O and A foot-and-mouth disease multi-epitope bivalent virus-like particle antigen, its preparation method and application

Abstract

The present invention discloses an O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigen, a preparation method thereof and an application thereof. The virus-like particle (VLP) antigen is obtained by respectively fusing the A-type and O-type foot-and-mouth disease multi-epitope antigen sequences to the N-terminus and C-terminus of the phage AP205. After immunizing animals with the vaccine prepared by formulating the VLP antigen with the ISA206VG adjuvant, it can induce high-level protective antibodies, and the immunized animals can resist the attack of O-type and A-type foot-and-mouth disease viruses, with 100% (5 / 5) protection. The present invention can prevent two serotypes of O-type and A-type foot-and-mouth disease by only producing one antigen, achieving the goal of "preventing two diseases with one injection" and solving the production process and cost problems that enterprises are most concerned about. Therefore, this vaccine can be said to be a new vaccine with broad prospects, and it will be of great significance for the purification and eradication of O-type and A-type foot-and-mouth disease in China and achieving the status of foot-and-mouth disease-free immunity.

Description

Technical Field

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

[0002] Foot-and-mouth disease is a major animal disease caused by foot-and-mouth disease virus, which affects the main economic livestock species such as pigs, cattle and sheep. The OIE lists it as an animal disease that must be notified, and China lists it as a class of major animal infectious diseases that need to be focused on for prevention and control. China adopts a strategy of combining vaccine immunization and culling to prevent and control foot-and-mouth disease. With the continuous enhancement of China's economic strength and the improvement of its international status, China has set the goal of purifying and eradicating foot-and-mouth disease. To achieve this goal, a safe, efficient and distinguishable foot-and-mouth disease new vaccine is an indispensable important technical means. Although distinguishable foot-and-mouth disease virus marker vaccines have been developed using molecular biology techniques, live viruses need to be used in the production process, which requires high-level production facilities and there is still a biosafety risk.

[0003] At present, foot-and-mouth disease in China is still mainly type O, and type A foot-and-mouth disease occurs from time to time. If traditional inactivated vaccines are used for prevention and control, it means that pathogens of two serotypes need to be operated for vaccine research and production, which not only increases the biological risk, but also increases the vaccine cost. Using reverse vaccinology technology, with protective antigens and / or antigenic epitopes as materials, developing environmentally friendly, biosafe and distinguishable high-efficiency genetic engineering subunit vaccines has become a new direction for vaccine research. At present, except for the synthetic peptide vaccine of foot-and-mouth disease type O in pigs, there is no foot-and-mouth disease subunit vaccine for epidemic prevention and control. Recently, the virus-like particle vaccine of foot-and-mouth disease type O developed by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences has obtained two first-class new veterinary drug certificates, marking that the foot-and-mouth disease subunit genetic engineering vaccine has entered a new era. This vaccine uses the capsid protein gene of foot-and-mouth disease virus as an element, expresses it in segments and assembles it into virus-like particles (VLP) in vitro. Summary of the Invention

[0004] The purpose of the present invention is to provide a multi-epitope bivalent virus-like particle antigen of foot-and-mouth disease type O and type A, a preparation method and an application thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical means:

[0006] In order to develop a new multi-epitope vaccine against foot-and-mouth disease that is biosafe, environmentally friendly, highly immunogenic, has a simple production process, and is not restricted by animal species. In the present invention, the protective antigen epitopes of foot-and-mouth disease virus serotype O and A are used as elements to design a multi-epitope tandem DNA. Using bacteriophage AP205 as a backbone and phage display technology, the protective antigen epitopes of foot-and-mouth disease virus are displayed on the surface of bacteriophage AP205, which is the bivalent VLP antigen of foot-and-mouth disease virus serotype O and A, and a bivalent VLP universal vaccine against foot-and-mouth disease virus serotype O and A is developed.

