Preparation of O-type foot-and-mouth disease virus recombinant protein and its nanoparticle vaccine
Patent Information
- Application Number
- CN202610872957.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-16
- Publication Date
- 2026-09-15
Smart Images
Figure CN122749698A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, specifically to the preparation of a recombinant protein of type O foot-and-mouth disease virus and its nanoparticle vaccine. Background Technology
[0002] Foot-and-mouth disease (FMD) is a major, highly contagious, and deadly infectious disease affecting cloven-hoofed animals. It is characterized by rapid spread, wide distribution, and difficulty in control, severely impacting global livestock development and causing enormous economic losses. Vaccination remains a crucial means of FMD control, with commercially available inactivated vaccines being the most widely used. However, with the rapid development of genetic engineering technology, significant progress has been made in the development of novel subunit vaccines, including recombinant protein vaccines, VLPs, marker vaccines, feedable vaccines, and epitope vaccines. Among these, epitope vaccines can tandem multiple dominant viral epitopes, covering antigens from different circulating strains and even different serotypes, showing great potential in the development of broad-spectrum multivalent vaccines and becoming a hot topic in novel vaccine research. However, traditional epitope vaccines generally suffer from weak immunogenicity, easy degradation in vivo, low delivery efficiency, and difficulty in inducing a strong immune response. Therefore, constructing efficient and suitable antigen delivery systems has become a key strategy for improving epitope vaccines.
[0003] Enc nanoparticles are protein nanocages discovered in bacteria in recent years that can self-assemble and form stable icosahedral structures. Larger than ferritin and possessing excellent thermal stability, they have been widely used in drug delivery, antigen display, and nanoreactor construction. With their stable spatial structure and efficient surface display capabilities, Enc nanoparticles provide an ideal antigen delivery and display platform for the development of FMD multi-epitope vaccines. Summary of the Invention
[0004] This invention targets the prevalent porcine type O FMDV strain MYA98 / BY / 2010 in my country, precisely designing and constructing the multi-epitope recombinant protein F9E. Furthermore, utilizing the SpyTag / SpyCatcher system, it is displayed on the surface of Enc self-assembled nanoparticles, thereby preparing a multi-epitope nanoparticle vaccine. The aim is to provide a theoretical basis, technical pathway, and candidate vaccine materials for developing safer, more efficient, and stable FMD multi-epitope nanoparticle vaccines. Specifically, it includes the following: In a first aspect, the present invention provides a recombinant FMDV protein, the amino acid sequence of which is shown in SEQ ID NO.1.
[0005] Preferably, the N-terminus of the FMDV recombinant protein is fused with a SpyTag tag, and the amino acid sequence of the FMDV recombinant protein is shown in SEQ ID NO.2.
[0006] In a second aspect, the present invention provides the application of the FMDV recombinant protein described in the first aspect above in the preparation of FMD vaccines.
[0007] Thirdly, the present invention provides a nanoparticle obtained by coupling the FMDV recombinant protein described in the first aspect with an Encapsulin protein; the amino acid sequence of the Encapsulin protein is shown in SEQ ID NO. 3.
[0008] Preferably, the C-terminus of the Encapsulin protein is fused with a SpyCatcher tag, the amino acid sequence of which is shown in SEQ ID NO.4.
[0009] Preferably, the molar ratio of the FMDV recombinant protein to the Encapsulin protein is 1:1.
[0010] Fourthly, the present invention provides the application of the nanoparticles described in the third aspect above in the preparation of FMD nanoparticle vaccines.
[0011] Fifthly, the present invention provides a method for preparing the nanoparticles described in the third aspect above, the method comprising the following steps: (1) Synthesize the gene fragment encoding the FMDV recombinant protein described in the first aspect above and the gene fragment encoding the Encapsulin protein; (2) The gene fragment encoding the FMDV recombinant protein described in the first aspect above and the gene fragment encoding the Encapsulin protein are respectively ligated into a prokaryotic expression vector to construct a recombinant expression plasmid; (3) The recombinant expression plasmid was transformed into Escherichia coli, and the FMDV recombinant protein and Encapsulin protein described in the first aspect were obtained after induction expression and purification. (4) The FMDV recombinant protein described in the first aspect above is mixed with Encapsulin protein at a molar ratio of 1:1 and coupled to obtain nanoparticles Enc-F9E.
[0012] Preferably, the gene fragment encoding the FMDV recombinant protein described in the first aspect is as shown in SEQ ID NO.5, and the gene fragment encoding the Encapsulin protein is as shown in SEQ ID NO.7.
[0013] Preferably, the gene fragment encoding the FMDV recombinant protein described in the first aspect is shown in SEQ ID NO.6, and the gene fragment encoding the Encapsulin protein is shown in SEQ ID NO.8.
[0014] In a sixth aspect, the present invention provides a nanoparticle vaccine for preventing FMDV infection, the nanoparticle vaccine comprising the FMDV recombinant protein described in the first aspect above, or the nanoparticles described in the third aspect above.
[0015] The beneficial effects of this invention are: (1) The present invention first provides an FMDV recombinant protein F9E with an amino acid sequence as shown in SEQ ID NO.1, and obtains a soluble recombinant protein F9E by co-expressing it in Escherichia coli using the molecular chaperone TF16; (2) A SpyTag tag was fused to the N-terminus of recombinant protein F9E to obtain recombinant protein ST-F9E. The insertion of the SpyTag tag did not disrupt the spatial conformation of the core antigen of recombinant protein F9E, nor did it have a significant effect on the solubility of recombinant protein F9E. A SpyCatcher tag was fused to the C-terminus of Enc protein to obtain recombinant protein Enc-SC, which can achieve efficient soluble expression in Escherichia coli. Recombinant protein ST-F9E and Enc-SC were mixed and coupled to form nanoparticles Enc-F9E. The Enc-F9E can self-assemble into structurally complete nanoparticles with uniform particle size and exhibits good storage stability. (3) Through cytotoxicity test, in vitro hemolysis test and in vivo histopathological examination of mice, it was confirmed that the Enc-F9E nanoparticles have good biosafety in both in vivo and in vitro, providing a safe and reliable basis for subsequent immunization applications; (4) The nanoparticle vaccine Enc-F9E can simultaneously induce potent humoral and cellular immune responses in mice: it can rapidly induce the body to produce high levels of neutralizing antibodies and specific IgG antibodies, activate the Th1 / Th2 mixed immune response dominated by Th2, promote germinal center response and Tfh, GC B cell proliferation, and help generate high-affinity antibodies; it can also significantly promote the proliferation of splenic lymphocytes and increase CD4. + CD8 + The proportion of T cells effectively induces the secretion of IL-4 (Th2) and IFN-γ (Th1) cytokines, thereby stimulating a cellular immune response dominated by Th1-type responses. (5) After immunizing fattening pigs with the Enc-F9E nanoparticle vaccine, the levels of specific blocking antibodies and neutralizing antibodies were induced to be no significantly different from those of the inactivated vaccine group, and the humoral immune response level was comparable to that of the inactivated vaccine. It also showed a more potent specific cellular immune response level, providing 100% protection against FMDV O / BYA98 / BY / 2010 strain challenge (5 / 5), and significantly reducing the viral load of FMDV in the blood, heart and spleen, thus possessing the potential to become a novel and highly effective FMD vaccine. Attached Figure Description
[0016] Figure 1 Construction and identification results of recombinant expression plasmid pColdII-F9E; where M represents the DNA molecular weight standard; 1 represents pColdII-F9E; 2 represents... Hin d III and Bam pColdII-F9E after HI digestion.
