Lysis of red blood cells: Resuspend the cells with an appropriate amount of red blood cell lysis buffer and place in a 37°C water bath to lyse the red blood cells for no more than 3 minutes. After lysis, add washing buffer to the same volume and centrifuge at 800 rpm for 7 minutes.

[0091] (6) Washing: Resuspend the cells in washing solution and centrifuge (700 rpm, 5 min). Repeat the washing process two to three times.

[0092] (7) Resuspending and plating: Resuspend the cells in RPMI 1640 complete medium, adjust the cell number, and plate the cells for culture in a 37°C incubator.

[0093] The specific method of IFN-γ ELISPOT method is as follows:

[0094] (1) Dilute the coating antibody (MAb pIFNγ-I) with sterile PBS to a concentration of 10 μg / mL.

[0095] (2) Take a 96-well ELISpot plate (MABTECH product), add 50 μL 70% (v / v) ethanol to each well to pretreat the bottom membrane of the plate, and incubate at room temperature for 2 min.

[0096] (3) Discard the ethanol, add 200 μL of sterile water to each well, and wash the cell plate 5 times, each time for 10 min.

[0097] (4) Add 100 μL of diluted coating antibody to each well, seal the plate with tape and incubate at 4°C overnight.

[0098] (5) Discard the coated antibody and add 200 μL sterile PBS to each well to wash the plate five times, each time for 10 min.

[0099] (6) Add 200 μL of RPMI-1640 medium containing 10% (v / v) fetal bovine serum (FBS) to each well for blocking and incubate at room temperature for 1 h.

[0100] (7) PBMCs were diluted to a concentration of 5.0×10 6 100 μL of cell suspension was plated in the ELISPOT plate wells. The stimulants required for the experiment were added at the same time, and 3 replicate wells were set for all samples.

[0101] (8) The treated cells were cultured in a 5% CO2, 37°C incubator for 36 h.

[0102] (9) Discard the supernatant and cells, add 200 μL PBS to each well, and clean the plate 5 times, each time for 10 min.

[0103] (10) Take an appropriate amount of biotinylated detection antibody (Biotinylated MAb PAN), dilute the detection antibody to a concentration of 0.5 μg / mL with PBS containing 0.5% FBS (PBS-0.5% FBS), add 100 μL to each well, and incubate at room temperature for 2 h.

[0104] (11) Discard the liquid in the wells after incubation and wash the plate five times with PBS, each time for 10 min.

[0105] (12) Take an appropriate amount of AKP-labeled streptavidin (Streptavidin-Alkaline Phosphatase), dilute it 1000 times with PBS-0.5% FBS, add 100 μL to each well, and incubate at room temperature for 1 h.

[0106] (13) After incubation, discard the liquid in the wells and wash the plate five times with PBS, each time for 10 min.

[0107] (14) Filter the BCIP / NBT (BCIP / NBT Chromogen) substrate using a 0.45 μm filter membrane, add 100 μL to each well, and incubate in the dark at room temperature for 5-15 min until spots are formed at the bottom of the wells.

[0108] (15) Rinse the wells with deionized water to terminate the reaction and dry at room temperature.

[0109] (16) The number of spot-forming cells (SFC) in each well was analyzed using an AID Immunospot Series I Analyzer. The results were recorded as spot-forming units: IFN-γ SFC / well, with each spot representing a cell secreting IFN-γ antibody.

[0110] 1.3 IFN-γ ELISPOT screening and identification

[0111] To test the ability of synthetic short peptides to stimulate IFN-γ secretion by PBMCs of challenged pigs, 5 x 10 5 PBMC cells were added with a final concentration of 10 μg / mL synthetic short peptide solution, and 10 μg / mL Con A was set as the positive control. After incubation in a 37°C incubator for 36 hours, the test was performed according to the steps in 1.2 and the data were recorded.

[0112] 1.4 T cell epitope conservation analysis

[0113] All sequence information was obtained from the NCBI database. Figure 3 The accession number of each representative PRRSV strain is provided in . Multiple sequence alignment analysis and visualization were performed using the Jalview version 2.11.3.3 application (https: / / www.jalview.org / ).

[0114] Results: PBMCs were collected and isolated at 28 and 42 days after infection and the secretion of PRRSV-specific IFN-γ was determined by ELISpot method. The results showed that the secretion of PRRSV-specific IFN-γ at 42 dpi was significantly higher than that at 28 dpi ( Figure 1 ), indicating that the cellular immune response in the challenged pigs was at its peak at this time, which was more conducive to the analysis of T cell epitopes. Therefore, the PBMCs used for subsequent synthetic short peptide stimulation were collected and separated 42 dpi after the challenge. Through IFN-γ ELISpot screening, 7 short peptides that could induce IFN-γ production in multiple challenged pigs were identified from 58 synthetic short peptides, with a reaction rate between 27.3% and 72.7%, indicating that these 7 short peptides are potential T cell epitopes related to IFN-γ production ( Figure 2and Table 2). Multiple sequence alignment analysis showed that the 7 potential epitopes identified were highly conserved or had only individual amino acid mutations among different American PRRSV strains ( Figure 3 ).

[0115] Table 2 Statistics of PRRSV-specific T cell responses to synthetic peptides detected by IFN-γ ELISPOT

[0116]

[0117] Example 2: Design and production of recombinant proteins based on PRRSV FJ1402 structural protein epitopes

[0118] 1. Experimental materials:

[0119] pFastBac TM Dual vector, E. coli DH10Bac engineered bacteria, Sf9 and Hi5 cells are all conventional materials in this field preserved in this laboratory. His-tag was purchased from Yifeixue Biotechnology Co., Ltd. Lipofectamine 3000 is a product of Invitrogen. Sheep anti-swine IgG-HRP and sheep anti-mouse IgG-HRP were purchased from Shanghai Youningwei Biotechnology Co., Ltd. IB905 serum-free insect cell culture medium is a product of Gibco. Protein purification prepacked columns Hiscap Smart6FF and Precap STarm Strepactin were purchased from Changzhou Tiandirenhe Biotechnology Co., Ltd. Other conventional reagents were domestically produced or imported analytical grade. Gene synthesis was completed by Nanjing GenScript Biotechnology Co., Ltd.

[0120] (II) Experimental methods:

[0121] 1.1 Target gene design and synthesis

[0122] Based on the T cell epitopes obtained by analysis in Example 1 and the publicly known epitope T cell and B cell epitope information, the three structural proteins GP3, GP4 and GP5 were selected as the target proteins for the design of recombinant antigens. By analyzing the potential signal peptides and transmembrane region positions of the three structural proteins GP3, GP4 and GP5, combined with the protein structure predicted by Alphafold, the regions rich in a large number of T cell epitopes and B cell epitopes in the protein were selected to be recombined into new recombinant antigens. The three recombinant antigens constructed based on GP3, GP4 and GP5 were named GP3m, GP4m and GP5m, respectively. In order to achieve the purpose of in vitro self-assembly based on the Spycatcher-Spytag system, the three recombinant antigens constructed were fused to the spycatcher003 protein through a flexible linker to form a recombinant protein, and the GP67 secretion signal peptide was added to the N-terminus of the protein, and the His and Strep tags were added to the C-terminus of the protein ( Figure 4 The fusion proteins were named GP3m-spycatcher, GP4m-spycatcher and GP5m-spycatcher, and the relevant sequence information is shown below. The corresponding gene sequences were optimized according to insect cell preference and connected to pFastBac TM Under the PH promoter of the Dual vector, gene codon optimization, synthesis and plasmid construction were completed by Nanjing GenScript Biotechnology Co., Ltd.

