CN109536461B

CN109536461B - O-type foot-and-mouth disease virus mutant strain and preparation method and application thereof

Info

Publication number: CN109536461B
Application number: CN201811406871.8A
Authority: CN
Inventors: 白兴文; 刘在新; 卢曾军; 包慧芳; 李平花; 李冬; 孙普; 付元芳; 陈应理; 曹轶梅; 马雪青; 李坤; 张婧; 陈冬冬; 宫晓华
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2018-11-23
Filing date: 2018-11-23
Publication date: 2020-07-03
Anticipated expiration: 2038-11-23
Also published as: CN109536461A

Abstract

The invention provides an O-type foot-and-mouth disease virus mutant strain, a preparation method and application thereof, belonging to the technical field of vaccine candidate strains. The O type foot-and-mouth disease virus mutant strain takes rHN strain as a mother strain, and the following amino acids in the G-H ring of VP3 protein are mutated: aspartic acid 173 is mutated to asparagine, valine 174 to glutamic acid and asparagine 179 to cysteine. The virus mutant is genetically stable and acquires the ability of caveolin-mediated infection of CHO-K1 cells. The results show that compared with the maternal virus strain, the cross protection capability of the virus mutant strain for inducing neutralizing antibodies of foot-and-mouth disease viruses generated by organisms is remarkably improved, and the excellent broad-spectrum antigen is shown. The vaccine prepared by inactivating the virus mutant strain can be used for preventing the infection of the O-type foot-and-mouth disease virus.

Description

O-type foot-and-mouth disease virus mutant strain and preparation method and application thereof

Technical Field

The invention belongs to the technical field of vaccine candidate strains, and particularly relates to an O-type foot-and-mouth disease virus mutant strain, and a preparation method and application thereof.

Background

Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious and rapidly remotely transmissible virulent infectious disease caused by FMDV (foot-and-mouth disease virus) which is a common disease of major domestic animals such as pigs, cows and sheep and other domestic and wild artiodactyls. The disease is one of animal infectious diseases which are legally reported by the world animal health Organization (OIE), and is listed as the first name of animal epidemic diseases in China [ Ministry of agriculture, No. 1125, 2008 ]. Since the first time of 1546, the foot-and-mouth disease is recorded in more exact characters, the disease still has a long-term wide epidemic in the world and is extremely harmful. Although foot-and-Mouth disease has low lethality to adult susceptible animals (< 5%), the disease has extremely strong infectivity, the morbidity can reach 100%, as the production performance of the affected animals is reduced, the import and export international trade of animal products is limited, serious direct economic loss and indirect economic loss can be caused, and the latter is often tens of times or even hundreds of times of the former (Rowlands DJ. For example, in 2001, economic losses due to Foot and Mouth Disease in the United kingdom exceed 80 billion pounds, and even the annual meeting is highly selective, thus postponing time (Knowles NJ, et al. Outbreak of Foot-and-Foot Disease Virus surgery in the UK used by a miniature line. vet Rec.2001,148: 258-. Therefore, the disease not only seriously threatens the sustainable and healthy development of agriculture and animal husbandry and even the whole national economy, but also can cause serious negative social effects, and is called as the political and economic disease.

China is a big country in the breeding industry and is also one of countries with serious foot and mouth disease harm. According to the statistical data of Ministry of agriculture, the annual loss of the industry chain is estimated to be billions of yuan, and the prevention and control situation is severe (Puhua, the review of the economic research of animal disease prevention and control, the agricultural economic problem 2006,6: 61-64). Nowadays, international trade is increasingly frequent, the prevention, control, purification and eradication of foot-and-mouth disease in China play a key role in developing agricultural economy, guaranteeing food safety, sanitation and health and maintaining national reputation, and have important significance in promoting the control of the foot-and-mouth disease in the world.

The Foot-and-Mouth disease virus is the first discovered animal-derived virus, belongs to the Foot-and-Mouth disease (sore) virus genus of the family of small (micro) RNA viruses, and comprises seven serotypes O, A, C, Asia1 and SAT 1-3, wherein each serotype is rarely subjected to immunological cross protection, and the antigenicity of different subtypes of the same serotype is different, and the cross protection power of the subtypes is different (Brown F. the History of research in Foot-and-moon disease. Virus Res.2003,91: 3-7). The type O, A and type Asia1 Foot-and-Mouth Disease viruses are historically or currently popular in China, wherein the type O Foot-and-Mouth Disease Virus has the greatest threat, the type A Virus is the second, and no Asia1 clinical cases exist after 5 months in 2009 (Bai X, et al. evolution and molecular epidemic of Foot-and-molecular Disease Virus in Chinese Sci Bull.2011,56: 2191-2201). At present, the O type foot-and-mouth disease virus isolated in China also comprises the Chinese ancient classic (Cathay), Panasia-1 (Panasia-1) pedigree, Myanmar 98(Mya98) pedigree, India 2001d (IND2001d) branch (Liuxiangtao, etc.) besides the historical representative strain Xinjiang Aksu virus.

