Method for improving assembly efficiency and heat-resistant stability of foot-and-mouth disease virus-like particles through Foldon tripolymer fusion

Abstract

The invention discloses a method for improving assembly efficiency and heat-resistant stability of foot-and-mouth disease virus-like particles through Fldon trimer fusion, which comprises the following steps: introducing a hydrophobic Fldon peptide fragment to the N end of O-type foot-and-mouth disease virus VP2 protein, seamlessly fusing the O-type foot-and-mouth disease virus VP1 gene, the O-type foot-and-mouth disease virus VP3 gene and the Fldon VP2 gene with a small ubiquitination modified protein gene respectively, and assembling the expressed fusion protein to obtain the foot-and-mouth disease virus-like particles. In the assembling process of the VLPs, the foldon peptide is self-assembled to form a trimer compound, the assembling efficiency of the VLPs is improved through the interaction force among pentamers of an auxiliary assembling precursor, and in addition, the formation of the foldon trimer compound on the inner side of the cubic axis of the VLPs is like a lock catch, so that the stability of the VLPs is enhanced. The establishment of the method is beneficial to reducing the production cost of the VLPs vaccine and promoting the development of a novel vaccine technology, and has important significance on disease control.

Description

Technical Field

[0001] The present invention relates to a method for assembling foot-and-mouth disease virus-like particles, and in particular to a method for improving the assembly efficiency and heat stability of foot-and-mouth disease virus-like particles through Foldon trimer fusion. The present invention belongs to the field of medical technology. Background Art

[0002] Foot-and-mouth disease (FMD) is a highly contagious disease that primarily affects even-toed ungulates such as pigs, cattle, and sheep. FMDV belongs to the Picornaviridae family and has seven serotypes, with no cross-protection between types. During the FMDV life cycle, three other antigenic forms are formed: a protomer (5S) formed by the structural proteins VP0, VP3, and VP1; a pentamer (12S) formed by the aggregation of five protomers; and an empty capsid (75S) formed by the aggregation of twelve pentamers. VP1, VP2, and VP3 are located on the surface of the viral capsid, while the VP4 protein, tightly bound to RNA, is located within the viral capsid, forming the internal components of the virion. FMD virus-like particles (VLPs) vaccines are a new type of genetically engineered vaccine, following the traditional inactivated vaccine. They are morphologically identical or similar to intact virions but do not contain viral genetic material and are therefore non-infectious. During the assembly process of VLPs, the formation of stable intermediate structures (protomers (5S) and pentamer units (12S)) is crucial for the final formation of stable icosahedral VLPs. At the same time, by optimizing the structure of the intermediate assembly unit, the assembly efficiency of VLPs can be improved, which has important practical significance for the large-scale production of VLPs vaccines.

[0003] The C-terminal domain (Foldon) of the T4 phage fibrin is a small, 27-amino acid domain (amino acid residues 457 to 483). As an artificial trimerization domain, Foldon provides a stable trimerization platform, enabling efficient fibrin trimerization and promoting the proper assembly and formation of fibrin trimers. During the life cycle of the T4 phage, fibrin serves as both a structural and chaperone protein within its head. As a key component of trimerization and folding, the foldon peptide ensures that fibrin properly performs its roles in phage assembly and infection. Foldon itself consists of a trimeric β-hairpin propeller, which is stable at low pH. Even under acidic conditions, the foldon trimer dissociates into the monomeric A-state, which retains the structural integrity and stability of the β-hairpin. In summary, foldon plays a multifaceted role in the fibrin trimer structure. It not only participates in trimer formation and stability but also plays a crucial role in the proper folding and biological function of fibrin.

