O-type foot-and-mouth disease virus multi-epitope virus-like particle nano antigen as well as preparation method and application thereof

Abstract

The invention discloses an O-type foot and mouth disease virus multi-epitope virus-like particle nano antigen as well as a preparation method and application thereof. The nano antigen is obtained by sequentially connecting antigen epitopes of representative strains O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017 and O / HKN / 2007 of three topological types of the O-type foot and mouth disease virus in series, introducing a T cell epitope of a foot and mouth disease virus 3A gene to a C terminal and finally inserting the T cell epitope into a cast mouse hepatitis B virus core antigen, and the amino acid sequence of the nano antigen is as shown in SEQ ID NO.6. An immunological test result shows that the nano antigen has good antigenicity, and a vaccine prepared from the nano antigen not only can induce generation of a high-level protective antibody, but also can protect immune animals from virus attack. In addition, the nano antigen has an immune protection effect on O-type foot-and-mouth diseases of pigs, cattle and sheep. The invention provides an effective technical means for preventing, controlling and purifying the foot-and-mouth disease.

Description

Technical Field

[0001] The present invention relates to an O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen, a preparation method thereof and an application thereof. The present invention belongs to the field of medical technology. Background Art

[0002] Foot-and-mouth disease (FMD) is an acute infectious disease caused by foot-and-mouth disease virus (FMDV). This disease mainly harms livestock farming, and can lead to production reduction of cloven-hoofed animals such as pigs, cattle, sheep, etc., thus causing serious economic losses. The World Organization for Animal Health (WOAH) lists it as a legally notifiable animal infectious disease, and it is a disease that must be quarantined for international trade of animals and animal products. China lists it as a Class I animal disease. It is also one of the important animal diseases that plague the healthy and sustainable development of China's pig, cattle and sheep breeding industries.

[0003] China mainly adopts comprehensive prevention and control measures of prevention first, combination of immunization and culling for foot-and-mouth disease prevention and control. Vaccine immunization is still the most effective and economical means for foot-and-mouth disease prevention and control in China at present. Traditional inactivated foot-and-mouth disease vaccines have made indelible contributions to the global effective prevention and control of foot-and-mouth disease, but their research and production require the use of live viruses, posing potential biosafety risks. Therefore, it is particularly necessary to develop a new type of foot-and-mouth disease vaccine with biosafety, environmental friendliness and differential diagnosis for disease purification and eradication. With the development and improvement of genetic engineering technology and molecular biology technology, the use of reverse vaccinology technology to develop genetic engineering vaccines, especially the research and development of virus-like particle (VLP) vaccines and multi-epitope virus-like particle vaccines, etc. has achieved remarkable results.

[0004] Virus-like particles (VLPs), which are similar to natural virus particles in morphological structure, are antigens with strong immunogenicity and biological activity. Compared with traditional vaccines, VLPs do not contain viral nucleic acids and have high safety. The assembled VLPs can correctly display target virus protective antigens at high density, and can trigger strong T cell and B cell immune response reactions after immunization. Since Merck successfully produced hepatitis B VLP antigen using Saccharomyces cerevisiae in 1986, the development of vaccines using VLPs as antigens or as drug delivery systems has become a research hotspot in the biomedical field, and VLPs are also considered to be the most likely new vaccines 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 pathogen VLP vaccines have been developed using virus self-assembled proteins as components, including hepatitis B VLP vaccine, foot-and-mouth disease virus VLP vaccine, porcine circovirus VLP vaccine, HPV VLP multivalent vaccine, etc. Nowadays, with the discovery of more and more pathogen protective antigen epitopes, multi-epitope VLP vaccines are designed with self-assembled VLP viral proteins or other natural self-assembled proteins as the backbone and antigen epitopes as components. This strategy has obvious advantages for the vaccine design research of pathogens with multiple serotypes, multiple subtypes, and no cross-immune protection between different topological strains within or between serotypes. Although extensive research has been carried out on foot-and-mouth disease multi-epitope VLP vaccines based on this theory and shown immune effects, the immune efficacy experiments are all based on data obtained from immunized experimental animals guinea pigs or mice, and cannot be extrapolated to the immune effects of large animals such as pigs, cattle, and sheep, and have not been applied in practice yet. However, displaying a few antigen epitopes (1-2 types of viruses) with VLP can neither solve the genetic diversity and adaptability of highly variable pathogens nor the immunological polymorphism of vaccine recipients; in addition, expressing in the form of inclusion bodies and assembling VLP in vitro may form new structures unrelated to the antigen.

