Method for preparing O-type foot-and-mouth disease virus multi-epitope virus-like particles through escherichia coli as well as product and application thereof

Abstract

The invention discloses a method for preparing O-type foot-and-mouth disease virus multi-epitope virus-like particles through escherichia coli as well as a product and application thereof. The method comprises the following steps: inserting nucleic acid for coding O-type foot-and-mouth disease virus multi-epitope virus-like particles into a prokaryotic expression vector, then converting the prokaryotic expression vector into escherichia coli competent, and carrying out protein expression and purification, wherein the sequence of the nucleic acid for coding the O-type foot and mouth disease virus multi-epitope virus-like particle is as shown in SEQ ID NO. 5. The O-type foot and mouth disease virus multi-epitope VLP antigen is designed by taking HBc protein as a skeleton and all topological representative strain antigen epitopes of O-type foot and mouth disease virus separated in China as elements and introducing T cell epitopes, the antigen spectrum is wider, auxiliary T cell epitopes are introduced, rapid immune response can be excited in an early stage, and the recombinant antigen is soluble in expression, so that the recombinant antigen can be used for preparing the O-type foot and mouth disease virus multi-epitope VLP antigen for the O-type foot and mouth disease virus. And only the soluble antigen needs to be purified, so that the purification step is simplified, the antigen loss is reduced, and industrial production can be realized.

Description

Technical Field

[0001] The present invention relates to a method for preparing virus-like particles, in particular to a method for preparing multi-epitope virus-like particles of foot-and-mouth disease virus serotype O by Escherichia coli, and also relates to the multi-epitope virus-like particles of foot-and-mouth disease virus serotype O prepared by this method and their application in preventing foot-and-mouth disease virus serotype O infection. The present invention belongs to the field of biotechnology. 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 the reduction of cloven-hoofed animals such as pigs, cattle, and sheep, thus causing serious economic losses. The World Organization for Animal Health (WOAH) lists it as a 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 farming industries.

[0003] China mainly adopts comprehensive prevention and control measures for foot-and-mouth disease prevention, combining prevention with immunization and culling. 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 global effective foot-and-mouth disease prevention and control, but their research and production require the use of live viruses, which pose 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. The research and development of genetic engineering vaccines using reverse vaccinology technology, especially virus-like particle (VLP) vaccines and multi-epitope virus-like particle vaccines, have achieved remarkable results.

[0004] Virus-like particles are similar to natural virus particles in morphological structure and have immunogenicity similar to that of whole pathogen antigens. It is worth mentioning that VLP does not contain viral nucleic acid, has high safety, and has a simple production process. In addition, the developed multi-epitope VLP antigen can display protective antigens of multiple pathogens at high density, achieving the purpose of protecting multiple diseases with one VLP antigen. These advantages mean that VLP vaccines are the most likely new vaccines to replace inactivated vaccines for the prevention and control and eradication of animal and human infectious diseases in the future.

[0005] With the discovery of more and more protective antigen epitopes of pathogens, it has become a research hotspot in the biomedical field to design multi-epitope VLP vaccines using the viral proteins of self-assembled VLPs or other natural self-assembled proteins as the backbone and antigen epitopes as the components. This strategy has obvious advantages for the design and research of vaccines against 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 immunogenicity experiments are all based on data obtained from immunized experimental animals such as guinea pigs or mice, and cannot be extrapolated to the immunization effects of large animals such as pigs, cattle, and sheep, and it has not been applied in practice so far. In addition, displaying a small number of antigen epitopes (1-2 kinds of viruses) with VLPs can neither solve the genetic diversity and adaptability of highly variable pathogens nor the polymorphism of the vaccinated individuals; moreover, the recombinant antigen is expressed in the form of inclusion bodies and assembled into VLPs after in vitro renaturation, which may form new structures unrelated to the antigen, and these disadvantages limit the practical application of VLP vaccines.

