CN111773383B

CN111773383B - O-type foot-and-mouth disease subunit vaccine and preparation method and application thereof

Info

Publication number: CN111773383B
Application number: CN202010630218.0A
Authority: CN
Inventors: 茹毅; 刘华南; 张贵财; 杨帆; 李丹; 郭建宏; 何继军; 张娇燕; 李亚军; 马坤; 伍春平; 郝荣增; �田宏; 张克山; 曹伟军; 马旭升; 党文; 靳野
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2021-02-02
Anticipated expiration: 2040-07-03
Also published as: CN111773383A

Abstract

The invention relates to the technical field of molecular biology, in particular to an O-type foot-and-mouth disease subunit vaccine with a cross protection effect, and a preparation method and application thereof. The invention firstly discovers a strain with wide cross neutralization function on the O-type foot-and-mouth disease epidemic virus in China unexpectedly in the screening through a serum cross neutralization test; the three structural proteins VP0, VP3 and VP1 of the strain are optimally designed on the basis of amino acid sequences, and a single plasmid is screened out to simultaneously express the three structural proteins in escherichia coli in a high-efficiency, uniform and soluble manner by virtue of small ubiquitin-like fusion protein (SUMO), and the three virus structural proteins are successfully self-assembled in vitro; the finally obtained target protein accounts for about 30-35% of the total protein of the thallus, and the yield of the target protein after purification can reach 150mg/L at most. The foot-and-mouth disease vaccine prepared by the three foot-and-mouth disease virus structural proteins has the minimum full protective immune dose as low as 20 mu g/head, and has wide cross protection effect on a plurality of O-type foot-and-mouth disease epidemic viruses in China.

Description

O-type foot-and-mouth disease subunit vaccine and preparation method and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to an O-type foot-and-mouth disease subunit vaccine and a preparation method and application thereof.

Background

Foot and Mouth Disease Virus (FMDV) belongs to the family of picornaviridae, the genus of Foot and Mouth Disease Virus, and virions have no envelope and are icosahedral symmetric, and consist of structural proteins VP0, VP3 and VP1, wherein the VP0 protein consists of VP4 and VP 2. The virus mainly infects worldwide virulent infectious diseases of artiodactyls such as pigs, cattle, sheep and the like, has the characteristics of fast replication, high contact infection, strong pathogenicity and the like, and can cause pandemics particularly in swinery. The outbreak of foot-and-mouth disease can limit the trade of animals and products thereof, which causes serious economic loss and social influence, and the world animal health Organization (OIE) classifies the outbreak as a legal compulsory disease, and the department of agriculture in China also classifies the outbreak as an animal infectious disease. FMDV is subject to variation, is a natural attribute, and is also a major cause of long-term foot-and-mouth disease epidemic and is difficult to control and decontaminate. The foot-and-mouth disease virus has seven serotypes (O, A, Asia1, SAT1, SAT2, SAT3 and C type), and no cross protection reaction exists among the serotypes. The foot-and-mouth disease epidemic history in China is long, and currently, O type and A type are mainly epidemic, and O type is mainly used. According to the national foot-and-mouth disease reference laboratory monitoring, the type-O foot-and-mouth disease epidemic virus in China mainly has 4 lineages (linkage), including the classical topological (Cathay), Panasia (Panasia) lineages, Myanmar 98(Mya98) pedigree and India 2001(Ind-2001) lineages in China, and the like, and a plurality of epidemic strains exist simultaneously, thereby bringing great pressure to the prevention and control of the foot-and-mouth disease in China.

In China, a comprehensive prevention and control strategy mainly based on prevention is adopted to prevent and control the foot-and-mouth disease, and the inoculation of inactivated vaccines to animals susceptible to the foot-and-mouth disease is the most common immunization measure. The inactivated vaccine is prepared by a large amount of amplification, inactivation, emulsification and other processes of foot-and-mouth disease field strains through a virus culture system, and plays an important role in controlling the pandemic of foot-and-mouth disease in China. However, the traditional inactivated vaccine has the problems of short immunity duration, high virus dispersing risk due to incomplete virus inactivation, high production cost and the like in the production and preparation process. More importantly, the existing foot-and-mouth disease epidemic strains in China are complex and variable, and the traditional inactivated vaccines are difficult to deal with. Taking the O-type Mya98 strain as an example, the strain is introduced into China from 2010, and causes the foot-and-mouth disease pandemic in China. The O-type foot-and-mouth disease vaccine represents a strain (O/Mya98/BY/2010) which should be born and is applied in the field for nearly 10 years. Under the action of multiple factors such as immune pressure, continuous introduction of foreign strains and the like, the O-type Mya98 virus is subjected to rapid mutation, the difference of the VP1 nucleotide sequences of the existing O-type Mya98 strain and the 2010 early-epidemic strain (such as vaccine strain O/Mya98/BY/2010) is nearly 10%, the antigenic relationship (r value) between the epidemic strain and the vaccine strain is reduced, and even the virus with the r value lower than 0.3 is found, so that the virus is beyond the OIE immune matching range.

In the face of the simultaneous existence of a plurality of epidemic strains and the formation and occurrence of antigen variation strains, which cause poor matching performance of vaccine strains, and cannot provide high-efficiency cross protection for the 4 pedigree O-type epidemic variation strains at the same time, the problem needs to be effectively solved.

In terms of virus characteristics, the type O foot-and-mouth disease vaccine has the problems of unstable antigen and low immune efficacy compared with the type a and the type Asia1, and how to improve the immune efficacy, antigen matching and cross protection of the type O foot-and-mouth disease vaccine is important. No vaccine with wide cross neutralization protection on O-type foot-and-mouth disease epidemic virus in China is reported in the prior literature or patent.