[0006] The inventor of the present invention has also designed a multi-epitope VLP antigen of foot-and-mouth disease virus type O using HBc protein as the backbone (CN116041547A), but it is expressed in the form of inclusion bodies in Escherichia coli, and the antigen needs to be denatured and renatured to assemble VLP. Although the VLP antigen is obtained, since the renaturation process cannot ensure that the protein can be assembled into VLP with the correct structure, the yield is not high, the production cost is increased, and the complex production process is not conducive to industrial production. In addition, the early designed foot-and-mouth disease multi-epitope VLP antigen only contains the antigen epitopes of 1 strain of foot-and-mouth disease virus type O and the antigen epitopes of 1 strain of foot-and-mouth disease virus type A. There are 3 topological types of foot-and-mouth disease virus type O and 2 branches formed by 1 topological type of type A identified in China. This multi-epitope vaccine can no longer meet the actual needs. In addition, the inventor of the present invention has also designed multi-epitope VLP monovalent vaccines and A, O multi-epitope VLP bivalent vaccines against foot-and-mouth disease virus type O and type A prevalent in China based on the self-assembled protein of phage AP205. It can protect animals against virulent attack, but the yield of soluble VLP is relatively low, and endotoxin needs to be removed during purification, resulting in a high production cost.

[0007] To address the issue that the research on epitope-based VLP vaccines is still in the laboratory stage, and the common key technologies restricting vaccine industrialization, such as the stable and efficient assembly of antigenic epitope VLPs, antigen broad-spectrum, and production processes, have not been resolved. The inventor of the present invention used the HBc protein that can be highly soluble expressed and assembled into VLPs in eukaryotic and prokaryotic expression systems as a backbone, and used the antigenic epitopes of all topological representative strains of foot-and-mouth disease virus type O isolated in China as elements. A series of optimizations were carried out on the epitope length, tandem order, linker composition and its length, etc. using bioinformatics software, and its structure prediction and immunological function evaluation were carried out. Multiple VLPs with different structural forms were designed, and recombinant antigens were expressed and purified. Through structure identification, immunological function evaluation and immune efficacy tests, a recombinant expression plasmid and its cell line that can secrete and express the foot-and-mouth disease virus type O multi-epitope VLP antigen in CHO cells were screened out. The expressed VLP antigen has regular and uniform morphology under the electron microscope and can protect animals from virus infection. More importantly, compared with the early foot-and-mouth disease virus type O multi-epitope VLP antigen (CN116041547A), the VLP is secreted and expressed, has better morphological structure and homogeneity, higher yield, and a simpler production process, which is conducive to large-scale production. This is of great significance for the prevention, control and purification of foot-and-mouth disease. Summary of the Invention

[0008] The object of the present invention is to provide a foot-and-mouth disease virus type O multi-epitope virus-like particle nano-antigen, its preparation method and application.

[0009] To achieve the above object, the present invention adopts the following technical means:

[0010] First, the present invention discloses an O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen. The O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen is obtained by sequentially concatenating the antigenic epitopes of representative strains of 3 topological types of O-type foot-and-mouth disease virus, namely O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017, and O / HKN / 2007, to form an epitope tandem structure of 5 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 is introduced at the C-terminus of OB5 and connected 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 is inserted between the 78th and 81st positions of the amino acid sequence of the core protein of Woodchuck hepatitis B virus through a linker.

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

[0012] Among them, preferably, 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.

[0013] Among them, preferably, the O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen is obtained by secreting and expressing it after transferring its encoding gene into CHO cells.

[0014] Among them, preferably, the CHO cell is a CHO-S cell.

