O-type foot-and-mouth disease virus polyepitope virus-like particle nanogen and preparation method and application thereof

Abstract

The application discloses an O-type foot-and-mouth disease virus multi-epitope virus-like particle nano-antigen and a preparation method and application thereof. The nano-antigen is obtained by sequentially connecting antigen epitopes of three topological representative strains O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017 and O / HKN / 2007 of the O-type foot-and-mouth disease virus in sequence, introducing a T cell epitope of a 3A gene of the foot-and-mouth disease virus at a C terminal, and finally inserting a woodchuck hepatitis B virus core antigen, and an amino acid sequence of the nano-antigen is shown as SEQ ID NO. 6. Immunological test results show that the nano-antigen has good antigenicity, a vaccine prepared from the nano-antigen can not only induce high-level protective antibodies, but also protect immunized animals against virus attack. In addition, the nano-antigen has immunoprotective effects on the O-type foot-and-mouth diseases of pigs, cattle and sheep. The application provides an effective technical means for preventing and controlling and purifying the foot-and-mouth disease.

Description

Technical Field

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

[0002] Foot-and-mouth disease (FMD) is an acute infectious disease caused by the foot-and-mouth disease virus (FMDV). This disease primarily affects livestock farming, leading to reduced production in cloven-hoofed animals such as pigs, cattle, and sheep, resulting in severe economic losses. The World Organization for Animal Health (WOAH) lists it as a notifiable animal infectious disease and it is a mandatory quarantine disease for international customs clearance of animals and animal products. In my country, it is classified as a Class A animal disease.

[0003] my country primarily employs a comprehensive prevention and control strategy for foot-and-mouth disease (FMD), combining prevention with immunization and culling. Vaccination remains the most effective and economical means of FMD control in my country. Traditional inactivated FMD vaccines have made indelible contributions to global FMD control, but their research and production require the use of live viruses, posing potential biosafety risks. Therefore, developing a novel, biosafe, environmentally friendly, and diagnostically sound FMD vaccine for disease eradication is crucial. The development and refinement of genetic engineering and molecular biology technologies, along with the use of reverse vaccinology techniques to develop genetically engineered vaccines, particularly virus-like particle (VLP) vaccines and multi-epitope VLP vaccines, have yielded remarkable achievements.

[0004] Virus-like particles (VLPs), morphologically and structurally similar to natural virus particles, possess strong immunogenicity and biologically active antigens. Compared to traditional vaccines, VLPs do not contain viral nucleic acid, resulting in higher safety. The assembled VLPs can accurately and densely display the protective antigens of the target virus, triggering a strong T-cell and B-cell immune response after immunization. Since Merck successfully produced hepatitis B VLP antigen using Saccharomyces cerevisiae in 1986, developing vaccines or using VLPs as drug delivery systems has become a hot research topic in the biomedical field. VLPs are also considered the most likely new type of vaccine to replace traditional inactivated vaccines in the future.

[0005] With the development of theories and technologies in molecular biology, bioinformatics, molecular immunology, reverse vaccinology, and structural vaccinology, various VLP vaccines for pathogens have been developed using viral self-assembled proteins as components, including hepatitis B VLP vaccines, foot-and-mouth disease virus VLP vaccines, porcine circovirus VLP vaccines, and HPV VLP multivalent vaccines. Currently, with the discovery of more and more protective epitopes for pathogens, multi-epitope VLP vaccines are being designed using viral proteins from self-assembled VLPs or other naturally occurring self-assembled proteins as the backbone and epitopes as components. This strategy has significant advantages in vaccine design research for pathogens with multiple serotypes and subtypes, and where cross-immunoprotection cannot be provided between or within different topological strains of different serotypes. Although extensive research has been conducted on foot-and-mouth disease multi-epitope VLP vaccines based on this theory, demonstrating immunization effects, immunogenicity experiments are based on data obtained from immunized experimental animals such as guinea pigs or mice, and cannot be extrapolated to immunization effects in larger animals such as pigs, cattle, and sheep, and has not yet been applied in practice. However, using VLPs to display a limited number of antigenic epitopes (1-2 viruses) cannot address the genetic diversity and adaptability of highly variable pathogens, nor can it address the immunological polymorphism of vaccinated individuals; furthermore, expressing VLPs in the form of inclusion bodies and assembling them in vitro may result in new, antigen-independent structures.