[0007] The results showed that the tandem epitopes of foot-and-mouth disease virus serotype O and A were successfully displayed on the surface of the phage, and its morphology and size were consistent with the expected size, which was about 43 nm VLP; the recombinant VLP antigen could have a strong immune reaction with the standard positive sera of foot-and-mouth disease virus serotype O and A, and there was no obvious difference in immunoreactivity from the inactivated antigen of FMDV, indicating that the antigen epitopes of foot-and-mouth disease virus serotype O and A were correctly displayed and could exert their normal immunological functions. The vaccine prepared by combining the recombinant VLP antigen with ISA206 VG adjuvant could induce high levels of protective antibodies after immunizing animals, and the immunized animals could resist the attack of foot-and-mouth disease virus serotype O and A, with 100% (5 / 5) protection.

[0008] Based on the above research, the present invention provides a bivalent virus-like particle antigen of foot-and-mouth disease virus serotype O and A. The virus-like particle antigen is obtained by fusing the multi-epitope antigen sequences of foot-and-mouth disease virus serotype A and O to the N-terminus and C-terminus of bacteriophage AP205 through the spacer sequences GGGGSGGGGS and GGSGGS respectively, and is named AB2-GGGGSGGGGS-AP205-GGSGGS-OB5. Among them, the multi-epitope antigen of foot-and-mouth disease virus serotype A is formed by connecting the protective antigen epitopes of the VP1 regions of two foot-and-mouth disease virus strains A / GDMM / 2013 and AF72 of serotype A through a flexible spacer GS, forming an A / GDMM / 2013-GS-AF72 chimeric gene structure, named AB2; the multi-epitope antigen of foot-and-mouth disease virus serotype O is formed by sequentially connecting the protective antigen epitopes of the VP1 regions of the representative strains of 5 topotypes of serotype O, O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, O / XJPS / CHA / 2017 and the Cathay type through a flexible spacer GS, forming an O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-Cathay multi-epitope tandem structure form, named OB5.

[0009] Among them, preferably, the amino acid sequence of OB5 is shown in SEQ ID NO.2; the amino acid sequence of AB2 is shown in SEQ ID NO.4.

[0010] Among them, preferably, the amino acid sequence of the virus-like particle antigen is as shown in SEQ ID NO.6.

[0011] The nucleic acid encoding the O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigen is also within the protection scope of the present invention. Preferably, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO.5.

[0012] Furthermore, the present invention also provides a method for preparing the O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigen, comprising the following steps:

[0013] (1) Design of the multi-epitope gene of foot-and-mouth disease virus of type O

[0014] According to the amino acid sequences encoded by the VP1 genes of the representative strains of 5 topotypes of type O, namely O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, O / XJPS / CHA / 2017 and the Cathay type, the linear antigenic epitopes of the protective antibodies in the VP1 region of foot-and-mouth disease virus are selected, a flexible spacer GS is introduced between adjacent epitopes, and then 5 epitopes are sequentially connected to form a tandem structure of O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-Cathay multi-epitopes, named OB5;

[0015] (2) Design of the multi-epitope gene of foot-and-mouth disease virus of type A

[0016] The protective antigenic epitopes in the VP1 regions of 2 foot-and-mouth disease virus strains A / GDMM / 2013 and AF72 of type A are connected through a flexible spacer GS to form an A / GDMM / 2013-GS-AF72 chimeric gene structure, named AB2;

[0017] (3) Construction of recombinant expression plasmid, protein expression and purification

[0018] The AB2 and OB5 sequences designed above were respectively fused to the N-terminus and C-terminus of phage AP205, and two spacer sequences GGGGSGGGGS and GGSGGS were introduced between the three genes to form the AB2-GGGGSGGGGS-AP205-GGSGGS-OB5 structural form. At the same time, specific restriction enzyme sites BamH 1 and Xho l were introduced at the 5'-end and 3'-end of this artificial gene. After artificial synthesis, it was inserted into the pET-28a(+) vector linearized with BamH1 and Xho l to construct the recombinant expression plasmid pET-28 / AB2 / AP205 / OB5 recombinant expression plasmid. It was transformed into JM109 competent cells, and positive recombinant expression plasmids were identified by resistance screening, double enzyme digestion and sequence analysis.

