O-type foot-and-mouth disease virus polyepitope biomimetic nano self-assembled virus-like particles, and preparation method and application thereof

Abstract

The application discloses O-type foot-and-mouth disease virus polyepitope biomimetic nano self-assembly virus-like particles and a preparation method and application thereof. The O-type foot-and-mouth disease virus polyepitope biomimetic nano self-assembly virus-like particles are obtained by self-assembly of a recombinant protein SPC-B4T and 2STAP205 in vitro; wherein the recombinant protein SPC-B4T is obtained by sequentially connecting SpyCatcher, antigen epitopes of four topological representative strains of O-type and T cell epitopes of 3A in sequence; and the recombinant protein 2STAP205 is obtained by connecting genes of SpyTag to both ends of a phage AP205 gene through flexible spacers respectively. In addition, the application also prepares the virus-like particles into vaccines, and it is found that the prepared vaccines have good immunization efficacy, are a new type of vaccine with wide prospects, can provide material reserves and technical support for O-type foot-and-mouth disease prevention and control in China, and can generate great economic benefits and important social benefits.

Description

Technical Field

[0001] This invention relates to a virus-like particle, its preparation method, and its application, and particularly to a multi-epitope biomimetic nano-self-assembled virus-like particle of type O foot-and-mouth disease virus, its preparation method, and its application. This invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Foot-and-mouth disease (FMD) is a major animal disease caused by the foot-and-mouth disease virus (FMDV), affecting the main economic livestock breeds of pigs, cattle, and sheep. The OIE lists it as a notifiable animal disease, and my country classifies it as a Class A animal infectious disease requiring key prevention and control. Due to its high infectivity, significant economic damage, and the variability in its epidemic dynamics, it receives high attention from countries worldwide. Safe, highly effective, and diagnostically sound new FMD vaccines are indispensable technological tools. Although marker-controlled inactivated FMD vaccines can be obtained through genetic manipulation, the entire research and production process still requires the use of live viruses, necessitating high-level research facilities and production workshops, and presenting potential biosafety risks.

[0003] Given the challenges of biosafety risks, high costs, and complex vaccine components in the research and production of inactivated foot-and-mouth disease (FMD) vaccines, developing environmentally friendly, biosafety-compliant, and diagnostically accurate high-efficiency genetically engineered subunit vaccines using reverse vaccinology technology, based on the protective antigens and / or epitopes of the pathogen, has become a hot topic in vaccine research and development. However, most current genetically engineered vaccines focus on protective antigens, expressing recombinant proteins through eukaryotic / prokaryotic expression systems to obtain relatively simple forms such as recombinant protein mixtures and recombinant fusion proteins; they also include expressing complete viral capsid proteins or segmented recombinant proteins assembled in vitro into VLPs. However, these protein expression forms have many drawbacks and limitations. For example, recombinant proteins expressed as single genes or multiple genes cannot guarantee their correct natural antigenic structure, affecting immunization efficacy. It is worth mentioning that the foot-and-mouth disease virus-like particles (VLPs) developed by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences have obtained a Class I new veterinary drug certificate. This vaccine uses memory segmented expression of the virus-encoded capsid protein and in vitro assembly of VLPs.

[0004] In order to develop a novel genetically engineered subunit vaccine for type O foot-and-mouth disease that is biosafe, environmentally friendly, highly effective in immunogenicity, and simple in production process, this invention proposes a multi-epitope biomimetic nano-self-assembled virus-like particle for type O foot-and-mouth disease virus, its preparation method, and its application. Summary of the Invention

[0005] The purpose of this invention is to provide O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled virus-like particles, their preparation methods, and applications.