The foot-and-mouth disease virus structural protein is responsible for assembling virus capsid, determines antigen specificity and is an important antigen component of the virus. Similar to other viruses of picornaviridae, after three structural proteins of foot-and-mouth disease virus, VP0, VP3 and VP1, are mixed in vitro, a part of them will assemble by themselves to form empty capsids (virus-like particles), which have the same or similar morphology and structure as real virus particles, and maintain the spatial conformation of virus particles, but do not contain virus nucleic acids, cannot replicate, and do not have infectivity. The foot-and-mouth disease virus empty capsid contains the specific epitope of the virus, can simulate the infection of the natural virus to host cells, effectively stimulates the organism to generate strong immune response, and is a safer and more effective vaccine candidate. The preparation of the foot-and-mouth disease virus empty capsid by the genetic engineering technology is reported in partial documents, and the current commonly used genetic engineering expression system of the foot-and-mouth disease virus empty capsid is an escherichia coli expression system, has the characteristics of low cost, fast cell growth, large-scale amplification and the like, but the expression product of the foot-and-mouth disease virus empty capsid lacks necessary post-translational modification, is easy to form inclusion bodies and causes poor biological activity. Research has shown that the solubility of foot-and-mouth disease virus structural protein can be improved by introducing small ubiquitin-like fusion protein (SUMO) into an escherichia coli expression system. For example, patent (CN101914501B) discloses that SUMO is introduced into foot-and-mouth disease structural proteins VP0, VP1 and VP3, and expressed by three plasmids, respectively, to construct Asia1 type foot-and-mouth disease virus-like particles; in the patent (CN104404074B), small ubiquitin-like fusion protein (SUMO) is introduced to construct co-expression plasmids of SUMO-VP0, SUMO-VP1 and SUMO-VP3, so that Asia1 type foot-and-mouth disease virus-like particles are obtained, but the expression efficiency is low. The highest yield of the purified Asia1 type foot-and-mouth disease virus structural protein prepared by an escherichia coli expression system only reaches about 20mg/L, and the quantity of generated fully-protected immune antigen at least needs 50 mu g/head, thereby greatly limiting the value and the prospect of commercial application.

Disclosure of Invention

The invention firstly develops the high-efficiency foot-and-mouth disease subunit vaccine with wide cross protection effect aiming at the 4 main pedigree epidemic viruses of the O-type foot-and-mouth disease in China.

Firstly, through a serum cross neutralization test, a strain O/17002 preserved in a foot-and-mouth disease reference laboratory of China is unexpectedly found to have a wide cross neutralization effect on the foot-and-mouth disease epidemic virus type O in China, and the strain is used as a vaccine antigen source strain; the amino acid sequences of three structural proteins VP0, VP3 and VP1 of an O/17002 strain are taken as a basis for optimization design, so that the degradation of the virus structural protein VP3 by host cells is avoided, the inhibition of the virus structural protein VP1 on a natural immune signal channel of the host cells is avoided, and the stability and the antigenicity of the vaccine antigen are improved; the inventor also discovers that the expression efficiency of three structural proteins of a foot-and-mouth disease virus strain O/17002 is not uniform in single expression for the first time, and even if the soluble tag protein is fused with the structural proteins VP0, VP3 and VP1 for expression, the problem of nonuniform expression amount of the three structural proteins VP0, VP3 and VP1 cannot be effectively improved, so that the virus empty capsid assembly efficiency is seriously influenced, the invention solves the technical problem for the first time, screens out three structural proteins which are simultaneously expressed efficiently, uniformly and in a soluble way in escherichia coli by a single plasmid, and the yield of the target protein after purification can reach 150mg/L to be more than 7 times of the highest yield reported by the existing literature; the foot-and-mouth disease vaccine prepared by the three foot-and-mouth disease virus structural proteins has the minimum full protective immune dose as low as 20 mu g/head, and has wide cross protection effect on O-type foot-and-mouth disease epidemic virus in China.

The specific invention content is as follows:

the invention aims to provide an application of O type foot-and-mouth disease virus strain O/17002 in preparation of an O type foot-and-mouth disease subunit vaccine, wherein the amino acid sequence of the O/17002 structural protein VP0 is shown as SEQ ID NO.1, the amino acid sequence of the O/17002 structural protein VP3 is shown as SEQ ID NO.3, and the amino acid sequence of the O/17002 structural protein VP1 is shown as SEQ ID NO. 5.

Another purpose of the invention is to provide an O type foot-and-mouth disease vaccine prepared according to the structural proteins VP0, VP3 and VP1 sequence of O type foot-and-mouth disease virus strain O/17002.

Aiming at the technical problem of lower immune protection of foot-and-mouth disease virus-like particles in the prior art, the invention also aims to provide an O-type foot-and-mouth disease virus structural protein composition, wherein the O-type foot-and-mouth disease virus structural protein composition consists of structural proteins VP0, VP3 and VP1 of O-type foot-and-mouth disease virus O/17002, the amino acid sequence of the structural protein VP0 is shown as SEQ ID NO.1, the amino acid sequence of the structural protein VP3 is shown as SEQ ID NO.3, and the amino acid sequence of the structural protein VP1 is shown as SEQ ID NO. 5.

Preferably, the nucleotide sequence for coding the structural protein VP0 is shown in SEQ ID NO.2, the nucleotide sequence for coding the structural protein VP3 is shown in SEQ ID NO.4, and the nucleotide sequence for coding the structural protein VP1 is shown in SEQ ID NO. 6.

Preferably, the 118 th lysine of the structural protein VP3 is mutated into arginine, and the amino acid sequence of the mutated structural protein VP3 is shown as SEQ ID NO. 7; and/or the lysine at position 210 of the structural protein VP1 is mutated into arginine, and the amino acid sequence of the mutated structural protein VP1 is shown as SEQ ID NO. 9; the strategy of mutating lysine 118 of the structural protein VP3 to arginine and/or mutating lysine 210 of the structural protein VP1 to arginine improves the in vivo stability and protective efficacy of the aftosa subunit vaccine.

Preferably, the nucleotide sequence for coding the mutated structural protein VP3 is shown in SEQ ID NO.8, and the nucleotide sequence for coding the mutated structural protein VP1 is shown in SEQ ID NO. 10.

The invention also aims to provide application of the O-type foot-and-mouth disease virus structural protein composition in preparation of a foot-and-mouth disease vaccine.

It is another object of the present invention to provide a vaccine for preventing foot and mouth disease infection, which includes a foot and mouth disease virus structural protein composition.

Preferably, the vaccine further comprises an adjuvant.

Preferably, the adjuvant is one or more of chemical immune adjuvant, microbial immune adjuvant, plant immune adjuvant and biochemical immune adjuvant.