[0006] The inventors previously designed a multi-epitope VLP antigen for type O foot-and-mouth disease virus (FMDV) using HBc protein as a backbone (CN116041547A). However, it was expressed as inclusion bodies in E. coli, requiring denaturation and renaturation to assemble into VLPs. Although VLP antigens were obtained, the renaturation process could not guarantee that the protein would assemble into structurally correct VLPs, resulting in low yields and increased production costs. The complex production process was also detrimental to industrial-scale production. Furthermore, the early-designed multi-epitope VLP antigen for FMDV only contained the epitopes of one strain of type O FMDV and one strain of type A FMDV. However, the identified type O FMDV in my country has three topotypes, and the type A topotype has formed two branches. This multi-epitope vaccine could no longer meet practical needs. In addition, the inventors also designed a monovalent multi-epitope VLP vaccine and a bivalent A / O multi-epitope VLP vaccine based on the phage AP205 self-assembly protein, targeting the prevalent type O and type A FMDV in my country. It can protect animals from highly toxic attacks, but the yield of soluble VLPs is low, purification requires the removal of endotoxins, and the production cost is high.

[0007] To address the fact that epitope-based VLP vaccine research is still in the laboratory stage and has not yet solved common key technologies hindering vaccine industrialization, such as stable and efficient assembly of antigenic epitope VLPs, broad antigen spectrum, and production processes, the inventors utilized HBc protein, which can be expressed and assembled into VLPs at a high level in eukaryotic and prokaryotic expression systems, as a backbone. Using antigenic epitopes from all topological representative strains of type O foot-and-mouth disease virus isolated in my country as components, bioinformatics software was used to optimize epitope length, tandem sequence, linker composition and length, and to perform structural prediction and immunological functional evaluation. Various VLPs with different structural forms were designed, and recombinant antigens were expressed and purified. Through structural identification, immunological functional evaluation, and immunogenicity assays, a recombinant expression plasmid and its cell line capable of secreting and expressing type O foot-and-mouth disease virus multi-epitope VLP antigens in CHO cells were screened. The expressed VLP antigens showed regular and uniform morphology under electron microscopy and could protect animals from viral infection. More importantly, compared with the early type O foot-and-mouth disease virus multi-epitope VLP antigen (CN116041547A), VLP is secreted expression, with better morphology, structure and homogeneity, higher yield and simpler production process, which is conducive to large-scale production. This is of great significance for the prevention and eradication of foot-and-mouth disease. Summary of the Invention

[0008] The purpose of this invention is to provide a multi-epitope virus-like particle nanoantigen of type O foot-and-mouth disease virus, its preparation method, and its application.

[0009] To achieve the above objectives, the present invention employs the following technical means:

[0010] First, this invention discloses a multi-epitope virus-like particle nanoantigen of type O foot-and-mouth disease virus. The multi-epitope virus-like particle nanoantigen of type O foot-and-mouth disease virus is formed by sequentially tandemly selecting the antigenic epitopes of representative strains of three topotypes of type O foot-and-mouth disease virus: O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017, and O / HKN / 2007, to form a tandem structure of five strains: O / Tibet / CHA / 99-O / Mya98 / BY / 2010-OZK / 93-O / XJPS / CHA / 2017-O / HKN / 2007, named OB5, and its amino acid sequence is shown in SEQ ID. As shown in NO.2; then, the T-cell epitope of the foot-and-mouth disease virus 3A gene was introduced into the C-terminus of OB5, and linked with GS spacers to form an antigen fragment containing 5 B-cell epitopes and 1 T-cell epitope, named OB5T; finally, the obtained antigen fragment OB5T was inserted into the amino acid sequence of the core protein of woodchuck hepatitis B virus between positions 78 and 81 through a linker.

[0011] Preferably, the amino acid sequence of the antigen fragment OB5T is shown in SEQ ID NO.4; the linker sequence is GGGGSGGGG; and the GenBank accession number of the prairie hepatitis B virus core protein encoding gene is J02442.

[0012] Preferably, the amino acid sequence of the O-type foot-and-mouth disease virus multi-epitope virus-like particle antigen is as shown in SEQ ID NO.6.

[0013] Preferably, the type O foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen is obtained by transferring its encoding gene into CHO cells and then having it secreted and expressed by the CHO cells.

[0014] Preferably, the CHO cells are CHO-S cells.

[0015] The nucleic acid encoding the O-type foot-and-mouth disease virus multi-epitope virus-like particle antigen is also within the scope of protection of this invention. Preferably, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.5.