[0006] The present inventors once designed a foot-and-mouth disease virus multi-epitope VLP antigen using the core protein (HBc) of Woodchuck hepatitis B virus as the backbone, but it was expressed in the form of inclusion bodies in Escherichia coli, and the antigen needed to be denatured and renatured to assemble VLPs. Although the VLP antigen was obtained, the renaturation process could not ensure that the protein could be assembled into VLPs with correct structures. In addition, the early-designed foot-and-mouth disease multi-epitope VLP antigen only contained the antigen epitopes of 1 strain of foot-and-mouth disease virus type O and 1 strain of foot-and-mouth disease virus type A, while there are 3 topological types of foot-and-mouth disease virus type O identified in China, and this multi-epitope vaccine can no longer meet the actual needs. Based on the self-assembled protein of phage AP205, a multi-epitope VLP vaccine was designed for foot-and-mouth disease virus type O prevalent in China. The trial-produced vaccine can protect animals against the attack of virulent strains, but the yield of soluble VLPs is relatively low. The results of electron microscopy and DLS show that the VLPs are of different sizes, and gradient centrifugation is required to achieve homogeneity, with a complex process and high production cost.

[0007] In order to solve the common key technologies restricting vaccine industrialization such as the stable and efficient assembly of antigen epitope VLPs, antigen broad-spectrum, and antigen homogeneity. The present inventors used bioinformatics and immunoinformatics technologies, used the HBc protein as the backbone, used the antigen epitopes of all topological type representative strains of foot-and-mouth disease virus type O isolated in China as the components, and introduced Th cell epitopes at the same time, and designed a foot-and-mouth disease virus type O multi-epitope VLP vaccine, which can realize industrial production and practical application, and is of great significance for the prevention, control, and purification of foot-and-mouth disease. Summary of the Invention

[0008] One of the objectives of the present invention is to provide a method for preparing O-type foot-and-mouth disease virus multi-epitope virus-like particles through Escherichia coli.

[0009] Another objective of the present invention is to provide the O-type foot-and-mouth disease virus multi-epitope virus-like particles prepared by this method and their application in preventing O-type foot-and-mouth disease virus infection.

[0010] To achieve the above objectives, the present invention adopts the following technical means:

[0011] A method for preparing O-type foot-and-mouth disease virus multi-epitope virus-like particles through Escherichia coli according to the present invention, the method comprising inserting the nucleic acid encoding the O-type foot-and-mouth disease virus multi-epitope virus-like particles into a prokaryotic expression vector to obtain a recombinant expression plasmid, and then transforming the recombinant expression plasmid into Escherichia coli competent cells for protein expression and purification steps, wherein the sequence of the nucleic acid encoding the O-type foot-and-mouth disease virus multi-epitope virus-like particles is as shown in SEQ ID NO.5.

[0012] Among them, preferably, the prokaryotic expression vector is pET-30a(+).

[0013] Among them, preferably, the method comprises the following steps:

[0014] (1) Design of O-type foot-and-mouth disease virus multi-epitope antigen gene

[0015] According to the VP1 gene coding sequences of the representative strains O / Tibet / CHA / 99, O / Mya98 / BY / 2010, OZK / 93, O / XJPS / CHA / 2017 and O / HKN / 2007 of 3 topotypes of O-type, the 135-160bp segment sequences of the VP1 gene coding region are selected and concatenated in sequence, and a GS spacer is introduced between adjacent epitopes to form an epitope concatenated structure of 5 strains:

[0016] O / Tibet / CHA / 99 - O / Mya98 / BY / 2010 - OZK / 93 - O / XJPS / CHA / 2017 - O / HKN / 2007, named OB5, and its coding nucleotide sequence is as shown in SEQ ID NO.1; the T cell epitope of the foot-and-mouth disease virus 3A gene is introduced at the C-terminus of OB5 and connected with GS to form an O-type foot-and-mouth disease virus multi-epitope antigen containing 5 B cell epitopes and 1 T cell epitope, named OB5T, and its coding nucleotide sequence is as shown in SEQ ID NO.3;

[0017] (2) Construction of recombinant expression plasmid of O-type foot-and-mouth disease virus multi-epitope chimeric gene