Aiming at the technical problem of low expression efficiency of foot-and-mouth disease virus-like particles in the prior art, the invention provides a preparation method of an O-type foot-and-mouth disease virus structural protein composition, which comprises the following steps:

(1) designing a gene sequence THS for coding a fusion tag protein, wherein T is a nucleotide sequence of a translation initiation region, H is a nucleotide sequence for coding a protein containing a histidine tag, and S is a nucleotide sequence for coding a protein containing saccharomyces cerevisiae small ubiquitin-like modification (SUMO);

(2) respectively connecting the gene sequence THS of the encoding fusion tag protein in the step (1) with genes encoding O-type aftosa epidemic strain structural proteins VP0, VP3 and VP1 in series to form three sections of fusion gene sequences THS-VP0, THS-VP3 and THS-VP 1; the gene sequence THS of the coded fusion tag protein is respectively connected in series with the genes of the coded O-type aftosa epidemic strain structural proteins VP0, VP3 and VP1, so that the expression efficiency of the virus structural protein is improved;

(3) cloning the gene sequences of the three sections of fusion target proteins coded in the step (2) into a prokaryotic expression vector pET30a by a molecular cloning technology to obtain a recombinant expression plasmid pET-FMDV-VP 031; cloning genes encoding O-type aftosa epidemic strain structural proteins VP0, VP3 and VP1 into the same expression vector, so that the equivalent expression of the three structural proteins can be realized, and the three structural proteins can be self-assembled in vitro;

(4) transforming the recombinant expression plasmid pET-FMDV-VP031 in the step (3) into escherichia coli to obtain a genetic engineering bacterium, fermenting and culturing the genetic engineering bacterium, and inducing and expressing foot-and-mouth disease virus structural proteins VP0, VP3 and VP1 with fusion protein labels by IPTG;

(5) after the thalli of the genetic engineering bacteria are crushed, supernatant fluid is recovered, and the mixture of foot-and-mouth disease virus structural proteins VP0, VP3 and VP1 with fusion tag protein is obtained by affinity chromatography separation and purification;

(6) and (3) after the fusion tag protein in the mixture in the step (5) is removed by enzyme digestion, separating and purifying by affinity chromatography to obtain a mixture of foot-and-mouth disease virus structural proteins VP0, VP3 and VP 1. The two-step affinity chromatography purification avoids the molecular sieve chromatography purification with higher cost and improves the recovery rate of the antigen protein.

Preferably, the amino acid sequence of the structural protein VP0 in the step (2) is shown in SEQ ID NO.1, the amino acid sequence of the structural protein VP3 is shown in SEQ ID NO.3, and the amino acid sequence of the structural protein VP1 is shown in SEQ ID NO. 5.

Preferably, the 118 th lysine of the structural protein VP3 is mutated into arginine, and the amino acid sequence of the mutated structural protein VP3 is shown as SEQ ID NO. 7; and/or the lysine at position 210 of the structural protein VP1 is mutated into arginine, and the amino acid sequence of the mutated structural protein VP1 is shown as SEQ ID NO. 9; the strategy of mutating the 118 th lysine of the structural protein VP3 into arginine and/or mutating the 210 th lysine of the structural protein VP1 into arginine improves the in vivo stability and protective efficacy of the foot-and-mouth disease genetic engineering vaccine.

Preferably, the nucleotide sequence of THS in step (1) is shown in SEQ ID NO. 11.

The invention has the beneficial effects that: firstly, the invention provides the O-type foot-and-mouth disease virus strain which has high immune efficiency and good antigen matching property and can effectively protect a plurality of field epidemic strains for the first time; introducing fusion tag protein based on the sequences of the structural protein genes VP0, VP3 and VP1 for encoding the strain, and simultaneously expressing three structural proteins in escherichia coli in an efficient, uniform and soluble manner through a single plasmid; combining a purification process of two-step affinity chromatography, the finally obtained target protein accounts for about 30-35% of the total protein of the thalli, and the yield of the target protein after purification can reach 150mg/L at most, which is more than 7 times of the highest yield reported by the existing literature; the construction method is simple, the chromatographic purification of the molecular sieve with higher cost is avoided, complex processes such as ultracentrifugation and the like are not needed, the recovery rate of the two-step affinity purification is close to 60 percent, and the preparation and purification of the foot-and-mouth disease virus structural protein in an industrialized scale are easy to realize; fourthly, the 118 th lysine of the structural protein VP3 is mutated into arginine, and/or the 210 th lysine of the structural protein VP1 is mutated into arginine, so that the degradation of the virus structural protein VP3 by TBK1 in host cells is avoided, the inhibition of the virus structural protein VP1 on the natural immune signal channel of the host cells is avoided, and the stability and the protective efficacy of the vaccine are improved; the minimal dose of the obtained foot-and-mouth disease genetic engineering vaccine can be as low as 20 mug/head, and the vaccine has wide cross protection effect on O-type foot-and-mouth disease epidemic virus in China.

Drawings

FIG. 1: the foot-and-mouth disease virus structural protein of the fused small ubiquitin-like modified protein (SUMO) label obtained by the invention is subjected to SDS-PAGE identification result; wherein M is a molecular weight Marker; 1 is whole pre-induction bacteria; 2 is induced whole bacteria; 3 is supernatant after the lysis of the whole bacteria after induction, and the sample loading amount is 5 mu L;

FIG. 2: the foot-and-mouth disease virus structural protein with the SUMO label purified by the affinity chromatography is subjected to SDS-PAGE identification result after the SUMO enzyme digestion; wherein M is a molecular weight Marker; 1 is a foot-and-mouth disease virus structural protein with SUMO label after affinity chromatography purification; 2, after the SUMO enzyme is digested, the foot-and-mouth disease virus structural protein without the SUMO label is loaded in an amount of 5 mu L;

FIG. 3: the foot-and-mouth disease virus structural protein without the SUMO label is subjected to SDS-PAGE identification result after affinity chromatography purification; wherein M is a molecular weight Marker; the O/FMDV is three purified structural proteins of O-type foot-and-mouth disease virus, and the sample loading amount is 5 mu L;

FIG. 4: the foot-and-mouth disease virus structural protein obtained by the invention is observed by a transmission electron microscope after self-assembly;