[0004] Research on the modification and application of FMDV VLPs is continuously making new progress. In terms of VLP modification, researchers have attempted to improve the specific recognition and presentation capabilities of VLPs through chimeric technology. To enhance the stability of VLPs, researchers have also conducted amino acid modification studies. However, there is currently no modification strategy that can significantly improve the self-assembly efficiency of FMDV VLPs while also enhancing their stability. In view of this, the present invention aims to provide a structure-based modification strategy to improve the assembly efficiency and stability of O-type FMDV VLPs. This will help further enhance the safety, immunogenicity, and production efficiency of VLP vaccines, and fundamentally contribute to the comprehensive prevention and control of foot-and-mouth disease in my country. Summary of the Invention

[0005] The object of the present invention is to provide a method for improving the assembly efficiency, stability and immunogenicity of O-type FMDV VLPs.

[0006] The present invention introduces a hydrophobic foldon peptide at the N-terminus of FMDV VP2. During the VLP assembly process, the foldon peptide self-assembles into a trimer complex, which aids the interaction between the assembly precursor pentamers and improves the assembly efficiency of the VLPs. Furthermore, the formation of the foldon trimer complex on the inner side of the VLP's tertiary axis, resembling a "lock," enhances the stability of the VLPs. The establishment of this modification strategy is conducive to reducing the production cost of VLP vaccines, promoting the development of new vaccine technologies, and has important significance for disease control.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The present invention provides a method for improving the assembly efficiency and heat stability of foot-and-mouth disease virus-like particles by Foldon trimer fusion, wherein the method comprises the following steps:

[0009] (1) A hydrophobic Foldon peptide was introduced into the N-terminus of the VP2 protein of the O-type foot-and-mouth disease virus through a linker to obtain the FoldonVP2 gene fragment. The VP1 gene, VP3 gene, and FoldonVP2 gene of the O-type foot-and-mouth disease virus were seamlessly fused with the small ubiquitination protein gene and cloned into the pET-28a vector to obtain the recombinant vectors pET / SUMO-FoldonVP2, pET / SUMO-VP1, and pET / SUMO-VP3;

[0010] (2) The recombinant vectors pET / SUMO-FoldonVP2, pET / SUMO-VP1, and pET / SUMO-VP3 obtained in step (1) are transformed into Escherichia coli respectively and identified, and the correctly identified Escherichia coli containing the recombinant vectors pET / SUMO-FoldonVP2, pET / SUMO-VP1, and pET / SUMO-VP3 are inoculated into LB culture medium containing ampicillin, kanamycin, and chloramphenicol, and after induction expression and purification, a mixed solution containing the fusion proteins SUMO-VP1, SUMO-FoldonVP2, and SUMO-VP3 is obtained;

[0011] (3) Adding SUMO enzyme to the mixed solution of the purified fusion proteins SUMO-VP1, SUMO-FoldonVP2, and SUMO-VP3, placing the solution in a dialysis bag for enzyme digestion and assembly reaction, and obtaining the enzyme digestion and assembly product after the assembly is completed;

[0012] (4) The collected enzyme-digested assembly products are purified by size exclusion chromatography (SEC) to obtain purified foot-and-mouth disease virus-like particles.

[0013] Among them, preferably, in step (1), the nucleotide sequence of the FoldonVP2 gene fragment is as shown in SEQ ID NO: 3, the nucleotide sequence of the O-type foot-and-mouth disease virus VP1 gene is as shown in SEQ ID NO: 4, the nucleotide sequence of the O-type foot-and-mouth disease virus VP3 gene is as shown in SEQ ID NO: 5, and the nucleotide sequence of the small ubiquitination modified protein gene is as shown in SEQ ID NO: 6.

[0014] Wherein, preferably, in step (2), the induced expression and purification include the following steps: the Escherichia coli strains containing the recombinant vectors pET / SUMO-FoldonVP2, pET / SUMO-VP1 and pET / SUMO-VP3 were inoculated into LB medium containing ampicillin, kanamycin and chloramphenicol at a 1% v / v inoculation volume, and cultured at 37°C and 220 r / min until OD 600 When the value was 0.6-0.8, the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mmol / L, and the expression was induced at 16°C and 200 r / min for 16 hours; after induction, the cells were collected by centrifugation at 4000 r / min for 30 minutes, the cells were resuspended in ice bath lysis solution, ultrasonically disrupted, and centrifuged at 10,000×g for 30 minutes, the supernatant was taken, the precipitate was discarded, and the supernatant was purified by chromatography to obtain recombinant proteins SUMO-VP1, SUMO-FoldonVP2 and SUMO-VP3. The ice bath lysis solution was 20 mM Tris-HCl, pH 7.4, containing 500 mM NaCl and 20 mM imidazole.