[0123] GP3m-spycatcher amino acid sequence (SEQ ID NO.1):

[0124] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFASNATFCFWFPLVRGNFSFELMVNYTVCPLCPTRQAAAEILEPGKSFWCRIGHDRCSESDHDELGFMVPPGLSSEGHLTSVYAWLAFLSFSYTAQFHPEIFGIGNVSQVYVDIKHQLICAVHDGDNATLPR HDNISAVFQTYYQHQVDGGNWFHGGSGGSGGSGGSVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGGSHHHHHHHHWSHPQFEK*

[0125] GP3m-spycatcher nucleotide sequence (SEQ ID NO.2):

[0126]

[0127] The amino acid sequence of GP4m-spycatcher (SEQ ID NO.3):

[0128] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAACKPCFSSSLSDIKTNTTSAASSVVLQDISCLRHGNPPFAAFRKIPQCRAAIGTPVYITVTANVTDENYLHSSDLLMLSSCLFYASEMSEKGFKVIFGNVSGIVAVCVNFTSYVQHVKEFTQRSLVVDHVRLLHGGSGGSGGSGGSVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGGSHHHHHHHHWSHPQFEK*

[0129] The nucleotide sequence of GP4m-spycatcher (SEQ ID NO.4):

[0130] ATGCTGCTCGTGAATCAGTCTCACCAAGGATTTAATAAGGAACATACTAGCAAAATGGTATCAGCAATTGTACTGTACGTGTTACTCGCCGCGGCAGCCCATTCGGCCTTCGCGGCCTGTAAGCCGTGTTTCAGTTCGAGTCTTTCAGACATCAAGACCAACACCACCAGCGCAGCAAGTTCCGTTGTCCTCCAAGACATCAGCTGTCTTAGGCATGGCAACCCGCCCTTTGCGGCGTTTCGCAAAATTCCTCAATGTCGCGCGGCAATAGGAACACCCGTCTACATCACTGTCACAGCCAACGTGACGGATGAGAATTATTTGCATTCCTCTGACCTTCTCATGCTCTCTTCTTGCCTTTTCTACGCTTCTGAAATGAGCGAAAAGGGATTTAAGGTGATATTTGGCAATGTGTCAGGCATTGTGGCTGTGTGTGTCAACTTTACCAGCTATGTCCAACATGTCAAGGAGTTTACCCAGCGATCCTTAGTGGTCGACCATGTGCGGCTGCTTCACGGTGGCAGCGGGGGCTCGGGCGGGTCTGGTGGCAGTGTGACAACATTATCTGGACTTTCGGGTGAGCAGGGCCCTAGTGGGGACATGACGACTGAAGAAGACTCAGCAACGCATATAAAGTTTAGTAAGAGAGATGAAGATGGTCGGGAACTCGCCGGTGCTACCATGGAGCTGCGTGATAGCTCCGGAAAAACCATTTCAACGTGGATCTCGGACGGACATGTAAAAGACTTCTACTTGTACCCAGGGAAGTATACTTTTGTAGAGACTGCAGCTCCCGACGGCTATGAGGTGGCGACGCCGATCGAGTTCACAGTTAATGAAGACGGCCAAGTCACCGTTGATGGAGAAGCGACTGAGGGGGATGCCCACACCGGGGGCTCCCACCATCACCATCACCATCACCATTGGAGCCACCCGCAGTTCGAAAAGTGA

[0131] GP5m-spycatcher amino acid sequence (SEQ ID NO.5):

[0132] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFANASSNSSSHLQLIYNLTICELNGTDWLSTKFGGSGGSGGSGGSTVSTAGYYHKRYGGSGGSGGSGGSRYTNFLLDTKGKLYRWRSPVIIEKGGKVDVEGHLIDLKTVVLDGSAATPVTKISAEQWGRPGGSGGSGGSGGSVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGGSHHHHHHHHWSHPQFEK*

[0133] GP5m-spycatcher nucleotide sequence (SEQ ID NO.6):

[0134] ATGCTGCTCGTGAATCAGTCTCACCAAGGATTTAATAAGGAACATACTAGCAAAATGGTATCAGCAATTGTACTGTACGTGTTACTCGCCGCGGCAGCCCATTCGGCCTTCGCGAACGCTAGCTCAAATAGTTCCTCTCACTTACAACTGATTTATAACTTGACCATTTGCGAACTGAACGGTACAGATTGGCTGAGCACCAAATTTGGTGGTAGTGGCGGCTCCGGTGGTAGCGGCGGCTCTACGGTTAGCACCGCTGGCTATTACCATAAGCGCTATGGTGGCTCGGGAGGCTCGGGCGGTTCCGGAGGCAGCCGTTACACCAACTTCTTGCTGGACACCAAGGGCAAATTGTACCGCTGGCGTAGCCCGGTCATCATTGAGAAAGGCGGTAAGGTGGATGTTGAGGGTCACCTTATCGATCTGAAGACTGTGGTGCTCGACGGCTCCGCGGCAACCCCGGTAACCAAGATCTCTGCCGAACAGTGGGGTCGTCCGGGTGGCAGCGGGGGCTCGGGCGGGTCTGGTGGCAGTGTGACAACATTATCTGGACTTTCGGGTGAGCAGGGCCCTAGTGGGGACATGACGACTGAAGAAGACTCAGCAACGCATATAAAGTTTAGTAAGAGAGATGAAGATGGTCGGGAACTCGCCGGTGCTACCATGGAGCTGCGTGATAGCTCCGGAAAAACCATTTCAACGTGGATCTCGGACGGACATGTAAAAGACTTCTACTTGTACCCAGGGAAGTATACTTTTGTAGAGACTGCAGCTCCCGACGGCTATGAGGTGGCGACGCCGATCGAGTTCACAGTTAATGAAGACGGCCAAGTCACCGTTGATGGAGAAGCGACTGAGGGGGATGCCCACACCGGGGGCTCCCACCATCACCATCACCATCACCATTGGAGCCACCCGCAGTTCGAAAAGTGA

[0135] 1.2 Transformation of synthetic plasmid into DH10Bac and transposition screening

[0136] (1) DH10Bac fresh bacterial solution inoculated with 50 μg / mL K + , 3 mL LB containing 10 μg / mL tetracycline resistance, shake at 37°C for 2 h;

[0137] (2) After removal, collect the cells at 12000 rpm for 1 min;

[0138] (3) Add 1 mL of 4°C pre-cooled CaCl2 (0.1 M) to resuspend the cells and place on ice for 30 min;

[0139] (4) Collect the cells at 4°C, 12,000 rpm for 1 min, add 100 μl of 4°C pre-cooled CaCl2 (0.1 M) to resuspend the cells, and place on ice for later use;

[0140] (5) Add pFastBac TM Dual recombinant plasmid 1 μl, flick to mix, and place on ice for 30 min;

[0141] (6) Heat shock in a 42°C water bath for 90 seconds and immediately remove from heat and place on ice for 3 minutes;

[0142] (7) Add 800 μl of SOC resuscitation solution and resuscitate on a shaker at 37°C for 4 h.

[0143] (8) Take 100 μl of bacterial solution + 400 μl of LB dilution (5-fold dilution), and then take out 100 μl of the diluted bacterial solution to plate;

[0144] (9) Before plating the bacterial solution, take out the triple-antibody plate stored at 4°C, warm it up in a 37°C incubator for 30 min, add 40 μl X-gal (20 mg / mL), 10 μl IPTG, and 50 μl LB to each plate (protected from light), and place it at 37°C for 30 min before use.