FIG. 5: the expression efficiency SDS-PAGE identification result of the foot-and-mouth disease virus structural protein composition; wherein M is a molecular weight Marker; 1 and 2 are Re-O/17002/VP3 whole bacteria before induction and supernatant after lysis of whole bacteria after induction; 3 and 4 are Re-O/17002/VP1 whole bacteria before induction and supernatant after lysis of whole bacteria after induction; 5 and 6 are Re-O/17002/VP3+ VP1 whole bacteria before induction and supernatant after lysis of whole bacteria after induction; 7 and 8 are supernatants of whole bacteria before induction and after lysis of whole bacteria after induction of O/17002, and the sample loading amount is 5 mu L;

FIG. 6: neutralizing antibody response results of the genetically engineered vaccine immunized animals before and after optimization of foot and mouth disease virus structural proteins;

FIG. 7: genetic engineering vaccine immune pig PD (PD) optimized by foot-and-mouth disease virus structural protein₅₀Neutralizing antibody response results in the assay.

Detailed Description

The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention. The conditions used in the examples may be further adjusted according to the conditions of the particular manufacturer, and the conditions not specified are generally the conditions in routine experiments.

The experiments described in the following examples obtain biosafety permits and foot and mouth disease laboratory activity permits:

according to the related requirements of biological safety 3-level laboratory (BSL-3) and the related biological safety of foot-and-mouth disease, the Lanzhou veterinary research institute has reported step by step through biological safety committee of Lanzhou veterinary research institute, ethical committee of experimental animals, biological safety committee of Chinese agricultural scientific institute, ethical committee of experimental animals of Lanzhou veterinary research institute and biological safety committee of Lanzhou veterinary research institute, and the permission of developing highly pathogenic FMDV pathogen and animal research is obtained by the department of agriculture, and the permission is filed by the department of agriculture and rural area, and meets the requirements of national biological safety level.

Foot-and-mouth disease type O epidemic BY/2010(Mya98), 13152(Mya98), 14064(Mya98), 15126(Panasia), 16045(Mya98), 17016(Mya98), 17002(Mya98), 17009(Ind-2001), 17042(Ind-2001) and 18074(Cathay) are from national foot-and-mouth disease reference laboratories.

Description and explanation of the related terms in the present invention:

the term "E.coli expression system" means a system consisting of E.coli (strain) derived from commercially available sources, exemplified but not limited thereto: BL21(DE3), BL21(DE3) pLysS, B834(DE3), BLR (DE3), JM109, XL1Blue, ER2566, Rosetta, GI698, preferably BL21(DE 3).

The term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell to obtain expression of the genetic material element carried by the vector in the host cell. By way of example, the carrier includes: a plasmid; bacteriophage; cosmids, etc.

The foot-and-mouth disease virus structural proteins VP3, VP1 and VP0 (which are gene fusion of VP4 and VP 2) are subjected to tandem co-expression, and the tandem co-expression refers to that a plurality of genes are inserted into the same vector for co-expression. Tandem coexpression sequences include, but are not limited to, VP3-VP1-VP0, which can be various combinations between VP3, VP1, VP0 and various possible combinations between VP1, VP2, VP3, VP4, which can be, for example, the tandem sequence of VP1-VP3-VP0, the tandem sequence of VP3-VP0-VP1, the tandem sequence of VP1-VP0-VP3, the tandem sequence of VP3-VP1-VP2-VP4, the tandem sequence of VP4-VP2-VP3-VP1, etc., preferably the tandem sequence of VP3-VP1-VP 0.

The term "vaccine" refers to a biological agent capable of providing a protective response in an animal, wherein the vaccine has been delivered and is not capable of causing serious disease. The vaccine of the invention is a subunit vaccine which is combined by the structural proteins VP0, VP3 and VP1 of O type foot-and-mouth disease virus strain O/17002 and is genetically engineered.

The vaccine of the present invention, further optionally comprises one or more adjuvants, excipients, carriers and diluents. The adjuvant can be any suitable adjuvant, chemical immune adjuvants such as aluminum hydroxide, Freund's adjuvant, mineral oil, span, etc.; microbial immune adjuvants such as mycobacteria, BCC, lipopolysaccharide, muramyl dipeptide, cytopeptide, fat-soluble waxy D, and short corynebacterium; the plant immunologic adjuvant is polysaccharides extracted from plant or large fungi, such as pachyman, carthamus tinctorius polysaccharide, Chinese herbal medicine, etc. And biochemical immune adjuvants such as thymosin, transfer factor, interleukin, etc. Preferred adjuvants may be nano-adjuvant biological adjuvants, interleukins, interferons, etc.

The vaccine of the invention can also be used for combined vaccines, such as combined vaccines with other vaccines of pigs, but the emphasis is on live attenuated vaccines, especially on integration of viral genes, such as bivalent vaccine, trivalent vaccine, and the like. The combination vaccine may comprise a plurality of attenuated non-swine fever viruses of different genotypes, such that a cross-protective immune response against the plurality of non-swine fever virus genotypes may be induced.

The administration of the vaccines of the present invention may be by any convenient route, for example, intramuscular injection, intranasal, oral, subcutaneous, transdermal and vaginal routes. The attenuated vaccines of the present invention are preferably administered intramuscularly. The vaccine may be administered after a prime-boost regimen. For example, after a first vaccination, the subject may receive a second booster administration after a period of time (e.g., about 7, 14, 21, or 28 days). Typically, the booster is administered at the same or a lower dose than the prime dose. In addition, a third booster immunization may be performed, for example 2-3 months, 6 months or a year after immunization.

EXAMPLE 1 foot-and-mouth disease Virus O/17002 serum Cross-neutralization assay

The invention unexpectedly discovers a strain O/17002 which has wide cross-neutralization function on the O-type foot-and-mouth disease epidemic virus in China, from the foot-and-mouth disease epidemic strains preserved in a national foot-and-mouth disease reference laboratory (the depending unit is Lanzhou veterinary research institute of Chinese agricultural academy of sciences).