[0016] Secondly, this invention also proposes a method for preparing and purifying the O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen, comprising the following steps:

[0017] (1) Synthesis of the gene for the multi-epitope VLP antigen of type O foot-and-mouth disease virus

[0018] Based on the VP1 gene sequences of representative strains of the three O-type topologies, O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017, and O / HKN / 2007, linear antigenic epitopes of protective antibodies against foot-and-mouth disease virus (FMDV), specifically the VP1 gene coding region (segment 135-160), were selected and sequentially tandemly. GS spacers were introduced between adjacent epitopes to form a tandem epitope structure of five strains: O / Tibet / CHA / 99-O / Mya98 / BY / 2010-OZK / 93-O / XJPS / CHA / 2017-O / HKN / 2007, named OB5. Its encoding nucleotide sequence is shown in SEQ ID. As shown in NO.1; a T-cell epitope of the foot-and-mouth disease virus 3A gene was introduced into the C-terminus of OB5 and linked with a GS spacer to form an antigen fragment containing 5 B-cell epitopes and 1 T-cell epitope, named OB5T, whose encoding nucleotide sequence is shown in SEQ ID NO.3;

[0019] (2) Construction of OB5T-Hbc chimeric recombinant expression plasmid

[0020] The OB5T encoding nucleotide sequence obtained in step (1) was inserted between nucleotides 234-240 of the core protein encoding gene of marmot hepatitis B virus using a linker to obtain a chimeric gene encoding the multi-epitope VLP antigen of type O foot-and-mouth disease virus, named OB5T-HBc. The linker sequence is GGGGSGGGG. The codons of the chimeric gene OB5T-HBc were optimized based on the codon bias of CHO cells. The nucleotide sequence of the optimized OB5T-HBc chimeric gene is shown in SEQ ID NO.5. EcoRI and NotRI restriction sites were introduced at the Nʹ- and Cʹ-termini of the OB5T-HBc sequence, respectively, and a stop codon was introduced at the N-terminus of the NotRI sequence. Then, it was inserted into pcDNA3.1(+) to construct the recombinant expression plasmid pcDNA3.1 / OB5T-HBc.

[0021] (3) Expression and purification of multi-epitope VLP antigen of type O foot-and-mouth disease virus

[0022] The recombinant expression plasmid pcDNA3.1 / OB5T-HBc was transfected into CHO-S cells. The cells were then cultured on a track shaker at 37°C and 8% CO2. The cell supernatant was collected 3-10 days after transfection and centrifuged at 4°C and 4000g for 30 min to obtain the O-type foot-and-mouth disease virus multi-epitope VLP antigen, which was named CHO-VLP.

[0023] (4) Purification of multi-epitope VLP antigen of type O foot-and-mouth disease virus

[0024] Purification was performed using a sucrose density gradient centrifugation method. The specific procedure was as follows: a sucrose density gradient of 15% w / v, 25% w / v, 35% w / v, and 45% w / v was prepared using TNE solution. The samples were centrifuged at 35,000 r / min for 3 h at 4 °C. Samples were collected sequentially from the top layer in fractions of 0.5 mL. All samples were then analyzed using a UV spectrophotometer at 280 nm. nm and 260 nm The antigen content was detected and calculated, and the sample was stored at -20°C.

[0025] Preferably, the GenBank accession number for the core protein encoding gene of the marmot hepatitis B virus is J02442.

[0026] Furthermore, this invention also proposes the application of the aforementioned O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen in the preparation of drugs for preventing O-type foot-and-mouth disease virus infection.

[0027] Preferably, the drug is a virus-like particle vaccine.

[0028] Finally, the present invention also proposes an O-type foot-and-mouth disease multi-epitope virus-like particle vaccine, wherein the vaccine contains the O-type foot-and-mouth disease virus multi-epitope virus-like particle antigen and adjuvant as described in any of the above.

[0029] Preferably, the adjuvant is ISA201 VG.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] This invention discloses an O-type foot-and-mouth disease virus-like particle (VLP) nanoantigen, its preparation method, and its application. The O-type foot-and-mouth disease virus multi-epitope VLP is prepared by sequentially tandemly selecting antigenic epitopes from representative strains of three topotypes of O-type foot-and-mouth disease virus: O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017, and O / HKN / 2007, and inserting them between nucleotides 234-240 of the gene encoding the core protein (HBc) of marmot hepatitis B virus, forming a chimeric DNA of O-type foot-and-mouth disease virus multi-epitope DNA and the HBc gene. The recombinant protein is expressed through secretion by CHO cells, thus forming the O-type foot-and-mouth disease virus-like particle (VLP) nanoantigen. Immunological experiments show that this VLP nanoantigen has good antigenicity and is not significantly different from inactivated O-type foot-and-mouth disease virus antigen, and can replace inactivated foot-and-mouth disease virus antigen in the establishment of detection methods. Animal efficacy experiments showed that the vaccine made with this VLP nanoantigen not only induced high levels of protective antibodies but also protected immunized animals against viral attack. Furthermore, the VLP nanoantigen also elicited an immune response with positive sera from cattle and sheep immunized with an inactivated type O foot-and-mouth disease virus vaccine, with the response intensity being indistinguishable from that of positive swine sera. In addition, the epitopes displayed by the VLP nanoantigen cover all type O topotypes isolated in my country, and the HBc cells displaying the antigenic epitopes do not exhibit species bias. Therefore, the VLP vaccine developed in this invention provides immunoprotection against type O foot-and-mouth disease in pigs, cattle, and sheep.