[0145] (10) Take the diluted plate and spread it with gentamicin / tetracycline / K + / IPTG / X-gal plates were placed in a 37°C incubator for 40 h, then placed in a 4°C refrigerator for 4-6 h, and blue-white spots were observed;

[0146] (11) Select the three-zone line of the white plate next to the blue spot (containing gentamicin / tetracycline / K + / IPTG / X-gal), placed in a 37°C incubator for 40 h, then placed in a 4°C refrigerator for 4-6 h, and observed for blue-white spots;

[0147] (12) Select white spots and add 50 μg / mL K + , 10 μg / mL tetracycline, 7 μg / mL gentamicin resistance LB, at 37°C for 20 h, and then taken out to prepare for Bacmid extraction.

[0148] 1.3 Recombinant Bacmid Extraction

[0149] (1) The white spot amplified bacteria collected in the 2 mL EP tube were centrifuged at 12000 g for 1 min and the supernatant was discarded;

[0150] (2) Add 300 μl of solution I (with RNase A) to thoroughly resuspend all the precipitate;

[0151] (3) Add 300 μl of Solution II, gently invert 4-6 times to completely lyse the bacteria, and let stand at room temperature for 3 min;

[0152] (4) Add 400 μl of solution III and gently invert 6-10 times to mix thoroughly. White floccules can be seen. Centrifuge at 15,000 g for 10 min and take 800 μl of the supernatant into a new 2 mL EP tube;

[0153] (5) Slowly add 800 μl of 4°C precooled isopropanol to the supernatant, mix by inversion, let stand on ice for 10 min, centrifuge at 15,000 g for 10 min at 4°C, and discard the supernatant;

[0154] (6) Resuspend the precipitate in 1 mL of 70% ethanol precooled at 4°C, centrifuge at 15,000 g for 10 min, and discard the supernatant;

[0155] (7) The precipitate was resuspended in 500 μl of 70% ethanol precooled at 4°C, centrifuged at 15,000 g for 10 min, and the supernatant was discarded;

[0156] (8) After drying at room temperature for 5 min, add 50 μl of 60°C preheated ddH2O to dissolve;

[0157] (9) For short-term use, store at 4°C; for long-term storage, store at -40°C.

[0158] 1.4 Obtaining and Propagating Recombinant Baculovirus

[0159] (1) Sf9 cells were plated in 24-well plates and cultured at 28°C until the cell confluence reached 80%-85% and then transfected;

[0160] (2) Mix 5 μL of recombinant Bacmids with 2 μL of P3000, add IB905 nutrient solution to make up to 25 μL, and mix well;

[0161] (3) Thoroughly mix 2 μL Lip3000 and 23 μL IB905 nutrient solution;

[0162] (4) Mix the two mixtures thoroughly and let stand at room temperature for 5 minutes;

[0163] (5) Slowly add the mixture into the cell plate, 50 μL per well, and use the untransfected empty cell group as a control.

[0164] (6) Observe the cells under a microscope 5-7 days after transfection. It can be observed that the cells become significantly larger and rounder, the permeability increases, the adhesion ability decreases, and there is obvious floating. Collect the culture medium and centrifuge it at 12000 rpm at 4°C for 5 minutes to obtain the P0 virus solution;

[0165] (7) The P0 virus solution was inoculated into Sf9 cells at a ratio of 1:1000. When the lesions were obvious about 3-4 days after infection, the nutrient solution was collected and centrifuged to obtain the P1 virus solution. The P2 virus solution obtained by subculturing and proliferation was used for recombinant protein expression.

[0166] 1.5 Recombinant protein purification and identification

[0167] The P2 virus solution was inoculated into Hi5 cells to express the recombinant protein, and the cell culture fluid was collected by centrifugation 48-60 hours after infection. After clarification, the target protein secreted in the Hi5 cell culture supernatant was collected and purified by Hiscap Smart 6FF column, and then purified by Precap STarm Strepactin column. The purified protein was concentrated, separated, and then frozen for storage.

[0168] Another portion of the protein was used for Coomassie Brilliant Blue staining and Western blot identification.

[0169] (1) preparing protein gel, wherein the acrylamide concentration of the upper layer of the stacking gel is 5%, and the acrylamide concentration of the lower layer of the separation gel is 10%;

[0170] (2) Add the prepared sample to the protein gel lane, 25 μL / well; adjust the voltage to 80 V until the sample enters the separation gel and then adjust the voltage to 120 V;

[0171] (3) Coomassie Brilliant Blue staining or transfer: Stain the gel with Coomassie Brilliant Blue for 60 minutes at room temperature, and then decolorize it with 45% methanol and 10% glacial acetic acid in distilled water until a clear background appears and record it. Alternatively, place a sponge, NC membrane, protein gel, and sponge soaked in transfer buffer on the transfer apparatus in this order, and transfer at 23V constant voltage for 38 minutes;

[0172] (4) Blocking: The transferred NC membrane was placed in 5% skim milk at 37°C for 2 h, and then washed three times with PBST, each time for 10 min;

[0173] (5) Primary antibody incubation: His-tag (1:1000) and PRRSV-positive pig serum (1:200) were diluted with PBST, respectively, and incubated on the NC membrane at 37°C for 2 h. The NC membrane was then washed three times with PBST, each time for 10 min.

[0174] (6) Secondary antibody incubation: The NC membrane was incubated with goat anti-mouse IgG-HRP (1:10000) or goat anti-swine IgG-HRP (1:10000) diluted in PBST at 37°C for 1 h, and then the NC membrane was washed three times with PBST, each time for 10 min;

[0175] (7) Exposure: Open the exposure instrument in advance to precool it, add an appropriate amount of ECL chemiluminescent liquid to the NC film for color development, and then expose it for color development.

[0176] Results: Soluble recombinant protein was successfully secreted and expressed in the insect cell-baculovirus system, and high-purity protein was prepared by two-step affinity chromatography purification. The purified protein was detected by SDS-PAGE, and Coomassie Brilliant Blue staining showed a single and pure protein band in each lane. Antibody incubation also confirmed that the size of the purified protein was consistent with the expected size and had good reactivity with infected pig serum ( Figure 5 ).

[0177] Example 3: Construction and production of ferritin nanoparticles

[0178] (I) Experimental materials: Escherichia coli BL21 engineered bacteria are conventional materials in this field preserved in this laboratory. LB medium, antibiotics and protein expression inducer IPTG used for bacterial culture were purchased from Sigma and Sangon Biotech (Shanghai) Co., Ltd. Q Focurose HPR anion exchange resin prepacked column was purchased from Wuhan Huiyan Biotechnology Co., Ltd. Superdex 200Increase 10 / 300GL gel filtration prepacked column was purchased from GE HealthCare. 50kDa ultrafiltration tube was purchased from Merck Millipore. Other reagents were domestic analytical grade. Ferritin nanoparticle gene codon optimization, synthesis and plasmid construction were completed by Nanjing GenScript Biotechnology Co., Ltd.

[0179] (II) Experimental methods:

[0180] 1.1 Target gene design and synthesis

[0181] The amino acid sequence of ferritin is derived from Pyrococcus furiosus. In order to achieve the purpose of in vitro self-assembly based on the Spycatcher-Spytag system, the constructed SpyTag003 tag is fused to the N-terminus of ferritin through a flexible linker ( Figure 6The fusion protein was named SpyTag-Ferritin, and its corresponding gene sequence was optimized according to the preference of E. coli and connected to the pET29b+ expression vector. Gene codon optimization, synthesis and plasmid construction were completed by Nanjing GenScript Biotechnology Co., Ltd.