Performing amplification propagation on a strain O/17002 through suspension culture of BHK21 cells, obtaining viruses, determining the toxin value, performing BEI inactivation, emulsifying with an ISA 206 adjuvant, and preparing a vaccine; the vaccine is used for immunizing pigs to obtain the hyperimmune positive serum of the strain O/17002. The obtained hyperimmune serum is respectively subjected to virus neutralization tests with O/Mya98, O/Panasia, O/Ind-2001 and O/Cathay4 main epidemic variant strains stored in a national foot and mouth disease reference laboratory, wherein the specific strains comprise the following 10 strains: BY/2010(Mya98), 13152(Mya98), 14064(Mya98), 15126(Panasia), 16045(Mya98), 17016(Mya98), 17002(Mya98), 17009(Ind-2001), 17042(Ind-2001) and 18074(Cathay), and the cross-neutralization effect is judged according to the r value (r is less than or equal to 1), wherein the higher the r value is, the better the cross-neutralization effect is. The experimental results are shown in Table 1, and serum cross-neutralization tests show that the O-type foot-and-mouth disease virus strain O/17002 has a wide cross-neutralization effect on the O-type foot-and-mouth disease epidemic virus in China.

TABLE 1O/17002 serum Cross-neutralization test results

The amino acid sequences of the structural proteins VP0, VP3 and VP1 of the O/17002 strain and the gene sequences encoding the structural proteins VP0, VP3 and VP1 are obtained by whole genome sequencing of the O/17002 strain.

The amino acid sequence of the structural protein VP0 of the O/17002 strain is shown in SEQ ID NO.1, and the nucleotide sequence of the coding structural protein VP0 is shown in SEQ ID NO. 2; the amino acid sequence of the structural protein VP3 of the O/17002 strain is shown in SEQ ID NO.3, and the nucleotide sequence of the coding structural protein VP3 is shown in SEQ ID NO. 4; the amino acid sequence of the structural protein VP1 of the O/17002 strain is shown in SEQ ID NO.5, and the nucleotide sequence of the coding structural protein VP1 is shown in SEQ ID NO. 6.

EXAMPLE 2 preparation of foot-and-mouth disease Virus structural protein composition

1. Construction of foot-and-mouth disease virus structural protein recombinant expression vector

(1) Designing a gene sequence THS for coding a fusion tag protein, wherein the gene sequence THS is composed of the following elements in series connection, wherein T is a nucleotide sequence of a translation initiation region, H is a nucleotide sequence for coding a protein containing a histidine tag, and S is a nucleotide sequence for coding a protein containing saccharomyces cerevisiae small ubiquitin-like modification (SUMO); the nucleotide sequence of THS is shown in SEQ ID NO. 11.

(2) The THS gene sequence is respectively connected with structural protein genes VP0, VP3 and VP1 of an encoding O/17002 strain in series in sequence to form three-segment fusion gene sequences THS-VP0, THS-VP3 and THS-VP 1. Wherein the genes VP0, VP3 and VP1 for encoding the structural proteins of the O/17002 strain comprise the following four cases:

coding structural proteins VP0, VP3 and VP1 of an O/17002 strain without any treatment;

secondly, mutating the structural protein gene VP3 encoding the O/17002 strain, wherein the mutation is as follows: the codon of 118 th lysine of the encoding structural protein VP3 in the gene VP3 is mutated into the codon of encoding arginine, the nucleotide sequence of the mutated gene VP3 is shown in SEQ ID NO.8, and the encoding O/17002 strain structural protein VP0 and VP1 genes are not treated;

③ mutating the gene VP1 encoding the structural protein of the O/17002 strain, wherein the mutation is as follows: the codon of 210 th lysine of the encoding structural protein VP1 in the gene VP1 is mutated into the codon of encoding arginine, the nucleotide sequence of the mutated gene VP1 is shown as SEQ ID NO.10, and the encoding O/17002 strain structural protein VP0 and VP3 genes are not treated;

fourthly, carrying out mutation on genes encoding the structural proteins VP1 and VP3 of the O/17002 strain, wherein the mutation is as follows: the codon of 210 th lysine of the structural protein VP1 coded in the gene VP1 is mutated into the codon coding arginine, the codon of 118 th lysine of the structural protein VP3 coded in the gene VP3 is mutated into the codon coding arginine, the nucleotide sequence of the mutated VP3 gene is shown as SEQ ID No.8, the nucleotide sequence of the mutated VP1 gene is shown as SEQ ID No.10, and the structural protein gene VP0 coded with O/17002 strain is not treated;

(3) synthesizing three sections of optimized fusion gene fragments by Huada gene biotechnology limited, cloning the fragments into the same pET30a vector according to the sequence of VP0, VP3 and VP1 by a molecular cloning technology, and obtaining a recombinant expression plasmid pET-FMDV-VP031 after identifying the sequence to be correct;

(4) the pET-FMDV-VP031 plasmid is transformed into competent Escherichia coli BL21(DE3), spread on a kanamycin-resistant solid LB medium, and cultured at 37 ℃ for 10-12 hours to make a single colony clear. Single colonies were picked up in 4mL tubes containing a liquid LB medium resistant to kanamycin, cultured at 37 ℃ for 12 hours with shaking at 220 rpm, and 1mL of the resulting suspension was stored at-80 ℃.

2. Prokaryotic expression of foot-and-mouth disease virus structural protein

(1) Taking out the Escherichia coli strain with the recombinant plasmid pET-FMDV-VP031 from a refrigerator at-80 ℃, inoculating 50mL LB liquid culture medium with kanamycin resistance, culturing at 250rpm and 37 ℃ for about 12 hours, then transferring into 1LLB liquid culture medium, culturing at 37 ℃, adding IPTG with the final concentration of 0.5mM after OD600 value reaches 0.6-0.8, and inducing protein expression overnight at 16 ℃. The foot-and-mouth disease virus structural protein SDS-PAGE identification result of the fusion small ubiquitin-like modified protein (SUMO) label is shown in figure 1, wherein M is a molecular weight Marker, 1 is whole bacteria before induction, 2 is whole bacteria after induction, 3 is supernatant after the whole bacteria are cracked, and the sample loading amount is 5 mu L. Experimental results show that the foot-and-mouth disease virus structural protein with the SUMO label can be dissolved and co-expressed in escherichia coli, and the target protein accounts for about 30% -35% of the soluble total protein of thalli.