[0032] Furthermore, this invention utilizes a CHO system to express recombinant antigens in a secretory form. The VLP nanoantigens are approximately 32 nm in size, similar to natural virus particles. Therefore, purification of this antigen only requires continuous gradient centrifugation of the culture medium supernatant to obtain the recombinant antigen, eliminating the need for endotoxin removal. This not only reduces the loss of recombinant antigens but also simplifies the production process, enabling industrial-scale production. Compared to the earlier type O foot-and-mouth disease virus multi-epitope VLP antigen (CN116041547A), the type O foot-and-mouth disease virus-like particle nanoantigen of this invention is expressed secretoriously, exhibiting better morphology and homogeneity, higher yield, and a simpler production process, which is beneficial for large-scale production. This is of great significance for the prevention and eradication of foot-and-mouth disease. Attached Figure Description

[0033] Figure 1 Western blot results of type O foot-and-mouth disease virus multiepitope VLP nanoantigen CHO-VLP in culture supernatant at different time points after CHO transfection;

[0034] Among them, 1 is protein Maker, and 2-12 are CHO-VLP in the supernatant of the culture medium from day 0 to day 10 after transfection with CHO;

[0035] Figure 2Western blot results for CHO-VLP, a multi-epitope VLP nanoantigen of type O foot-and-mouth disease virus;

[0036] A: Western blot results of CHO-VLP and porcine O-type foot-and-mouth disease positive serum; B: Western blot results of CHO-VLP and O-type foot-and-mouth disease VP1 protein GH ring monoclonal antibody; where 1 is protein marker and 2 is purified CHO-VLP.

[0037] Figure 3 DLS results for the purification of the multi-epitope VLP nanoantigen CHO-VLP of type O foot-and-mouth disease virus;

[0038] Figure 4 Transmission electron microscopy results for the purification of the CHO-VLP multi-epitope nanoantigen of type O foot-and-mouth disease virus;

[0039] Figure 5 The results of the reaction between porcine foot-and-mouth disease virus type O multiepitope VLP nanoantigen CHO-VLP and positive and negative serum of porcine foot-and-mouth disease virus type O;

[0040] Figure 6 The results show the reactivity of the O-type foot-and-mouth disease virus multi-epitope VLP nanoantigen CHO-VLP with positive sera from pigs, cattle, and sheep. Detailed Implementation

[0041] To illustrate the above-mentioned objectives and features of the present invention in detail, the specific embodiments of the present invention will be further described below with reference to the accompanying drawings. However, the present invention is not limited to the following embodiments.

[0042] Example 1: Synthesis and expression of the multi-epitope VLP nanoantigen chimeric gene of type O foot-and-mouth disease virus.

[0043] 1. Design of chimeric gene DNA for O-type foot-and-mouth disease virus multi-epitope VLP antigen

[0044] Based on the amino acid sequences encoded by the VP1 gene of the representative strains of the three O-type topologies (O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017, and O / HKN / 2007), the linear antigenic epitopes of the protective antibodies against foot-and-mouth disease virus, namely the sequence of the VP1 gene coding region 135-160, were selected and sequentially tandemly. GS spacers were introduced between adjacent epitopes to form a tandem epitope structure of 5 strains: O / Tibet / CHA / 99-O / Mya98 / BY / 2010-OZK / 93-O / XJPS / CHA / 2017-O / HKN / 2007, named OB5. Its encoded nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. In addition, a T-cell epitope of the foot-and-mouth disease virus 3A gene was introduced into the C-terminus of OB5 and linked with GS to form an antigen fragment containing 5 B-cell epitopes and 1 T-cell epitope, named OB5T, whose encoding nucleotide sequence is shown in SEQ ID NO.3 and amino acid sequence is shown in SEQ ID NO.4.