[0182] SpyTag-Ferritin amino acid sequence (SEQ ID NO.7):

[0183] MRGVPHIVMVDAYKRYKGGGGSGGGGSGGGGSRMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE*

[0184] SpyTag-Ferritin nucleotide sequence (SEQ ID NO.8):

[0185] ATGAGGGGAGTTCCCCACATAGTAATGGTCGACGCGTACAAGCGCTATAAGGGTGGTGGCGGCTCTGGTGGCGGTGGTTCTGGCGGAGGTGGCAGCCGTATGCTCAAGGCGTTGAATGATCAACTGAACCGCGAATTATATAGCGCATACCT GTATTTCGCTATGGCTGCCTACTTCGAAGATCTGGGTCTTGAGGGTTTCGCGAACTGGATGAAAGCCCAGGCAGGAAGAGATCGGCCACGCACTGCGCTTTTATAACTACATTTATGATCGTAATGGCCGTGTGGAATTGGACGAAATTC CGAAGCCGCCAAAAGAATGGGAAAGCCCGCTGAAGGCGTTCGAGGCGGCGTACGAACATGAAAAATTCATCAGCAAATCCATTTACGAGTTGGCGGCTCTGGCGGAAGAGGAGAAGGACTACTCGACCCGTGCATTTCTGGAGTGGTTTATC AACGAACAGGTCGAGGAGGAGGCCAGCGTTAAAAAAATCCTGGACAAACTGAAGTTCGCGAAAGATTCCCCGCAAATTTTGTTTATGCTGGACAAGGAGCTGAGCGCGCGTGCTCCGAAGCTGCCGGGTTTGCTGATGCAGGGTGGCGAATAA

[0186] 1.2 Preparation of ferritin nanoparticles

[0187] The constructed recombinant pET29b+ expression plasmid was introduced into BL21 competent cells, and a single positive colony was picked and cultured overnight in 3 mL LB medium containing 50 μg / mL kanamycin at 37°C and 200 rpm. The culture was then amplified in 300 mL LB medium containing 50 μg / mL kanamycin at 37°C and 200 rpm until OD 600Reach 0.6, then add IPTG with a final concentration of 1mM to induce expression for 8 hours. Harvest the bacterial culture precipitate and resuspend it with Tris buffer (20mM Tris, pH7.6), ultrasonically disrupt, and take the soluble supernatant after high-speed centrifugation and heat it to 72°C and maintain it for 15min to precipitate most of the E. coli autologous proteins. After high-speed centrifugation again, the supernatant is further purified by Q Focurose HPR anion exchange resin column. The eluted protein is concentrated by ultrafiltration and added to a Superdex 200Increase 10 / 300GL size exclusion column pre-equilibrated with 20mM Tris 50mM NaCl buffer (pH7.6). Use the same buffer for elution separation at a flow rate of 0.5mL / min.

[0188] 1.3 Identification of ferritin nanoparticles

[0189] Take some of the purified ferritin and boil it for identification by Coomassie Brilliant Blue staining.

[0190] Dynamic light scattering (DLS) analysis was performed using a Zetasizer Ultra instrument to characterize the hydrodynamic diameter and polydispersity index of SpyTag-Ferritin nanoparticles. The samples were loaded into reusable test tubes and measured at 25°C in triplicate. The average values ​​of the hydrodynamic diameter and polydispersity index of the samples were analyzed using the manufacturer's software (Malvern).

[0191] The morphology of SpyTag-Ferritin nanoparticles was observed using a transmission electron microscope (TEM). Briefly, 10 μL of the sample was dropped onto a silicon grid, immediately stained with 2% (w / v, g / 100 ml) uranyl acetate for 60 seconds, and dried. Images were taken using an HT7800 (Hitachi) at an accelerating voltage of 80 kV.

[0192] Results: The protein structure predicted by Alphafold online software showed that SpyTag was located on the surface of ferritin nanoparticles, which provided a structural basis for the assembly with SpyCatcher ( Figure 6 The total volume of the Superdex 200Increase 10 / 300GL size exclusion column was 24 mL, and the SpyTag-Ferritin nanoparticles were eluted at a volume of approximately 10 mL ( Figure 7 ). SDS-PAGE electrophoresis results ( Figure 7 ) showed that the position of the purified protein band was basically consistent with the predicted protein size (22.8 kDa). DLS detection results showed that the purified SpyTag-Ferritin nanoparticles had good dispersion and uniform size, with an average particle size of 12.43 ± 1.59 nm ( Figure 8 ). Transmission electron microscopy showed that the purified SpyTag-Ferritin had a uniform size, stable structure and morphology, and a nanoparticle structure with a diameter of about 10 nm ( Fig. 9 ).

[0193] Example 4: In vitro self-assembly and characterization of recombinant ferritin nanoparticles

[0194] (I) Experimental materials: Superdex 200 Increase 10 / 300 GL gel filtration prepacked column was purchased from GE Healthcare. The purified proteins used for assembly were from Examples 2 and 3. Other reagents were all domestic analytical grade.

[0195] (II) Experimental methods:

[0196] 1.1 Optimization of the ratio of recombinant protein-SpyCatcher and SpyTag-Ferritin in vitro assembly

[0197] As shown in the schematic diagram ( Fig.10 ), based on the rapid covalent bonding between SpyTag and SpyCatcher proteins, recombinant proteins can be easily loaded on the surface of nanoparticles, and recombinant nanoparticles can be produced simply and quickly. SpyTag-Ferritin and recombinant protein-SpyCatcher were mixed at a molar ratio of 1:0.5, 1:1, 1:2 and 1:3, and mixed overnight on a rotating shaker at 4°C. The binding efficiency was detected by SDS-PAGE protein electrophoresis.

[0198] 1.2 Characterization of recombinant nanoparticles after assembly

[0199] In the purification and characterization of recombinant protein-Ferritin nanoparticles, SpyTag-Ferritin and recombinant protein-SpyCatcher were mixed in a molar ratio of 1:3, mixed on a rotary shaker at 4°C overnight, concentrated by ultrafiltration, and loaded onto a Superdex 200Increase 10 / 300GL size exclusion column after high-speed centrifugation. The column was equilibrated and eluted with PBS buffer (pH 7.4) at a flow rate of 0.5 ml / min throughout the process, and the protein fractions of each peak were collected for further analysis.

[0200] Dynamic light scattering (DLS) analysis was performed using a Zetasizer Ultra instrument to characterize the hydrodynamic diameter and polydispersity index of the recombinant nanoparticles after assembly. The samples were loaded into reusable test tubes and measured at 25°C in triplicate. The average values ​​of the hydrodynamic diameter and polydispersity index of the samples were analyzed using the manufacturer's software (Malvern).

[0201] The morphology of the recombinant nanoparticles after assembly was observed using a transmission electron microscope (TEM). Briefly, 10 μL of the sample was dropped onto a silicon grid, immediately stained with 2% (w / v, g / 100 ml) uranyl acetate for 60 seconds, and dried. Images were taken at an accelerating voltage of 80 kV using a HT 7800 (Hitachi).