[0019] The above positive recombinant expression plasmid was transformed into BL21(DE3)pLysS by heat shock. A single colony was picked and inoculated into 5 ml of LB culture medium containing kanamycin, and cultured overnight at 37 °C and 220 rmp. The overnight culture was taken and added to the newly prepared sterile LB culture medium containing kanamycin at 1% (V / V), and cultured at 37 °C and 220 rmp. When OD 600 nm ≈ 0.4 - 0.6, 1 mM IPTG was added, and the culture was continued at 37 °C for 4 - 6 hours. The culture was harvested by centrifugation at 4000 rpm for 20 min. The precipitate was resuspended with protein lysate, sonicated, and the supernatant was collected by centrifugation at 20000 g for 20 min. The precipitate was discarded, and the protein was purified according to the Ni-NTA histidine purification column instruction manual to obtain the product.

[0020] Among them, preferably, the nucleotide sequence of OB5 is shown in SEQ ID NO.1; the nucleotide sequence of AB2 is shown in SEQ ID NO.3.

[0021] Among them, preferably, the nucleotide sequence of AB2-GGGGSGGGGS-AP205-GGSGGS-OB5 is shown in SEQ ID NO.5.

[0022] Furthermore, the present invention also provides the use of the O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigen in the preparation of vaccines for preventing O-type and A-type foot-and-mouth disease.

[0023] Furthermore, the present invention also provides an O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle vaccine, and the vaccine contains the O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigen and adjuvant of the present invention.

[0024] Among them, preferably, the adjuvant is ISA206 VG.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] The present invention provides a multi-epitope bivalent virus-like particle antigen for foot-and-mouth disease of type O and type A. This recombinant VLP antigen can have a strong immune reaction with the standard positive sera of foot-and-mouth disease viruses of type O and type A, and there is no obvious difference in immunoreactivity compared with the inactivated antigen of FMDV, indicating that the antigenic epitopes of foot-and-mouth disease viruses of type O and type A are correctly presented and can exert their normal immunological functions. The vaccine prepared by formulating this recombinant VLP antigen with ISA206 VG adjuvant can induce high levels of protective antibodies after immunizing animals, and the immunized animals can resist the attacks of foot-and-mouth disease viruses of type O and type A, with 100% (5 / 5) protection.

[0027] The multi-epitope bivalent VLP vaccine for foot-and-mouth disease of type O and type A developed by the present invention not only correctly presents the protective antigens of foot-and-mouth disease viruses of type O and type A and has good immunological functions, but also overcomes the restrictive problem that the use of animal species-specific carrier proteins leads to the vaccine being only applicable to specific species of animals. In addition, the tandem arrangement of antigenic epitopes of multiple strains solves the problem of the single antigen spectrum of a single foot-and-mouth disease virus strain and the inability to provide immune protection against antigenic variant strains. More importantly, producing only one antigen can prevent foot-and-mouth disease of two serotypes, type O and type A, in pigs, cattle and sheep, achieving the goal of "preventing two diseases with one injection" and solving the production process and cost issues that enterprises are most concerned about. Therefore, this vaccine can be said to be a new type of vaccine with broad prospects and will be of great significance for the purification and eradication of foot-and-mouth disease of type O and type A in China and achieving the status of foot-and-mouth disease-free immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the SDS-PAGE result of the multi-epitope bivalent VLP antigen for foot-and-mouth disease of type O and type A;

[0029] Figure 2 is the particle size of the multi-epitope bivalent VLP antigen for foot-and-mouth disease of type O and type A measured by dynamic light scattering under liquid conditions;

[0030] Figure 3 is the observation result of the multi-epitope bivalent VLP antigen for foot-and-mouth disease of type O and type A by transmission electron microscopy;

[0031] Figure 4 is the antigenicity result of the multi-epitope bivalent VLP antigen for foot-and-mouth disease of type O and type A. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0033] Preparation of O - type and A - type foot - and - mouth disease multi - epitope bivalent virus - like particle antigen in Example 1

[0034] 1. Design of multi - epitope gene of foot - and - mouth disease virus of O type