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

[0016] Secondly, the present invention also proposes a preparation and purification method for the O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen, including the following steps:

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

[0018] Based on the VP1 gene sequences of the representative strains of 3 topological types of serotype O, namely O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017, and O / HKN / 2007, linear antigenic epitopes of foot-and-mouth disease virus protective antibodies were selected, that is, the sequence of the 135-160 segment in the coding region of the VP1 gene. They were sequentially concatenated in order, and a GS spacer was introduced between adjacent epitopes to form an epitope tandem structure of 5 strains: O / Tibet / CHA / 99 - O / Mya98 / BY / 2010 - OZK / 93 - O / XJPS / CHA / 2017 - O / HKN / 2007, named OB5, and its encoding nucleotide sequence is shown in SEQ ID NO.1; the T cell epitope of the foot-and-mouth disease virus 3A gene was introduced at the C-terminus of OB5 and connected with a GS spacer to form an antigen fragment containing 5 B cell epitopes and 1 T cell epitope, named OB5T, and its encoding nucleotide sequence is shown in SEQ ID NO.3;

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

[0020] The OB5T encoding nucleotide sequence obtained in step (1) was inserted between the 234th and 240th nucleotides of the marmot hepatitis B virus core protein coding gene through a linker to obtain a chimeric gene encoding a multi-epitope VLP antigen of serotype O foot-and-mouth disease virus, named OB5T-HBc. The sequence of the linker is GGGGSGGGG; the codons of the chimeric gene OB5T-HBc were optimized according to 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 NotⅠ restriction enzyme sites were introduced at the N'-terminus and C'-terminus of the OB5T-HBc sequence respectively, and a stop codon was introduced at the N-terminus of the NotⅠ sequence, and then it was inserted into pcDNA3.1(+), and the recombinant expression plasmid pcDNA3.1 / OB5T-HBc was constructed;

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

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

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

[0024] Purification was carried out by sucrose density gradient centrifugation. The specific operation was as follows: sucrose density gradients of 15% w / v, 25% w / v, 35% w / v, and 45% w / v were prepared with TNE solution, and centrifuged at 35000 r / min for 3 h at 4 °C. Samples were collected sequentially from the top layer in 0.5 mL fractions. All samples were detected by ultraviolet spectrophotometer at 280 nm and 260 nm to calculate the antigen content, and stored at -20 °C.

[0025] Among them, preferably, the GenBank accession number of the marmot hepatitis B virus core protein coding gene is: J02442.

[0026] Furthermore, the present invention also proposes the application of the above-mentioned O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen in the preparation of a drug for preventing O-type foot-and-mouth disease virus infection.

[0027] Among them, 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, and the vaccine contains the O-type foot-and-mouth disease virus multi-epitope virus-like particle antigen described in any one of the above and an adjuvant.

[0029] Among them, preferably, the adjuvant is ISA201 VG.

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

[0031] The present invention discloses an O-type foot-and-mouth disease virus-like particle nanoantigen, its preparation method and application. The O-type foot-and-mouth disease virus multi-epitope virus-like particle antigen is prepared by sequentially concatenating the antigenic epitopes of representative strains of 3 topotypes of O-type foot-and-mouth disease virus, namely O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017 and O / HKN / 2007, and inserting the resulting sequence between nucleotides 234-240 of the gene encoding the core protein (HBc) of woodchuck hepatitis B virus, thereby forming a chimeric DNA of O-type foot-and-mouth disease virus multi-epitope DNA and HBc gene. The recombinant protein is secreted and expressed by CHO cells, which is the O-type foot-and-mouth disease virus-like particle nanoantigen. The results of immunological tests show that the VLP nanoantigen has good antigenicity, and there is no significant difference between it and the inactivated O-type foot-and-mouth disease virus antigen, so it can replace the inactivated foot-and-mouth disease virus antigen to establish a detection method. The results of the immunization efficacy experiment on animals show that the vaccine prepared with this VLP nanoantigen can not only induce the production of high levels of protective antibodies, but also protect the immunized animals against virus attack. In addition, this VLP nanoantigen can also react with the positive sera of cattle and sheep immunized with inactivated O-type foot-and-mouth disease virus vaccine, and the reaction intensity is no different from that of porcine positive sera. Moreover, the epitopes displayed by this VLP nanoantigen cover all O-type topotypes isolated in China, and HBc displaying the antigenic epitopes has no species preference. Therefore, the VLP vaccine developed in the present invention has immunoprotective effects against O-type foot-and-mouth disease in pigs, cattle and sheep.