[0202] Results: SDS-PAGE protein electrophoresis results showed that the SpyTag-Ferritin: recombinant protein-SpyCatcher coupling efficiency was the highest when the molar ratio was 1:2-1:3 ( Fig.11 ), which can ensure that all ferritin nanoparticles are consumed while leaving as little recombinant protein-SpyCatcher as possible. In order to further confirm whether the assembled protein can maintain the properties of soluble protein particles, the assembled protein was analyzed using a Superdex 200Increase 10 / 300GL size exclusion column. At the same time, in order to ensure that all SpyTag-Ferritin is coupled to the recombinant protein-SpyCatcher, an appropriate excess of recombinant protein-SpyCatcher is added. The SEC analysis results showed that the recombinant nanoparticles assembled with excess recombinant protein-SpyCatcher showed two main peaks, one peak was in the front, which was the multimeric protein peak, and the SDS-PAGE test results showed that the protein was coupled to the protein; the other peak was relatively backward, and the SDS-PAGE test results showed that the peak was the added excess recombinant protein-SpyCatcher ( Fig.12 ). The protein of the first peak outflowed by SEC was taken for DLS detection and transmission electron microscopy observation. The DLS detection results showed that the purified recombinant nanoparticles had good dispersion and uniform size. The average particle size of GP3-Fe was 31.39±1.57nm, the average particle size of GP4-Fe was 25.86±2.09nm, and the average particle size of GP5-Fe was 21.62±3.93nm ( Fig.13 ). Transmission electron microscopy showed that the recombinant nanoparticles were uniformly shaped and nearly round particles with larger diameters than those of unassembled SpyTag-Ferritin and rough edges ( Fig.14 ), indicating that the recombinant protein was successfully loaded. Example 5: Evaluation of the immune characteristics of recombinant ferritin nanoparticles in mice

[0203] (I) Experimental materials: Horseradish peroxidase-labeled goat anti-mouse IgG was purchased from Shanghai Union Biotech Co., Ltd.; ELISA colorimetric substrate TMB was purchased from Shanghai Biotech Co., Ltd.; 96-well ELISA plate was purchased from Corning Costar; aluminum gel adjuvant was purchased from Vertellus, USA. ICR female mice (6 weeks old) were purchased from the Center for Comparative Medicine, Yangzhou University. Fixation / permeabilization solution was purchased from BD. Mouse flow cytometry-related antibodies were purchased from BioLegend.

[0204] (II) Experimental methods:

[0205] 1.1 Mouse immunization protocol and procedures

[0206] Female ICR mice aged 6 weeks were randomly divided into cages, with 5 mice in each group. Purified recombinant protein-SpyCatcher monomer protein (5 μg), recombinant protein-Ferritin (equivalent to 5 μg recombinant protein-SpyCatcher component) or recombinant protein-Ferritin mixture (GP3m-Fe, GP4m-Fe and GP5m-Fe were mixed in a mass ratio of 1:1:1, named FeCocktail) was used in combination with aluminum gel adjuvant to prepare vaccines, and 0.1 mL of the prepared vaccine was injected into the thigh muscle of each mouse each time. Each mouse was immunized twice (on the 0th day and the 21st day respectively). Blood was collected from the eye sockets on the 21st day and the 42nd day after the first immunization. After the blood was allowed to coagulate at room temperature, it was centrifuged at 4000 rpm and 4°C for 10 minutes, and the supernatant was collected.

[0207] 1.2ELISA to detect specific antibody levels in serum

[0208] (1) Coating: Dilute the purified GP3m, GP4m, and GP5m proteins expressed in Escherichia coli inclusion bodies to 1 μg / mL in carbonate buffer (pH 9.6), add 100 μL per well of the ELISA plate, and coat at 37°C for 2 h;

[0209] (2) Washing: Discard the liquid in the wells, add 200 μL PBST to each well, and shake and wash three times;

[0210] (3) Blocking: Add 200 μL of 5% (w / v, g / 100 ml) skim milk to each well and block for 2 h at 37°C, then wash;

[0211] (4) Serum incubation: The separated mouse serum was diluted at a 2-fold ratio of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, respectively. 100 μL of the diluted serum to be tested was added to each well, and a standard positive and negative serum control was set. The cells were incubated at 37°C for 1 h and then washed.

[0212] (5) Enzyme-labeled antibody incubation: HRP-labeled goat anti-mouse IgG antibody was diluted 1:1000 in PBST as the secondary antibody, 100 μL / well, incubated at 37°C for 45 min, and then washed;

[0213] (6) Substrate color development: Add TMB color development solution (100 μL / well) in dark place and color at 37°C for 10 min.

[0214] (7) Stopping: Add 50 μL of stop solution to each well, read OD450nm with a microplate reader, and calculate the P / N value.

[0215] (8) Result determination: The maximum serum dilution with a P / N value greater than 2.1 is used as the ELISA titer of the serum

[0216] 1.3 Flow cytometry to measure cellular immune response

[0217] (1) After killing the mice, immerse them in 75% anhydrous ethanol and remove the spleen of the mice aseptically in a clean bench;

[0218] (2) Place the spleen in a six-well plate pre-filled with 5 mL of 0.5 mM EDTA·Na2-PBS and a 70 μM cell strainer;

[0219] (3) Grind gently with a syringe rubber pad and collect the filtrate into a 15 mL centrifuge tube;

[0220] (4) Centrifuge at 400 g for 5 min and discard the supernatant;

[0221] (5) Resuspend in 5 mL of red blood cell lysis buffer and lyse at room temperature for 3-5 min;

[0222] (6) Add 5 mL of 0.5 mM EDTA·Na2-PBS to terminate lysis, incubate at 400 g for 5 min, and discard the supernatant;

[0223] (7) Resuspend the cells in 1 mL of 10% FBS-1640, count and plate into a 24-well plate, with 5 x 10 cells per well. 6 indivual;

[0224] (8) Set up a positive control stimulation group, an experimental antigen stimulation group (recombinant protein), a single staining group, and a blank cell control group; for the positive control stimulation group, add the positive control stimulus and Brefeldin A inhibition simultaneously, and treat for a total of 5 hours; for the experimental antigen stimulation group (recombinant protein), first add antigen stimulation for 12-16 hours and then add Brefeldin A for 4 hours, for a total of 6 hours; the blank control group does not receive stimulation treatment, but adds Brefeldin A.

[0225] (9) After stimulation, the cells were collected and transferred to a 1.5 mL eppendorf tube and centrifuged at 400 g for 5 min at 4°C.

[0226] (10) Dilute TruStain FcX with staining buffer TM PLUS, resuspend cells in 50 μL / tube, vortex to mix, and incubate at 4°C for 10 min; centrifuge at 400 g for 5 min at 4°C and discard the supernatant;

[0227] (11) Use staining buffer to dilute anti-mouse CD3, anti-mouse CD4 and rat anti-mouse CD8a, resuspend the cells at 50 μL / tube, vortex to mix, and incubate at 4°C in the dark for 30 min;

[0228] (12) Resuspend in 1 mL of 0.9% BSA-PBS, centrifuge at 400 g for 5 min at 4°C, discard the supernatant, and wash twice;

[0229] (13) Resuspend in 200 μL Fixation / permeabilization solution, 4°C, 20 min; centrifuge and discard the supernatant; then resuspend in 0.5 mL 1x Perm / Wash TM Resuspend in buffer, centrifuge at 500 g for 3 min, discard the supernatant, and wash twice;

[0230] (14) Use 1x Perm / Wash TM Buffer was used to dilute anti-mouse IL-4, anti-mouse IL-2 and anti-mouse IFN-γ to 2 μL per tube of cells, and cells were resuspended in 50 μL / tube, vortexed to mix, and incubated at 4°C in the dark for 30 min;

[0231] (15) Resuspend in 1 mL of 0.9% BSA-PBS, centrifuge at 400 g for 5 min at 4°C, discard the supernatant, and wash twice; add 300 μL of 0.9% BSA-PBS to each tube to resuspend the cells and load on the analyzer for detection.