[0045] 2. Construction of OB5T-Hbc chimeric recombinant expression plasmid

[0046] The OB5T DNA fragment designed above was inserted between nucleotides 234-240 (i.e., amino acids 78-81) of the core protein encoding gene of prairie hepatitis B virus (GenBank accession number: J02442) using a linker (GGGGSGGGG) sequence. A chimeric gene encoding the multi-epitope VLP antigen of type O foot-and-mouth disease virus was designed and named OB5T-HBc. The codons of the chimeric gene were optimized based on the codon bias of Chinese hamster ovary cells (CHO). The nucleotide sequence of the optimized OB5T-HBc chimeric gene is shown in SEQ ID NO. 5. To ensure directional insertion of this DNA, EcoRI and NotI restriction enzyme sites were introduced at the Nʹ- and Cʹ-termini of the OB5T-HBc chimeric gene, respectively, and a stop codon was introduced at the N-terminus of the NotI sequence to ensure the integrity of the recombinant antigen. This was then inserted into pcDNA3.1(+) to construct the recombinant expression plasmid pcDNA3.1 / OB5T-HBc. The synthesis and sequencing verification were commissioned to Nanjing GenScript Co., Ltd.

[0047] 3. Cell Culture

[0048] One vial of CHO-S cells was removed from liquid nitrogen, thawed in a 37°C water bath, and then all cells were transferred to a container containing 30 mL of ExpiCHO. TMSterile ventilated Erlenmeyer flasks (125 mL) of expression medium (Gibco) were placed in an incubator with 8% CO2 and 80% relative humidity at 37°C and 120 rpm for 3 days. Afterward, the viable cell density and survival rate percentage were measured. Cells were considered viable when the survival rate was greater than 90% and the cell density reached 4–6 × 10⁻⁶. 6 When the number of live cells / mL is 2–3 × 10⁻⁶, follow the dosage of 2– 7 Passage culture at an inoculation density of 1 live cells / mL.

[0049] 4. Plasmid extraction

[0050] The recombinant expression plasmid pcDNA3.1 / OB5T-HBc was transformed into DH5α competent cells using a heat shock method. The cells were then inoculated into LAB solid medium (Amp+) and cultured overnight at 37°C. Single colonies were selected and inoculated into LB medium (Amp+) and cultured overnight at 37°C and 220 rpm. The overnight culture was then inoculated into 300 mL of fresh LB medium (Amp+) at a 1:1000 ratio and cultured for another 16 h. Plasmids (Novizan) were extracted according to the instructions of the endotoxin-free plasmid extraction kit and sequenced for verification by Shanghai Sangon Biotech Co., Ltd.

[0051] 5. Expression and purification of type O foot-and-mouth disease virus multi-epitope VLP nanoantigen (CHO-VLP)

[0052] Transfection method: On the day of transfection, measure the CHO-S viable cell density and survival rate percentage. The CHO-S cell density should reach approximately 7 × 10⁻⁶ cells / year. 6 –1×10 7 CHO-S cells should be kept at a viable cell / mL density of 95–99% before further transfection. Dilute CHO-S cells to a final density of 6 × 10⁶ cells / mL using fresh, preheated (37°C) ExpiCHO™ expression medium. 6 1 live cells / mL, mix well. Adjust the final recombinant expression plasmid pcDNA3.1 / OB5T-HBc concentration to 0.5–1.0 μg / mL culture volume. Dilute the recombinant expression plasmid pcDNA3.1 / OB5T-HBc and ExpiFectamine™ CHO reagent separately using cold OptiPRO™ medium, gently pipetting 2–3 times to mix. Add the diluted ExpiFectamine™ CHO reagent to the diluted recombinant expression plasmid pcDNA3.1 / OB5T-HBc and mix well. Incubate the ExpiFectamine™ CHO and plasmid complex at room temperature for 2 minutes, then transfer the mixture to a culture flask, gently agitating the flask during addition. Incubate the cells on a track shaker (37°C, 8% CO2).

[0053] Supernatants from cultured cells were collected from days 0 to 10 post-transfection and centrifuged at 4°C and 4000g for 30 min. 60 µl of supernatant from each sample was mixed with 20 µl of 4×SDS-PAGE Protein Loading buffer, boiled in water for 10 min, and then subjected to SDS-PAGE electrophoresis. The mixture was transferred to a PVDF membrane, blocked with PBS containing 5% skim milk powder for 2 h, and washed three times with TBST. An appropriate amount of porcine foot-and-mouth disease type O positive serum (1:500) was added, and the mixture was incubated overnight at 4°C, followed by three TBST washes for 5 min each. HRP-labeled goat anti-porcine IgG antibody (1:5000) was added, and the mixture was incubated at room temperature for 1 h, followed by three TBST washes for 5 min each. Chemiluminescence reagent (Luminol) was added to detect recombinant protein expression.