[0032] In addition, the present invention uses the CHO system to express the recombinant antigen in a secreted form. The size of the VLP nanoantigen is about 32 nm, similar to natural virus particles. Therefore, when purifying this antigen, only continuous gradient centrifugation of the culture medium supernatant is required to obtain the recombinant antigen, without the process of removing endotoxin. This not only reduces the loss of the recombinant antigen, but also simplifies the production process, enabling industrial production. Compared with the early O-type foot-and-mouth disease virus multi-epitope VLP antigen (CN116041547A), the O-type foot-and-mouth disease virus-like particle nanoantigen of the present invention is secreted and expressed, with better morphological structure and homogeneity, higher yield, and simpler production process, which is conducive to large-scale production and is of great significance for the prevention, control and purification of foot-and-mouth disease. Brief Description of the Drawings

[0033] Figure 1 WB results of O-type foot-and-mouth disease virus multi-epitope VLP nanoantigen CHO-VLP in the culture medium supernatant at different time points after transfection of CHO;

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

[0035] Figure 2WB results of the O-type foot-and-mouth disease virus multi-epitope VLP nano-antigen CHO-VLP;

[0036] Among them, A: WB results of CHO-VLP with porcine O-type foot-and-mouth disease positive serum; B: WB results of CHO-VLP with monoclonal antibody against the G-H loop of O-type foot-and-mouth disease VP1 protein; among them, 1 is the protein Maker, and 2 is the purified CHO-VLP;

[0037] Figure 3 DLS results of the purified O-type foot-and-mouth disease virus multi-epitope VLP nano-antigen CHO-VLP;

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

[0039] Figure 5 Reaction results of the O-type foot-and-mouth disease virus multi-epitope VLP nano-antigen CHO-VLP with porcine O-type foot-and-mouth disease virus positive and negative sera;

[0040] Figure 6 Reactivity results of the O-type foot-and-mouth disease virus multi-epitope VLP nano-antigen CHO-VLP with positive sera of pigs, cattle, and sheep. Specific implementation manners

[0041] To elaborate the above objects and features of the present invention in detail, the specific implementation manners of the present invention will be further described below in conjunction with the accompanying drawings of the specification, but the present invention is not limited to the following examples.

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

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

[0044] Based on the amino acid sequences encoded by the VP1 genes of the representative strains of 3 topotypes of serotype O (O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017, and O / HKN / 2007), the linear antigenic epitopes of foot-and-mouth disease virus protective antibodies were selected, namely the sequence of the 135-160 segment in the coding region of the VP1 gene. They were sequentially concatenated, and a GS spacer was introduced between adjacent epitopes to form an epitope concatenated structure of 5 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 NO.1, and the amino acid sequence is shown in SEQ ID NO.2. In addition, the T cell epitope of the foot-and-mouth disease virus 3A gene was introduced at 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. Its encoding nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

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

[0046] The DNA fragment of OB5T designed above was inserted between the 234th and 240th nucleotides (i.e., between the 78th and 81st amino acids of the amino acid sequence) of the core protein coding gene of woodchuck hepatitis B virus (GenBank accession number: J02442) through the linker (GGGGSGGGG) sequence to design a chimeric gene encoding the multi-epitope VLP antigen of serotype O foot-and-mouth disease virus, named OB5T-HBc. The codons of the chimeric gene were optimized according to the codon bias of Chinese hamster ovary (CHO) cells. The nucleotide sequence of the optimized OB5T-HBc chimeric gene obtained by synthesis is shown in SEQ ID NO.5. For the directional insertion of this DNA, EcoRⅠ and NotⅠ restriction enzyme sites were introduced at the N'-terminus and C'-terminus 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. Then it was inserted into pcDNA3.1(+), and the recombinant expression plasmid pcDNA3.1 / OB5T-HBc was constructed. It was commissioned to Nanjing Genscript Co., Ltd. for synthesis and sequencing verification