[0015] Wherein, preferably, in step (3), the volume ratio of the mixed solution of fusion proteins SUMO-VP1, SUMO-FoldonVP2 and SUMO-VP3 to SUMO enzyme is 100:1; the dialysis bag is an 8000 MWCO dialysis bag, and the dialysis bag is placed in an assembly buffer for dialysis at 4°C. The assembly buffer used is 20 mmol / L Tris-HCl containing 600 mmol / L NaCl and 2 mmol / L CaCl2, pH 8.0; 5 mmol / L DTT is added to the buffer 12 h before assembly, the entire buffer system should be placed on a stirrer, the buffer should be slightly stirred, and the enzyme-digested assembly product is collected after 36 hours.

[0016] Preferably, in step (4), the collected enzyme-digested assembly products are purified by size exclusion chromatography to produce two peaks, wherein the F1 peak is the O-type FMDV VLPs component fused with the Foldon polypeptide, named OPF VLPs.

[0017] Preferably, the method further comprises the step of finely separating the collected enzyme-digested assembly products by sucrose density gradient centrifugation, wherein the specific operation is as follows: 1 mL of 1 mg / mL OPF VLPs is centrifuged at 10,000 r / min for 10 min to remove the sediment, added to the top of a pre-prepared 150 g to 450 g / L sucrose density gradient, centrifuged at 35,000 r / min for 3 h in an ultracentrifuge, and the samples are separated step by step from top to bottom at 500 μL / tube, and the OD is measured using a UV spectrophotometer. 280 Plot the curves for each fraction.

[0018] Among them, preferably, the assembly efficiency of the method described is 78.5%, which is significantly higher than the assembly efficiency of the original foot-and-mouth disease virus-like particles of 19.65%. The original foot-and-mouth disease virus-like particles are prepared according to the method described in the patent application with application number 201010251915.1 and invention name "Foot-and-mouth disease virus-like particles and preparation method and use".

[0019] The foot-and-mouth disease virus-like particles prepared according to any of the above methods are also within the protection scope of the present invention.

[0020] Under room temperature conditions, the foot-and-mouth disease virus-like particles prepared according to any of the methods described above are extremely stable, and the proportion of remaining intact antigens can still reach more than 75% within one week, while the original foot-and-mouth disease virus-like particles are degraded to less than 20%. Under 37°C conditions, the stability of the foot-and-mouth disease virus-like particles prepared according to any of the methods described above is significantly stronger than that of the original foot-and-mouth disease virus-like particles. Within 1 hour, the proportion of remaining intact antigens in the original foot-and-mouth disease virus-like particles is degraded to less than 20%, while the foot-and-mouth disease virus-like particles prepared according to any of the methods described above can still reach more than 70% within 1 week.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention, based on the intercalation of Foldon trimers at the FMDV tertiary axis, enhances the assembly efficiency and stability of FMDV VLPs in a "lock-and-lock" manner. This helps address issues such as low VLP yield and instability in large-scale production, and is of great significance for improving vaccine performance, reducing costs, simplifying vaccine management, and advancing vaccine technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The recombinant protein was purified and analyzed by SDS-PAGE;

[0024] Wherein, M: protein molecular weight standard, 1-4: purified protein eluate;

[0025] Figure 2 This is the curve of the SEC separation and purification assembly product;

[0026] Figure 3 Recombinant VLPs were purified by SEC and analyzed by SDS-PAGE;

[0027] Wherein, M: protein molecular weight standard 1-4, F1 peak sample after SEC separation and purification; 5. SUMO protease; 6-10, F2 peak sample after SEC separation and purification; 11. assembly product after enzyme digestion;