[0232] Results: Compared with the non-nanosized soluble monomeric protein, the nanosized recombinant protein - Ferritin can induce a stronger humoral immune response, and the detectable antibody titer is significantly increased, especially the enhancement effect on the less immunogenic GP4m and GP5m proteins is more significant ( Fig.15 ), showing the strong immunogenicity of nanoproteins. Furthermore, in order to induce a more comprehensive immune response, it was investigated whether the cocktail vaccine (FeCocktail) prepared by mixing three recombinant proteins-Ferritin would affect the humoral immune response of each protein. The results showed that the antibody titers of each protein measured by indirect ELISA after FeCocktail immunization were not significantly different from those after immunization with the corresponding recombinant protein-Ferritin, indicating that the cocktail vaccine is feasible ( Fig.15 ). However, even though the cocktail vaccine induced a strong humoral immune response in mice after two immunizations, we did not detect any neutralizing antibodies.

[0233] Three weeks after the mice were boosted, single cell suspensions were isolated from the mouse spleen and stimulated with immune proteins. The ability of specific cell types to secrete cytokines was measured by flow cytometry. The results showed that the nano-FeCocktail induced CD3 + CD4 + The percentage and absolute number of cells secreting IL-4 and IFN-γ cytokines increased significantly ( Fig.16 ). This phenomenon demonstrates the ability of nano-FeCocktail to induce a balanced Th1 and Th2 immune response after immunization. PRRSV-specific cytotoxic T lymphocytes play an important role in virus clearance. Therefore, we also analyzed CD3 + CD8 + Compared with the monomeric cocktail protein, FeCocktail more effectively stimulated antigen-specific multifunctional CD8 + T cell responses, manifested by IFN-γ and IL-2 release from CD8 + The proportion of T cells increased significantly ( Fig.16 ).

[0234] Example 6 Evaluation of the protective effect of recombinant ferritin nanoparticles on piglet immune challenge

[0235] (I) Experimental materials: PRRSV FJ1402 strain is a conventional strain isolated and preserved in this laboratory. Horseradish peroxidase-labeled sheep anti-swine IgG was purchased from Shanghai Youningwei Biotechnology Co., Ltd.; ELISA colorimetric substrate TMB was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; 96-well enzyme-linked immunosorbent assay plate was purchased from Corning Costar; aluminum gel adjuvant was purchased from Vertellus, USA. IFN-γ ELISPOT kit was purchased from Swedish MABTECH, porcine lymphocyte separation fluid was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., fetal bovine serum and RPMI1640 were purchased from GIBCO, ELISPOT reading only was purchased from German AID ELISPOT READER, and ConA was purchased from Sigma. Fixation / permeabilization solution and porcine flow cytometry-related antibodies were purchased from BD. 30 PRRSV, ASFV, CSFV and PCV2 nucleic acid and antibody negative piglets were purchased from farmers in Rugao County, Jiangsu Province. Other conventional reagents were of analytical grade.

[0236] (II) Experimental methods:

[0237] 1.1 Experimental plan and procedures for piglet immune protection

[0238] Thirty healthy piglets (6-8 kg) with negative nucleic acid and antibodies to PRRSV, ASFV, CSFV and PCV2 were divided into six groups, with five pigs in each group. Purified recombinant protein-Ferritin (equivalent to 50 μg recombinant protein-SpyCatcher component) or recombinant protein-Ferritin mixture (GP3m-Fe, GP4m-Fe and GP5m-Fe mixed in a mass ratio of 1:1:1, named FeCocktail) was used in combination with aluminum gel adjuvant to make the vaccine. Each piglet was injected with 1 mL of the prepared vaccine into the neck muscle each time. After 21 days, the same dose of vaccine was used for booster immunization, and blood was collected at the 3rd and 6th weeks after the first immunization. After standing at room temperature for blood coagulation, the blood was centrifuged at 4000 rpm and 4°C for 10 minutes, and the supernatant was collected. Three weeks after the booster immunization, 2 mL was injected into the neck muscle and 2 mL was dropped into the nose for 10 minutes. 5 TCID 50 / mL FJ1402 F5 virus liquid. After the virus attack, the pigs were isolated and raised, and the body temperature was measured at a fixed time every morning. The pigs' body conditions were observed and recorded. Blood was collected and serum was separated on the 7th, 14th and 21st days for viremia detection. All pigs were killed on the 21st day after the virus attack, and the pathological changes of various tissues were observed.

[0239] 1.2ELISA to detect specific antibody levels in serum

[0240] Same method as above

[0241] 1.3 Lymphocyte proliferation assay

[0242] (1) Lymphocyte separation was performed as above. The density of the separated lymphocytes was adjusted to 5×10 6 cells / mL and then added into a 96-well cell culture plate at 100 μL / well.

[0243] (2) For the negative control, three parallel wells were prepared and serum-free RPMI-1640 culture medium was added. Another group of three parallel wells with only nutrient solution but no cells were set up.

[0244] (3) For the positive control, three parallel wells were prepared and stimulated by adding ConA at a final concentration of 10 μg / mL.

[0245] (4) The experimental group was stimulated by adding immune protein at a final concentration of 10 μg / mL.

[0246] (5) Culture in a 5% CO2, 37°C constant temperature cell culture incubator for 68 h.

[0247] (6) Add 10 μL of enhanced CCK-8 solution to each well and continue incubating in the incubator for 4 h. Measure the absorbance at 450 nm using an ELISA reader.

[0248] (7) Calculate the stimulation index: Stimulation index SI = OD value of the stimulation well - background OD value / OD value of the negative control well - background OD value

[0249] 1.4 IFN-γ ELISPOT

[0250] Same method as above

[0251] 1.5 Flow cytometry to measure cellular immune response

[0252] Same method as above

[0253] 1.6 Body temperature changes and clinical symptom scoring during the challenge period

[0254] After the piglets were challenged with the virus, they were kept in isolation, and their body temperature was recorded daily. Their clinical symptoms, including hair, skin, eating, diarrhea, breathing, coughing, and mental state, were observed. The scoring items were mental state, breathing, and coughing. The score for each item was an integer between 0 and 4, with 0 points for normal conditions, 1 point for extremely mild symptoms, 2 points for mild symptoms, 3 points for severe symptoms, and 4 points for death. Each indicator was scored every day after the challenge and the sum of the indicators was calculated. Finally, the average values ​​of each group were statistically analyzed.

[0255] 1.7 Recording of visual lesions of piglets’ organs after infection and preparation of pathological sections

[0256] Scores were assigned according to the proportion of each lobe: 10 points for the apical lobe and cardiac lobe, 27.5 points for the diaphragmatic lobe, and 5 points for the accessory lobe, with a total score of 100. Scores were given according to the degree of lesions in each part. The larger the lesion area, the higher the score. The sum of all parts was the final score of the lung. After three independent scorings, the lung scores of each group were statistically analyzed.

[0257] The specific method for making pathological sections is as follows:

[0258] (1) Fixation: Take 1cm×2cm 2 Small pieces of lung tissue were fixed in 4% paraformaldehyde for more than 24 h.