[0047] 3. Cell culture

[0048] Take out 1 vial of CHO-S cells from liquid nitrogen. After thawing the cells in a 37 °C water bath, transfer all the cells to a container containing 30 mL of ExpiCHO TMA sterile vented conical flask (125 mL) of expression medium (Gibco) was placed in an incubator at 8% CO2, 80% relative air humidity, cultured at 37 °C and 120 rpm for 3 days, and then the viable cell density and percentage of survival rate were measured. When the survival rate was greater than 90% and the cell density reached 4 - 6×10 6 viable cells / mL, subculture was carried out at an inoculation density of 2 - 3×10 7 viable cells / mL.

[0049] 4. Plasmid extraction

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

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

[0052] Transfection method: On the day of transfection, the viable cell density and percentage of survival rate of CHO-S cells were measured. The CHO-S cell density should reach about 7×10 6 –1×10 7 viable cells / mL, and the survival rate reached 95 - 99% before transfection could continue. The CHO-S cells were diluted to a final density of 6×10 TM viable cells / mL with fresh pre-warmed ExpiCHO 6 expression medium, and the cells were mixed evenly. The final concentration of the recombinant expression plasmid pcDNA3.1 / OB5T-HBc was 0.5 - 1.0 μg / mL of the culture volume. The recombinant expression plasmid pcDNA3.1 / OB5T-HBc and ExpiFectamine TM CHO reagent were respectively diluted with cold OptiPRO TM medium, and gently pipetted 2 - 3 times to mix evenly. The diluted ExpiFectamine TM CHO reagent was added to the diluted recombinant expression plasmid pcDNA3.1 / OB5T-HBc, and mixed evenly. The ExpiFectamine TM CHO and plasmid complex was incubated at room temperature for 2 minutes, and then the mixture was transferred to the culture flask, and the culture flask was gently shaken during the addition process. The cells were cultured on an orbital shaker (37 °C, containing 8% CO2).

[0053] Collect the culture cell supernatants at 0 - 10 days after transfection respectively, and centrifuge at 4000 g for 30 min at 4°C. Pipette 60 μl of the supernatant of each sample and mix it with 20 μl of 4×SDS-PAGE Protein Loading buffer. After boiling in water for 10 min, perform SDS-PAGE electrophoresis, and transfer it to a PVDF membrane. Block it with PBS containing 5% non-fat milk powder for 2 h, and wash it 3 times with TBST. Add an appropriate amount of porcine foot-and-mouth disease type O positive serum (1:500), incubate overnight at 4°C, wash it 3 times with TBST, 5 min each time. Add HRP-labeled goat anti-pig IgG antibody (1:5000), incubate at room temperature for 1 h, wash it 3 times with TBST, 5 min each time, add chemiluminescent reagent (luminol), and detect the expression of the recombinant protein.

[0054] The results showed that the expressed foot-and-mouth disease virus type O multi-epitope VLP nano-antigen could be recognized by foot-and-mouth disease virus type O positive serum and the monoclonal antibody against the G-H loop of foot-and-mouth disease virus type O VP1 protein. Its size was about 34 kDa, which was consistent with the expected size. Moreover, the antigen could be detected in the culture supernatant 72 hours after transfection. With the extension of the culture time, the expression level gradually increased. The protein expression level was the highest at 8 days, and a high level of secreted antigen could still be detected in the supernatant on the 10th day ( Figure 1 ).