[0006] To achieve the above objectives, the present invention employs the following technical means:

[0007] This invention utilizes the protective antigenic epitopes of type O foot-and-mouth disease virus (FMDV) as components, employing the SpyTag / SpyCatcher system—a naturally occurring self-assembling protein molecule—combined with phage display technology, to design two recombinant protein expression plasmids. These recombinant proteins are expressed using E. coli and assembled in vitro into multi-epitope VLP nanoantigens of type O FMDV. Results show that this invention successfully constructed prokaryotic recombinant expression plasmids, expressing, purifying, and assembling biomimetic nanovirus-like particle antigens with morphological similarities to natural virus particles. These antigens possess the same antigenicity as the inactivated type O FMDV antigen, suggesting that this antigen can be used to replace the inactivated FMDV antigen for monitoring and evaluating FMDV vaccine-induced immunity antibodies. This not only simplifies the antigen preparation process of FMDV antibody detection kits and reduces costs but also provides biosafety. Animal immunization experiments show that when this recombinant VLP antigen is combined with ISA206VG adjuvant to prepare a vaccine, it induces high levels of protective antibodies in immunized animals, with even higher levels and longer duration of protection after booster immunization. Immune animals showed 100% (5 / 5) protection after challenge with a virulent strain.

[0008] Based on the above research, this invention proposes a multi-epitope biomimetic nano-self-assembled virus-like particle for type O foot-and-mouth disease virus. The multi-epitope biomimetic nano-self-assembled virus-like particle for type O foot-and-mouth disease virus is obtained by in vitro self-assembly of recombinant proteins SPC-B4T and 2STAP205.

[0009] The recombinant protein SPC-B4T is obtained by sequentially tandemly connecting the antigenic epitopes of SpyCatcher, the representative strains of the four O-type topologies O / Tibet / CHA / 99, O / Mya98, O / HN / CHA / 93, and O / XJPS / CHA / 2017, and the T-cell epitope of 3A, with the spacer sequence GS introduced between adjacent antigenic epitopes, and the spacer sequence GGGGSGGGGS introduced between the SpyCatcher gene and the O / Tibet / CHA / 99 antigenic epitope gene. The connection order is SpyCatcher-O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-3A.

[0010] The recombinant protein 2STAP205 is obtained by linking the SpyTag gene to both ends of the phage AP205 gene through flexible spacers GSGTAGGGSGS and GGSGGSG, respectively, in the following linking order: SpyTag-GSGTAGGGSGS-AP205-GGSGGSG-SpyTag.

[0011] Preferably, the amino acid sequence of the recombinant protein SPC-B4T is shown in SEQ ID NO.2, and the amino acid sequence of the recombinant protein 2STAP205 is shown in SEQ ID NO.4.

[0012] Furthermore, this invention also proposes a method for preparing the aforementioned O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled virus-like particles, comprising the following steps:

[0013] (1) Synthesis of SpyCatcher and O-type foot-and-mouth disease virus multi-epitope chimeric DNA

[0014] The antigenic epitopes of SpyCatcher, O / Tibet / CHA / 99, O / Mya98, O / HN / CHA / 93, and O / XJPS / CHA / 2017, and the T-cell epitope of 3A were sequentially tandemly. A spacer sequence GS was introduced between adjacent antigenic epitopes, and GGGGSGGGGS was introduced between the SpyCatcher gene and the O / Tibet / CHA / 99 antigenic epitope gene. This resulted in the designed chimeric DNA: SpyCatcher-O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-3A. The chimeric DNA gene was optimized for codon bias using biological software, and BamH1 and Xho1 specific restriction enzyme sites were introduced at the 5′- and 3′- ends of the gene, named SPC-B4T. The nucleotide sequence of the optimized chimeric DNA gene is shown in SEQ ID NO.1.

[0015] (2) Synthesis of the SpyTag-phage AP205 chimeric gene

[0016] The SpyTag gene was linked to both ends of the phage AP205 gene via flexible spacers GSGTAGGGSGS and GGSGGSG, respectively, forming the SpyTag-GSGTAGGGSGS-AP205-GGSGGSG-SpyTag structure. The chimeric gene was optimized for codon bias using biological software, and BamH1 and Xho1 specific restriction sites were introduced at the 5′- and 3′- ends of the gene, named 2×SpyTag / AP205. The nucleotide sequence of the optimized chimeric gene is shown in SEQ ID NO.3.