[0035] According to the amino acid sequences encoded by the VP1 genes of representative strains of 5 topotypes of O type (O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, O / XJPS / CHA / 2017 and Cathay type), linear antigenic epitopes of protective antibodies in the VP1 region of foot - and - mouth disease virus were selected. A flexible spacer GS was introduced between adjacent epitopes, and then 5 epitopes were sequentially linked to form a tandem structure of O / Tibet / CHA / 99 - O / Mya98 - O / HN / CHA / 93 - O / XJPS / CHA / 2017 - Cathay multi - epitopes, named OB5. Its nucleotide sequence is:

[0036] AAGTACTCCGCACCTCAAAACCGGCGAGGTGACTCGGGTCCTCTCGCG GCGAGACTCGCTGCACAGGGTTCTAAGTACTCCACAGGTAATGCAGGCAGA CGGGGTGATCTAGGGTCTCTTGCGGCGAGGGTCGCCGCACAG (shown in SEQ ID NO.1).

[0037] The encoded amino acid sequence is:

[0038] KYSAPQNRRGDSGPLAARLAAQGSKYSTGNAGRRGDLGSLAARVAAQ (shown in SEQ ID NO.2).

[0039] 2. Design of multi - epitope gene of foot - and - mouth disease virus of A type

[0040] The protective antigenic epitopes in the VP1 regions of 2 foot - and - mouth disease virus strains A / GDMM / 2013 and AF72 of A type were linked by a flexible spacer GS to form an A / GDMM / 2013 - GS - AF72 chimeric gene structure, named AB2. Its nucleotide sequence is:

[0041] AAGTATGACGAGAGCCCCGTGACCAATGTGAGAGGTGACCTGCAAGTGTTGGCCCAGAAGGCGGCAAGAACGGGTTCTAAATACGCCGGGGGCTCACTGCCCAACGTGAGAGGCGATCTCCAAGTGCTGGCTCAGAAGGCGGCGAGGCCGGGTAGCAAGTACAGTGACGCCCGCGTGAGCAACGTGAGGGGTGACCT TCAAGTGTTGGCTCAGAAGGCAGAAAGAGCTGGTTCAAAGTATGGCGAGGGCGCTGTGACCAACGTGAGGGGTGACTTGCAAGTGTTGGCTCAGAAGGCAGCAAGAACGGGGAGTAAGTACGGTGACGCCAGCACTAACAACGTGAGAGGTGACCTTCAAGTGTTGGCTAAGAAGGCAGAAAGAGCT (shown in SEQ ID NO.3), and the encoded amino acid sequence is:

[0042] KYDESPVTNVRGDLQVLAQKAARTGSKYAGGSLPNVRGDLQVLAQKAA RPGSKYSDARVSNVRGDLQVLAQKAERAGSKYGEGAVTNVRGDLQVLAQKA ARTGSKYGDASTNNVRGDLQVLAKKAERA (shown in SEQ ID NO.4).

[0043] 3. Construction, protein expression and purification of recombinant expression plasmid

[0044] Fuse the above-designed AB2 and OB5 to the N-terminus and C-terminus of phage AP205 respectively, design multi-epitope chimeric DNAs of foot-and-mouth disease type O and foot-and-mouth disease type A, and introduce two spacer sequences GGGGSGGGGS and GGSGGS between the three genes to form the structure of AB2-GGGGSGGGGS-AP205-GGSGGS-OB5, and its nucleotide sequence is:

[0045] AAGTACTCCGCACCTCAAAACCGGCGAGGTGACTCGGGTCCTCTCGCGGCGAGACTCGCTGCACAGGGTTCTAAGTACTCCACAGGTAATGCAGGCAGACGGGGTGATCTAGGGTCTCTTGCGGCGAGGGTCGCCGCACAGGGCGGTGGCGGTAGCGGTGGCGGTGGCAGTATGGCAAATAAGCCAATGCAACCGATCACATCTACAGCAAATAAAATTGTGTGGAGTGATCCAACTCGTTTATCAACTACATTTTCAGCAAGTCTGTTACGCCAACGTGTTAAAGTTGGTATAGCCGAACTGAATAATGTTTCAGGTCAATATGTATCTGTTTATAAGCGTCCTGCACCTAAACCGGAAGGTTGTGCAGATGCCTGTGTCATTATGCCGAATGAAAACCAATCCATTCGCACAGTGATTTCAGGGTCAGCCGAAAACTTGGCTACCTTAAAAGCAGAATGGGAAACTCACAAACGTAACGTTGACACACTCTTCGCGAGCGGCAACGCCGGTTTGGGTTTCCTTGACCCTACTGCGGCTATCGTATCGTCTGATACTACTGCTGGGGGTTCCGGGGGTTCCGGTAAGTATGACGAGAGCCCCGTGACCAATGTGAGAGGTGACCTGCAAGTGTTGGCCCAGAAGGCGGCAAGAACGGGTTCTAAATACGCCGGGGGCTCACTGCCCAACGTGAGAGGCGATCTCCAAGTGCTGGCTCAGAAGGCGGCGAGGCCGGGTAGCAAGTACAGTGACGCCCGCGTGAGCAACGTGAGGGGTGACCTTCAAGTGTTGGCTCAGAAGGCAGAAAGAGCTGGTTCAAAGTATGGCGAGGGCGCTGTGACCAACGTGAGGGGTGACTTGCAAG TGTTGGCTCAGAAGGCAGCAAGAACGGGGAGTAAGTACGGTGACGCCAGCACTAACAACGTGAGAGGTGACCTTCAAGTGTTGGCTAAGAAGGCAGAAAGAGCT(shown in SEQ ID NO.5).

[0046] Specific restriction enzyme sites BamH I and Xho I were introduced at the 5'-end and 3'-end of the artificial gene. After artificial synthesis, it was inserted into the pET-28a(+) vector linearized with BamH I and Xho I to construct the recombinant expression plasmid pET-28 / AB2 / AP205 / OB5 recombinant expression plasmid. JM109 competent cells were transformed, and positive recombinant expression plasmids were identified by resistance screening, double digestion, and sequence analysis.

[0047] The above positive recombinant expression plasmid was transformed into BL21(DE3)pLysS by heat shock. A single colony was picked and inoculated into 5 ml of LB culture medium containing kanamycin, and cultured overnight at 37 °C and 220 rmp. The overnight culture was taken and added to the newly prepared sterile LB culture medium (kan+) at 1% (V / V), and cultured at 37 °C and 220 rmp. When OD 600 nm ≈ 0.4 - 0.6, 1 mM IPTG was added, and the culture was continued at 37 °C for 4 - 6 hours. The culture was harvested by centrifugation at 4000 rpm for 20 min. The precipitate was resuspended with protein lysis buffer (20% of the original culture volume), sonicated (ice bath), and centrifuged at 20000 g for 20 min to collect the supernatant (4 °C), and the precipitate was discarded. The protein was purified according to the Ni-NTA His purification column instruction manual. The SDS-PAGE result showed that the protein size was 37 KDa, Figure 1 . Its amino acid sequence is:

[0048] KYSAPQNRRGDSGPLAARLAAQGSKYSTGNAGRRGDLGSLAARVAAQGGGGSGGGGSMANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTAGGSGGSGKYDESPVTNVRGDLQVLAQKAARTGSKYAGGSLPNVRGDLQVLAQKAARPGSKYSDARVSNVRGDLQVLAQKAERAGSKYGEGAVTNVRGDLQVLAQKAARTGSKYGDASTNNVRGDLQVLAKKAERA (shown in SEQ ID NO.6).

[0049] 4. Morphology and size identification of O-type and A-type foot-and-mouth disease multi-epitope VLP antigens

[0050] The morphology of the VLP purified by 15% - 50% sucrose gradient was observed by transmission electron microscopy, and the size was measured; the particle size under liquid conditions was measured by dynamic light scattering. The results are asFigure 2 , 3 As shown in 3 , the results showed that the recombinant protein was a typical VLP structure, and the vast majority of VLP particles had a diameter of 43 nm (81.9%).