[0054] The results showed that the expressed type O foot-and-mouth disease virus (FMDV) multi-epitope VLP nanoantigen could be recognized by FMDV type O positive serum and GH loop monoclonal antibody against type O FMDV VP1 protein. The nanoantigen was approximately 34 kDa, consistent with the expected size. It was detectable in the culture supernatant 72 hours after transfection, and the expression level gradually increased with increasing culture time, reaching its maximum on day 8. High levels of secreted antigen were still detectable in the supernatant on day 10. Figure 1 ).

[0055] In addition, sucrose density gradients of 15% w / v, 25% w / v, 35% w / v, and 45% w / v were prepared using TNE solution. These gradients were centrifuged at 35,000 r / min for 3 h at 4 °C. Samples were collected sequentially from the top layer, with each fraction consisting of 0.5 mL fractions (one sample). A total of 24 fractions were collected, and all samples were analyzed by UV spectrophotometer at 280 nm. nm and 260 nm The antigen content was detected and calculated, and stored at -20℃ for later use. The purified type O foot-and-mouth disease virus multi-epitope VLP nanoantigen was subjected to Western blotting (WB) experiments with porcine type O positive serum (1:500) and / or a laboratory-prepared monoclonal antibody targeting the GH ring, the major antigenic epitope of type O foot-and-mouth disease virus VP1. The results showed that the purified type O foot-and-mouth disease virus multi-epitope VLP nanoantigen could induce an immune response with type O foot-and-mouth disease virus positive serum and the monoclonal antibody targeting the GH ring antigenic epitope of type O foot-and-mouth disease virus VP1 protein, indicating that the antigenic epitope was correctly displayed. Figure 2 The purified type O foot-and-mouth disease virus multi-epitope VLP nanoantigen was named CHO-VLP, and its amino acid sequence is shown in SEQ ID NO.6.

[0056] 6. Identification of the morphology and size of O-type foot-and-mouth disease virus multi-epitope VLP nanoantigen (CHO-VLP)

[0057] The cell culture supernatant was collected and filtered through a 0.22 μm filter. 80 g / L PEG6000 and 40 g / L NaCl were added to the solution, and the mixture was stirred thoroughly overnight at 4°C. The overnight solution was centrifuged at 8000 rpm for 30 min, the supernatant was discarded, and the precipitate was resuspended in PBS (pH 7.4). The precipitate was transferred to a centrifuge tube containing 30% sucrose (RNase-free) and centrifuged at 35000 rpm for 3 h. The supernatant was collected and stored at 2–8°C for later use.

[0058] Sucrose density gradients of 15% w / v, 25% w / v, 35% w / v, and 45% w / v were prepared using TNE solution. The samples were centrifuged at 35,000 r / min for 3 h at 4 °C. Samples were collected sequentially from the top layer in fractions of 0.5 mL each, for a total of 24 fractions. All samples were analyzed using a UV spectrophotometer at 280 nm. nm and 260 nm The antigen content was detected, calculated, and stored at -20℃ for later use.

[0059] Dynamic light scattering (DLS) was used to determine the particle size and distribution under liquid conditions. The DLS results showed that the particle size of the O-type foot-and-mouth disease virus multi-epitope nanoparticles (VLPs) was approximately 32 nm, and they exhibited good homogeneity. Figure 3 The sample, purified by sucrose density gradient, was dropped onto a carbonized 300-mesh copper grid for adsorption for 10 minutes, then negatively stained in 1% phosphotungstic acid solution for 30 seconds. Excess stain was removed, and the sample was dried. The morphology and structure of the type O foot-and-mouth disease virus multi-epitope VLP nanoantigen were observed using transmission electron microscopy. The results showed that the type O foot-and-mouth disease virus multi-epitope VLP nanoantigen exhibited typical VLP structural characteristics under transmission electron microscopy, and the particle size was consistent with the dynamic light scattering results. Figure 4 ).

[0060] Example 2: Immunological Function Identification of Multi-Epitope VLP Nanoantigen of Type O Foot-and-Mouth Disease Virus

[0061] To verify the immunomodulatory activity of the O-type foot-and-mouth disease virus multi-epitope VLP nanoantigen (CHO-VLP) prepared in Example 1, 96-well ELISA plates were coated with O-type foot-and-mouth disease virus inactivated antigen (100 μl / well, 1 μg / mL) and purified CHO-VLP recombinant antigen, respectively, and incubated overnight at 4°C. The plates were then blocked with PBST (pH 7.4) containing 5% skim milk powder at 37°C for 2 h, washed three times with PBST, and 100 μl of 1:100 diluted porcine O-type foot-and-mouth disease virus positive serum was added per well. Porcine foot-and-mouth disease negative serum was also included as a control. The plates were incubated at 37°C for 45 min, washed three times with PBST, and 100 μl of 1:5000 diluted HRP-labeled goat anti-porcine IgG was added per well. The plates were incubated at 37°C for 30 min, washed three times with PBST, and 100 μl of TMB chromogenic solution was added per well. The plates were incubated at 37°C for 10-15 min, and the reaction was terminated with 2M concentrated H2SO4. The OD was then measured. 450nm The absorbance value.