[0017] Figure 2 Construction and identification results of recombinant expression plasmid pColdII-ST-F9E; where M represents DNA molecular weight standard; 1 represents pColdII-ST-F9E; 2 represents... Hin d III and Bam pColdII-ST-F9E after HI enzyme digestion.
[0018] Figure 3 Construction and identification results of recombinant expression plasmid pET28a-Enc; where M represents the DNA molecular weight standard; 1 represents pET28a-Enc; 2 represents... Hind III and Nco pET28a-Enc after Ⅰ.
[0019] Figure 4 Construction and identification results of recombinant expression plasmid pET28a-Enc-SC; where M represents DNA molecular weight standard; 1 represents pET28a-Enc-SC; 2 represents... Hind III and Nco I. pET28a-Enc-SC after enzyme digestion.
[0020] Figure 5 Results of induction and identification of recombinant proteins F9E and ST-F9E; where A represents the induction and identification of recombinant protein F9E; B represents the induction and identification of recombinant protein ST-F9E; M represents the molecular weight standard of the protein; 1, 2: supernatant and precipitate after lysis of uninduced bacterial cells, respectively; 3, 4: supernatant and precipitate after lysis of bacterial cells induced by IPTG, respectively; 5, 6: supernatant and precipitate after lysis of bacterial cells induced by simultaneous IPTG and arabinose, respectively.
[0021] Figure 6 Results of induction expression identification of recombinant proteins Enc and Enc-SC; where A represents the induction expression identification of recombinant protein Enc; B represents the induction expression identification of recombinant protein Enc-SC; M represents the molecular weight standard of the protein; 1 and 2 are the supernatant and precipitate after lysis of uninduced bacterial cells, respectively; 3 and 4 are the supernatant and precipitate after lysis of induced bacterial cells, respectively.
[0022] Figure 7SDS-PAGE analysis results of purified F9E and ST-F9E recombinant proteins; where A is the purified F9E recombinant protein; B is the purified ST-F9E recombinant protein; M is the protein molecular weight standard; 1 is the precipitate after cell disruption; 2 is the supernatant after cell disruption; 3 is the flow-through sample after loading; 4 is the eluted sample in buffer containing 80 mM imidazole; 5 is the eluted sample in buffer containing 160 mM imidazole; 6 is the eluted sample in buffer containing 300 mM imidazole; 7 is the eluted sample in buffer containing 500 mM imidazole.
[0023] Figure 8 SDS-PAGE analysis results of purified Enc and Enc-SC recombinant proteins; where A is the purified Enc recombinant protein; B is the purified Enc-SC recombinant protein; M is the protein molecular weight standard; 1 is the precipitate after cell disruption; 2 is the centrifuged precipitate after treatment at 80℃; 3 is the supernatant after centrifugation after treatment at 80℃; 4 is flow-through 1; 5 is flow-through 2; 6 is flow-through 3.
[0024] Figure 9 SDS-PAGE identification results of the optimized conjugation ratio of Enc-SC to ST-F9E recombinant protein; where A is the SDS-PAGE identification image after conjugation of Enc-SC and ST-F9E; M is the protein molecular weight standard; 1 is the conjugation ratio of 1:0.5; 2 is the conjugation ratio of 1:1; 3 is the conjugation ratio of 1:1.5; 4 is the conjugation ratio of 1:2; 5 is the conjugation ratio of 1:2.5; 6 is the conjugation ratio of 1:3; 7 is the ST-F9E recombinant protein; 8 is the Enc-SC recombinant protein; B is the SDS-PAGE identification image of the conjugated product after purification by Capto Core 400 chromatography column; M is the protein molecular weight standard; 1 is the ST-F9E protein; 2 is the Enc-SC protein; 3 is the Enc-F9E protein.
[0025] Figure 10 Western blot (WB) results of recombinant proteins; where M is the molecular weight standard of the protein; A-1 is the WB analysis of ST-F9E recombinant protein with His antibody; A-2 is the WB analysis of Enc-SC recombinant protein with His antibody; A-3 is the WB analysis of Enc-F9E recombinant protein with His antibody; B-1 is the WB analysis of ST-F9E recombinant protein with positive serum; B-2 is the WB analysis of Enc-SC recombinant protein with positive serum; B-3 is the WB analysis of Enc-F9E recombinant protein with positive serum.
[0026] Figure 11 Results of transmission electron microscopy (TEM) analysis of recombinant nanoparticle proteins.
[0027] Figure 12 Results of dynamic light scattering (DLS) analysis of recombinant nanoparticle proteins.
[0028] Figure 13 The results of the storage stability analysis of Enc-F9E nanoparticles; where A is the SDS-PAGE identification of Enc-F9E nanoparticle protein at different storage temperatures; and B is the particle size detection of Enc-F9E nanoparticle protein at different storage temperatures.
[0029] Figure 14 Results of hemolytic activity analysis of Enc-F9E nanoparticles; where A represents porcine erythrocytes and B represents mouse erythrocytes.
[0030] Figure 15 Cytotoxicity analysis results of Enc-F9E nanoparticles.
[0031] Figure 16 Results of tissue pathological toxicity analysis of Enc-F9E nanoparticles.
[0032] Figure 17 Measurement of anti-Enc antibody levels in mouse immune serum.
[0033] Figure 18 Detection of IgG antibodies in mouse immune serum.
[0034] Figure 19 Neutralizing antibody titers in mouse immune serum.
[0035] Figure 20 Body temperature detection in pigs after immunization.