(2) Adjusting pH electrode of fermentation tank (German Saedolis CT5-2 fermentation tank), preparing 4L culture medium, placing in the fermentation tank, sterilizing at 121 deg.C for 30min, adjusting dissolved oxygen electrode, taking the non-aerated state after sterilization as zero point, and taking the initial stirring speed before aeration and non-inoculation at fermentation time as 100 rpm.

(3) The next day, 400mL of seed solution was inoculated into a fermentor, the temperature was 37 ℃, the pH was 7.0, the stirring speed and aeration were manually adjusted, and dissolved oxygen was maintained at 40% or more. Feeding was performed, and 50% glucose was fed at a rate of 30 mL/hr. The dissolved oxygen in the fermentation tank is controlled to be 30-40% by adjusting the rotating speed. When the culture was carried out until the bacterial concentration reached about OD600, the culture temperature was lowered to 16 ℃ and IPTG was added to a final concentration of 0.5mM for induction culture for 12 hours. The final bacterial liquid concentration OD600 was about 45, about 300g of the cells were collected by centrifugation.

3. Affinity chromatography purification of foot-and-mouth disease virus structural protein with SUMO label

The cells were resuspended in a proportion of 1g of the cells to 10mL of a lysate (20mM Tris, 20mM imidazole, 400mM NaCl, pH7.5), and the cells were disrupted 2 times at 700bar pressure using a homogenizer. The supernatant was centrifuged at 30,000g for 1 hour, and the supernatant was detected by 12% SDS-PAGE electrophoresis, filtered through a 0.45 μm pore filter and purified by a nickel affinity column (HisTrap FF, GEHealthcare Life Sciences).

Buffer solution: 20mM Tris, 0.4M NaCl, pH 8.0;

eluent: 20mM Tris, 0.4M NaCl, 500mM imidazole, pH 8.0.

The sample is 1.4L of Escherichia coli cell supernatant which is filtered by a filter membrane with the aperture of 0.45 mu m and crushed by a homogenizer.

The elution procedure was: after the sample was run through, the buffer eluted the hybrid proteins and the eluent eluted the SUMO-tagged foot and mouth disease virus structural protein (VP0, VP3, VP1) products.

mu.L of the sample purified by the method of this example was taken, 5. mu.L of 5 Xloading Buffer was added and mixed, and 5. mu.L was electrophoresed in 12% SDS-PAGE after being subjected to metal bath at 100 ℃ for 10 min. Then, dyeing with Coomassie brilliant blue to display an electrophoresis strip, and performing SDS-PAGE identification on the obtained affinity chromatography-purified foot-and-mouth disease virus structural protein with the SUMO label after the SUMO enzyme digestion to obtain a result shown in figure 2, wherein M is a molecular weight Marker, 1 is the affinity chromatography-purified foot-and-mouth disease virus structural protein with the SUMO label, 2 is the foot-and-mouth disease virus structural protein without the SUMO label after the SUMO enzyme digestion, and the sample loading amount is 5 mu L; SDS-PAGE identification results show that the fusion target protein with the expected size is obtained by purification; and performing gray scanning analysis on the SDS-PAGE identification result, wherein the concentration ratio of SUMO VP0, SUMO VP3 and SUMO VP1 is 1: 1.02: 1.06, the content of the three fusion proteins is uniform.

4. Affinity chromatography purification of foot-and-mouth disease virus capsid protein without SUMO label

Taking the elution samples of the four foot and mouth disease virus structural proteins with the SUMO labels in the step 3, carrying out enzyme digestion for 12 hours at 4 ℃ by using SUMO enzyme, carrying out flow-through on the solution containing the foot and mouth disease virus structural proteins after enzyme digestion by using a nickel column (HisTrap FF, GE Healthcare Life Sciences), and collecting flow-through liquid. The SUMO tag is bound to a nickel column, and the structural proteins VP0, VP1 and VP3 of the foot-and-mouth disease virus without the SUMO tag are in flow-through fluid.

20 mu L of the foot-and-mouth disease structural protein sample purified by the method of the embodiment is taken, 5 mu L of 5 × Loading Buffer is added for mixing, 5 mu L of the mixture is respectively taken for electrophoresis on 12% SDS-PAGE after metal bath at 100 ℃ for 10 min. And then displaying an electrophoresis band by using Coomassie brilliant blue staining, wherein the electrophoresis result is shown in figure 3, wherein M is a molecular weight Marker, O/FMDV is a purified O-type foot-and-mouth disease virus structural protein without a fusion tag, and the loading amount is 5 mu L. SDS-PAGE identification results show that target protein without the fusion tag with expected size is obtained through purification; the SDS-PAGE identification result is subjected to gray-scale scanning analysis, and the concentration ratio of VP0, VP3 and VP1 is about 1: 1.03: 1.12, the content of three structural proteins is uniform, and the purity is more than 90%. The concentration of the protein of interest after purification to remove the SUMO tag was approximately 1.12mg/mL as determined by BCA. By adopting the production process, about 120mg of purified protein can be obtained per liter of fermentation liquor, and the yield reaches 120mg/L, which is much higher than the yield of the purified protein reported in the current literature.

5. Morphological detection of foot-and-mouth disease virus structural protein self-assembly

Collecting the flow-through liquid containing the foot-and-mouth disease virus structural proteins VP0, VP1 and VP3 in the step 4 in an assembly buffer (50mM Tris-HCl, 500mM NaCl, pH7.6), and observing the self-assembly of the foot-and-mouth disease virus structural proteins by a transmission electron microscope after overnight at 4 ℃, wherein the apparatus is an FEI transmission electron microscope. After hydrophilization treatment, 1% UF is dyed for 20 seconds, and the foot-and-mouth disease virus structural protein composition is fixed on an ultrathin carbon copper net for electron microscope observation. As shown in FIG. 4, it can be seen by transmission electron microscopy that a large number of particles with a radius of about 20nm are uniform in size, present a hollow morphology, and similar to natural foot and mouth disease virus particles, the three structural proteins of the virus are successfully self-assembled, and the amino acid point mutations of the VP3 and VP1 structural proteins do not affect the self-assembly of the O/17002 virus structural proteins. The prepared foot-and-mouth disease virus structural protein compositions are respectively named as O/17002(VP0, VP3 and VP1 genes are not mutated), Re-O/17002/VP3(VP3 gene mutation), Re-O/17002/VP1(VP1 gene mutation) and Re-O/17002/VP3+ VP1(VP3 and VP1 genes are mutated at the same time).