[0017] (3) Gene cloning and recombinant protein expression and purification

[0018] SPC-B4T, 2×SpyTag / AP205, and pET-28a(+) were digested with BamH1 and Xhol enzymes, purified, and recovered. They were then inserted into pET-28a(+) linearized with the same enzymes to construct recombinant expression plasmids pET-28 / SPC-B4T and pET-28 / 2×SpyTag / AP205. These plasmids were then transformed into JM109 competent cells. Positive recombinant expression plasmids were identified by resistance selection, double enzyme digestion, and sequence analysis.

[0019] The two positive recombinant expression plasmids were transformed into BL21(DE3)pLysS by heat stimulation. Single clones were selected and inoculated into 5 ml of LB medium containing kanamycin and cultured overnight at 37°C and 220 rpm. The overnight culture was added to freshly prepared sterile LB medium containing kanamycin at 1% (V / V) and cultured at 37°C and 220 rpm until OD600nm≈0.4-0.6. Then, under sterile conditions in a laminar flow hood, 0.4 mM IPTG was added and expression was induced at 37°C for 4-6 hours. The culture was harvested by centrifugation at 2000 rpm for 30 min. Protein lysis buffer was added at 20% of the original culture volume, and the mixture was sonicated. The supernatant was collected by centrifugation at 20000 g for 20 min, and the precipitate was discarded. The protein was purified according to the instructions of the Ni-NTA histidine purification column and named SPC-B4T and 2STAP205, respectively.

[0020] (4) Assembly of O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled virus-like particles

[0021] The SPC-B4T and 2STAP205 proteins obtained by affinity chromatography were mixed at a mass ratio of 2-5:1 and assembled into a biomimetic nanoantigen (VLP) at room temperature or 4°C.

[0022] Preferably, the amino acid sequence of the recombinant protein SPC-B4T is shown in SEQ ID NO.2, and the amino acid sequence of the recombinant protein 2STAP205 is shown in SEQ ID NO.4.

[0023] Furthermore, this invention also proposes the use of the aforementioned O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled virus-like particles in the preparation of a vaccine for the prevention of O-type foot-and-mouth disease.

[0024] Furthermore, the present invention also proposes a multi-epitope biomimetic nano-self-assembled virus-like particle vaccine for type O foot-and-mouth disease virus, wherein the vaccine contains the multi-epitope biomimetic nano-self-assembled virus-like particles for type O foot-and-mouth disease virus described in the present invention and an adjuvant.

[0025] Preferably, the adjuvant is ISA206.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] This invention utilizes a self-assembled protein system and phage display technology to successfully develop biomimetic nano-self-assembled virus-like particles with multiple epitopes of type O foot-and-mouth disease virus (FMDV). This significantly enhances the antigenicity and immunogenicity of FMDV epitopes, and its antigenic base is indistinguishable from that of the whole FMDV antigen. Furthermore, this invention has also prepared a vaccine from these biomimetic nano-self-assembled virus-like particles. Results show that the prepared vaccine has good immunogenicity; a single dose can protect immunized animals against virulent FMDV infection. The vaccine can be widely used for immunization and prevention of disease in important economic livestock species such as pigs, cattle, and sheep. It is a promising new vaccine that will provide material reserves and technical support for the prevention and control of type O FMDV in my country, generating significant economic and social benefits. Attached Figure Description

[0028] Figure 1 SDS-PAGE results of multi-epitope biomimetic nano-VLP assembly for foot-and-mouth disease type O;

[0029] Where: 1 is the result of mixing SPC-B4T and 2STAP205 proteins, 2 is SPC-B4T protein, and 3 is 2STAP205 protein;

[0030] Figure 2 To observe the morphology of multi-epitope biomimetic nano-VLPs for type O foot-and-mouth disease using transmission electron microscopy;

[0031] Figure 3 The particle size of three batches of O-type foot-and-mouth disease multi-epitope biomimetic nano-VLP vaccine was determined by dynamic scattering.

[0032] Figure 4 The results show the antigenicity of multi-epitope biomimetic nanoparticles (VLPs) for type O foot-and-mouth disease virus.