[0036] Figure 21 Detection of protective blocking antibodies in porcine serum.
[0037] Figure 22 Detection of neutralizing antibodies in porcine serum.
[0038] Figure 23 Viral load detection in immunized pigs after challenge with the virus.
[0039] Figure 24 IHC analysis of the heart and spleen of immunized pigs after viral challenge. Detailed Implementation
[0040] The above-described solution will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The implementation conditions used in the embodiments may be further adjusted according to the conditions of specific manufacturers, and the implementation conditions not specified are generally those in routine experiments.
[0041] The experiments described in the following examples obtained biosafety clearance and foot-and-mouth disease laboratory activity permits.
[0042] Unless otherwise specified, the materials and methods used in the following examples are commercially available or conventional methods.
[0043] BALB / c mice (6-9 weeks old) were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; BHK-21 cells, PK-15 cells, and FMDV (O / MYA98 / BY / 2010 strain) were preserved by the State Key Laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; the inactivated vaccine was a bivalent inactivated vaccine for porcine type O and type A FMD (Re-O / MYA98 / JSCZ / 2013 strain + Re-A / WH09 strain), purchased from Zhongnong Weite Biotechnology Co., Ltd.; the O-type FMD liquid-phase blocking ELISA antibody detection kit was purchased from Lanzhou Veterinary Research Biotechnology Co., Ltd.
[0044] Example 1 Soluble expression of recombinant protein 1. Design and construction of recombinant expression plasmids for F9E, ST-F9E, Enc, and Enc-SC Based on the gene sequence of type O FMDV strain MYA98 / BY / 2010 (accession number: JN998085.1) obtained from the NCBI database, a recombinant protein F9E with the amino acid sequence shown in SEQ ID NO.1 was designed. Furthermore, a SpyTag tag was fused to the N-terminus of F9E to obtain the recombinant protein ST-F9E with the amino acid sequence shown in SEQ ID NO.2. The secondary structure, hydrophilicity, antigenic index, and surface accessibility of the designed recombinant protein were predicted using the Protean module of DNAStar 7.1 software. Simultaneously, online bioinformatics tools such as SignalP-6.0, TMHMM, and Protein-Sol were used to analyze and evaluate its signal peptide, transmembrane domain, and soluble expression potential. The results showed that the insertion of the SpyTag tag had little effect on the physicochemical properties of the F9E protein, did not disrupt the spatial conformation of the F9E protein core antigen, and had no significant effect on the solubility of the F9E recombinant protein, demonstrating that both F9E and ST-F9E recombinant proteins have the potential for soluble expression in Escherichia coli.
[0045] Subsequently, the codon-optimized F9E and ST-F9E gene sequences (as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively) were cloned into the pColdII expression vector to construct recombinant plasmids pColdII-F9E and pColdII-ST-F9E, respectively. Simultaneously, the Enc protein sequence (amino acid sequence as shown in SEQ ID NO.3, gene sequence as shown in SEQ ID NO.7) was cloned into the pET28a expression vector to construct the recombinant plasmid pET28a-Enc. A SpyCatcher tag (amino acid sequence as shown in SEQ ID NO.4, gene sequence as shown in SEQ ID NO.8) was further fused to the C-terminus of the Enc protein and cloned into the pET28a vector to obtain the recombinant plasmid pET28a-Enc-SC. The obtained recombinant plasmids were subjected to double enzyme digestion and sequencing to verify the correctness of their construction. The results are as follows: Figures 1 to 4 As shown, the recombinant protein sequence has no mutations or frameshifts, and the target band size is consistent with the theoretical value. Sequencing verification confirmed that the above recombinant expression plasmid was successfully constructed.
[0046] 2. Induced expression of recombinant proteins Place 100 μL of competent *E. coli* cells BL21 on ice for 20 min to allow them to thaw slowly. Under aseptic conditions, pipette 2 μL of recombinant plasmid into the BL21 and incubate on ice for 20-30 min. Then, transfer the centrifuge tube containing the plasmid and competent cell mixture to a float and heat-shock it in a 42°C water bath for 60-90 s. Immediately afterward, place it on ice for 2-5 min. In a laminar flow hood, add an appropriate amount of liquid culture medium to the centrifuge tube and incubate at 37°C and 220 rpm for 1 h. Finally, pipette 200 μL of the bacterial culture into LB solid medium (containing the appropriate antibiotic), gently spread the culture using a disposable spreader, and invert the petri dish in a 37°C incubator for overnight incubation.
[0047] Single colonies of *E. coli* (BL21) containing the pTF16 plasmid were picked and incubated overnight at 37°C and 220 rpm in 5 mL LB broth. The next day, they were transferred to fresh LB broth and incubated for further growth. When the OD... 600The culture was stopped when the pH value was approximately 0.8. The bacterial cells were collected, washed twice with PBS, and then placed in a clean bench with pre-chilled 100 mmol / mL CaCl2 solution. After incubating on ice for 20 min, the cells were centrifuged at 6000 rpm for 10 min at 4°C. The supernatant was discarded, and the cells were resuspended in 30 mL of CaCl2 solution. After incubating on ice for 30 min, the cells were centrifuged at 6000 rpm for 20 min at 4°C. The supernatant was discarded again, and the cells were resuspended in 5 mL of ice-cold CaCl2 solution. The solution was aliquoted into 2 mL sterile centrifuge tubes (100 µL each) and stored at -80°C for later use. The prepared competent bacteria were named BL21-pTF16.
[0048] Recombinant plasmids pColdII-F9E and pColdII-ST-F9E were transformed into *E. coli* BL21-pTF16. Simultaneously, plasmids pET28a-Enc and pET28a-Enc-SC were transformed into *E. coli* BL21. Single positive colonies were picked and cultured overnight at 37°C and 220 rpm in 5 mL of LB broth. The culture was then inoculated at a 1:1000 ratio into 50 mL of LB broth and cultured at 37°C and 220 rpm for 5 h. IPTG was then added to a final concentration of 0.4 mM, and the culture was induced overnight at 25°C. For *E. coli* BL21-pTF16 containing the pTF16 plasmid, arabinose was added simultaneously with IPTG to induce the expression of the molecular chaperone protein, at a final concentration of 0.75 g / L. Centrifuge at 5000 r / min for 15 min at 4℃ to collect bacterial cells, discard the supernatant, and resuspend the precipitate in 30 mL of Binding Buffer (this buffer is prepared in 1 L system containing 8 g NaCl, 0.2 g KCl, and Na2HPO4). 1.44 g of 12H2O and KH2PO4 Add 0.24 g of 2H₂O and 1.36 g of imidazole, and adjust the pH to 8.0 after all solutes have fully dissolved. Resuspend the bacterial culture to ensure complete dissolution. Pour the resuspended bacterial culture into a 100 mL glass beaker, place it on ice, and sonicate it for 10 min (5 s for 5 s, 5 s for 2 seconds) to ensure complete lysis. Centrifuge the lysed bacterial culture at 13000 r / min for 30 min at 4 °C, collect the supernatant, and lyse the precipitate with 300 μL of 8 mol / L urea at room temperature for 30 min. Perform SDS-PAGE analysis on appropriate amounts of the supernatant and precipitate to identify the results.