[0259] (2) Trimming: Trim the tissue block into smaller pieces, keep the cut surface flat, and place it in an embedding box. Fix the tissue block again for 8 hours. In order to remove residual formaldehyde, rinse the tissue block continuously with running water for more than 12 hours.

[0260] (3) Dehydration: Dehydrate the tissue blocks with gradient alcohol, placing them in 70% alcohol, 80% alcohol, and 90% alcohol for 2 h each, then remove them and place them in 95% alcohol for 1 h. Finally, use anhydrous ethanol to perform final dehydration on the tissue blocks.

[0261] (4) Transparent and wax immersion: After dehydration, use xylene to transparentize the tissue block. The time for transparentization should be determined according to the characteristics of different organs and tissues. Open the incubator in advance to melt the liquid paraffin, clamp the transparent tissue block and place it in the melted paraffin, and immerse it in wax at 65℃ for 3 hours.

[0262] (5) Embedding: Before embedding, raise the temperature of the incubator to 70°C, pour the melted paraffin into the embedding frame, and then neatly place the tissue blocks. After the paraffin solidifies, trim the blocks.

[0263] (6) Sectioning: Freeze the tissue block in a -20°C refrigerator for 1 h before sectioning. Adjust the slicer to 3 mm thickness and rotate the slicer at a constant speed until the ideal tissue wax slice is cut. Use a slide dipped in 60% alcohol to transfer the tissue slice into 45°C warm water to fully unfold it. Take a new slide and gently stick it on the surface of the tissue block to remove it from the water. Place the slide in a 37°C incubator to bake overnight.

[0264] (7) Hematoxylin-eosin staining: First, immerse the sections in xylene for 20 min to remove the paraffin wax. Then, immerse the sections in alcohol / xylene, anhydrous ethanol, 95% alcohol, 85% alcohol, 75% alcohol, and distilled water for 4 min each. After staining in hematoxylin stain for 6 min, wash off the excess stain with running water. After immersing the slides in 1% hydrochloric acid alcohol for 5 s, rinse with running water for 5-10 min, immerse in 90% alcohol, quickly remove, and immerse in 1% eosin for 4 s. After staining, dehydrate with anhydrous ethanol and immerse in xylene again for 20 min.

[0265] (8) Sealing: Add a drop of neutral resin to the tissue and cover it with a coverslip. Be careful not to create bubbles. After the process is completed, place the coverslip at room temperature to dry.

[0266] (9) Observation and shooting: Use 40 digital slices were taken and recorded by scanning microscope.

[0267] 1.8 Determination of viral load in serum and lungs of piglets after challenge

[0268] After pretreatment of serum and lung samples, viral RNA was extracted according to the RNA Kit I extraction kit operation steps, and reverse transcription was performed. SYBR Green real-time PCR was used to detect the PRRSV nucleic acid content in serum and lungs. Viral RNA was extracted according to the OMEGA RNA Kit I extraction kit operation steps, as follows:

[0269] (1) Sample pretreatment, serum: collect blood from the anterior vena cava of pigs on days 7, 14, and 21 after infection, centrifuge at 4°C, 4,000 g for 5 min, and collect the supernatant. Lung: weigh 1 g of fresh lung tissue in a 5 ml centrifuge tube, chop it with sterile scissors, add 3 ml of pre-cooled sterile PBS, use a homogenizer to obtain tissue homogenate, freeze-thaw three times after homogenization, and collect the supernatant after centrifugation.

[0270] (2) Take 200 μL of serum and lung homogenate supernatant and place them in an EP tube. Add 350 μL of TRK Lysis Buffer. Vortex and mix thoroughly. Add to RNA Homogenizer Spin Column and centrifuge at 12,000 rpm for 2 min.

[0271] (3) Add 700 μL of 70% anhydrous ethanol to the collection tube, shake and mix, transfer to the Mini Column, centrifuge at 10,000 g for 1 min, discard the liquid, and repeat this step to centrifuge the entire mixture through the column.

[0272] (4) Pipette 500 μL RNA Wash Buffer I and add it to the Mini Column. Centrifuge at 10,000 g for 1 min and discard the liquid.

[0273] (5) Add 500 μL RNA Wash Buffer II to the column, centrifuge at 10,000 g for 1 min, and discard the liquid.

[0274] (6) Repeat step (5).

[0275] (7) Transfer the Mini Column to a new EP tube and leave it uncovered in a ventilated place to dry for 3 min to promote ethanol volatilization.

[0276] (8) Add 30 μL of 70°C preheated DEPC water to the column, let it stand for 2 min, and centrifuge at 13,000 rpm for 1 min to collect the eluate, which is the extracted RNA.

[0277] (9) Reverse transcription: Add 8 μL of extracted RNA template and 2 μL of 5×qRT Super Mix to a PCR tube, mix well and centrifuge. Reaction procedure: 50°C, 15 min; 85°C, 5 s. The product is viral cDNA.

[0278] Then, the recombinant plasmid stored in the laboratory was used to prepare the standard, the plasmid concentration was measured and converted into the copy number, and double distilled water was used to dilute 10 times to 10 2 ~10 8 copies / μL is used as a positive standard. Plasmid copy number = 6.02×10 23 ×[Plasmid concentration (ng / μL) × 10 -9 ] / (full length of plasmid × 660). The mRNA content of PRRSV in serum and lung was determined by real-time fluorescence quantitative PCR (Real-Time PCR). The reaction system was: SBYR Green Master Mix 10μL, ddH2O7.8μL, cDNA 1μL, ROX20.4μL, upstream and downstream primers 0.4μL each, totaling 20μL.

[0279] The primer sequences for Real-Time PCR were as follows: upstream primer, 5'-AAACCAGTCCAGAGGCAAGG-3'; downstream primer, 5'-TCAGTCGCAAGAGGGAAATG-3'.

[0280] The reaction procedure was 95℃5min, 95℃10s, 60℃31s, and 40 cycles. Each group of samples was repeated 3 times, and a standard and negative control were set up. Finally, a standard curve was drawn based on the Ct value of the standard, and the Ct value of each sample was used to calculate the corresponding viral copy number.

[0281] Results: ELISA test results showed that after two immunizations, the nanoparticle vaccine effectively induced an immune response in piglets, and the levels of GP3 and GP5 protein antibodies increased significantly, but no GP4 protein antibodies were detected ( Fig.17 ). Lymphocyte proliferation experiments also showed that GP3m-Fe and FeCocktail proteins can effectively activate lymphocyte proliferation after immunization ( Fig.18 ).

[0282] IFN-γ has been reported to be a key cytokine involved in the control and clearance of PRRSV infection in pigs. Therefore, PBMCs were isolated by collecting anticoagulated blood from piglets three weeks after booster immunization, and the production of PRRSV-specific IFN-γ was detected after stimulation with purified PRRSV FJ1402 virus particles at a final concentration of 10 μg / mL during in vitro culture. The results of ELISPOT test showed that FeCocktail protein combined with Alum adjuvant could effectively induce the production of PRRSV-specific IFN-γ after immunization, and the production of these IFN-γ was mainly contributed by the two proteins GP3m-Fe and GP5m-Fe ( Fig.19 ). The results of flow cytometry also confirmed the results of ELISPOT. FeCocktail protein combined with Alum adjuvant induced the highest level of PRRSV-specific IFN-γ in piglets, which was mainly produced by functional CD3 + CD4 + and CD3 + CD8 + Secretion production, no role of γδT cells in the production of PRRSV-specific IFN-γ was observed ( Fig. 20 ).