[0033] Figure 5 The immune response results of the O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled VLP antigen to positive serum of pigs infected with four lineages of O-type FMDV were significantly better than those of the single-strain O-type FMDV inactivated antigen.

[0034] Figure 6To induce an immune response between bovine and ovine positive serum immunized with type O foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled VLP antigen and type O FMDV inactivated vaccine;

[0035] Figure 7 The results of specific antibody detection 7 days after immunization with the O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled VLP antigen. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0037] Example 1: Preparation of multi-epitope biomimetic nano-self-assembled virus-like particles of foot-and-mouth disease virus type O

[0038] 1. Design of SpyCatcher and O-type foot-and-mouth disease virus multi-epitope chimeric DNA

[0039] Based on the amino acid sequences encoding the VP1 gene of representative strains of the four O topologies (O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, and O / XJPS / CHA / 2017), linear antigenic epitopes of protective antibodies against foot-and-mouth disease virus (FMDV) and T-cell epitopes (amino acid 21-35) of the conserved non-structural protein 3A of FMDV were selected. The antigenic epitopes of SpyCatcher, O / Tibet / CHA / 99, O / Mya98, O / HN / CHA / 93, and O / XJPS / CHA / 2017, and the T-cell epitope of 3A were sequentially tandem. To prevent the epitopes from being tandemly cascaded... A new epitope was formed, and a spacer sequence GS was introduced between adjacent antigenic epitopes. GGGGSGGGGS was introduced between the SpyCatcher gene and the O / Tibet / CHA / 99 antigenic epitope gene, resulting in the designed chimeric DNA: SpyCatcher-O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-3A. Codon preference optimization was performed on the chimeric DNA gene using bioinformatics software, and BamH1 and Xho1 specific restriction enzyme sites were introduced at the 5′ and 3′ ends of the gene, naming it SPC-B4T. The nucleotide sequence of the optimized chimeric DNA gene is as follows:

[0040] GCCATGGTTGATACCTTATCAGGTTTATCAAGTGAGCAAGGTCAGTCCGGTGATATGACAATTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAGGACGGCAAAGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGACAAGTGAAAGATTTCTACCTGTATCCAGGAAAATATACATTTGTCGAAACCGCAGCACCAGACGGTTATGAGGTAGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCAAAGCAACTAAAGGTGACGCTCATATTGGCGGTGGCGGTAGCGGTGGCGGTGGCAGTAAGTATGACGAGAGCCCCGTGACCAATGTGAGAGGTGACCTGCAAGTGTTGGCCCAGAAGGCGGCAAGAACGGGTTCTAAATACGCCGGGGGCTCACTGCCCAACGTGAGAGGCGATCTCCAAGTGCTGGCTCAGAAGGCGGCGAGGCCGGGTAGCAAGTACAGTGACGCCCGCGTGAGCAACGTGAGGGGTGACCTTCAAGTGTTGGCTCAGAAGGCAGAAAGAGCTGGTTCAAAGTATGGCGAGGGCGCTGTGACCAACGTGAGGGGTGACTTGCAAGTGTTGGCTCAGAAGGCAGCAAGAACGGGGAGTGCAGCAATTGAATTCTTTGAGGGGATGGTCCACGACTCCATCAAG (shown in SEQ ID NO.1)

[0041] The encoded amino acid sequence is:

[0042] AMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGGGSGGGG SKYDESPVTNVRGDLQVLAQKAARTGSKYAGGSLPNVRGDLQVLAQKAARPGSKYSDARVSNVRGDLQVLAQKAERAGSKYGEGAVTNVRGDLQVLAQKAARTGSAAIEFFEGMVHDSIK(SEQ ID NO.2 shown)

[0043] 2. Design of the SpyTag-phage AP205 chimeric gene

[0044] The SpyTag gene was linked to both ends of the AP205 phage gene using flexible spacers GSGTAGGGSGS and GGSGGSG, respectively, forming the SpyTag-GSGTAGGGSGS-AP205-GGSGGSG-SpyTag structure. Codon preference optimization was performed on the chimeric gene using bioinformatics software, and BamH1 and Xho1 specific restriction enzyme sites were introduced at the 5′ and 3′ ends of the gene, naming it 2×SpyTag / AP205. The optimized chimeric gene nucleotide sequence is as follows:

[0045] ATGGGCAGCAGCCATCATCATCATCACGGGAGTGGCGCCCACATCGTGATGGTGGACGCCTACAAGCCGACGAAGGGGAGTGGCACTGCAGGAGGTGGCAGCGGAAGTATGGCAAATAAGCCAATGCAACCGATCACATCTACAGCAAATAAAATTGTGTGGAGTGATCCAACTCGTTTATCAACTACATTTTCAGCAAGTCTGTTACGCCAACGTGTTAAAGTTGGTATAGCCGAACTGAATAATGTTTCAGGTCAATATGTATCTGTTTATAAGCGTCCTGCACCTAAACCGGAAGGTTGTGCAGATGCCTGTGTCATTATGCCGAATGAAAACCAATCCATTCGCACAGTGATTTCAGGGTCAGCCGAAAACTTGGCTACCTTAAAAGCAGAATGGGAAACTCACAAACGTAACGTTGACACACTCTTCGCGAGCGGCAACGCCGGTTTGGGTTTCCTTGACCCTACTGCGGCTATCGTATCGTCTGATACTACTGCTGGGACAGCAAGTGGTGGCAGCGGAGGCTCTGGGGCCCACATCGTGATGGTGGACGCCTACAAGCCGACGAAG(shown in SEQ ID NO.3)

[0046] The encoded amino acid sequence is:

[0047] MGSSHHHHHGSGAHIVMVDAYKPTKGSGTAGGGSGSMANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTAGTASGGSGGSGAHIVMVDAYKPTK(shown in SEQ ID NO.4)

[0048] 3. Gene cloning, expression and purification of its recombinant protein

[0049] SPC-B4T, 2×SpyTag / AP205, and pET-28a(+) were digested with BamH1 and Xhol enzymes, purified, and recovered. They were then inserted into pET-28a(+) linearized with the same enzymes to construct recombinant expression plasmids pET-28 / SPC-B4T and pET-28 / 2×SpyTag / AP205. These plasmids were then transformed into JM109 competent cells. Positive recombinant expression plasmids were identified by resistance selection, double enzyme digestion, and sequence analysis.

[0050] The two positive recombinant expression plasmids were transformed into BL21(DE3)pLysS using heat-activated transformation. Single clones were selected and inoculated into 5 ml of LB medium containing kanamycin and cultured overnight at 37°C and 220 rpm. The overnight culture was added at 1% (V / V) to freshly prepared sterile LB medium (kan+) and cultured at 37°C and 220 rpm until OD600nm≈0.4-0.6. Under sterile conditions in a laminar flow hood, 0.4 mM IPTG was added and expression was induced at 37°C for 4-6 hours. The culture was harvested by centrifugation at 2000 rpm for 30 min. Protein lysis buffer was added at 20% of the original culture volume, and the mixture was sonicated (ice bath, 30 min). The supernatant was collected by centrifugation at 20000g for 20 min (4°C), and the precipitate was discarded. The proteins were purified according to the Ni-NTA histidine purification column instructions and named SPC-B4T and 2STAP205, respectively.

[0051] 4. Assembly and identification of O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled virus-like particles (VLPs).

[0052] The SPC-B4T and 2STAP205 proteins purified by affinity chromatography were mixed at a mass ratio of 2:1 and assembled into nanoparticles (VLPs) at room temperature or 4°C. The assembly efficiency at different times and concentrations was analyzed by SDS-PAGE electrophoresis. Nanoparticles (VLPs) were purified using a 15%–50% sucrose gradient, and their morphology and size were observed by transmission electron microscopy. Results are as follows: Figure 1-3 As shown, the results indicate that both proteins can be assembled into biomimetic nanoantigens (VLPs) through a self-assembly system, regardless of whether the conditions are room temperature or 4°C. After purification by sucrose gradient, transmission electron microscopy results show that the recombinant antigen is in VLP form. Dynamic scattering determination shows that the particle size of three batches of O-type foot-and-mouth disease multi-epitope biomimetic nanoVLP vaccines is about 200 nm.