[0055] In addition, prepare sucrose density gradients of 15% w / v, 25% w / v, 35% w / v, and 45% w / v with TNE solution, centrifuge at 35000 r / min for 3 h at 4°C. Collect samples sequentially from the top layer at 0.5 mL per fraction (for 1 sample), and collect a total of 24 samples. All samples were detected by ultraviolet spectrophotometer at 280 nm and 260 nm to calculate the antigen content, and store them at -20°C for later use. Perform WB experiments on the purified foot-and-mouth disease virus type O multi-epitope VLP nano-antigen with porcine foot-and-mouth disease type O positive serum (1∶500) and / or the monoclonal antibody against the main antigenic epitope G-H loop of foot-and-mouth disease virus type O prepared in the laboratory. The results showed that the purified foot-and-mouth disease virus type O multi-epitope VLP nano-antigen could have an immune reaction with foot-and-mouth disease virus type O positive serum and the monoclonal antibody against the G-H loop antigenic epitope of foot-and-mouth disease virus type O VP1 protein, indicating that the antigenic epitopes were correctly displayed ( Figure 2 ). The purified foot-and-mouth disease virus type O multi-epitope VLP nano-antigen was named CHO-VLP, and its amino acid sequence was as shown in SEQ ID NO.6.

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

[0057] After collecting the above cell culture supernatant and filtering it through a 0.22 μm filter, add 80 g / L of PEG6000 and 40 g / L of NaCl to the liquid, and stir well overnight at 4°C. Stir the overnight solution, centrifuge at 8000 r / min for 30 min, discard the supernatant, and resuspend the precipitate with PBS (pH 7.4). Transfer it into a centrifuge tube containing 30% sucrose (RNase-free), and centrifuge at 35000 r / min for 3 h. Collect the upper liquid and store it at 2 - 8°C for later use.

[0058] Prepare sucrose density gradients of 15% w / v, 25% w / v, 35% w / v, and 45% w / v with TNE solution, centrifuge at 4°C and 35000 r / min for 3 h. Take 0.5 mL as one fraction (for one sample), collect samples sequentially from the top layer, and collect a total of 24 samples. All samples are detected by ultraviolet spectrophotometer at 280 nm and 260 nm to calculate the antigen content, and store it at -20°C for later use.

[0059] Dynamic light scattering (DLS) was used to measure the particle size and distribution under liquid conditions. The DLS results showed that the particle size of the multi-epitope nano-VLP of foot-and-mouth disease virus type O was about 32 nm, and the homogeneity was good ( Figure 3 ). Drop the sample purified by sucrose density gradient onto a 300-mesh copper grid treated by carbonization and adsorb for 10 minutes, place it in 1% phosphotungstic acid solution for negative staining for 30 s, suck out the excess staining solution, and observe the morphological structure of the multi-epitope VLP nano-antigen of foot-and-mouth disease virus type O using a transmission electron microscope after drying. The results showed that the multi-epitope VLP nano-antigen of foot-and-mouth disease virus type O had typical VLP structural characteristics under the transmission electron microscope, and the particle size was consistent with the dynamic light scattering results ( Figure 4 ).

[0060] Example 2. Immunological function identification of the multi-epitope VLP nano-antigen of foot-and-mouth disease virus type O

[0061] To verify the immunological 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 inactivated O-type foot-and-mouth disease virus antigen (100 μl / well, 1 μg / mL) and purified CHO-VLP recombinant antigen respectively, incubated overnight at 4°C, blocked with PBST (pH 7.4) containing 5% skim milk powder at 37°C for 2 h, washed three times with PBST, added with 1:100 diluted positive serum of swine O-type foot-and-mouth disease virus, 100 μl per well, and at the same time, negative serum of swine foot-and-mouth disease was set as a control; incubated at 37°C for 45 min, washed 3 times with PBST, added with 1:5000 diluted HRP-labeled goat anti-swine IgG, 100 μl per well, incubated at 37°C for 30 min, washed three times with PBST, added with TMB chromogenic solution, 100 μl per well, developed color at 37°C for 10 - 15 min, terminated the reaction with 2 M concentrated H2SO4, and measured the OD 450nm absorbance value.

[0062] The results showed that the CHO-VLP recombinant antigen could have a strong immune reaction with the positive serum of swine O-type foot-and-mouth disease, and its immunoreactivity had no significant difference from that of the inactivated O-type foot-and-mouth disease virus antigen, and it did not have an immune reaction with the negative serum of swine foot-and-mouth disease ( Figure 5 ). It fully demonstrated that the protective antigen epitopes of the O-type foot-and-mouth disease virus in the CHO-VLP recombinant antigen were correctly displayed and its immunological activity was fully exerted.