[0018] Insert the coding nucleotide sequence of the OB5T multi-epitope antigen designed in step (1) between nucleotides 234-240 of the coding gene of the Woodchuck hepatitis B virus core protein through a linker to design a multi-epitope chimeric gene of foot-and-mouth disease virus type O, named OB5T-HBc. The sequence of the linker is GGGGSGGGG. Then optimize the OB5T-HBc multi-epitope chimeric gene according to the codon bias of Escherichia coli. The nucleotide sequence of the optimized OB5T-HBc multi-epitope chimeric gene is shown in SEQ ID NO.5. Introduce NdeⅠ and XhoⅠ restriction enzyme sites at the N'-terminus and C'-terminus of the OB5T-HBc antigen gene respectively, and introduce a stop codon at the N-terminus of the Xho 1 sequence. After double digestion with NdeⅠ and XhoⅠ and purification, insert it into the DNA fragment of pET-30a(+) linearized with the same enzymes to construct a recombinant expression plasmid, named pET-30a / OB5T-HBc;

[0019] (3) Expression, purification and identification of recombinant antigen BL21-VLP

[0020] Transform the positive recombinant expression plasmid pET-30a / OB5T-HBc into BL21(DE3), pick a single colony and inoculate it into Kan + LB medium and culture it overnight at 37°C and 220 rpm. Take the overnight culture and inoculate it into fresh Kan + LB medium and culture it at 37°C and 220 rpm until OD 600 ≈0.4 - 0.6, add 0.5 mM IPTG and continue to culture for 6 - 8 hours. Then centrifuge at 8000 rpm for 10 min to harvest the cells. Resuspend the cells in pH 7.4 PBS at 20% of the original culture volume, sonicate on ice, centrifuge at 4°C and 10000 rpm for 30 min to harvest the supernatant, discard the precipitate, and purify the protein by sucrose density gradient centrifugation to obtain the foot-and-mouth disease virus type O multi-epitope virus-like particle, named BL21-VLP.

[0021] Among them, preferably, the GenBank accession number of the coding gene of the Woodchuck hepatitis B virus core protein (HBc) is: J02442.

[0022] Among them, preferably, the specific steps for purifying the protein by sucrose density gradient centrifugation are as follows: 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 rpm for 3 h, collect samples sequentially from the top layer at 0.5 mL per fraction, collect a total of 24 samples, detect all samples at 280 nm and 260 nm with an ultraviolet spectrophotometer, calculate the antigen content, and store at -20°C for later use.

[0023] The O-type foot-and-mouth disease virus multi-epitope virus-like particles prepared by the method described above are also within the protection scope of the present invention. Preferably, the amino acid sequence of the O-type foot-and-mouth disease virus multi-epitope virus-like particles is as shown in SEQ ID NO.6.

[0024] Furthermore, the present invention also proposes the application of the O-type foot-and-mouth disease virus multi-epitope virus-like particles in the preparation of drugs for preventing O-type foot-and-mouth disease virus infection.

[0025] Among them, preferably, the drug is a virus-like particle vaccine.

[0026] Still further, 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 particles and an adjuvant described in the present invention.

[0027] Among them, preferably, the adjuvant is ISA201VG.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] The present invention proposes a method for preparing O-type foot-and-mouth disease virus multi-epitope virus-like particles by Escherichia coli. The present invention uses the HBc protein as the backbone, uses the antigen epitopes of all topological type representative strains of O-type foot-and-mouth disease virus isolated in China as elements, and simultaneously introduces T cell epitopes to design the O-type foot-and-mouth disease virus multi-epitope VLP antigen. At the same time, through structure identification, immunological function evaluation and immune efficacy tests, a recombinant expression plasmid and its recombinant bacteria that can soluble express the O-type multi-epitope VLP antigen of foot-and-mouth disease virus in the Escherichia coli system are screened out. The expressed VLP antigen has a regular and homogeneous morphology under the electron microscope and can protect animals from virus infection. Compared with the early studied foot-and-mouth disease multi-epitope vaccine, the multi-epitope VLP vaccine designed by the present invention contains the antigen epitopes of 5 strains, has a wider antigen spectrum, introduces helper T cell epitopes, can stimulate a rapid immune response at an early stage, and the recombinant antigen is soluble expressed, overcoming the problem that the early designed foot-and-mouth disease HBc VLP cannot be soluble expressed in the Escherichia coli expression system and requires complex operations of denaturation and then renaturation. Only the soluble antigen needs to be purified, which simplifies the purification steps, reduces antigen loss, and can be industrially produced. Brief Description of the Drawings