[0053] 5. Immunological identification of recombinant multi-epitope VLPs

[0054] The antigenicity of a multi-epitope biomimetic nano-self-assembled VLP of type O foot-and-mouth disease virus was evaluated by ELISA. Specifically, 96-well ELISA plates were coated with inactivated type O foot-and-mouth disease virus antigen (100 μl / well, 1 μg / ml), a multi-epitope biomimetic nano-self-assembled VLP of type O foot-and-mouth disease virus (i.e., SPC-B4T / 2STAP205, 100 μl / well, 1 μg / ml), and pre-assembled SPC-B4T (100 μl / well, 1 μg / ml) and 2STAP205 (100 μl / well, 1 μg / ml), and incubated overnight at 4°C or for 2 hours at 37°C. The plates were then coated with a solution containing 5% dehydrogenase. Block the milk powder with PBST (pH 7.4) at 37°C for 1 hour; wash 3 times with PBST; add 100 μl / well of 1:100 diluted foot-and-mouth disease virus type O standard positive serum, and incubate at 37°C for 1 hour; wash 3 times with PBST; add 100 μl / well of 1:1000 diluted HRP-labeled rabbit anti-bovine IgG, and incubate at 37°C for 1 hour; wash 3 times with PBST; add TMB chromogenic solution, and react at 37°C for 10–15 min; terminate the reaction with 2M H2SO4, and measure OD. 450nm The absorbance values ​​were measured and the results were analyzed. The results showed that the O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled VLP developed in this invention could induce a strong immune response with O-type foot-and-mouth disease virus positive serum. Its antigenicity was no different from that of the inactivated O-type foot-and-mouth disease virus antigen (O / Mya98 strain inactivated antigen), but it was significantly superior to the unassembled O-type foot-and-mouth disease multi-epitope recombinant antigen. Furthermore, the 2STAP205 protein did not induce an immune response with O-type foot-and-mouth disease virus positive serum. Figure 4 ).

[0055] The above-described ELISA method was used to detect positive sera from pigs infected with the four lineages of foot-and-mouth disease virus, as well as positive sera from cattle and sheep immunized with type O FMD inactivated vaccine. Simultaneously, randomly selected single-lineage type O inactivated FMDV was used as the antigen detection serum. The results showed that the type O foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled VLP antigen developed in this invention could induce a strong immune response with positive sera from pigs infected with the four lineages of type O FMDV. Figure 5 It is worth mentioning that the O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled VLP antigen can also produce a strong immune response with the positive serum of cattle and sheep immunized with the O-type FMD inactivated vaccine, indicating that the recombinant VLP developed in this invention has a broad antigen spectrum and can be used to detect O-type FMDV infection or to detect specific antibodies in vaccine-immunized pigs, cattle, and sheep. Figure 6 ).

[0056] The above results fully demonstrate that the O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled VLP antigen of the present invention can be used to replace the whole virus inactivated antigen for serological detection and diagnosis of antibodies against O-type FMDV-infected animals or immunized animals. This helps to eliminate the biosafety risks and high costs associated with the production of virus inactivated antigens, and is of great significance.

[0057] Example 2: Preparation and Immunoprotective Experiment of Multi-epitope Biomimetic Nanoparticle Self-Assembled Virus-like Particle Vaccine for Foot-and-Mouth Disease Virus Type O

[0058] 1. Vaccine preparation:

[0059] The purified type O foot-and-mouth disease virus multi-epitope biomimetic nanoparticles (VLPs) from Example 1 were quantified using a Bio-Rad quantitative reagent kit and diluted to a concentration of 200 μg / ml with PBS. The mixture was then emulsified with oil adjuvant ISA206 VG (Seppic, France) at a ratio of 50 g:50 g to form a vaccine formulation (W / O / W), with each dose being 1 ml (containing 100 μg of type O foot-and-mouth disease virus multi-epitope biomimetic nanoparticles VLP antigen). Dynamic light scattering (DSL) analysis showed that the vaccine particle size was approximately 211 nm.

