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

Abstract

This invention discloses a multi-epitope virus-like particle antigen (VLP) of type O foot-and-mouth disease virus (FMDV), its preparation method, and its application. The type O FMDV VLP antigen is a recombinant antigen protein formed by sequentially tandemly connecting the antigenic epitopes of five representative strains of type O FMDV topotypes—O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, O / XJPS / CHA / 2017, and Cathay—with the SpyCather and bacteriophage AP205 genes, and then tandemly connecting them with the epitopes of the first four strains. This recombinant protein can self-assemble into a VLP antigen. Immunological experiments show that this VLP antigen has good antigenicity, and its antigenic reactivity with type O FMDV is not significantly different, making it a viable alternative to inactivated FMDV antigens in detection methods. Vaccines prepared using this VLP antigen not only induce high levels of protective antibodies but also protect immunized animals against viral challenge. Furthermore, it is not limited by animal species and provides immune protection against type O foot-and-mouth disease in pigs, cattle, and sheep.

Description

Technical Field

[0001] This invention relates to a Virus-like particles Antigens, their preparation methods, and applications, particularly involving a multi-epitope of type O foot-and-mouth disease virus. Virus-like particles Antigens, their preparation methods, and applications; this invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Foot-and-mouth disease (FMD) is a major animal disease caused by the foot-and-mouth disease virus (FMDV), affecting the main economic livestock breeds of pigs, cattle, and sheep. The OIE lists it as a notifiable animal disease, and my country classifies it as a Class A animal infectious disease requiring priority prevention. Safe, highly effective, and diagnostically reliable novel FMD vaccines are indispensable technological tools. Although marker-controlled inactivated FMD vaccines can be obtained through genetic manipulation, the entire research and production process still requires the use of live viruses and high-level production facilities, thus biosafety risks remain.

[0003] To address key scientific challenges in the research and production of inactivated foot-and-mouth disease (FMD) vaccines, such as biosafety risks, high costs, and complex vaccine components, reverse vaccinology technology has emerged as a new direction in vaccine research. This involves using protective antigens and / or epitopes of the pathogen as materials to develop environmentally friendly, biosafety-compliant, and highly effective genetically engineered subunit vaccines. However, most current genetically engineered vaccines focus on protective antigens, expressing recombinant proteins through eukaryotic / prokaryotic expression systems. These recombinant proteins come in various forms, including relatively simple forms such as recombinant protein mixtures and recombinant fusion proteins; they also include expressing complete viral capsid proteins or assembling segmented recombinant proteins in vitro into VLPs. However, these protein expression methods have several drawbacks. For example, recombinant proteins expressed as single or multiple genes may have compromised immunization efficacy if the correct antigen structure cannot be guaranteed. Notably, the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences has successfully obtained a VLP through segmented expression and in vitro assembly, and has received a Class I new veterinary drug certificate.

[0004] In order to develop a novel genetically engineered subunit vaccine for foot-and-mouth disease that is biosafe, environmentally friendly, and highly effective in producing immunogenicity, improve the immunogenicity of epitope vaccines, and solve key technical problems in large-scale production, this invention uses protective antigenic epitopes of foot-and-mouth disease virus as elements and utilizes natural self-assembled protein molecules of organisms as a backbone to design and develop biomimetic nano-VLP antigens and their vaccines with structures and sizes close to those of the natural pathogen. Summary of the Invention

[0005] The purpose of this invention is to provide a multi-epitope method for type O foot-and-mouth disease virus. Virus-like particles Antigens, their preparation methods, and applications.

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

[0007] This invention utilizes phage surface display technology, using protective antigenic epitopes of type O foot-and-mouth disease virus (FMDV) as elements and bacteriophage AP205 as a backbone, to design a multi-epitope tandem DNA of FMDV type O. This DNA is then fused with the AP205 gene to construct a prokaryotic recombinant expression plasmid, expressing the multi-epitope VLP recombinant protein, and developing a biomimetic nano-VLP antigen and vaccine for FMDV type O. This invention successfully constructed the prokaryotic recombinant expression plasmid, expressed, purified, and assembled the biomimetic nano-VLP (multi-epitope VLP). ELISA results showed that the recombinant VLP possessed good antigenicity, and its immunoreactivity was not significantly different from that of the inactivated FMDV antigen, suggesting that this multi-epitope VLP can be used as a detection antigen to replace the inactivated FMDV antigen for the detection of FMDV type O antibodies and the development of related kits. More importantly, the vaccine prepared by combining this recombinant VLP with ISA 206 VG adjuvant induced high levels of protective antibodies in immunized animals, with even higher antibody levels and a longer duration after booster immunization, providing 100% (5 / 5) protection after challenge.