[0063] In addition, 96-well ELISA plates were coated with the antigen O-type foot-and-mouth disease virus multi-epitope VLP nanoantigen developed by the present invention and inactivated O-type FMDV antigen respectively, and the positive sera of pigs, cattle, and sheep immunized with O-type FMDV were detected according to the above method. The results showed that the O-type foot-and-mouth disease virus multi-epitope VLP nanoantigen CHO-VLP developed by the present invention could have a strong immune reaction with the positive sera of pigs, cattle, and sheep, and there was no obvious difference from the inactivated O-type foot-and-mouth disease virus antigen, which was superior to the O-type foot-and-mouth disease multi-epitope VLP vaccine developed with the AP205 backbone disclosed in the publication number CN116041547A ( Figure 6 ).

[0064] Example 3. Preparation and immunopotency experiment of O-type foot-and-mouth disease virus multi-epitope VLP vaccine

[0065] 1. Vaccine preparation

[0066] After quantifying the purified O-type foot-and-mouth disease virus multi-epitope VLP nano-antigen (CHO-VLP) in Example 1 by the BCA method, it was diluted to 200 μg / ml and 100 μg / ml with PBS, and emulsified into a vaccine preparation (W / O / W) by adding the water-in-oil-in-water adjuvant ISA201 VG (Seppic, France) at a ratio of 50 g:50 g. Each dose contained 100 μg / ml and 50 μg / ml VLP antigen respectively.

[0067] 2. Immune efficacy test

[0068] Twelve pigs weighing about 30 kg, with O-type foot-and-mouth disease virus antibody <1:4 (liquid-phase blocking ELISA) and negative 3ABC protein antibody (negative by 3ABC antibody chemiluminescence kit) were selected. Five pigs were inoculated intramuscularly with 1 ml of each of the two different formulations of the vaccine, and two non-immunized pigs were used as negative controls. Blood samples were collected before immunization and 28 days after immunization, and the sera were separated for detecting specific antibody IgG. Twenty-eight days after immunization, together with the two controls, they were infected with a virulent strain of O-type foot-and-mouth disease virus according to the national standard and observed for 10 days. The results showed that the multi-epitope VLP vaccine of O-type foot-and-mouth disease virus could induce high levels of foot-and-mouth disease-specific antibodies in immunized pigs and protect against O-type foot-and-mouth disease virus infection after challenge (Table 1). In addition, there were no phenomena such as swelling, redness, and fever at the injection site of the vaccinated animals, nor any other adverse reactions. The appetite was normal and the mental state was good, confirming that the vaccine was very safe.

[0069] Table 1 Results of the immune efficacy experiment of the multi-epitope VLP vaccine of O-type foot-and-mouth disease virus in pigs

[0070]

[0071]

Claims

1. A multi-epitope virus-like particle nanoantigen of type O foot-and-mouth disease virus (FMDV), characterized in that: The O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen is prepared by selecting antigenic epitopes of three representative strains of the O-type foot-and-mouth disease virus topological types, O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017 and O / HKN / 2007, and sequentially connecting them in series to form an epitope series 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, the T cell epitope of the foot-and-mouth disease virus 3A gene was introduced into the C-terminus of OB5 and connected 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 78th-81th position of the core protein amino acid sequence of the woodchuck hepatitis B virus through a linker.

2. The multi-epitope virus-like particle nanoantigen of O-type foot-and-mouth disease virus according to claim 1, characterized in that: The amino acid sequence of the antigen fragment OB5T is shown in SEQ ID NO.4; the sequence of the linker is GGGGSGGGG; the GenBank accession number of the woodchuck hepatitis B virus core protein encoding gene is: J02442.

3. The multi-epitope virus-like particle nanoantigen of O-type foot-and-mouth disease virus according to claim 1, characterized in that: 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.

4. The multi-epitope virus-like particle nanoantigen of O-type foot-and-mouth disease virus according to claim 1, characterized in that: The O-type foot-and-mouth disease virus multi-epitope virus-like particle nanoantigen is obtained by transferring its encoding gene into Chinese hamster ovary cells (CHO) and secreting and expressing it by the CHO cells. Preferably, the CHO cells are CHO-S cells.