Example 3 expression efficiency of foot-and-mouth disease Virus structural protein composition

According to the method described in the example 2, the 4 foot-and-mouth disease virus structural protein compositions O/17002, Re/O/17002/VP3, Re/O/17002/VP1, Re/O/17002/VP3+ VP1 are subjected to prokaryotic expression identification, the expression efficiency of different foot-and-mouth disease virus structural protein compositions is analyzed through SDS-PAGE, the experimental result is shown in FIG. 5, and the single mutation of the gene of the foot-and-mouth disease virus structural protein VP3 or VP1 and the simultaneous mutation of the structural proteins VP3 and VP1 do not significantly affect the expression efficiency of the foot-and-mouth disease virus structural protein compositions.

The 4 aftosa virus structural protein compositions O/17002, Re/O/17002/VP3, Re/O/17002/VP1 and Re/O/17002/VP3+ VP1 were purified and prepared according to the method described in example 2. The BCA method is used for determining the purification yield of different compositions, and the results are shown in Table 2, and the purification yield of the 4-foot-and-mouth disease virus structural protein composition is between 120 and 150mg/L, which are all far higher than the highest yield reported in the prior literature.

TABLE 2 purified yield of foot and mouth disease virus structural protein compositions

Structural protein combinationsArticle (A)	O/17002	Re/O/17002/VP3	Re/O/17002/VP1	Re/O/17002/VP3+VP1
					Yield of target protein after purification	150mg/L	130mg/L	125mg/L	120mg/L

Example 4 detection of neutralizing antibody response in animals immunized with subunit vaccine for foot and mouth disease

After vaccines are prepared by using the foot-and-mouth disease virus structural protein compositions O/17002, Re/O/17002/VP3, Re/O/17002/VP1 and Re/O/17002/VP3+ VP1 obtained in example 3 with the same antigen dose, pig body immunization is carried out, blood is collected respectively on 0, 7, 14, 21 and 28 days after immunization, and serum is separated for neutralizing antibody titer detection. The test results are shown in FIG. 6, the O-type foot-and-mouth disease virus structural protein compositions O/17002, Re/O/17002/VP3, Re/O/17002/VP1 and Re/O/17002/VP3+ VP1 constructed by the structural protein genes VP0, VP3 and VP1 of the O/17002 strain can generate a high-level neutralizing antibody after animals are immunized, wherein the O-type foot-and-mouth disease virus structural protein compositions Re/O/17002/VP3, Re/O/17002/VP1 and Re/O/17002/VP3+ VP1 constructed by mutating the structural protein genes VP3 and VP1 of the O/17002 strain respectively or simultaneously can generate a high-level neutralizing antibody after animals are immunized, and the in vivo stability and the protective efficacy of the O-type foot-and-mouth disease gene engineering vaccine are improved.

Example 5 foot and mouth disease subunit vaccine immune serum cross-neutralization assay

The foot-and-mouth disease subunit vaccine prepared by Re/O/17002/VP3+ VP1 is used for immunizing pigs to obtain the hyperimmune positive serum of the foot-and-mouth disease subunit vaccine. The obtained hyperimmune serum is respectively subjected to virus neutralization tests with 4 main epidemic variant strains such as O/Mya98, O/Panasia, O/Ind-2001, O/Catay and the like stored in national foot and mouth disease reference laboratories, wherein the specific strains comprise the following 10 strains: BY/2010(Mya98), 13152(Mya98), 14064(Mya98), 15126(Panasia), 16045(Mya98), 17016(Mya98), 17002(Mya98), 17009(Ind-2001), 17042(Ind-2001) and 18074(Cathay), and the cross-neutralization effect is judged according to the r value (r is less than or equal to 1), wherein the higher the r value is, the better the cross-neutralization effect is. The test results are shown in table 3, and the serum cross-neutralization test results show that the O-type foot-and-mouth disease subunit vaccine provided by the invention has a wide cross-neutralization effect on the O-type foot-and-mouth disease epidemic virus in China.

TABLE 3 foot-and-mouth disease subunit vaccine serum Cross-neutralization test results

Example 6 foot-and-mouth disease genetic engineering vaccine animal immunization challenge PD₅₀Evaluation of

Selecting experimental pigs with screened foot-and-mouth disease virus antibody negativity, and 10 weeks old. The foot-and-mouth disease virus structural protein composition Re-O/17002/VP3+ VP1 prepared in example 2 is mixed with an equal amount of 206 adjuvant, the immunization mode is intramuscular injection, and the immunization dose is divided into three gradients which are respectively 20, 60 and 180 mu g per head. Collecting the neck venous blood after immunization at 0, 7, 14, 21 and 28 days respectively, separating serum, and storing for detection. The result of neutralizing antibody response is shown in FIG. 6, the vaccine prepared from the foot-and-mouth disease virus structural protein composition Re-O/17002/VP3+ VP1 produces neutralizing antibody response after immunizing pig, and the antibody peak is reached 28 days after immunization; after the lowest dose of 20 mu g/head part is immunized, the organism can be induced to generate a high-titer neutralizing antibody, and the O-type foot-and-mouth disease in China represents epidemic strains O/17016 and 5.5SID₅₀The dose is used for counteracting the virus, and full protection is provided, so that the genetic engineering vaccine prepared by the foot-and-mouth disease virus structural protein composition is proved to have high-efficiency immune protection.

The O/FMDV genetic engineering vaccine (foot-and-mouth disease virus structural protein composition Re-O/17002/VP3+ VP1 mixed with an equal amount of 206 adjuvant) developed by the invention is evaluated by a pig body immune challenge test, and a high-titer neutralizing antibody can be induced to generate by using 20 mu g/head immune dose. The animals of the control group 3 all suffered from the disease on the 4 th day after being challenged by the foot-and-mouth disease O-type epidemic virus 17016; after the animals of 3 immunization groups with different doses are attacked by the O-type foot-and-mouth disease virus 17016, one animal does not attack the virus, the O-type foot-and-mouth disease representative virus O/17016 attacking complete protection of the foot-and-mouth disease virus in China is realized, the foot-and-mouth disease virus structural protein composition is proved to have excellent immune protection, and the results are shown in table 4.