[0008] Based on the above research, this invention proposes a multi-epitope virus-like particle antigen for type O foot-and-mouth disease virus. This antigen is derived from the linear antigenic epitopes of protective antibodies against five representative strains of type O foot-and-mouth disease virus topotypes: O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, O / XJPS / CHA / 2017, and Cathay type foot-and-mouth disease virus. These epitopes are sequentially tandem, with GS spacers introduced between adjacent epitopes, forming a tandem structure of five strains: O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-Cathay, named OB5. Then, O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-Cathay is further tandemly constructed. The antigenic epitopes of et / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, and O / XJPS / CHA / 2017 are sequentially tandem to form a tandem epitope structure of 4 strains: O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017, named OB4. Finally, the designed OB5 multi-epitope tandem gene is tandemly linked with the SpyCather and bacteriophage AP205 genes using the spacer sequence GGGGS, and then linked with OB4 using the GGSGGSGGSG spacer sequence to form the OB5-SpyCather-AP205-OB4 recombinant antigen protein. This recombinant protein self-assembles into virus-like particle antigen.

[0009] Preferably, the amino acid sequence of OB5 is shown in SEQ ID NO.2, and the amino acid sequence of OB4 is shown in SEQ ID NO.4.

[0010] Preferably, the Virus-like particles The amino acid sequence of the antigen is shown in SEQ ID NO.6.

[0011] The O-type foot-and-mouth disease virus multiepitope encoding described above Virus-like particles The nucleic acid of the 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.

[0012] Furthermore, this invention also proposes a method for preparing the O-type foot-and-mouth disease virus multi-epitope. Virus-like particles The method for using antigens includes the following steps:

[0013] (1) Foot-and-mouth disease virus type O multi-epitope chimeric DNA design:

[0014] Linear antigenic epitopes of protective antibodies against five representative strains of type O foot-and-mouth disease virus (FMDV) topotypes—O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, O / XJPS / CHA / 2017, and Cathay type FMDV—were selected and sequentially tandemly, with GS spacers introduced between adjacent epitopes to form a tandem epitope structure of five strains: O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-Cathay, named OB5. Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.1. As shown in NO.2; then the antigenic epitopes of O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, and O / XJPS / CHA / 2017 are sequentially tandem to form the epitope tandem structure of the 4 strains: O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017, named OB4, whose nucleotide sequence is shown in SEQ ID NO.3 and whose encoded amino acid sequence is shown in SEQ ID NO.4;

[0015] (2) Construction of OB5-SpyCather-AP205-OB4 chimeric gene recombinant expression plasmid

[0016] The designed OB5 multi-epitope tandem gene was sequentially tandemly with the SpyCather and AP205 phage genes using the spacer sequence GGGGS, and then tandemly with OB4 using the GGSGGSGGSG spacer sequence to form the OB5-SpyCather-AP205-OB4 chimeric gene DNA. Codon preference optimization was performed on the chimeric DNA using biological software, and Nco 1 and Xho 1 specific restriction enzyme sites were introduced at the 5' and 3' ends of the chimeric DNA, respectively. A GS spacer was introduced between the two restriction enzyme sites and the chimeric gene. The entire chimeric gene was named OB5S25OB4. Nco 1 and Xho 1 specific restriction enzyme sites were then introduced at the 5' and 3' ends of the chimeric DNA. After digesting OB5S25OB4 with enzymes, the DNA fragments were purified and inserted into the pET-28a(+) expression plasmid linearized with the same enzyme. The two target DNA fragments were ligated with T4 ligase to construct a prokaryotic recombinant expression plasmid for the O-type foot-and-mouth disease multi-epitope gene, named pET-28 / OB5S25OB4. Positive recombinant expression plasmids were identified by resistance selection, double enzyme digestion, and sequence analysis.