[0030] Figure 1 For the expression identification of recombinant multi-epitope VLP antigen BL21-VLP;

[0031] Among them, 1 is the protein Marker, and 2, 3, 4, and 5 are respectively before induction, after induction, supernatant after induction, and precipitate after induction;

[0032] Figure 2 For the WB results of purified recombinant multi-epitope VLP antigen BL21-VLP with type O foot-and-mouth disease virus positive serum and type O monoclonal antibody;

[0033] 1 is the protein Marker, and 2 is the purified recombinant multi-epitope VLP antigen BL21-VLP;

[0034] Figure 3 For the determination result of the particle size DLS of recombinant multi-epitope VLP antigen BL21-VLP in liquid;

[0035] Figure 4 For the transmission electron microscopy result of recombinant multi-epitope VLP antigen BL21-VLP;

[0036] Figure 5 For the reaction results of recombinant multi-epitope VLP antigen BL21-VLP, inactivated antigen of type O foot-and-mouth disease virus, and AP205VLP (O4S25) with type O foot-and-mouth disease virus positive sera of pigs, cattle, and sheep;

[0037] Figure 6 For the cellular immune response after immunizing pigs with recombinant multi-epitope VLP antigen BL21-VLP and AP205VLP (O4S25). Detailed Embodiments

[0038] The following further describes the present invention in combination with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the described embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, the details and forms of the technical solutions of the present invention can be modified or replaced, but such modifications or replacements all fall within the protection scope of the present invention.

[0039] Example 1 Preparation of Multi-epitope Virus-like Particles of Type O Foot-and-Mouth Disease Virus by Escherichia coli

[0040] 1. Design of multi-epitope antigen gene of type O foot-and-mouth disease virus

[0041] Based on the amino acid sequences encoded by the VP1 genes of the representative strains of 3 topological types of serotype O (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, namely the sequence of the 135-160 segment in the amino acid coding region of the VP1 gene. They were concatenated in sequence, 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 encoded nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. In addition, a T cell epitope of the foot-and-mouth disease virus 3A gene was introduced at the C-terminus of OB5 and connected with GS to form a multi-epitope antigen of serotype O foot-and-mouth disease virus containing 5 B cell epitopes and 1 T cell epitope, named OB5T. Its encoded nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0042] 2. Construction of recombinant expression plasmid of multi-epitope chimeric gene of serotype O foot-and-mouth disease virus

[0043] The nucleotide sequence encoding the OB5T multi-epitope antigen 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 gene encoding the woodchuck hepatitis B virus core protein (GenBank accession number: J02442) using linker (GGGGSGGGG) to design a multi-epitope chimeric gene of serotype O foot-and-mouth disease virus, named OB5T-HBc. According to the codon bias of Escherichia coli, the codons of the OB5T-HBc antigen gene were optimized. The nucleotide sequence of the optimized OB5T-HBc multi-epitope chimeric gene is shown in SEQ ID NO.5. To insert this multi-epitope chimeric gene directionally, NdeⅠ and XhoⅠ restriction enzyme sites were introduced at the N'-terminus and C'-terminus of the DNA respectively, and a stop codon was introduced at the N-terminus of the Xho 1 sequence to ensure the integrity of the recombinant antigen. The chimeric DNA designed and synthesized above was double-digested with NdeⅠ and XhoⅠ, purified, and then inserted into the DNA fragment of pET-30a(+) linearized with the same enzymes to construct a recombinant expression plasmid, named pET-30a / OB5T-HBc, which was synthesized by Nanjing Genscript Co., Ltd. and verified by sequencing.