[0049] After induction of F9E and ST-F9E recombinant protein expression, the SDS-PAGE identification results are as follows: Figure 5As shown: Without IPTG induction, neither recombinant protein was expressed (lanes 1 and 2). With IPTG induction alone, trace amounts of F9E and ST-F9E proteins were expressed in the supernatant, invisible to the naked eye (lane 3). When induced simultaneously by arabinose and IPTG, a clear band of the molecular chaperone TF16 protein (approximately 55 kDa) and bands of F9E and ST-F9E proteins (approximately 26 kDa and 28 kDa, respectively) were visible in the lysed bacterial supernatant (lane 5), consistent with expected sizes. These results indicate that the two recombinant proteins are solublely expressed in *E. coli*, and their expression levels are significantly enhanced with the synergistic effect of the molecular chaperone.
[0050] After induction of Enc and Enc-SC recombinant protein expression, the SDS-PAGE identification results are as follows: Figure 6 As shown, both Enc and Enc-SC recombinant proteins were expressed in *E. coli* BL21 cells (supernatant and precipitate) regardless of IPTG induction, with band sizes of approximately 35 kDa and 55 kDa, respectively. However, compared to uninduced cells, the expression in the supernatant was significantly increased in induced cells. These results indicate that Enc and Enc-SC recombinant proteins can achieve efficient soluble expression in *E. coli*.
[0051] 3. Chromatographic purification and identification of recombinant proteins (1) F9E and ST-F9E recombinant proteins were prepared using Ni column affinity chromatography. The specific methods and steps are as follows: Cell disruption: Resuspend the collected bacterial cells in Binding Buffer and add 0.01% totipotent nuclease to remove most of the nucleic acid from the bacterial culture. After the cells are completely dissolved, homogenize them using a homogenizer (pressure 700-1000 bar). Centrifuge the disrupted bacterial culture at 4°C and 17000 r / min for 2 h. After centrifugation, collect the supernatant and filter it through a 0.45 μm filter to prevent clogging of the chromatography column.
[0052] Column equilibration: After connecting the column to the protein chromatography system, rinse with 5 column volumes of deionized water, and then equilibrate with 4 column volumes of Binding Buffer.
[0053] Protein loading: Load the filtered supernatant at a flow rate of 10 mL / min, ensuring that the column pressure does not exceed 0.2 Mbar.
[0054] Protein elution: After sample loading, the chromatography column was washed with 5 column volumes of deionized water. Then, the column was washed with gradient washes containing 2 column volumes of elution buffers containing 40 mM imidazole, 80 mM imidazole, 160 mM imidazole, 300 mM imidazole and 500 mM imidazole, respectively. The protein was collected and the results were identified by SDS-PAGE.
[0055] Concentration and desalting: F9E and ST-F9E recombinant proteins were concentrated and desalted using an ultrafiltration membrane with a pore size of 10 kDa, and the results were identified by SDS-PAGE.
[0056] SDS-PAGE identification results are as follows: Figure 7 As shown, the F9E recombinant protein obtained a high-purity target band with a molecular weight of approximately 26 kDa in a buffer containing 300 mM and 500 mM imidazole, consistent with the theoretical value. The ST-F9E recombinant protein obtained a high-purity target band with a molecular weight of approximately 28 kDa in a buffer containing 160 mM and 300 mM imidazole, also consistent with the theoretical value. These results demonstrate that the F9E and ST-F9E recombinant proteins were successfully prepared in this application.
[0057] (2) Enc and Enc-SC nanoparticle proteins were prepared using Capto Core 400 multimode binding chromatography medium. The specific methods and steps are as follows: Cell disruption: The collected bacterial cells were resuspended in PBS (imidazolium-free) until completely dissolved, and then homogenized. The disrupted bacterial solution was centrifuged at 17,000 r / min for 2 h at 4°C, and the supernatant was collected.
[0058] Boiling to remove impurities: Take the supernatant after centrifugation, boil at 80℃ for 30 min, centrifuge at 17000 r / min for 1 h at 4℃, collect the supernatant, boil at 80℃ for 30 min again, centrifuge at 17000 r / min for 1 h, and collect the supernatant.
[0059] Column equilibration: Rinse the column with 5 column volumes of deionized water, then equilibrate with 5 column volumes of desalting buffer (PBS, pH 8.0).
[0060] Sample loading and collection: The filtered supernatant was loaded at a flow rate of 10 mL / min, and the flow-through samples were collected in segments. The results were identified by SDS-PAGE.
[0061] Concentration: Enc and Enc-SC recombinant proteins were concentrated using an ultrafiltration membrane with a pore size of 10 kDa.
[0062] SDS-PAGE identification results as follows Figure 8As shown, Enc and Enc-SC proteins of different purities were purified from the flow-through buffer, with molecular weights of approximately 35 kDa (Enc) and 55 kDa (Enc-SC), respectively, consistent with theoretical predictions. These results demonstrate the successful preparation of recombinant Enc and Enc-SC proteins in this application. Finally, the target band with higher purity in lane 4 was selected for subsequent experiments.
[0063] In summary, this embodiment successfully constructed four prokaryotic expression vectors: pColdII-F9E, pColdII-ST-F9E, pET28a-Enc, and pET28a-Enc-SC. Using the molecular chaperone TF16, they were co-expressed in *E. coli*, and combined with nickel column affinity chromatography, soluble recombinant proteins F9E and ST-F9E were obtained. Enc and Enc-SC nanoparticle proteins were then purified using Capto Core 400 multimode binding chromatography.
[0064] Example 2 Preparation of Enc-F9E nanoparticles 1. Conjugation of ST-F9E recombinant protein with Enc-SC nanoparticle protein Enc-SC nanoparticle protein and ST-F9E recombinant protein were mixed at molar ratios of 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, and 1:3, and ligated overnight at 4°C and 100 r / min. The ligation efficiency was determined by SDS-PAGE to identify the optimal coupling ratio of Enc-SC to ST-F9E, and the resulting coupled protein was named Enc-F9E. Protein concentration was simultaneously determined using a BCA assay kit.