[0017] (3) Recombinant protein expression and purification

[0018] The positive recombinant expression plasmid was transformed into BL21(DE3)pLysS using heat stimulation. Single colonies were selected and inoculated with an appropriate amount of LB broth containing kanamycin, and cultured overnight at 37°C and 220 rpm. The overnight culture was then added at 1% (V / V) to freshly prepared sterile LB broth containing kanamycin and cultured at 37°C and 220 rpm. When the OD... 600 When the concentration is approximately 0.4-0.6, add 0.5 mM IPTG and continue culturing for 4-6 hours. Centrifuge at 4000 rpm for 20 min to harvest the culture. Add protein lysis buffer at 20% of the original culture volume, sonicate to disrupt, centrifuge at 20000g for 20 min to collect the supernatant, and discard the precipitate. Purify the protein according to the instructions of the Ni-NTA histidine purification column. This protein is the O-type foot-and-mouth disease virus multi-epitope virus-like particle antigen, named OB5S25OB4.

[0019] 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.

[0020] Furthermore, this invention also proposes the aforementioned O-type foot-and-mouth disease virus multi-epitope theory. Virus-like particles Antigens in the preparation of prevention of type O foot-and-mouth disease Uses in vaccines.

[0021] Furthermore, this invention also proposes a multi-epitope method for foot-and-mouth disease type O. Virus-like particle vaccine, the vaccine described Contains The aforementioned O-type foot-and-mouth disease virus multi-epitope Virus-like particles Antigens and adjuvants.

[0022] Among them, the preferred The adjuvant is ISA206 VG.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] This invention successfully expressed a multi-epitope recombinant protein of type O foot-and-mouth disease virus (FMDV) using the E. coli system. This protein can self-assemble into a VLP (virtual protein molecule). Immunological experiments showed that this VLP antigen has good antigenicity, and its antigenic reactivity with type O FMDV is not significantly different. It can be used as a detection antigen to replace the inactivated FMDV antigen in establishing a detection method. The vaccine made using this VLP antigen not only induces high levels of protective antibodies but also protects immunized animals against viral attack. More importantly, this vaccine is not species-specific and provides immunoprotection against type O FMD in pigs, cattle, and sheep. It is a promising new vaccine that will generate significant economic and social benefits. Attached Figure Description

[0025] Figure 1 SDS-PAGE results of recombinant protein of type O foot-and-mouth disease virus;

[0026] Figure 2 The particle size and dispersion of type O foot-and-mouth disease virus multi-epitope VLP in liquid state;

[0027] Figure 3 To observe the results of multi-epitope VLP of type O foot-and-mouth disease using transmission electron microscopy;

[0028] Figure 4 Results for the antigenicity of recombinant protein OB5S25OB4;

[0029] Figure 5 This is the result of an immunogenicity test of a multi-epitope VLP vaccine for type O foot-and-mouth disease.

[0030] Figure 6 A comparative diagram showing the immune responses of type O foot-and-mouth disease multi-epitope VLP antigen, MEO-Fc recombinant antigen, and type O FMDV single-strain inactivated antigen to bovine type O FMDV positive serum.

[0031] Figure 7 This is a comparison of the immune antibody levels between 50 μg of type O foot-and-mouth disease multi-epitope VLP antigen and 100 μg of type O foot-and-mouth disease multi-epitope VLP. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

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

[0034] 1. Foot-and-mouth disease virus type O multi-epitope chimeric DNA design:

[0035] Based on the amino acid sequences encoded by the VP1 gene of the representative strains of the five topotypes of type O (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 against foot-and-mouth disease virus were selected and sequentially tandemly, with GS spacers introduced between adjacent epitopes, forming a tandem epitope structure of the five strains: O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-Cathay, named OB5, with the following nucleotide sequence:

[0036] AAATACGATGAGAGCCCGGTGACGAACGTCCGCGGTGACCTTCAGGTGTTGGCGCAGAAGGCGGCGCGTACGGGTAGCAAGTACGCAGGTGGTTCACTGCCGAACGTACGGGGCGACCTGCAGGTCCTCGCGCAAAAGGCTGCGCGTCCGGGTTCCAAGTACAGCGACGCTCGTGTTAGCAATGTCAGAGGCGA CCTGCAAGTTTTGGCGCAGAAGGCCGAGCGTGCAGGTAGTAAGTACGGCGAAGGTGCCGTTACCAACGTGGCGTGGTGACTTGCAGGTACTGGCCCAAAAAGCGGCGCGTACCGGTTCCAAGTACGGTGACGCAAGCACGAACAACGTGCGCGGCGACTTACAAGTGCTGGCGAAAAAAGCCGAGCGCGCT (SEQ ID NO.1 shown)

[0037] The encoded amino acid sequence is as follows:

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

[0039] The antigenic epitopes of the first four strains were sequentially tandem to form a tandem epitope structure of four strains: O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017, named OB4, with the following nucleotide sequence:

[0040] AAATACGATGAGAGCCCGGTGACGAACGTCCGCGGTGACCTTCAGGTGTTGGCGCAGAAGGCGGCGCGTACGGGTAGCAAGTACGCAGGTGGTTCACTGCCGAACGTACGGGGCGACCTGCAGGTCCTCGCGCAAAAGGCTGCGCGTCCGGGTTC CAAGTACAGCGACGCTCGTGTTAGCAATGTCAGAGGCGACCTGCAAGTTTTGGCGCAGAAGGCCGAGCGTGCAGGTAGTAAGTACGGCGAAGGTGCCGTTACCAACGGTGCGTGGTGACTTGCAGGTACTGGCCCAAAAAGCGGCGCGTACC (SEQ ID NO.3 shown)

[0041] The encoded amino acid sequence is as follows:

[0042] KYDESPVTNVRGDLQVLAQKAARTGSKYAGGSLPNVRGDLQVLAQKAARPGSKYSDARVSNVRGDLQVLAQKAERAGSKYGEGAVTNVRGDLQVLAQKAART (shown in SEQ ID NO.4)

[0043] 2. Construction of the OB5-SpyCather-AP205-OB4 chimeric gene recombinant expression plasmid

[0044] The designed OB5 multi-epitope tandem gene was sequentially tandemly linked with the SpyCather and AP205 phage genes using the spacer sequence GGGGS. The tandem DNA was then tandemly linked with OB4 using the GGSGGSGGSG spacer sequence to form the OB5-SpyCather-AP205-OB4 chimeric gene DNA. Codon preference optimization was performed on the chimeric DNA using biological software, and Nco1 and Xho1 specific restriction enzyme sites were introduced at the 5' and 3' ends of the chimeric DNA, respectively. A GS spacer was introduced between the two restriction enzyme sites and the chimeric gene. The entire chimeric gene was named OB5S25OB4, and its nucleotide sequence is as follows:

[0045]

[0046] After digesting OB5S25OB4 with Nco 1 and Xho 1 respectively, the DNA fragment was purified and inserted into the pET-28a(+) expression plasmid linearized with the same enzyme. The two target DNA fragments were ligated with T4 ligase to construct a prokaryotic recombinant expression plasmid for the O-type foot-and-mouth disease multi-epitope gene, named pET-28 / OB5S25OB4. The positive recombinant expression plasmid was identified by resistance selection, double enzyme digestion and sequence analysis.

[0047] 3. Recombinant protein expression and purification

[0048] The positive recombinant expression plasmid was transformed into BL21(DE3)pLysS using heat stimulation. Single colonies were selected and inoculated with an appropriate amount of LB broth (Kan+), and cultured overnight at 37°C and 220 rpm. The overnight culture was then added at 1% (V / V) to freshly prepared sterile LB broth (Kan+), and cultured at 37°C and 220 rpm. When OD... 600 When the protein concentration reaches approximately 0.4-0.6, add 0.5 mM IPTG and continue culturing for 4-6 hours. Harvest the culture by centrifugation at 4000 rpm for 20 min. Add protein lysis buffer at 20% of the original culture volume, sonicate (on ice), and centrifuge at 20000 g for 20 min to collect the supernatant (4℃). Discard the precipitate. Purify the protein according to the instructions for the Ni-NTA histidine purification column. This protein is the type O foot-and-mouth disease multi-epitope biomimetic nanoantigen (VLP), named OB5S25OB4, with the following amino acid sequence:

[0049] KYDESPVTNVRGDLQVLAQKAARTGSKYAGGSLPNVRGDLQVLAQKAARPGSKYSDARVSNVRGDLQVLAQKAERAGSKYGEGAVTNVRGDLQVLAQKAARTGSKYGDASTNNVRGDLQVLAKKA ERAGGGGSAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGG GGSMANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTA AIVSSDTTAGGSGGSGGSGKYDESPVTNVRGDLQVLAQKAARTGSKYAGGSLPNVRGDLQVLAQKAARPGSKYSDARVSNVRGDLQVLAQKAERAGSKYGEGAVTNVRGDLQVLAQKAART (SEQ ID NO.6 shown)

[0050] SDS-PAGE results ( Figure 1 The results showed that the recombinant protein was about 52 kDa in size and was expressed in a soluble form, meaning that the expressed protein directly self-assembled into a VLP.

[0051] 4. Identification of type O foot-and-mouth disease multiepitope VLP

[0052] VLPs were purified using a 15%–50% sucrose gradient. The morphology and size of the nanoantigens were observed using transmission electron microscopy. Dynamic light scattering (DSL) was used to determine the particle size and distribution under liquid conditions. DSL results showed that the particle size of the O-type foot-and-mouth disease multi-epitope VLPs was approximately 43 nm. Figure 2 Transmission electron microscopy revealed that the O-type foot-and-mouth disease multi-epitope recombinant protein exhibited a typical VLP structure, consistent with the size DSL results. Figure 3 ).

[0053] 5. Immunological identification of recombinant proteins

[0054] 96-well ELISA plates were coated with inactivated foot-and-mouth disease (FMD) type O virus antigen (100 μl / well, 1 μg / ml) and FMD type O virus multi-epitope VLP antigen (100 μl / well, 1 μg / ml), respectively, and incubated overnight at 4°C or for 2 hours at 37°C. The plates were then blocked with PBST (pH 7.4) containing 5% skim milk powder at 37°C for 1 hour. The plates were washed three times with PBST. 100 μl / well of 1:100 diluted FMD type O positive serum was added, and the plates were incubated at 37°C for 1 hour. The plates were washed three times with PBST. 100 μl / well of 1:1000 diluted HRP-labeled rabbit anti-bovine IgG was added, and the plates were incubated at 37°C for 1 hour. The plates were washed three times with PBST. TMB chromogenic solution was added, and the plates were reacted at 37°C for 10–15 min. The reaction was terminated with 2M H₂SO₄, and the OD was measured. 450nm The absorbance values ​​were measured. The results showed that the O-type multiepitope VLP of foot-and-mouth disease virus possessed good antigenicity, and its immunoreactivity was slightly higher than that of the inactivated O-type foot-and-mouth disease virus antigen. Figure 4 Using this established method, immunoreactivity was detected in sera from animals infected with the above five strains, as well as in positive sera from pigs, cattle, and sheep immunized with type O FMDV inactivated vaccine. The results showed that all positive sera against the five strains exhibited a strong immunological reaction with the type O foot-and-mouth disease virus multi-epitope VLP antigen. Figure 5 The results show that the protective antigenic epitopes of each strain were correctly displayed and had a broad spectrum.

[0055] Furthermore, using the previously developed MEO-Fc antigen (described in patent application CN108273054A, entitled "Porcine Foot-and-Mouth Disease Virus Type O and Type A Fc Polypeptide Bivalent Vaccine and its Preparation Method and Application"), the O-type foot-and-mouth disease multi-epitope VLP antigen and the O-type FMDV inactivated antigen developed in this invention, respectively coated 96-well ELISA plates, and positive sera from randomly selected O-type FMDV-immunized pigs, cattle, and sheep were tested according to the above method. The results showed that the O-type foot-and-mouth disease multi-epitope VLP antigen developed in this invention could generate a strong immune response with bovine O-type FMDV positive sera, which was significantly superior to the MEO-Fc recombinant antigen and the O-type FMDV single-strain inactivated antigen. Figure 6 This indicates that the antigen can be widely used for detecting type O FMDV infection and / or immune serum antibodies in pigs, cattle, sheep, etc.