[0044] 3. Expression, purification and identification of recombinant antigen BL21-VLP

[0045] The positive recombinant expression plasmid pET-30a / OB5T-HBc was transformed into BL21(DE3) by heat shock. A single colony was picked and inoculated into an appropriate amount of LB medium (Kan+), and cultured overnight at 37°C and 220 rpm. The overnight culture was taken and inoculated into fresh LB medium (Kan+) at 1% (V / V), and cultured at 37°C and 220 rpm until OD 600 ≈0.4 - 0.6, then 0.5 mM IPTG was added and the culture was continued for 6 - 8 hours. The cells were harvested by centrifugation at 8000 rpm for 10 min, resuspended in PBS (pH 7.4) at 20% of the original culture volume, sonicated on ice, and the supernatant was harvested by centrifugation at 4°C and 10,000 rpm for 30 min. The precipitate was discarded, and the protein was purified by sucrose density gradient centrifugation to obtain the multi-epitope virus-like particle of foot-and-mouth disease virus type O, named BL21-VLP, and the amino acid sequence is shown in SEQ ID NO.6.

[0046] Among them, the specific steps for purifying the protein by sucrose density gradient centrifugation are as follows: 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 35,000 rpm for 3 h, collect samples sequentially from the top layer at 0.5 mL per fraction (for 1 sample), a total of 24 samples were collected. All samples were detected at 280 nm and 260 nm by ultraviolet spectrophotometer, the antigen content was calculated, and stored at -20°C for later use.

[0047] The purified recombinant antigen was identified by SDS-PAGE. The results showed that the size of the recombinant antigen BL21-VLP was about 34 kDa, which was consistent with the expected size, and the content of the soluble recombinant antigen BL21-VLP was significantly increased ( Figure 1 ). The WB results showed that the recombinant antigen BL21-VLP could not only specifically immunoreact with the positive serum of foot-and-mouth disease virus type O, but also immunoreact with the monoclonal antibody against the main antigenic epitope (G-H loop) of foot-and-mouth disease virus type O ( Figure 2 ). It indicated that the expressed recombinant antigen BL21-VLP could correctly display the antigenic epitope.

[0048] 4. Identification of the morphology and structure of the recombinant antigen BL21-VLP

[0049] The purified BL21-VLP was measured for the particle size and distribution under liquid conditions by dynamic light scattering; its morphology and size were observed by transmission electron microscopy. The dynamic light scattering results showed that the particle size of BL21-VLP was about 32 nm, and the size homogeneity was good ( Figure 3 ). Under the transmission electron microscope, BL21-VLP was a typical VLP structure, and the size was consistent with the DLS results ( Figure 4 ).

[0050] Immunological Identification of Recombinant Antigen BL21-VLP in Example 2

[0051] Coat a 96-well ELISA plate with inactivated antigen of Foot-and-Mouth Disease virus type O (1 μg / mL), purified recombinant antigen BL21-VLP, and early-developed Foot-and-Mouth Disease virus type O VLP (named O4S25, prepared according to the method described in the patent application with the publication number CN116041547A), 100 μl / well, and incubate overnight at 4°C. Block with PBST (pH 7.4) containing 5% skim milk powder at 37°C for 2 h, wash three times with PBST, add porcine, bovine, and ovine positive sera against Foot-and-Mouth Disease virus type O diluted 1:100, 100 μl / well, and at the same time add corresponding negative control sera for Foot-and-Mouth Disease, and incubate at 37°C for 45 min. Wash three times with PBST, add HRP-labeled anti-animal species IgG diluted 1:5000, 100 μl / well, and incubate at 37°C for 30 min. Wash three times with PBST, add TMB chromogenic solution, 100 μl / well, and develop color at 37°C for 10 - 15 min. Terminate the reaction with 2 M H2SO4 and measure the OD 450nm absorbance value. The results show that the recombinant antigen BL21-VLP can have a strong immune reaction with porcine, bovine, and ovine positive sera against Foot-and-Mouth Disease virus type O, but not with negative sera for Foot-and-Mouth Disease; more importantly, the immunoreactivity of the recombinant antigen BL21-VLP is significantly better than that of inactivated antigen of Foot-and-Mouth Disease virus type O and O4S25, fully indicating that the protective antigen of Foot-and-Mouth Disease virus type O is correctly displayed and its immunological activity is fully exerted, significantly improving the immunological activity( Figure 5 ).