5. A nucleic acid encoding the multi-epitope virus-like particle antigen of type O foot-and-mouth disease virus according to any one of claims 1 to 4, preferably, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

5.

6. A method for preparing and purifying the multi-epitope virus-like particle nanoantigen of O-type foot-and-mouth disease virus according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Synthesis of genes for multi-epitope VLP antigens of type O foot-and-mouth disease virus According to the VP1 gene sequences of representative strains of the three O-type topological types, O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017 and O / HKN / 2007, the linear antigenic epitopes of protective antibodies of foot-and-mouth disease virus, i.e., the 135-160 segment sequence of the VP1 gene coding region, were selected and sequentially connected in series, and GS spacers were introduced between adjacent epitopes to form the epitope tandem structure of the five strains: O / Tibet / CHA / 99-O / Mya98 / BY / 2010-OZK / 93-O / XJPS / CHA / 2017-O / HKN / 2007, named OB5, whose encoding nucleotide sequence is shown in SEQ ID NO.1; the T cell epitope of the foot-and-mouth disease virus 3A gene was introduced into the C-terminus of OB5, and connected with a GS spacer to form an antigen fragment containing 5 B cell epitopes and 1 T cell epitope, named OB5T, and its 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) is inserted into the nucleotides 234-240 of the woodchuck hepatitis B virus core protein encoding gene through a linker to obtain a chimeric gene encoding a multi-epitope VLP antigen of type O foot-and-mouth disease virus, which is named OB5T-HBc, and the sequence of the linker is GGGGSGGGG; the codons of the chimeric gene OB5T-HBc are optimized and designed according to the codon preference of CHO cells, and the nucleotide sequence of the optimized OB5T-HBc chimeric gene is shown in SEQ ID NO.5, EcoRⅠ and NotⅠ restriction sites are introduced at the N′-terminus and C′-terminus of the OB5T-HBc sequence, respectively, and a stop codon is introduced at the N-terminus of the NotⅠ sequence, and then the chimeric gene is inserted into pcDNA3.1(+) to construct a recombinant expression plasmid pcDNA3.1 / OB5T-HBc; (3) Expression and purification of multi-epitope VLP antigen of foot-and-mouth disease virus type O The constructed recombinant expression plasmid pcDNA3.1 / OB5T-HBc was transfected into CHO-S cells, and the cells were cultured on an orbital shaker at 37°C and 8% CO2; the supernatant of the cultured cells 3-10 days after transfection was collected and centrifuged at 4°C and 4000g for 30 minutes to obtain the multi-epitope VLP antigen of type O foot-and-mouth disease virus, which was named CHO-VLP; (4) Purification of multi-epitope VLP antigens of foot-and-mouth disease virus type O Sucrose density gradient centrifugation was used for purification. The specific operation was as follows: 15% w / v, 25% w / v, 35% w / v, and 45% w / v sucrose density gradients were prepared with TNE solution, centrifuged at 4°C and 35000 r / min for 3 h, and 0.5 mL was taken as one fraction. Samples were collected from the top layer in sequence. All samples were measured by UV spectrophotometer at 280 nm and 260 nm Detect, calculate the antigen content, and store at -20℃.

7. The method according to claim 6, characterized in that The GenBank accession number of the woodchuck hepatitis B virus core protein encoding gene is: J02442.

8. Use of the multi-epitope virus-like particle nanoantigen of O-type foot-and-mouth disease virus according to any one of claims 1 to 4 in the preparation of a drug for preventing infection with O-type foot-and-mouth disease virus.

9. The use according to claim 8, characterized in that The medicine is a virus-like particle vaccine.

10. An O-type foot-and-mouth disease multi-epitope virus-like particle vaccine, characterized in that: The vaccine contains the multi-epitope virus-like particle antigen of type O foot-and-mouth disease virus according to any one of claims 1 to 4 and an adjuvant, preferably, the adjuvant is ISA201 VG.