[0065] The results were obtained by SDS-PAGE electrophoresis. Figure 9 As shown in Figure A, when the molar ratio of Enc-SC to ST-F9E recombinant protein is 1:1, the concentration of the target band (Enc-F9E) of the conjugated product is the highest, indicating that the conjugation efficiency is the highest at this time, which is the optimal reaction ratio.
[0066] To further remove uncoupled free protein and other contaminating proteins and obtain a single-band Enc-F9E recombinant protein, the above coupling product (1:1) was further purified using a Capto Core 400 chromatography column. The purification results are as follows. Figure 9 As shown in lane B (3), a target band of approximately 83 kDa is visible on the gel, consistent with the expected theoretical value. Most of the free protein ST-F9E and other contaminating proteins were removed. This result demonstrates the successful preparation of highly pure Enc-F9E nanoparticle recombinant protein.
[0067] The concentrations of recombinant proteins F9E, ST-F9E, Enc-SC, and Enc-F9E were determined using a BCA kit. A standard curve was established and plotted using bovine serum albumin (BSA) as a standard, yielding a linear regression equation of 0.4480x + 0.1142 with a correlation coefficient R² = 0.9978. This indicates a good linear relationship between absorbance at 592 nm and protein concentration within the range of 0–1.0 mg / mL. Based on the standard curve, the concentrations of recombinant proteins F9E, ST-F9E, Enc-SC, and Enc-F9E were calculated to be 0.45 mg / mL, 0.68 mg / mL, 0.78 mg / mL, and 0.70 mg / mL, respectively. These results indicate that all four recombinant proteins have high concentrations sufficient for subsequent experiments.
[0068] WB results are as follows Figure 10 As shown, the purified ST-F9E, Enc-SC recombinant proteins, and the conjugated Enc-F9E nanoparticles all specifically bound to mouse anti-histidine (His-tag) monoclonal antibody, with single target bands observed at molecular weights of approximately 28 kDa, 55 kDa, and 83 kDa, indicating the successful preparation of three recombinant antigen proteins with intact His tags. Simultaneously, both the ST-F9E protein and the Enc-F9E multi-epitope nanoparticles specifically reacted with FMDV-positive serum, exhibiting the expected specific bands, while the Enc-SC group showed no obvious signal and did not react with FMDV serum. This indicates that the specific reaction exhibited by Enc-F9E originates from the F9E protein displayed on its surface, rather than the non-specific binding of the Enc-SC vector itself. These results demonstrate that the F9E protein has been successfully displayed on the surface of Enc nanoparticles using the SpyTag / SpyCatcher system, and the constructed Enc-F9E nanoparticles possess good immunoreactivity.
[0069] 2. TEM and DLS characterization of Enc-F9E nanoparticles The assembly status and particle size of Enc, Enc-SC, and Enc-F9E granule proteins were observed and measured using TEM and DLS, respectively. The specific steps are as follows: TEM observation: 4 μL of 0.1 mg / mL recombinant protein was applied to the front side of a copper grid. After adsorption for 20 s, the surface was blotted dry with filter paper. Next, 4 μL of 2% phosphotungstic acid was added, and the surface was negatively stained for 20 s. The surface was then blotted dry again with filter paper. After the copper grid had completely dried at room temperature, TEM observation was performed.
[0070] TEM results as follows Figure 11As shown, ST-F9E, Enc-SC, and Enc-F9E nanoparticles all achieved efficient self-assembly. Under electron microscopy, a large number of structurally complete, uniformly sized, and clearly defined particles were observed, without obvious aggregation or agglomeration. This confirms that the three types of nanoparticles, Enc, Enc-SC, and Enc-F9E, have good dispersibility and assembly stability. It also confirms that the self-assembly process of Enc nanoparticles is not interfered with by the SpyTag / SpyCatcher system, whether coupled with the SpyCatcher tag or the SC-F9E recombinant protein, exhibiting good assembly characteristics.
[0071] DLS assay: Add recombinant protein to a cuvette at a final concentration of 0.1 mg / mL and perform DLS assay according to the instructions of the Zetasizer Nano nanoparticle size potentiometric analyzer.
[0072] DLS analysis results are as follows: Figure 12 As shown, the average particle size of the natural Enc nanoparticles is 28.62 nm. The Enc-SC nanoparticles with the SpyCatcher structure increase in size to 38.74 nm, while the Enc-F9E nanoparticles, after conjugation with the recombinant protein ST-F9E, further increase in size to 44.11 nm. These results demonstrate that the insertion of the SpyCatcher tag increases the particle size of Enc nanoparticles and further validate that the SpyTag / SpyCatcher system achieves precise conjugation of multi-epitope recombinant proteins with Enc nanoparticles.
[0073] 3. Storage stability assessment of Enc-F9E nanoparticles Five mL of Enc-F9E nanoparticles were stored at 4°C, -20°C, and -80°C for six months. Subsequently, their purity and degradation were analyzed by SDS-PAGE, and particle size changes were detected using a nanoparticle size potentiometric analyzer. The storage stability of the Enc-F9E nanoparticles was comprehensively evaluated.
[0074] The results are as follows Figure 13 As shown, no obvious impurities were generated in the Enc-F9E particle protein at the three storage temperatures; the bands were clear and uniform, indicating that the Enc-F9E protein did not undergo significant degradation. The particle size of the Enc-F9E nanoparticles also did not change significantly when stored at 4℃, -20℃, and -80℃. These results demonstrate that Enc-F9E nanoparticles possess good storage stability.
[0075] 4. Safety evaluation of Enc-F9E nanoparticles (1) Hemolysis test of Enc-F9E nanoparticles Fresh anticoagulated blood was collected from healthy mice and pigs, diluted 4-fold with physiological saline, centrifuged at 4000 r / min for 4 min, the supernatant was discarded, and the mixture was washed repeatedly 3-5 times until the supernatant was clear and transparent. Red blood cells were collected and resuspended to a hematocrit of 2%. Three groups were set up: Enc-F9E nanoparticle group, negative control (PBS), and positive control (1% Triton X-100). The Enc-F9E nanoparticle group was supplemented with Enc-F9E nanoparticle protein at final concentrations of 12.5, 25, 50, 100, 200, and 400 μg / mL. The negative control group received an equal volume of PBS, and the positive control group received an equal volume of 1% Triton X-100. The mixture was incubated in a 37℃ water bath for 1 h, centrifuged at 4000 r / min for 4 min, and the color change of the supernatant was observed. 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 450 nm using a microplate reader. Hemolysis rate (%) = (Enc-F9E nanoparticle group OD) 450 - Negative control group OD 450 ) / (Positive control group OD 450 - Negative control group OD 450 ()×100%, a hemolysis rate of less than 5% is considered to be without hemolysis.