[0052] Preparation and Immunopotency Experiment of Multiepitope Virus-Like Particle Vaccine against Foot-and-Mouth Disease virus type O in Example 3

[0053] 1. Vaccine Preparation

[0054] After quantifying the purified multiepitope virus-like particle antigen BL21-VLP of Foot-and-Mouth Disease virus type O in Example 1 by BCA method, dilute it with PBS to 200 μg / ml and 100 μg / ml, and emulsify it into a vaccine preparation (W / O / W) by adding 50 g:50 g of water-in-oil-in-water adjuvant ISA201VG (Seppic, France). Each dose contains 100 μg / ml and 50 μg / ml of antigen respectively, which is the BL21-VLP vaccine, and store it at 2 - 8°C for standby.

[0055] 2. Immunopotency Test

[0056] Twelve healthy pigs weighing about 30 kg, negative for foot-and-mouth disease virus antibodies and 3ABC antibodies, were selected. They were immunized with two different doses of the trial vaccine, with 5 pigs in each group, and inoculated intramuscularly at 1 ml / head. The remaining 2 pigs were used as non-immunized controls. Blood samples were collected before immunization and 28 days after immunization to isolate sera for detecting specific antibody IgG. At the same time, 28 days after immunization, together with 2 control pigs under the same conditions, they were challenged with foot-and-mouth disease virus type O according to the national standard and observed for 10 days. The results showed that the multi-epitope VLP vaccine against foot-and-mouth disease virus type O could induce high levels of foot-and-mouth disease-specific antibodies and completely protect pigs against foot-and-mouth disease virus type O infection (Table 1). In addition, the serum INF-γ level of the antigen developed in this invention was significantly higher than that of the antigen O4S25 developed earlier after immunizing animals. There were no phenomena such as swelling, redness, or fever at the injection site of the animals, nor any other adverse reactions. The appetite was normal and the mental state was good, confirming that the vaccine was very safe.

[0057] Table 1

[0058]

[0059] Based on the above results, compared with the foot-and-mouth disease type O multi-epitope VLP antigen O4S25 designed by the inventor earlier with AP205 as the backbone, the foot-and-mouth disease type O multi-epitope VLP antigen BL21-VLP developed in this invention with HBc as the backbone has significant immunological activity. In addition to inducing strong humoral immunity, it also induces strong cellular immune responses, that is, it can induce a comprehensive immune response. Moreover, it overcomes the problem that the early-designed foot-and-mouth disease VLP cannot be solubly expressed in the Escherichia coli expression system and requires a complex operation process of denaturation and then renaturation. Only the soluble antigen needs to be purified, which simplifies the purification steps, reduces antigen loss, and can be industrially produced.

Claims

1. A method for preparing multi-epitope virus-like particles of type O foot-and-mouth disease virus by using Escherichia coli, characterized in that: The method comprises inserting a nucleic acid encoding multi-epitope virus-like particles of O-type foot-and-mouth disease virus into a prokaryotic expression vector to obtain a recombinant expression plasmid, then transforming the recombinant expression plasmid into competent Escherichia coli, and performing protein expression and purification steps, wherein the sequence of the nucleic acid encoding multi-epitope virus-like particles of O-type foot-and-mouth disease virus is shown as SEQ ID NO.

5.