TABLE 4 immune challenge results of O/FMDV genetic engineering vaccine

And (4) surface note:

+: typical vesicular lesions appear in the snout part of the pig;

left front, right back: typical vesicular lesions appear on the left forehoof and the right hind hoof of the pig;

four-hoof: typical blister-like lesions appear in all four pigs.

The above examples are only for illustrating the technical idea and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Sequence listing

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> O type foot-and-mouth disease subunit vaccine, preparation method and application thereof

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Ala Leu Asp Asn Thr Thr Asn Pro Thr Ala Tyr His Lys Ala Pro Leu

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Arg Gly Asp Leu Gln Val Leu Ala Pro Lys Ala Ala Arg Pro Leu Pro

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Thr Ser Phe Asn Tyr Gly Ala Ile Lys Ala Thr Arg Val Thr Glu Leu

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210

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aaagtcacac caaaaggccc aataaatgta ctggacctga tgcaggcccc cccccacact 180

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attattgagg ctcacagaga acagattggt ggt 393

Claims

1. An O-type foot-and-mouth disease vaccine prepared according to the sequences of O/17002 structural proteins VP0, VP3 and VP1 is characterized in that the amino acid sequence of the O/17002 structural protein VP0 is shown as SEQ ID No.1, the amino acid sequence of the O/17002 structural protein VP3 is shown as SEQ ID No.3, and the amino acid sequence of the O/17002 structural protein VP1 is shown as SEQ ID No. 5.

2. The O-type foot-and-mouth disease virus structural protein composition is characterized by consisting of three virus structural proteins VP0, VP3 and VP1, wherein the amino acid sequence of the structural protein VP0 is shown as SEQ ID NO.1, the amino acid sequence of the structural protein VP3 is shown as SEQ ID NO.3, and the amino acid sequence of the structural protein VP1 is shown as SEQ ID NO. 5.

3. The structural protein composition of the type O foot-and-mouth disease virus of claim 2, wherein the nucleotide sequence encoding the structural protein VP0 is shown in SEQ ID No.2, the nucleotide sequence encoding the structural protein VP3 is shown in SEQ ID No.4, and the nucleotide sequence encoding the structural protein VP1 is shown in SEQ ID No. 6.

4. The structural protein composition of type O foot-and-mouth disease virus according to claim 2, wherein lysine at position 118 of the structural protein VP3 is mutated to arginine, and the amino acid sequence of the mutated structural protein VP3 is represented by SEQ ID No. 7; and/or the 210 th lysine of the structural protein VP1 is mutated into arginine, and the amino acid sequence of the mutated structural protein VP1 is shown as SEQ ID NO. 9.

5. The composition of structural proteins of foot-and-mouth disease virus type O according to claim 4, wherein the nucleotide sequence encoding the mutated structural protein VP3 is shown as SEQ ID No.8, and the nucleotide sequence encoding the mutated structural protein VP1 is shown as SEQ ID No. 10.

6. Use of the type O foot and mouth disease virus structural protein composition according to any one of claims 2 to 5 in the preparation of a foot and mouth disease vaccine.

7. A vaccine for the prevention of foot and mouth disease virus infection, comprising the foot and mouth disease virus structural protein composition of any one of claims 2 to 5.

8. The vaccine of claim 7, further comprising an adjuvant.

9. The vaccine of claim 8, wherein the adjuvant is one or more of a chemical immune adjuvant, a microbial immune adjuvant, a plant immune adjuvant and a biochemical immune adjuvant.

10. A method for preparing an O-type Oriental foot Virus structural protein composition, said method comprising the steps of:

(1) designing and encoding a fusion tag protein gene sequence THS, wherein T is a translation initiation region nucleotide sequence, H is a nucleotide sequence encoding a histidine tag, and S is a nucleotide sequence encoding a small ubiquitin-like modified protein (SUMO) containing saccharomyces cerevisiae;

(2) respectively connecting the fusion tag protein gene sequence THS in the step (1) with genes for coding O-type aftosa epidemic strain structural proteins VP0, VP3 and VP1 in series to form three sections of fusion target protein gene sequences THS-VP0, THS-VP3 and THS-VP 1; the amino acid sequence of the structural protein VP0 is shown as SEQ ID NO.1, the amino acid sequence of the structural protein VP3 is shown as SEQ ID NO.3, and the amino acid sequence of the structural protein VP1 is shown as SEQ ID NO. 5;

(3) cloning the three sections of fusion target protein gene sequences in the step (2) into a prokaryotic expression vector pET30a simultaneously by a molecular cloning technology to obtain a recombinant expression plasmid pET-FMDV-VP 031;

(4) transforming the recombinant expression plasmid pET-FMDV-VP031 in the step (3) into escherichia coli to obtain a genetic engineering bacterium, performing fermentation culture on the genetic engineering bacterium, and performing induced expression on foot-and-mouth disease virus structural proteins VP0, VP3 and VP1 with fusion tag proteins;

(6) and (3) after the fusion tag protein in the mixture in the step (5) is removed by enzyme digestion, separating and purifying by affinity chromatography to obtain a mixture of foot-and-mouth disease virus structural proteins VP0, VP3 and VP 1.

11. The method according to claim 10, wherein the nucleotide sequence encoding the structural protein VP0 is shown in SEQ ID No.2, the nucleotide sequence encoding the structural protein VP3 is shown in SEQ ID No.4, and the nucleotide sequence encoding the structural protein VP1 is shown in SEQ ID No. 6.

12. The method according to claim 10, characterized in that the lysine at position 118 of the structural protein VP3 is mutated to arginine, and the amino acid sequence of the mutated structural protein VP3 is shown in SEQ ID No. 7; and/or the 210 th lysine of the structural protein VP1 is mutated into arginine, and the amino acid sequence of the mutated structural protein VP1 is shown as SEQ ID NO. 9.

13. The method of claim 12, wherein the nucleotide sequence encoding the mutated structural protein VP3 is represented by SEQ ID No.8 and the nucleotide sequence encoding the mutated structural protein VP1 is represented by SEQ ID No. 10.

14. The method of any one of claims 10 to 13, wherein the nucleotide sequence of THS in step (1) is set forth in SEQ ID No. 11.