[0060] 2. Immunopotency test:

[0061] Healthy BABL / c mice were immunized with the vaccine prepared above, with each mouse receiving a subcutaneous injection (0.2 ml) at multiple sites. A booster immunization with the same dose was administered intramuscularly 14 days after the initial immunization. Simultaneously, an unassembled multi-epitope recombinant antigen (20 μg / 0.2 ml, SPC-B4T and 2STAP205 mass ratio of 2:1) was administered. Blood samples were collected weekly before and after immunization, and serum specific antibody titers were detected using the ELISA method described above. Results showed that 7 days after immunization with the O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled VLP antigen, serum specific antibodies in immunized animals significantly increased, while the unassembled multi-epitope recombinant protein only began to show a significant increase after the booster, indicating that the recombinant VLP has superior immunogenicity. Figure 7 ).

[0062] The experimental pigs weighed approximately 40 kg and had an O-type foot-and-mouth disease virus antibody ratio <1:4 (liquid-phase blocking ELISA result) and were negative for 3ABC protein antibodies (3ABC antibody chemiluminescence kit result). Five pigs were inoculated intramuscularly with the O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled VLP vaccine of this invention at a dose of 1 ml per pig (containing 100 μg of multi-epitope VLP antigen). Twenty-eight days post-immunization, all immunized pigs, along with three unimmunized control pigs under identical conditions, were administered the vaccine according to national standards using 1000 PID... 50Pigs were challenged with 3 ml of type O foot-and-mouth disease virus (O / Mya98 / BY2010 strain) and observed for 10 consecutive days. Results showed that the type O foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled VLP vaccine induced high levels of foot-and-mouth disease specific antibodies after immunization. According to the kit evaluation criteria, a ratio ≥1:64 indicated 99% protection. After booster immunization with this vaccine, serum specific antibody levels were not lower than 1:128; 5 / 5 protection.

[0063] Table 1. Experimental results of the efficacy of the type O foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled VLP vaccine in swine.

[0064] Group Antibody titer (LPB-ELISA) Protection rate (%) VLP+ISA206 1:3601:3601:5121:5121:512 5 / 5(100％) Comparison <1:4<1:4<1:4 0 / 3(0)

[0065] Furthermore, the vaccinated animals did not show redness, swelling, or fever at the injection site, nor did they experience any adverse reactions. Their appetite was normal, and their mental state was good, confirming that the vaccine is very safe.

Claims

1. A biomimetic nano-self-assembled virus-like particle with multiple epitopes for type O foot-and-mouth disease virus, characterized in that, The aforementioned O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled virus-like particles were obtained by in vitro self-assembly of recombinant proteins SPC-B4T and 2STAP205. The recombinant protein SPC-B4T is obtained by sequentially tandemly connecting the antigenic epitopes of SpyCatcher, the representative strains of the four O-type topologies O / Tibet / CHA / 99, O / Mya98, O / HN / CHA / 93, and O / XJPS / CHA / 2017, and the T-cell epitope of 3A, with a spacer sequence GS introduced between adjacent antigenic epitopes, and a spacer sequence GGGGSGGGGS introduced between the SpyCatcher gene and the O / Tibet / CHA / 99 antigenic epitope gene. The connection sequence is SpyCatcher-O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-3A. The amino acid sequence of the recombinant protein SPC-B4T is shown in SEQ ID NO.

2. The recombinant protein 2STAP205 was obtained by linking the SpyTag gene to both ends of the phage AP205 gene using flexible spacers GSGTAGGGSGS and GGSGGSG, respectively, in the following linking order: SpyTag-GSGTAGGGSGS-AP205-GGSGGSG-SpyTag; the amino acid sequence of the recombinant protein 2STAP205 is shown in SEQ ID NO.

4.