2. The method according to claim 1, characterized in that The prokaryotic expression vector is pET-30a(+).

3. The method according to claim 1, characterized in that The method comprises the following steps: (1) Design of multi-epitope antigen gene of O-type foot-and-mouth disease virus According to the VP1 gene coding 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 135-160bp segment sequence of the VP1 gene coding region was selected and serially connected in sequence, and GS spacers were introduced between adjacent epitopes to form epitope serial structures of the five strains: O / Tibet / CHA / 99-O / Mya98 / BY / 2010-OZK / 93-O / XJPS / CHA / 2017-O / HKN / 2007, named OB5, the encoding nucleotide sequence of which 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 GS was used to connect to form an O-type foot-and-mouth disease virus multi-epitope antigen containing 5 B cell epitopes and 1 T cell epitope, named OB5T, the encoding nucleotide sequence of which is shown in SEQ ID NO.3; (2) Construction of recombinant expression plasmid of multi-epitope chimeric gene of O-type foot-and-mouth disease virus The coding nucleotide sequence of the OB5T multi-epitope antigen designed in step (1) is inserted into the nucleotides 234-240 of the core protein coding gene of the woodchuck hepatitis B virus through a linker to design an O-type foot-and-mouth disease virus multi-epitope chimeric gene, which is named OB5T-HBc. The sequence of the linker is GGGGSGGGG; then, the OB5T-HBc multi-epitope chimeric gene is optimized according to the codon preference of Escherichia coli. The nucleotide sequence of the optimized OB5T-HBc multi-epitope chimeric gene is shown in SEQ ID NO.

5. NdeⅠ and XhoⅠ restriction sites are introduced at the N′-terminus and C′-terminus of the OB5T-HBc antigen gene, respectively, and a stop codon is introduced at the N-terminus of the Xho1 sequence. After double digestion with NdeⅠ and XhoⅠ and purification, a pET-30a(+) DNA fragment linearized with the same enzyme is inserted to construct a recombinant expression plasmid, which is named pET-30a / OB5T-HBc; (3) Expression, purification and identification of recombinant antigen BL21-VLP The positive recombinant expression plasmid pET-30a / OB5T-HBc was transformed into BL21 (DE3), and a single colony was selected to inoculate Kan + LB medium, 37 ℃, 220rpm overnight culture, take the overnight culture, inoculate 1% (V / V) into fresh Kan + LB medium, 37°C, 220 rpm, culture until OD 600 When the pH value was ≈0.4-0.6, 0.5 mM IPTG was added and culture was continued for 6-8 hours. The bacteria were harvested by centrifugation at 8000 rpm for 10 min, and pH 7.4 PBS was added according to 20% of the original culture volume to resuspend, and ultrasonically disrupted on ice. The supernatant was harvested by centrifugation at 4°C and 10000 rpm for 30 min, and the precipitate was discarded. The protein was purified by sucrose density gradient centrifugation to obtain multi-epitope virus-like particles of type O foot-and-mouth disease virus, named BL21-VLP.

4. The method according to claim 1, characterized in that The GenBank accession number of the woodchuck hepatitis B virus core protein encoding gene is: J02442.

5. The method according to claim 1, characterized in that The specific steps of protein purification by sucrose density gradient centrifugation are as follows: 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 35,000 rpm for 3 hours, collect samples in sequence starting from the top layer with 0.5 mL as one fraction, and collect 24 samples in total. All samples are detected by UV spectrophotometer at 280nm and 260nm, the antigen content is calculated, and the samples are stored at -20°C for future use.

6. The O-type foot-and-mouth disease virus multi-epitope virus-like particle prepared according to the method according to any one of claims 1 to 5, wherein the amino acid sequence of the O-type foot-and-mouth disease virus multi-epitope virus-like particle is shown in SEQ ID NO.

6.

7. Use of the multi-epitope virus-like particles of O-type foot-and-mouth disease virus according to claim 6 in the preparation of a drug for preventing infection with O-type foot-and-mouth disease virus.

8. The use according to claim 7, characterized in that The medicine is a virus-like particle vaccine.

9. An O-type foot-and-mouth disease multi-epitope virus-like particle vaccine, characterized in that: The vaccine contains the multi-epitope virus-like particles of O-type foot-and-mouth disease virus according to claim 6 and an adjuvant.

10. The O-type foot-and-mouth disease multi-epitope virus-like particle vaccine according to claim 9, characterized in that: The adjuvant is ISA201VG.

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