[0028] Figure 4 Western blotting analysis of F1 peak samples after SEC separation and purification;

[0029] Wherein, M: protein molecular weight standard, 1. recombinant protease before cleavage sample, 2. recombinant protease after cleavage sample, 3-6. SEC separation and purification of F1 peak sample;

[0030] Figure 5 WT VLPs and OPF VLPs were purified by 150 g to 450 g / L sucrose density gradient;

[0031] Figure 6 for DLS analysis of recombinant OPF VLPs;

[0032] Figure 7 Transmission electron microscopy analysis of recombinant OPF VLPs. DETAILED DESCRIPTION

[0033] The present invention will be described in further detail below in conjunction with the examples and accompanying drawings, but the examples of the present invention are merely illustrations of the inventive method and are not intended to limit the remainder of the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0034] Example 1 Establishment of a method for improving the assembly efficiency and heat stability of foot-and-mouth disease virus-like particles by Foldon trimer fusion

[0035] 1. Construction of recombinant plasmid

[0036] The present invention introduces a hydrophobic Foldon peptide segment at the N-terminus of the VP2 protein of the O-type foot-and-mouth disease virus (O / MYA98 / BY / 2010), and links the Foldon peptide gene (shown in SEQ ID NO: 1) to the 5' end of the VP2 gene (shown in SEQ ID NO: 2) with a "(GGGGS)2" linker, and names it as the FoldonVP2 gene (shown in SEQ ID NO: 3). GenScript Corporation seamlessly fuses the VP1 (shown in SEQ ID NO: 4) and VP3 (shown in SEQ ID NO: 5) genes of the O-type foot-and-mouth disease virus (O / MYA98 / 2021) and the FoldonVP2 gene with the small ubiquitination protein gene (shown in SEQ ID NO: 6), respectively, and clones them into the pET-28a vector using Sal I / BamH I and HindIII / Xho I endonucleases to obtain pET / SUMO-FoldonVP2, pET / SUMO-VP1, and pET / SUMO-VP3 plasmids.

[0037] 2. Expression and purification of recombinant fusion proteins

[0038] (1) Transformation of recombinant vectors pET / SUMO-FoldonVP2, pET / SUMO-VP1, and pET / SUMO-VP3

[0039] 1 μL of pET / SUMO-FoldonVP2, pET / SUMO-VP1, and 1 μL of pET / SUMO-VP3 were added to 100 μL of Escherichia coli BL21 (DE3) competent cells, gently flicked to mix, incubated on ice for 30 min, in a 42°C water bath for 1.5 min, and immediately incubated on ice for 2 min. 1 mL of LB medium was added and the cells were cultured in a shaker at 37°C for 40 min. The bacterial solution was spread on an LB plate (containing ampicillin, kanamycin, and chloramphenicol) and cultured at 37°C overnight. The next day, several positive clones were picked from the plate and inoculated into 5 mL of liquid LB medium (containing ampicillin, kanamycin, and chloramphenicol), cultured in a shaker at 37°C at 220 rpm overnight, and 15% sterile glycerol was added to each cell and frozen at -80°C to obtain E. coli seed banks containing pET / SUMO-FoldonVP2, pET / SUMO-VP1, or pET / SUMO-VP3, respectively.

[0040] (2) Expression and purification of recombinant fusion protein

[0041] The strains containing pET / SUMO-FoldonVP2, pET / SUMO-VP1 or pET / SUMO-VP3 that were successfully expressed were inoculated into LB medium containing ampicillin, kanamycin and chloramphenicol at a 1% v / v inoculum and cultured at 37°C and 220 r / min until OD 600 When the value is about 0.6 to 0.8, add the inducer isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1mmol / L, and induce expression at 16°C and 200r / min for 16 hours. After culture induction, collect the bacteria by centrifugation at 4000r / min for 30 minutes. Resuspend the bacteria in ice bath lysis solution (20mM Tris-HCl, 500mM NaCl, 20mM imidazole, pH7.4), ultrasonically disrupt the bacteria, and centrifuge at 10,000×g for 30 minutes. Take the supernatant and discard the precipitate. After chromatographic purification of the supernatant, a mixed solution of recombinant proteins SUMO-VP1, SUMO-FoldonVP2 and SUMO-VP3 was obtained, which was identified by SDS-PAGE. The results are as follows. Figure 1 shown.