2. A method for preparing the O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled virus-like particles as described in claim 1, characterized in that, Includes the following steps: (1) Synthesis of SpyCatcher and O-type foot-and-mouth disease virus multi-epitope chimeric DNA The antigenic epitopes of SpyCatcher, O / Tibet / CHA / 99, O / Mya98, O / HN / CHA / 93, and O / XJPS / CHA / 2017, and the T-cell epitope of 3A were sequentially tandemly. A spacer sequence GS was introduced between adjacent antigenic epitopes, and GGGGSGGGGS was introduced between the SpyCatcher gene and the O / Tibet / CHA / 99 antigenic epitope gene. This resulted in the designed chimeric DNA: SpyCatcher-O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-3A. Codon preference optimization was performed on the chimeric DNA gene using bioinformatics software, and BamH1 and Xho1 specific restriction enzyme sites were introduced at the 5' and 3' ends of the gene, naming it SPC-B4T. The nucleotide sequence of the optimized chimeric DNA gene is shown in SEQ ID. As shown in NO.1; the amino acid sequence of the recombinant protein SPC-B4T is shown in SEQ ID NO.2; (2) Synthesis of the SpyTag-phage AP205 chimeric gene The SpyTag gene was linked to both ends of the phage AP205 gene via flexible spacers GSGTAGGGSGS and GGSGGSG, respectively, forming the SpyTag-GSGTAGGGSGS-AP205-GGSGGSG-SpyTag structure. The chimeric gene was optimized for codon bias using biological software, and BamH1 and Xho1 specific restriction sites were introduced at the 5´- and 3´- ends of the gene, named 2×SpyTag / AP205. The nucleotide sequence of the optimized chimeric gene is shown in SEQ ID NO.

3. (3) Gene cloning and recombinant protein expression and purification SPC-B4T and 2×SpyTag / AP205 were digested with BamH1 and Xhol enzymes, respectively, purified and recovered, and then inserted into pET-28a(+) linearized with the same enzymes to construct recombinant expression plasmids pET-28 / SPC-B4T and pET-28 / 2×SpyTag / AP205. These plasmids were then transformed into JM109 competent cells, and positive recombinant expression plasmids were identified by resistance selection, double enzyme digestion and sequence analysis. The two positive recombinant expression plasmids pET-28 / SPC-B4T and pET-28 / 2×SpyTag / AP205 were transformed into BL21(DE3)pLysS using heat-activated transformation. Single clones were selected and inoculated into 5 ml of LB medium containing kanamycin and cultured overnight at 37°C and 220 rpm. The overnight culture was then added at 1% (V / V) to freshly prepared sterile LB medium containing kanamycin and cultured at 37°C and 220 rpm until OD200. 600nm When the concentration of protein is 0.4~0.6, 0.4 mM IPTG is added under aseptic conditions in a laminar flow hood, and expression is induced at 37℃ for 4~6 hours. The culture is harvested by centrifugation at 2000 rpm for 30 min, and protein lysis buffer is added at 20% of the original culture volume. The protein is then sonicated and centrifuged at 20000g for 20 min to collect the supernatant. The precipitate is discarded. The protein is purified according to the instructions of the Ni-NTA histidine purification column. The purified proteins are named SPC-B4T and 2STAP205, respectively. The amino acid sequence of the recombinant protein 2STAP205 is shown in SEQ ID NO.

4. (4) Assembly of O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled virus-like particles The SPC-B4T and 2STAP205 proteins obtained by affinity chromatography purification were mixed at a mass ratio of 2-5:1 and assembled at room temperature or 4°C. The SPC-B4T and 2STAP205 proteins were assembled into the O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled virus-like particles through the self-assembly system.

3. The use of the O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled virus-like particles as described in claim 1 in the preparation of a vaccine for the prevention of O-type foot-and-mouth disease.

4. A multi-epitope biomimetic nano-self-assembled virus-like particle vaccine for type O foot-and-mouth disease virus, characterized in that, The vaccine contains the O-type foot-and-mouth disease virus multi-epitope biomimetic nano-self-assembled virus-like particles as described in claim 1, and an adjuvant.

5. The virus-like particle vaccine as described in claim 4, characterized in that, The adjuvant is ISA206.

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