[0076] The results are as follows Figure 14 As shown, within the concentration range of 12.5-400 μg / mL, compared with the negative control group, the OD of the Enc-F9E nanoparticle group was significantly lower. 450 There was no significant difference (P>0.05). Red blood cells at the bottom of the centrifuge tubes were intact, and the supernatant was clear and colorless, with no hemolysis observed. The positive control group showed a significant hemolytic reaction, with an OD... 450 The concentrations were significantly higher than those of the Enc-F9E nanoparticle group (P<0.001), and the supernatant in the centrifuge tubes was red. These results indicate that the Enc-F9E nanoparticle protein had no significant hemolytic activity against mouse and pig erythrocytes, suggesting that this nanoparticle protein has good blood compatibility in vitro.
[0077] (2) Cytotoxicity assay of Enc-F9E nanoparticles BHK-21 (hamster kidney cells) and PK-15 (pig kidney epithelial cells) cells were cultured at 1×10⁻⁶ cells per cell line. 5Cells were seeded at a density of 100 cells / well in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. Three groups were set up: Enc-F9E nanoparticle group, negative control group, and blank group. The Enc-F9E nanoparticle group was treated with diluted Enc-F9E nanoparticle protein to final concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg / mL. The negative control group received complete culture medium, and the blank group received no treatment (cell-free). After 48 h of further incubation, 10 μL of CCK-8 solution was added to each well, and after incubation for 2–4 h, the OD was measured using a microplate reader. 450 Value. Cell viability (%) = (Enc-F9E nanoparticle group OD) 450 - Blank group OD 450 ) / (Negative control group OD 450 - Blank group OD 450 ) × 100%.
[0078] The results are as follows Figure 15 As shown, within a concentration range of 62.5-200 μg / mL, the cell viability of the Enc-F9E nanoparticle group remained above 90%, with no significant difference compared to the negative control group (P>0.05). This result indicates that the Enc-F9E nanoparticles did not exhibit significant cytotoxicity against either BHK21 or PK15 cells, suggesting good cell compatibility in vitro.
[0079] (3) Histopathological evaluation of Enc-F9E nanoparticles Six healthy BALB / c mice were randomly divided into an Enc-F9E group and a PBS group, with three mice in each group. Each mouse in the Enc-F9E group was immunized with 100 μg / 200 μL Enc-F9E protein, while each mouse in the PBS group was immunized with 200 μL PBS buffer. The mice were observed for 14 days post-immunization, and their mental status and clinical manifestations were recorded. After 14 days, the mice were euthanized, and the heart, liver, spleen, lungs, and kidneys were dissected and collected for gross morphology observation. The tissues were fixed in 4% paraformaldehyde for one week, followed by routine HE staining, and the organ tissue structure and cell morphology of each group were observed under a microscope.
[0080] Results of HE staining under an optical microscope: Figure 16 As shown, no obvious pathological damage such as cell degeneration, necrosis, hemorrhage, or inflammatory infiltration was observed in any organ of mice in the Enc-F9E group and the PBS control group. The overall tissue structure remained intact, and the cell morphology was normal. This result indicates that Enc-F9E nanoparticles have no significant histopathological toxicity to the major organs of mice.
[0081] In summary, this embodiment, based on SpyTag / SpyCatcher conjugation technology, determined the optimal conjugation ratio (1:1) of ST-F9E to Enc-SC. Western blot validation showed that Enc-F9E specifically binds to FMDV-positive serum, exhibiting good reactivity. TEM and DLS characterization results confirmed that Enc-F9E can self-assemble into structurally complete, uniformly sized nanoparticles and exhibit good storage stability. Cytotoxicity assays, in vitro hemolysis assays, and in vivo histopathological examination in mice confirmed that Enc-F9E nanoparticles have good safety both in vitro and in vivo, providing a safe and reliable basis for subsequent immunization applications.
[0082] Example 3: Immunogenicity evaluation of Enc-F9E nanoparticle vaccine in mice 1. Evaluation of immunogenicity in mice Female BALB / c mice aged 6-9 weeks were randomly divided into four groups: PBS group, Enc-SC group, ST-F9E group, and Enc-F9E group, with 6 mice in each group. After acclimatization for 3 days, recombinant protein and ISA201 adjuvant were emulsified at a mass ratio of 1:1, and primary and booster immunizations were administered on day 0 and day 21, respectively. Immunization was performed subcutaneously, with injection doses of 40 μg, 20 μg, and 60 μg in the Enc-SC, ST-F9E, and Enc-F9E groups, respectively. The PBS immunization group served as a negative control, and blood samples were collected from the orbital venous plexus at days 7, 14, 21, 28, 35, and 42 for antibody assay.
[0083] (1) Detection of mouse serum specific IgG antibody levels To investigate whether the Enc nanoparticle carrier itself can induce mice to produce specific antibodies against Enc, this example used an indirect ELISA method to measure the level of specific antibodies (coated with Enc protein) in mouse serum on day 35 post-immunization. The results are as follows: Figure 17 As shown, the PBS and ST-F9E groups did not produce specific antibodies against the Enc carrier, while the Enc-SC and Enc-F9E groups both produced high levels of specific antibodies (P<0.001). This result indicates that the Enc nanoparticles themselves have good immunoreactivity and exhibit a certain adjuvant effect.
[0084] To evaluate the induction of specific IgG antibodies against F9E by Enc-F9E nanoparticle protein, the levels of IgG-specific antibodies in mouse serum at 0, 7, 14, 21, 28, 35, and 42 days after immunization (F9E protein as the coating antigen) were measured using an indirect ELISA method. Results are as follows: Figure 18As shown, compared with the PBS group, both the ST-F9E and Enc-F9E groups induced mice to produce specific IgG antibodies against the F9E protein. With increasing immunization time, the antibody levels in both groups gradually increased, and the antibody level in the Enc-F9E group was consistently higher than that in the ST-F9E group, with a highly significant difference (P < 0.001). Further comparison with the Enc-SC group revealed a highly significant increase in the antibody levels induced by the ST-F9E and Enc-F9E proteins (P < 0.001), indicating that the Enc-SC group did not produce an antibody response against the F9E protein. These results demonstrate that Enc-F9E nanoparticles can effectively activate the mouse immune system, inducing the production of high levels of specific IgG antibodies against the F9E protein.