[0042] 3. Assembly, Purification and Identification of Recombinant VLPs

[0043] a. Add SUMOase (100:1 volume ratio) to a mixed solution of recombinant SUMO-VP1, SUMO-FoldonVP2, and SUMO-VP3 proteins and place the solution in an 8000 MWCO dialysis bag for enzymatic digestion and assembly. The bag was dialyzed at 4°C against assembly buffer (assembly buffer formulation: 20 mmol / L Tris-HCl, 600 mmol / L NaCl, 2 mmol / L CaCl2, pH 8.0, with 5 mmol / L DTT added only to the buffer for the first 12 hours). The entire buffer system should be placed on a stirrer for gentle agitation. The digestion and assembly products were collected after 36 hours.

[0044] b. The collected enzyme-digested assembly products were purified by size exclusion chromatography (SEC) and two peaks appeared. The results are as follows Figure 2 As shown, SDS-PAGE electrophoresis detected three relatively clear bands of VP2, VP3, and VP1. Figure 3 As shown, the F1 peak is the O-type FMDV VLPs component fused with Foldon polypeptide, named OPFVLPs, and the F2 peak is the separated unassembled protein.

[0045] c. After the F1 peak sample separated and purified by SEC was subjected to 12% SDS-PAGE, the protein was transferred to a nitrocellulose membrane and blocked with blocking solution (TBST, 5% skim milk powder solution) at room temperature for 1 hour. It was incubated with swine foot-and-mouth disease positive serum (1:2000 dilution) at 4°C overnight. After washing 3 times with TBST, rabbit anti-swine IgG labeled with horseradish peroxidase (1:5000 dilution) was added and incubated at room temperature for 1 hour. After washing 3 times with TBST, ECL luminescent solution was added. The reaction was protected from light for 1 minute and then exposed to light to observe the target band. The results are as follows. Figure 4 As shown, the results showed that OPF VLPs specifically bound to porcine anti-FMDV serum.

[0046] d. Finely separate the collected enzyme-digested assembly products using sucrose density gradient centrifugation. Take 1 mL of the 1 mg / mL sample and centrifuge at 10,000 rpm for 10 minutes to remove the sediment. Add it to the top of a pre-prepared 150g to 450g / L sucrose density gradient. Centrifuge at 35,000 rpm for 3 hours in an ultracentrifuge. Separate the sample step by step from top to bottom at 500 μL / tube. Measure the OD using a UV spectrophotometer. 280 The curves of each fraction were drawn. The results showed that the peak positions of OPF VLPs and original VLPs (WT VLPs, prepared according to the method described in the patent application with application number 201010251915.1, invention name "Foot-and-mouth disease virus-like particles and preparation method and use") were consistent. The results are as follows Figure 5 shown.

[0047] e. Dynamic light scattering (DLS) was used to measure the size of VLPs purified by sucrose density gradient centrifugation. It was found that OPF VLPs had a single component in solution and the hydration molecular dynamics diameter was 30-40 nm. Figure 6 The sample was negatively stained with 1% phosphotungstic acid (pH 7.4) and hollow particles of uniform size and shape were observed using a transmission electron microscope. Figure 7 .