[0283] According to the results of humoral immunity and cellular immune response determination after booster immunization, we challenged the FeCocktail protein immunization group and the PBS immunization group to observe the protective efficacy of FeCocktail protein after immunization. The body temperature, weight and clinical symptoms of the piglets were monitored for three weeks after the challenge. Three weeks later, the experimental pigs were killed to collect relevant tissues and organs for pathological section preparation and viral load determination. Multiple indicators were combined to more comprehensively evaluate the immune protection efficacy. The results showed that compared with the PBS immunization group, the piglets immunized with FeCocktail protein did not develop persistent fever during the three weeks of monitoring after the challenge, and the overall body temperature remained at a normal level (<40°C) ( Fig.21 ); The overall clinical symptoms of the FeCocktail protein immunization group were significantly alleviated compared with the PBS immunization group (mainly including appetite, mental state and respiratory status scores) ( Fig. 22 ); There was no significant difference in the daily weight gain of piglets immunized with FeCocktail protein during the challenge period compared with healthy piglets not challenged with the virus. On the contrary, the PBS immunized group was affected by symptoms such as fever and decreased appetite after the challenge, and the daily weight gain during the challenge period was significantly lower than that of healthy piglets not challenged with the virus ( Fig.23 ). All these clinical indicators show the positive effects of FeCocktail protein immunization.

[0284] Subsequently, the viral load in the serum of each group at different time points was detected. The results showed that FeCocktail protein immunization significantly reduced the viral load in the serum 7 and 14 days after the virus attack ( Fig.24 ). 21 days after the virus attack, no viral RNA was detected in the serum of the FeCocktail protein immunization group ( Fig.24 ). This result shows that FeCocktail protein immunization can accelerate the clearance of viruses in the blood circulation and shorten the duration of viremia compared with PBS immunization.

[0285] PRRSV specifically feeds on pig alveolar macrophages, causing lung damage, with widening of the lung interstitium as the main manifestation. Therefore, in order to evaluate the degree of lung pathological damage in piglets in each experimental group, some lung tissues were removed to prepare tissue sections and stain for observation. The results of histopathological staining showed that FeCocktail protein immunization significantly alleviated lung tissue damage, and the morphology of the lung interstitium did not change significantly ( Fig.25 and Fig.26 ), the lung pathological injury score was significantly lower than that of the PBS immunization group ( Fig. 27 ). The results of lung tissue viral load test showed that the lung viral load of the FeCocktail protein immunization group was significantly lower than that of the PBS immunization group after virus challenge ( Fig.28 ). All these results indicate that FeCocktail protein can effectively induce immune protection against homologous PRRSV in piglets.

[0286] In summary, we constructed an epitope-based PRRSV recombinant protein antigen, and successfully displayed the recombinant protein on the surface of ferritin nanoparticles with the help of the SpyCatcher-SpyTag system, and combined it with an aluminum gel adjuvant to form a new vaccine. The strong immunogenicity of this nanoparticle multi-epitope vaccine was confirmed in both mouse and piglet models. It can effectively and strongly induce specific cellular immune responses in vaccinated animals and give immune animals immune protection against homologous strains. The results of this study have laid a good theoretical foundation for the development of universal PRRSV vaccines in the future, and also provide a reference for the antigen selection and design of PRRSV subunit vaccines.

Claims

1. A method for preparing recombinant nanoparticle protein, characterized in that: The method comprises the following steps: (1) Designing recombinant antigenic proteins based on PRRSV structural protein epitopes; (2) The designed recombinant antigen protein was fused with the SpyCatcher003 protein through a flexible linker to form the recombinant antigen protein-SpyCatcher; (3) selecting the ferritin amino acid sequence from Pyrococcus furiosus, fusing the SpyTag003 tag to the N-terminus of the ferritin amino acid sequence through a flexible linker to obtain the SpyTag-ferritin recombinant protein, expressing the SpyTag-ferritin recombinant protein in large quantities and purifying it through ion exchange and size exclusion chromatography to obtain ferritin nanoparticles SpyTag-Ferritin; (4) Based on the SpyCatcher-SpyTag system, the recombinant antigen protein-SpyCatcher is loaded on the surface of ferritin nanoparticles SpyTag-Ferritin to obtain recombinant nanoparticle protein.

2. The method according to claim 1, characterized in that The recombinant antigen protein-SpyCatcher is at least one of GP3m-spycatcher, GP4m-spycatcher and GP5m-spycatcher; The GP3m-spycatcher has an amino acid sequence as shown in SEQ ID NO.1; The GP4m-spycatcher has an amino acid sequence as shown in SEQ ID NO.3; The GP5m-spycatcher has an amino acid sequence as shown in SEQ ID NO.5; The ferritin nanoparticle SpyTag-Ferritin has an amino acid sequence as shown in SEQ ID NO.7; The obtained recombinant nanoparticle protein is at least one of GP3m-Fe, GP4m-Fe and GP5m-Fe.

3. The method according to claim 1, characterized in that In step (4), the ferritin nanoparticles SpyTag-Ferritin and the recombinant antigen protein-SpyCatcher are mixed at a molar ratio of 1:0.5-1:3 and then self-assembled in vitro to obtain the recombinant nanoparticle protein.

4. The recombinant nanoparticle protein prepared by the method described in any one of claims 1 to 3.

5. A recombinant nanoparticle protein, characterized in that: The recombinant nanoparticle protein comprises: (I) a recombinant antigen protein-SpyCatcher, and (II) a ferritin nanoparticle SpyTag-Ferritin; the recombinant antigen protein-SpyCatcher is loaded on the surface of the ferritin nanoparticle SpyTag-Ferritin through a SpyCatcher-SpyTag system; The recombinant antigen protein-SpyCatcher is at least one of GP3m-spycatcher, GP4m-spycatcher and GP5m-spycatcher; The GP3m-spycatcher has an amino acid sequence as shown in SEQ ID NO.1; The GP4m-spycatcher has an amino acid sequence as shown in SEQ ID NO.3; The GP5m-spycatcher has an amino acid sequence as shown in SEQ ID NO.5; The ferritin nanoparticles have an amino acid sequence as shown in SEQ ID NO.7; The obtained recombinant nanoparticle protein is at least one of GP3m-Fe, GP4m-Fe and GP5m-Fe.

6. The recombinant nanoparticle protein according to claim 5, characterized in that The ferritin nanoparticle SpyTag-Ferritin and the recombinant antigen protein-SpyCatcher are mixed in a molar ratio of 1:0.5-1:3 and then self-assembled in vitro to obtain the recombinant nanoparticle protein.

7. Use of the recombinant nanoparticle protein according to any one of claims 4 to 6 in the preparation of a recombinant nanoparticle vaccine for porcine reproductive and respiratory syndrome virus.

8. A porcine reproductive and respiratory syndrome virus recombinant nanoparticle vaccine, characterized in that: Comprising the recombinant nanoparticle protein described in any one of claims 4-6.

9. The porcine reproductive and respiratory syndrome virus recombinant nanoparticle vaccine according to claim 8, characterized in that: The recombinant nanoparticle protein is combined with an aluminum gel adjuvant.

10. The porcine reproductive and respiratory syndrome virus recombinant nanoparticle vaccine according to claim 8 or 9, characterized in that: The recombinant nanoparticle protein is at least one of GP3m-Fe, GP4m-Fe and GP5m-Fe.

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