[0085] (2) Detection of neutralizing antibodies in mouse serum Serum neutralizing antibody levels are considered a key indicator for assessing the immunoprotective efficacy of FMD vaccines. To further evaluate the neutralizing protective capacity of immunized mice, serum samples were collected from mice 21 days after the first immunization and 14 days after the second immunization (35 days post-total immunization) for neutralizing antibody detection. Results are as follows: Figure 19 As shown, 21 days after the initial immunization, both ST-F9E and Enc-F9E proteins induced the production of neutralizing antibodies, with average Log2 neutralization titers of 3.3 and 7.33, respectively. 14 days after the second immunization, the neutralizing antibody titers in both groups further increased to 7 and 9.17 (the titer in the Enc-F9E group was approximately 4.5 times that of the ST-F9E group). Meanwhile, neither the PBS group nor the Enc-SC group produced neutralizing antibodies during the two immunizations. These results indicate that Enc-F9E can induce a significant neutralizing antibody response, providing potential resistance to type O FMDV, and that high levels of neutralizing antibodies are produced as early as 21 days after the initial immunization.
[0086] The above results indicate that the Enc-F9E nanoparticle vaccine induced a potent humoral immune response in mice, rapidly inducing high levels of neutralizing antibodies and specific IgG antibodies, and activating a Th1 / Th2 mixed immune response dominated by Th2, promoting germinal center responses and Tfh, GC, and B cell proliferation, which is conducive to the generation of high-affinity antibodies. Simultaneously, the Enc-F9E nanoparticle vaccine induced a high level of cellular immune response in mice, significantly promoting the proliferation of splenic lymphocytes and increasing CD4+. + CD8 + The proportion of T cells was increased, and the secretion of IL-4 (Th2) and IFN-γ (Th1) cytokines was effectively induced, thereby stimulating a cellular immune response dominated by Th1-type reactions.
[0087] Example 4: Evaluation of the immunoprotective efficacy of the Enc-F9E nanoparticle vaccine in pigs. 1. Preparation of Enc-F9E nanoparticle vaccine The Enc-F9E nanoparticle antigen was diluted to a concentration of 600 μg / mL using PBS buffer. It was then mixed with an equal mass of ISA 201 adjuvant and emulsified by uniform stirring at 31°C and 350 r / min. Next, it was incubated in a water bath at 20°C for 1 h to form a milky white, oil-in-water emulsion vaccine. If no stratification was observed after centrifugation, the vaccine emulsification was deemed successful and the vaccine could be used for subsequent immunization experiments.
[0088] 2. Immunization of pigs Fifteen healthy fattening pigs weighing 35–40 kg were randomly divided into three groups: an Enc-F9E immunization group, an inactivated vaccine group (IV group), and a PBS control group, with five pigs in each group and ear-tagged. Immunization was administered via intramuscular injection behind the ear, with a dose of 2 mL per pig. The total antigen dose for the Enc-7B2T immunization group was 600 μg. Body temperature was measured daily for 10 days post-immunization to observe the clinical response of the immunized pigs. Whole blood was collected at 0, 14, 21, and 28 days, and serum was separated for antibody detection. Simultaneously, anticoagulated blood was collected for peripheral blood mononuclear cell (PBMC) isolation.
[0089] The results are as follows Figure 20 As shown, pigs immunized in both the Enc-F9E group and the inactivated vaccine group experienced a slight increase in body temperature on day 1 post-immunization, which returned to normal naturally on day 3. No adverse reactions were observed at the injection site or in other parts of the body, and the immunized pigs were in good spirits. These results demonstrate that the prepared Enc-F9E nanoparticle vaccine has good safety in pigs.
[0090] 3. Detection of serum protective blocking antibodies Blood samples were collected from immunized pigs at 0, 14, 21, and 28 days, and serum was obtained by centrifugation at 6000 r / min. The titer of protective blocking antibodies in the serum was detected using a commercially available O-type LPB-ELISA antibody detection kit. Results are as follows: Figure 21 As shown, both the Enc-F9E immunization group and the IV group produced antibodies as early as 14 days post-immunization. Antibody levels gradually increased at 21 and 28 days post-immunization. Furthermore, at 21 and 28 days post-immunization, the blocking antibody levels in the IV group were slightly higher than those in the Enc-F9E group. Further analysis indicated no significant difference in the average antibody titers between the two groups (P>0.05). These results suggest that the Enc-F9E nanoparticle vaccine elicits a strong humoral immune response in pigs, and the protective blocking antibody titers it induces are comparable to those of inactivated vaccines.
[0091] 4. Serum neutralizing antibody titer determination Neutralizing antibodies were detected in cells from immunized pig serum at 0, 14, 21, and 28 days of gestation. The results are as follows: Figure 22 As shown, both the Enc-F9E and IV groups produced neutralizing antibodies 14 days post-immunization, with an initial neutralizing antibody titer of 1:8. Subsequently, with prolonged immunization time, the neutralizing antibody levels gradually increased. At 28 days, the highest neutralizing antibody titer in the IV group reached 1:256, while the highest in the Enc-F9E group reached 1:128. Simultaneously, the results also showed that the average antibody titer in the IV group was consistently slightly higher than that in the Enc-F9E immunization group during the immunization period, but the difference between the two groups was not statistically significant. These results indicate that the Enc-F9E nanoparticle vaccine elicited neutralizing antibody titers similar to those of inactivated vaccines in pigs, effectively neutralizing the FMDV O / MYA98 / BY / 2010 strain and providing effective antiviral protection.
[0092] 5. Pig challenge protection experiment Thirty days after immunization, the experimental pigs were transferred to the BSL-3 laboratory of the Lanzhou Veterinary Research Institute for challenge treatment. The Enc-F9E immunized group, IV group, and PBS control group were challenged with FMDV virus solution via intramuscular injection: each pig received 3 mL of FMDV virus solution (10... 3 PID 50 Within 10 days after the viral challenge, the body temperature of each group of pigs was measured daily, and their clinical symptoms were observed, with a focus on recording whether typical symptoms such as hoof blisters appeared.