[0048] 4. Assembly efficiency and stability testing of OPFVLPs

[0049] a. Detection of assembly efficiency of OPF VLPs

[0050] The protein of OPF VLPs purified by SEC was quantified by BCA quantitative method (see kit instructions), and the double antibody sandwich ELISA method was used to detect and quantify the OPF VLPs purified by SEC. The specific steps are as follows: 1.0 μg / ml coating antibody was coated with phosphate buffer at 4°C overnight; after washing with PBST, OPF VLPs were diluted to about 2 μg / ml, and then serially diluted 2-fold and incubated at room temperature for 45 minutes; after washing with PBST, 0.25 μg / ml detection antibody was added and incubated at room temperature for 45 minutes; after washing with PBST, 0.125 μg / ml enzyme-labeled secondary antibody was added and incubated at room temperature for 45 minutes, TMB was added for color development at room temperature for 1.5 minutes, and the color development was stopped, and the OD was detected by microplate reader. 450 The results showed that the assembly efficiency of the OPF VLPs sample was approximately 78.5%, significantly higher than the WT VLPs assembly efficiency of 19.65%, as shown in Table 1.

[0051] Table 1 Assembly efficiency calculation

[0052]

[0053] b. Thermal stability test of OPF VLPs

[0054] After sucrose density gradient separation and purification of OPF VLPs, they were diluted to 100 μg / mL and placed at room temperature (25°C). Samples were taken at regular intervals and the proportion of remaining intact antigens was detected by double-antibody sandwich ELISA. The results showed that at room temperature, OPF VLPs were extremely stable, with the proportion of remaining intact antigens still reaching more than 75% within a week, while WT VLPs degraded to less than 20%. The results are shown in Table 2. After being placed at 37°C, samples were taken at regular intervals and the proportion of remaining intact antigens was detected by double-antibody sandwich ELISA. The results showed that at 37°C, OPF VLPs were significantly more stable than WT VLPs. Within 1 hour, the proportion of remaining intact antigens in WT VLPs degraded to less than 20%, while OPF VLPs could still reach more than 70% within 1 week. The results are shown in Table 3.

[0055] Table 2 Ratio of remaining intact antigen at room temperature

[0056]

[0057] Table 3: Proportion of intact antigen remaining at 37°C

[0058]

Claims

1. A method for improving the assembly efficiency and heat stability of foot-and-mouth disease virus-like particles by Foldon trimer fusion, characterized in that: The method comprises the following steps: (1) A hydrophobic Foldon peptide was introduced into the N-terminus of the VP2 protein of the O-type foot-and-mouth disease virus through a linker to obtain the FoldonVP2 gene fragment. The VP1 gene, VP3 gene, and FoldonVP2 gene of the O-type foot-and-mouth disease virus were seamlessly fused with the small ubiquitination protein gene and cloned into the pET-28a vector to obtain the recombinant vectors pET / SUMO-FoldonVP2, pET / SUMO-VP1, and pET / SUMO-VP3; (2) The recombinant vectors pET / SUMO-FoldonVP2, pET / SUMO-VP1, and pET / SUMO-VP3 obtained in step (1) are transformed into Escherichia coli respectively and identified, and the correctly identified Escherichia coli containing the recombinant vectors pET / SUMO-FoldonVP2, pET / SUMO-VP1, and pET / SUMO-VP3 are inoculated into LB culture medium containing ampicillin, kanamycin, and chloramphenicol, and after induction expression and purification, a mixed solution containing the fusion proteins SUMO-VP1, SUMO-FoldonVP2, and SUMO-VP3 is obtained; (3) Adding SUMO enzyme to the mixed solution of the purified fusion proteins SUMO-VP1, SUMO-FoldonVP2, and SUMO-VP3, placing the solution in a dialysis bag for enzyme digestion and assembly reaction, and obtaining the enzyme digestion and assembly product after the assembly is completed; (4) The collected enzyme-digested assembly products are purified by size exclusion chromatography (SEC) to obtain purified foot-and-mouth disease virus-like particles.

2. The method according to claim 1, wherein In step (1), the nucleotide sequence of the FoldonVP2 gene fragment is shown in SEQ ID NO: 3, the nucleotide sequence of the O-type foot-and-mouth disease virus VP1 gene is shown in SEQ ID NO: 4, the nucleotide sequence of the O-type foot-and-mouth disease virus VP3 gene is shown in SEQ ID NO: 5, and the nucleotide sequence of the small ubiquitination modified protein gene is shown in SEQ ID NO:

6.