[0051] 5. Immunological function identification of O-type and A-type foot-and-mouth disease multi-epitope VLP antigens

[0052] 96-well ELISA plates were coated with inactivated antigens of O-type and A-type foot-and-mouth disease viruses (100 μl / well, 1 μg / ml) and purified O-type and A-type multi-epitope bivalent VLP antigens (100 μl / well, 1 μg / ml) respectively, and incubated overnight at 4°C or for 2 hours at 37°C; blocked with 5% skim milk powder (PBST, pH 7.4) at 37°C for 1 hour; washed 3 times with PBST; added O-type and A-type foot-and-mouth disease virus positive sera diluted 1:100 (100 μl / well) respectively; incubated at 37°C for 1 hour; washed 3 times with PBST; added HRP-labeled rabbit anti-bovine IgG diluted 1:1000 (100 μl / well, incubated at 37°C for 1 hour; washed 3 times with PBST; added TMB chromogenic solution, reacted at 37°C for 10 - 15 min, terminated the reaction with 2M H2SO4, and measured the absorbance value of OD 450nm , and analyzed the results. The results were as Figure 4 shown. The results showed that the O-type and A-type foot-and-mouth disease multi-epitope bivalent VLP antigens could have a strong immune reaction with O-type and A-type foot-and-mouth disease virus positive sera, and there was no significant difference in their immunoreactivity from the inactivated antigens of O-type and A-type foot-and-mouth disease viruses. The results fully demonstrated that the protective antigens of O-type and A-type foot-and-mouth disease viruses were correctly displayed and their immune activities were fully exerted.

[0053] Example 2 Preparation and immunoprotection test of O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle vaccine

[0054] 1. Vaccine preparation:

[0055] After quantification by Bradford method, the purified O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigens in Example 1 were diluted with PBS to 200 μg / ml, and emulsified into a vaccine preparation (W / O / W) with water-in-oil-in-water adjuvant ISA206 VG (Seppic, France) according to 50 g:50 g, with 1 ml per dose (containing 100 μg O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigens).

[0056] 2. Immunopotency test:

[0057] Select 16 pigs weighing about 40 kg, with anti-A foot-and-mouth disease virus antibody <1:4 (liquid-phase blocking ELISA result) and negative 3ABC protein antibody (3ABC antibody chemiluminescence kit result). Inoculate 10 pigs (including 100 μg of O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigen per dose) via the intramuscular route with the trial vaccine at 1 ml per dose. After 28 days of immunization, collect blood samples and isolate sera for specific antibody detection. At the same time, conduct a challenge protection test, specifically: among them, 5 immunized pigs together with 3 non-immunized control pigs with equal conditions are challenged with O-type foot-and-mouth disease virus according to the national standard; the other 5 immunized pigs together with 3 non-immunized controls are challenged with A-type foot-and-mouth disease virus, and observe for 10 days. The results show that the O-type and A-type foot-and-mouth disease multi-epitope bivalent VLP vaccine can induce high levels of foot-and-mouth disease specific antibodies after immunizing pigs (Table 1). Using liquid-phase blocking ELISA detection and according to the kit evaluation criteria, when ≥1:64, 99% protection. The levels of O-type and A-type foot-and-mouth disease specific antibodies in the immune sera are not lower than 1:128; after challenge, it can protect pigs from simultaneous infection with O-type and A-type foot-and-mouth disease viruses, with 5 / 5 protection for both types.

[0058] Table 1 Results of the immunoprotection test of O-type and A-type foot-and-mouth disease multi-epitope bivalent VLP antigen

[0059]

[0060] In addition, there were no phenomena such as swelling, fever, etc. at the injection site of the vaccinated animals, nor any other adverse reactions. The appetite was normal and the mental state was good, which confirmed that the vaccine was very safe.

Claims

1. O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigen, characterized in that, The virus-like particle antigen described above is obtained by fusing the multi-epitope antigen sequences of foot-and-mouth disease serotypes A and O to the N-terminus and C-terminus of phage AP205 through the spacer sequences GGGGSGGGGS and GGSGGS respectively, and is named AB2-GGGGSGGGGS-AP205-GGSGGS-OB5. Among them, the multi-epitope antigen of foot-and-mouth disease serotype A is formed by connecting the protective antigen epitopes in the VP1 regions of two foot-and-mouth disease virus strains A / GDMM / 2013 and AF72 of serotype A through the flexible spacer GS, forming the A / GDMM / 2013-GS-AF72 chimeric gene structure, named AB2; the multi-epitope antigen of foot-and-mouth disease serotype O is formed by sequentially connecting the protective antigen epitopes in the VP1 regions of the representative strains O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, O / XJPS / CHA / 2017 of 5 topotypes of serotype O and the Cathay type through the flexible spacer GS, forming the O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-Cathay multi-epitope tandem structure, named OB5; the amino acid sequence of the virus-like particle antigen described above is shown in SEQ ID NO.