[0062] The results showed that the CHO-VLP recombinant antigen could elicit a strong immune response with positive porcine foot-and-mouth disease (FMD) serum of type O, and its immunoreactivity was not significantly different from that of inactivated FMD virus antigen of type O. Furthermore, it did not elicit an immune response with negative porcine FMD serum. Figure 5 This fully demonstrates that the protective epitopes of type O foot-and-mouth disease virus in the CHO-VLP recombinant antigen were correctly displayed and its immune activity was fully exerted.

[0063] Furthermore, 96-well ELISA plates were coated with the O-type foot-and-mouth disease virus multi-epitope VLP nanoantigen and the O-type FMDV inactivated antigen developed in this invention, respectively. Positive sera from pigs, cattle, and sheep immunized with O-type FMDV were tested using the above method. The results showed that the O-type foot-and-mouth disease virus multi-epitope VLP nanoantigen CHO-VLP developed in this invention could induce a strong immune response with positive sera from pigs, cattle, and sheep, and was not significantly different from the O-type foot-and-mouth disease virus inactivated antigen. This was superior to the O-type foot-and-mouth disease multi-epitope VLP vaccine developed with the AP205 backbone disclosed in publication number CN116041547A. Figure 6 ).

[0064] Example 3: Preparation and Immunopotency Experiment of O-type Foot-and-Mouth Disease Virus Multiepitope VLP Vaccine

[0065] 1. Vaccine preparation

[0066] The purified type O foot-and-mouth disease virus multi-epitope VLP nanoantigen (CHO-VLP) from Example 1 was quantified using the BCA method and diluted with PBS to 200 µg / ml and 100 µg / ml. The mixture was then emulsified with water-in-oil-in-water adjuvant ISA201 VG (Seppic, France) at a ratio of 50 g to 50 g to form a vaccine formulation (W / O / W). Each dose contained 100 µg / ml and 50 µg / ml of VLP antigen, respectively.

[0067] 2. Immunopotency Test

[0068] Twelve pigs weighing approximately 30 kg were selected, exhibiting O-type foot-and-mouth disease virus antibody levels <1.4 (liquid-phase blocking ELISA) and negative for 3ABC protein antibodies (3ABC antibody chemiluminescence kit). Five pigs were each injected intramuscularly with 1 ml of two different vaccine formulations, with two unvaccinated pigs serving as negative controls. Blood samples were collected before immunization and 28 days post-immunization, and serum was separated for the detection of specific antibody IgG. Twenty-eight days post-immunization, the pigs, along with the two controls, were infected with a virulent strain of O-type foot-and-mouth disease virus according to national standards, and observed for 10 days. Results showed that the O-type foot-and-mouth disease virus multi-epitope VLP vaccine induced high levels of foot-and-mouth disease-specific antibodies in immunized pigs, providing protection against O-type foot-and-mouth disease virus infection after challenge (Table 1). Furthermore, no redness, swelling, or fever occurred at the injection site in the vaccinated animals, and no other adverse reactions were observed. The animals maintained normal appetite and good mental state, confirming the vaccine's safety.

[0069] .

Claims

1. A multi-epitope virus-like particle nanoantigen of type O foot-and-mouth disease virus (FMDV), characterized in that, The aforementioned O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen is formed by sequentially tandemly selecting the antigenic epitopes of representative strains of three topotypes of O-type foot-and-mouth disease virus: O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017, and O / HKN / 2007, to form a tandem epitope structure of five strains: O / Tibet / CHA / 99-O / Mya98 / BY / 2010-OZK / 93-O / XJPS / CHA / 2017-O / HKN / 2007, named OB5, and its amino acid sequence is shown in SEQ ID NO.2; Then, a T-cell epitope of the foot-and-mouth disease virus 3A gene was introduced into the C-terminus of OB5 and linked with a GS spacer to form an antigen fragment containing 5 B-cell epitopes and 1 T-cell epitope, named OB5T; finally, the obtained antigen fragment OB5T was inserted into the amino acid sequence of the core protein of woodchuck hepatitis B virus between positions 78 and 81 using a linker. The amino acid sequence of the O-type foot-and-mouth disease virus multi-epitope virus-like particle antigen is shown in SEQ ID NO.