3. The method according to claim 1, wherein In step (2), the induced expression and purification comprise the following steps: the Escherichia coli strains containing the recombinant vectors pET / SUMO-FoldonVP2, pET / SUMO-VP1 and pET / SUMO-VP3 were inoculated into LB medium containing ampicillin, kanamycin and chloramphenicol at a 1% v / v inoculation volume, and cultured at 37°C and 220 r / min until the OD 600 When the value was 0.6-0.8, the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mmol / L, and the expression was induced at 16°C and 200 r / min for 16 hours; after induction, the cells were collected by centrifugation at 4000 r / min for 30 minutes, the cells were resuspended in ice bath lysis solution, ultrasonically disrupted, and centrifuged at 10,000×g for 30 minutes, the supernatant was taken, the precipitate was discarded, and the supernatant was purified by chromatography to obtain recombinant proteins SUMO-VP1, SUMO-FoldonVP2 and SUMO-VP3. The ice bath lysis solution was 20 mM Tris-HCl, pH 7.4, containing 500 mM NaCl and 20 mM imidazole.

4. The method according to claim 1, wherein In step (3), the volume ratio of the mixed solution of fusion proteins SUMO-VP1, SUMO-FoldonVP2 and SUMO-VP3 to the SUMO enzyme is 100:1; the dialysis bag is an 8000MWCO dialysis bag, which is placed in an assembly buffer for dialysis at 4°C. The assembly buffer used is 20mmol / LTris-HCl containing 600mmol / L NaCl and 2mmol / L CaCl2, pH 8.0; 5mmol / L DTT is added to the buffer 12h before assembly, and the entire buffer system should be placed on a stirrer to gently stir the buffer. The enzyme-digested assembly product is collected after 36 hours.

5. The method according to claim 1, wherein In step (4), the collected enzyme-digested assembly products are purified by size exclusion chromatography to produce two peaks, of which the F1 peak is the O-type FMDV VLPs component fused with the Foldon polypeptide, named OPFVLPs.

6. The method according to claim 5, wherein The method also includes a step of finely separating the collected enzyme-digested assembly products by sucrose density gradient centrifugation. The specific operation is as follows: 1 mL of 1 mg / mL OPF VLPs is centrifuged at 10,000 rpm for 10 minutes to remove sediment, added to the top of a pre-prepared 150g to 450g / L sucrose density gradient, centrifuged at 35,000 rpm for 3 hours in an ultracentrifuge, and the samples are separated step by step from top to bottom at 500 μL / tube. The OD is measured using a UV spectrophotometer. 280 Plot the curves for each fraction.

7. The method according to any one of claims 1 to 6, wherein: The assembly efficiency of the method described is 78.5%, which is significantly higher than the assembly efficiency of the original foot-and-mouth disease virus-like particles of 19.65%. The original foot-and-mouth disease virus-like particles are prepared according to the method described in the patent application with application number 201010251915.1 and invention name "Foot-and-mouth disease virus-like particles and preparation method and use".

8. Foot-and-mouth disease virus-like particles prepared according to the method according to any one of claims 1 to 6.

9. The foot-and-mouth disease virus-like particle according to claim 8, wherein Under room temperature conditions, the foot-and-mouth disease virus-like particles prepared according to the method according to any one of claims 1 to 6 are extremely stable, and the proportion of remaining intact antigens can still reach more than 75% within one week, while the original foot-and-mouth disease virus-like particles are degraded to less than 20%. Under 37°C conditions, the stability of the foot-and-mouth disease virus-like particles prepared according to the method according to any one of claims 1 to 6 is significantly stronger than that of the original foot-and-mouth disease virus-like particles. Within 1 hour, the proportion of remaining intact antigens in the original foot-and-mouth disease virus-like particles is degraded to less than 20%, while the foot-and-mouth disease virus-like particles prepared according to the method according to any one of claims 1 to 6 can still reach more than 70% within 1 week.

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