6.

2. Nucleic acid encoding the multi-epitope bivalent virus-like particle antigen of foot-and-mouth disease serotypes O and A described in claim 1.

3. The nucleic acid according to claim 2, wherein, The nucleotide sequence of the nucleic acid described above is shown in SEQ ID NO.

5.

4. A method for preparing the O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigen according to claim 1, characterized in that, Comprising the following steps: (1) Design of the multi-epitope gene of foot-and-mouth disease virus serotype O According to the amino acid sequences encoded by the VP1 genes of the representative strains O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, O / XJPS / CHA / 2017 of 5 topotypes of serotype O and the Cathay type, select the linear antigen epitopes of the protective antibodies in the VP1 region of foot-and-mouth disease virus, introduce the flexible spacer GS between adjacent epitopes, and then sequentially connect the 5 epitopes to form the O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-Cathay multi-epitope tandem structure, named OB5, and the nucleotide sequence of OB5 is shown in SEQ ID NO.1; (2) Design of the multi-epitope gene of foot-and-mouth disease virus serotype A Connect the protective antigen epitopes in the VP1 regions of two foot-and-mouth disease virus strains A / GDMM / 2013 and AF72 of serotype A through the flexible spacer GS to form the A / GDMM / 2013-GS-AF72 chimeric gene structure, named AB2, and the nucleotide sequence of AB2 is shown in SEQ ID NO.3; (3) Construction, protein expression and purification of the recombinant expression plasmid The AB2 and OB5 sequences designed above were respectively fused to the N-terminus and C-terminus of phage AP205, and two spacer sequences GGGGSGGGGS and GGSGGS were introduced between the three genes to form the structure of AB2-GGGGSGGGGS-AP205-GGSGGS-OB5. At the same time, specific restriction sites BamH 1 and Xho l were introduced at the 5'-end and 3'-end of this artificial gene. After artificial synthesis, it was inserted into the pET-28a(+) vector linearized with BamH 1 and Xho l to construct the recombinant expression plasmid pET-28 / AB2 / AP205 / OB5. The recombinant expression plasmid was transformed into competent JM109 cells, and positive recombinant expression plasmids were identified by resistance screening, double digestion, and sequence analysis. Among them, the nucleotide sequence of AB2-GGGGSGGGGS-AP205-GGSGGS-OB5 is shown in SEQ ID NO.5; The above-mentioned positive recombinant expression plasmid was transformed into BL21(DE3)pLysS by heat excitation. A single colony was picked and inoculated into 5 ml of LB culture medium containing kanamycin, and cultured overnight at 37 °C and 220 rmp; The overnight culture was taken and added to the newly prepared sterile LB culture medium containing kanamycin at 1% (V / V), and cultured at 37 °C and 220 rmp. When OD 600nm ≈0.4 - 0.6, 1 mM IPTG was added, and the culture was continued at 37 °C for 4 - 6 hours. The culture was harvested by centrifugation at 4000 rpm for 20 min. The precipitate was resuspended with protein lysis buffer, sonicated, and centrifuged at 20000 g for 20 min to collect the supernatant. The precipitate was discarded, and the protein was purified according to the Ni-NTA histidine purification column instruction manual to obtain the target protein.

5. Use of the O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigen according to claim 1 in the preparation of a vaccine for preventing O-type and A-type foot-and-mouth disease.

6. A multi-epitope bivalent virus-like particle vaccine against foot-and-mouth disease of O and A types, characterized in that, The vaccine according to claim 1 contains the O-type and A-type foot-and-mouth disease multi-epitope bivalent virus-like particle antigen according to claim 1 and an adjuvant.

7. The virus-like particle vaccine according to claim 6, wherein, The adjuvant is ISA206VG.

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