[0056] The above results fully demonstrate that the O-type foot-and-mouth disease virus multi-epitope VLP antigen can not only be used as a detection antigen for serological detection and diagnosis, replacing the biosafety risks and high costs associated with producing whole-virus inactivated antigens, but also that vaccines developed using this antigen can be used for the prevention and control of O-type foot-and-mouth disease in pigs, cattle, and sheep, representing a broad-spectrum O-type FMD multi-epitope antigen.

[0057] Example 2: Multiepitope of type O foot-and-mouth disease virus Virus-like particles Vaccine preparation and immunoprotection testing

[0058] 1. Vaccine preparation

[0059] The purified type O foot-and-mouth disease virus multiepitope from Example 1 Virus-like particle antigen After quantification using the Bio-Rad quantitative kit, OB5S25OB4 was diluted with PBS to two concentration gradients of 200 μg / ml and 100 μg / ml. The mixture was then emulsified with oil adjuvant ISA 206 VG (Seppic, France) at a ratio of 50g:50g to form a vaccine (W / O / W). Each dose is 1 ml (containing 100 μg and 50 μg of multi-epitope VLP antigen).

[0060] 2. Immunopotency Test

[0061] The experimental pigs weighed approximately 40 kg and had O-type foot-and-mouth disease virus antibody <1:4 (liquid-phase blocking ELISA result) and were negative for 3ABC protein antibody. Multiepithelial antibodies containing 100 μg and 50 μg of O-type foot-and-mouth disease virus were used. VLP antigen Five pigs were immunized separately in each group, with each pig receiving 1 ml of vaccine intramuscularly. Twenty-eight days post-immunization, all immunized pigs, along with three unimmunized control pigs under identical conditions, were vaccinated according to national standards using 1000 PID (Polydioxanone Spectroradiol). 50 Pigs were challenged with 3 ml of type O foot-and-mouth disease virus (O / Mya98 / BY2010 strain) and observed for 10 consecutive days. Results showed that immunization of pigs with the type O foot-and-mouth disease biomimetic nanovaccine induced high levels of foot-and-mouth disease specific antibodies (Table 1), and 50 μg... VLP Antigen immune antibody levels and 100 μg VLP The antigens showed no significant difference, suggesting that low concentrations of antigens are sufficient to provide immune protection. Figure 7 According to the kit evaluation criteria, a ratio of ≥1:64 indicates 99% protection. After immunization of pigs with this vaccine, serum-specific antibody levels were not lower than 1:128; 5 / 5 protection was achieved.

[0062] ;

[0063] In addition, the vaccinated animals did not show redness, swelling, fever, or other adverse reactions at the injection site, and their appetite and mental state were normal, confirming that the vaccine is very safe.

Claims

1. Type O foot-and-mouth disease virus multi-epitope virus-like particle antigen, characterized in that, The aforementioned O-type foot-and-mouth disease virus (FMDV) multi-epitope virus-like particle antigen is derived from the linear antigenic epitopes of protective antibodies against five representative strains of the O-type FMDV topotypes: O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, O / XJPS / CHA / 2017, and Cathay-type FMDV. These epitopes are sequentially tandem, with GS spacers introduced between adjacent epitopes, forming a tandem epitope structure of five strains: O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-Cathay, named OB5. Then, O / Tibet / CHA / 99, O / Mya98 / BY / 2010, ... The antigenic epitopes of O / HN / CHA / 93 and O / XJPS / CHA / 2017 are sequentially tandem to form a tandem epitope structure of 4 strains: O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017, named OB4. Finally, the designed OB5 multi-epitope tandem gene is tandemly tandemly with the SpyCather and bacteriophage AP205 genes using the spacer sequence GGGGS, and then tandemly tandemly with OB4 using the GGSGGSGGSG spacer sequence to form the OB5-SpyCather-AP205-OB4 recombinant antigen protein. This recombinant protein self-assembles into virus-like particle antigen. The amino acid sequence of OB5 is shown in SEQ ID NO.2, and the amino acid sequence of OB4 is shown in SEQ ID NO.

4.

2. The O-type foot-and-mouth disease virus multi-epitope virus-like particle antigen as described in claim 1, characterized in that, The aforementioned sick Toxic particles The amino acid sequence of the antigen is shown in SEQ ID NO.

6.