[0093] The results are shown in Table 1. Pigs in the PBS group developed typical clinical symptoms of FMDV on day 3 post-challenge, mainly fever, accompanied by vesicular lesions, decreased appetite, and lethargy. Vesicles appeared on all four hooves and snouts of the experimental pigs (20-24#), with a clinical score of 5. In contrast, none of the 10 pigs in the Enc-F9E and IV groups showed typical clinical symptoms of FMDV; their body temperature remained within the normal range, and their appetite and mental state were normal, with a clinical score of 0. The protection rate for challenge was 100% in both groups. These results indicate that the Enc-F9E nanoparticle vaccine induces an effective humoral and cellular immune response, resisting FMDV invasion and demonstrating the same protective efficacy as the inactivated vaccine.
[0094] Table 1. Analysis of clinical symptoms and protection rates in immunized pigs after viral challenge.
[0095] Note: Clinical score (whether blisters are present on the hoof and snout): 0 points: no blisters; 1 point: blisters on the snout on one hoof; 2 points: blisters on both hooves or one hoof and snout; 3 points: blisters on three hooves or two hooves and snout; 4 points: blisters on four hooves or three hooves and snout; 5 points: blisters on all four hooves and snout.
[0096] 6. Detection of viral load in pigs On day 10 post-infection, anticoagulated blood, heart, spleen, liver, lung, and kidney tissues were collected from each group of pigs, and viral load was detected by RT-qPCR. Results are as follows: Figure 23 As shown, no significant viral copy numbers were detected in the liver, lung, and kidney tissues of any of the experimental groups, and there was no statistically significant difference between the groups (P>0.05). However, in the blood, heart, and spleen, the viral load in the PBS group was significantly higher than that in the Enc-F9E group and the inactivated vaccine group (P<0.001), and there was no significant difference between the Enc-F9E group and the inactivated vaccine group (P>0.05). These results indicate that the Enc-F9E nanoparticle vaccine has high immunogenicity, exhibits significant antiviral effects, effectively inhibits the replication and spread of FMDV in the blood, heart, and spleen, significantly reduces viral load, and thus enhances the body's ability to clear the virus.
[0097] 7. Immunohistochemistry The IHC experiment was performed by Kebo Biotechnology Co., Ltd., and the specific steps were as follows: heart and spleen tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, dewaxed, antigen retrieval, incubated with primary antibody, incubated with secondary antibody, DAB staining (3,3'-diaminobenzidine staining method), hematoxylin counterstaining, dehydrated and cleared, and finally mounted. After the sections dried, the results were observed under an optical microscope. The experimental group was considered positive for viral antigen by the presence of specific brownish-yellow staining, while the negative group showed no obvious specific staining. IHC analysis was performed on the hearts and spleens of immunized pigs. The results are as follows: Figure 24 As shown, in the heart and spleen, no obvious positive staining for viral antigen was observed in the negative control group, while a distinct brownish-yellow positive signal was observed in the viral control group (PBS), indicating that FMDV replicated extensively in the tissues after challenge. The positive signal was significantly reduced in both the Enc-F9E group and the inactivated vaccine group, and the staining intensity was similar between the two groups. These results demonstrate that the Enc-F9E nanoparticle vaccine can effectively inhibit viral replication and colonization in the heart and spleen, exhibiting good immunoprotective effects.
[0098] The above results indicate that after immunization of fattening pigs with the Enc-F9E nanoparticle vaccine, the levels of specific blocking antibodies and neutralizing antibodies induced showed a gradual increasing trend with immunization time, and there was no significant difference compared with the inactivated vaccine group, demonstrating a humoral immune response level comparable to that of the inactivated vaccine. The Enc-F9E nanoparticle vaccine can induce serum IL-4 and IFN-γ levels comparable to those of the inactivated vaccine, and can significantly enhance the proliferation capacity of porcine peripheral blood lymphocytes, the secretion levels of IL-2 and IFN-γ in PBMCs, and CD4+. + CD8 +The proportion of T lymphocytes showed a stronger level of specific cellular immune response; the Enc-F9E nanoparticle vaccine and the inactivated vaccine provided 100% protection against challenge with the FMDV O / BYA98 / BY / 2010 strain (5 / 5) and significantly reduced the viral load of FMDV in the blood, heart and spleen.
[0099] The above examples are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.
Claims
1. A recombinant FMDV protein, characterized in that, The amino acid sequence of the FMDV recombinant protein is shown in SEQ ID NO.
1.
2. The FMDV recombinant protein as described in claim 1, characterized in that, The FMDV recombinant protein has a SpyTag fused to its N-terminus, and the amino acid sequence of the FMDV recombinant protein is shown in SEQ ID NO.
2.
3. The use of the FMDV recombinant protein as described in claim 1 or 2 in the preparation of foot-and-mouth disease vaccine.
4. A nanoparticle, characterized in that, The nanoparticles are obtained by coupling the FMDV recombinant protein of claim 1 or 2 with the Encapsulin protein; the amino acid sequence of the Encapsulin protein is shown in SEQ ID NO.
3.
5. The nanoparticles as described in claim 4, characterized in that, The C-terminus of the Encapsulin protein is fused with a SpyCatcher tag, and its amino acid sequence is shown in SEQ ID NO.
4.
6. The nanoparticles as described in claim 5, characterized in that, The molar ratio of the FMDV recombinant protein to the Encapsulin protein is 1:
1.
7. The use of the nanoparticles as described in any one of claims 4-6 in the preparation of FMD nanoparticle vaccines.
8. The method for preparing nanoparticles according to any one of claims 4-6, characterized in that, The method includes the following steps: (1) Synthesize the gene fragment encoding the FMDV recombinant protein as described in claim 1 or 2 and the gene fragment encoding the Encapsulin protein; (2) The gene fragment encoding the FMDV recombinant protein as described in claim 1 or 2 and the gene fragment encoding the Encapsulin protein are respectively ligated into a prokaryotic expression vector to construct a recombinant expression plasmid; (3) Transform the recombinant expression plasmid into Escherichia coli, and obtain the FMDV recombinant protein and Encapsulin protein as described in claim 1 or 2 after induction expression and purification; (4) The FMDV recombinant protein described in claim 1 or 2 is mixed with Encapsulin protein in a molar ratio of 1:1 and coupled to obtain nanoparticles Enc-F9E.
9. The preparation method according to claim 8, characterized in that, The gene fragment encoding the FMDV recombinant protein of claim 1 or 2 is shown in SEQ ID NO. 5 or 6, and the gene fragment encoding the Encapsulin protein is shown in SEQ ID NO. 7 or 8.
10. A nanoparticle vaccine for the prevention of FMDV infection, characterized in that, The nanoparticle vaccine comprises the FMDV recombinant protein as described in claim 1 or 2, or the nanoparticles as described in any one of claims 4-6.