6.

2. The O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen as described in claim 1, characterized in that, The aforementioned type O foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen was obtained by transferring its encoding gene into Chinese hamster ovary cells (CHO) and then having it secreted and expressed by the CHO cells.

3. The O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen as described in claim 2, characterized in that, The CHO cells mentioned are CHO-S cells.

4. The nucleic acid encoding the O-type foot-and-mouth disease virus multi-epitope virus-like particle antigen as described in any one of claims 1-3.

5. The nucleic acid as described in claim 4, characterized in that, The nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

5.

6. A method for preparing and purifying O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Synthesis of the gene for the multi-epitope VLP antigen of type O foot-and-mouth disease virus Based on the VP1 gene sequences of representative strains of the three O-type topologies, O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017, and O / HKN / 2007, linear antigenic epitopes of protective antibodies against foot-and-mouth disease virus (FMDV), specifically the VP1 gene coding region (segment 135-160), were selected and sequentially tandemly. GS spacers were introduced between adjacent epitopes to form a tandem epitope structure of five strains: O / Tibet / CHA / 99-O / Mya98 / BY / 2010-OZK / 93-O / XJPS / CHA / 2017-O / HKN / 2007, named OB5. Its encoding nucleotide sequence is shown in SEQ ID. As shown in NO.1; a T-cell epitope of the foot-and-mouth disease virus 3A gene was introduced into the C-terminus of OB5 and linked with a GS spacer to form an antigen fragment containing 5 B-cell epitopes and 1 T-cell epitope, named OB5T, whose encoding nucleotide sequence is shown in SEQ ID NO.3; (2) Construction of OB5T-Hbc chimeric recombinant expression plasmid The OB5T encoding nucleotide sequence obtained in step (1) was inserted between nucleotides 234-240 of the core protein encoding gene of marmot hepatitis B virus using a linker to obtain a chimeric gene encoding the multi-epitope VLP antigen of type O foot-and-mouth disease virus, named OB5T-HBc. The linker sequence is GGGGSGGGG. The codons of the chimeric gene OB5T-HBc were optimized based on the codon bias of CHO cells. The nucleotide sequence of the optimized OB5T-HBc chimeric gene is shown in SEQ ID NO.

5. EcoRI and NotRI restriction sites were introduced at the Nʹ- and Cʹ-termini of the OB5T-HBc sequence, respectively, and a stop codon was introduced at the N-terminus of the NotRI sequence. Then, it was inserted into pcDNA3.1(+) to construct the recombinant expression plasmid pcDNA3.1 / OB5T-HBc. (3) Expression and purification of multi-epitope VLP antigen of type O foot-and-mouth disease virus The recombinant expression plasmid pcDNA3.1 / OB5T-HBc was transfected into CHO-S cells. The cells were then cultured on a track shaker at 37°C and 8% CO2. The cell supernatant was collected 3-10 days after transfection and centrifuged at 4°C and 4000g for 30 min to obtain the O-type foot-and-mouth disease virus multi-epitope VLP antigen, which was named CHO-VLP. (4) Purification of multi-epitope VLP antigen of type O foot-and-mouth disease virus Purification was performed using a sucrose density gradient centrifugation method. The specific procedure was as follows: a sucrose density gradient of 15% w / v, 25% w / v, 35% w / v, and 45% w / v was prepared using TNE solution. The samples were centrifuged at 35,000 r / min for 3 h at 4 °C. Samples were collected sequentially from the top layer in fractions of 0.5 mL. All samples were then analyzed using a UV spectrophotometer at 280 nm. nm and 260 nm The antigen content was detected and calculated, and the sample was stored at -20°C.

7. The method as described in claim 6, characterized in that, The GenBank accession number for the core protein encoding gene of the marmot hepatitis B virus is J02442.

8. The use of the O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen as described in any one of claims 1-3 in the preparation of a drug for preventing O-type foot-and-mouth disease virus infection.

9. The application as described in claim 8, characterized in that, The drug in question is a virus-like particle vaccine.

10. A type O foot-and-mouth disease multi-epitope virus-like particle vaccine, characterized in that, The vaccine contains the O-type foot-and-mouth disease virus multi-epitope virus-like particle antigen as described in any one of claims 1-3 and an adjuvant.

11. The type O foot-and-mouth disease multi-epitope virus-like particle vaccine as described in claim 10, characterized in that, The adjuvant is ISA201 VG.

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