3. Encoding the O-type foot-and-mouth disease virus multiepitopes as described in claim 1 or 2 Virus-like particles The nucleic acid of the antigen.

4. The nucleic acid as described in claim 3, characterized in that, The nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

5.

5. A method for preparing the O-type foot-and-mouth disease virus multi-epitope according to claim 1 or 2 Virus-like particles The method for antigens is characterized by, Includes the following steps: (1) Foot-and-mouth disease virus type O multi-epitope chimeric DNA design: Linear antigenic epitopes of protective antibodies against five representative strains of type O foot-and-mouth disease virus (FMDV) topotypes—O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, O / XJPS / CHA / 2017, and Cathay type FMDV—were selected and sequentially tandemly, with GS spacers introduced between adjacent epitopes to form a tandem epitope structure of five strains: O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017-Cathay, named OB5. Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.

1. As shown in NO.2; then the antigenic epitopes of O / Tibet / CHA / 99, O / Mya98 / BY / 2010, O / HN / CHA / 93, and O / XJPS / CHA / 2017 are sequentially tandem to form a tandem epitope structure of 4 strains: O / Tibet / CHA / 99-O / Mya98-O / HN / CHA / 93-O / XJPS / CHA / 2017, named OB4, whose nucleotide sequence is shown in SEQ ID NO.3 and whose encoded amino acid sequence is shown in SEQ ID NO.4; (2) Construction of OB5-SpyCather-AP205-OB4 chimeric gene recombinant expression plasmid The designed OB5 multi-epitope tandem gene was sequentially tandemly with the SpyCather and AP205 phage genes using the spacer sequence GGGGS, and then tandemly with OB4 using the GGSGGSGGSG spacer sequence to form the OB5-SpyCather-AP205-OB4 chimeric gene DNA. Codon preference optimization was performed on the chimeric DNA using biological software, and Nco 1 and Xho 1 specific restriction enzyme sites were introduced at the 5' and 3' ends of the chimeric DNA, respectively. A GS spacer was introduced between the two restriction enzyme sites and the chimeric gene. The entire chimeric gene was named OB5S25OB4. Nco 1 and Xho 1 specific restriction enzyme sites were then introduced at the 5' and 3' ends of the chimeric DNA. After digesting OB5S25OB4 with enzymes, the DNA fragments were purified and inserted into the pET-28a(+) expression plasmid linearized with the same enzyme. The two target DNA fragments were ligated with T4 ligase to construct a prokaryotic recombinant expression plasmid for the O-type foot-and-mouth disease multi-epitope gene, named pET-28 / OB5S25OB4. Positive recombinant expression plasmids were identified by resistance selection, double enzyme digestion, and sequence analysis. (3) Recombinant protein expression and purification The positive recombinant expression plasmid was transformed into BL21(DE3)pLysS using heat stimulation. Single colonies were selected and inoculated with an appropriate amount of LB broth containing kanamycin, and cultured overnight at 37°C and 220 rpm. The overnight culture was then added to freshly prepared sterile LB broth containing kanamycin at 1% (V / V), and cultured at 37°C and 220 rpm. When OD... 600 When the protein concentration is approximately 0.4-0.6, add 0.5 mM IPTG and continue culturing for 4-6 hours. Centrifuge at 4000 rpm for 20 min to harvest the culture. Add protein lysis buffer at 20% of the original culture volume, sonicate to disrupt the protein, centrifuge at 20000g for 20 min to collect the supernatant, and discard the precipitate. Purify the protein according to the instructions of the Ni-NTA histidine purification column. This protein is the O-type foot-and-mouth disease virus multi-epitope virus-like particle antigen, named OB5S25OB4.

6. The method as described in claim 5, 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.

7. The O-type foot-and-mouth disease virus multi-epitope as described in claim 1 or 2 Virus-like particles Antigens in the preparation of prevention of type O foot-and-mouth disease Uses in vaccines.

8. A multi-epitope variant of foot-and-mouth disease type O Virus-like particle vaccines, characterized in that, The vaccine described contains the claims. The O-type foot-and-mouth disease virus multiepitope described in 1 or 2 Virus-like particles Antigens and adjuvants.

9. As described in claim 8 Virus-like particle vaccines, characterized in that, The adjuvant is ISA206 VG.

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