Vaccine immunization is an important means for preventing foot and mouth disease. The immunological potency of Foot-and-Mouth disease vaccines depends primarily on the level of humoral immunity elicited by the vaccine virus, i.e., the ability of B cell epitopes to induce neutralizing antibodies (Barchrach HL. Immune and antiviral Responses to an Isolated Capsid Protein of Foot-and-mouse disease Virus. Immunol.1975,115: 1636-. In addition, the cell immunity mediated by the T cell epitope also plays an important role in the later stage of preventing foot and mouth disease virus infection (Liu Xing, etc. T cell epitope research overview of foot and mouth disease virus, Zhejiang agricultural science, 2010, (5): 1113-.

Foot-and-mouth disease vaccines of mature production technology in China comprise inactivated vaccines and synthetic peptide vaccines. Although synthetic peptide vaccines can circumvent the possibility of viral escape and the risk of virus shedding of inactivated vaccines, their vaccination in cattle does not result in complete immune protection, only trials in pigs have been successful [ ministry of agriculture, No. 437, 2004 ]. Furthermore, synthetic peptides have a narrow antigenic spectrum and limited immunity, and when new strains invade, immune failure may occur. At present (in the market), no genetic engineering vaccine for foot and mouth disease which can be compared with the 'high quality and low price' of inactivated vaccine exists (Liu is new, global foot and mouth disease prevention and control technology and pathogenic characteristic research overview, Chinese agricultural science 2015,48: 3547-3564). Several countries in the European Union and south America have effectively controlled foot-and-Mouth Disease through several decades of immunization with inactivated foot-and-Mouth Disease Virus vaccines (Sobrino F, et al. foot-and-Mouth Disease Virus: a Long Knownwn Virus, but a Current thread. vet Res.2001,32: 1-30). Although the foot-and-mouth disease virus has remarkable genetic variation characteristics, compared with the A type foot-and-mouth disease virus, the O type foot-and-mouth disease virus has smaller antigenic variation, and the vaccine strain can resist most of field epidemic strains. For example, the O-type Foot-and-mouth disease vaccine strains used in countries and regions of Europe and Asia are limited to O1Manisa and IND R2/75 (Asia) which belong to the same genus as the Pana-1 lineage and O1K, O1BFS (Europe) and O1Campos, O1Caseros (south America), whereas the O-type Foot-and-mouth disease vaccine production in our country is complicated with the species and components thereof (Qingge. Foot-and-mouth disease. Beijing: Chinese agricultural Press 2004), and this overproduction of intrinsic immune pressure and external environmental factors accelerates the selection of standard species of Foot-and-mouth disease Virus under naturally tolerated conditions, and is very detrimental to the purification and eradication of Foot-and mouth disease viruses (Woodbury, et al, analysis of Mixet Foutu-and-MothDisear disease Virus infection: region of Experimental, 201. environmental, 1994).

Intact 146S virions are the most effective antigens in inactivated aftosa vaccines, and invasion into the body requires the assistance of Antigen Presenting Cells (APC) such as Dendritic Cells (DC) and the delivery of MHC molecules to the surface of (lymphoid) cells, which are essential for the regulation of immune defences of the body (Bayty J, et al. interaction of Foot-and-Mouth Disease Virus with derived cells. trends Microbiol.2006,14: 346-347). The inactivated Foot-and-Mouth disease virus initiates and activates Immature bovine bone marrow Dendritic Cell Apoptosis series signaling pathways by binding the conserved Arginine-Glycine-Aspartic acid (RGD; positions 145-147, exemplified by form O) tripeptide motif in VP 1G-H loop to Integrin (Ins) on the Cell surface, possibly compromising immune response (Jin H, ethyl. indication of Immature Dentistic Cell Apoptosis by Foot and mouse disease Virus an Integrin Receptor for animal Viral Infection, Cell Biochem.2007,102:980 + 991). However, mature monocytic Dendritic cells are not susceptible to integrin-bound foot and Mouth Disease Virus Infection, and either the neutralized immune complex or the inactivated foot and Mouth Disease Virus still has the ability to recognize immune cells (Robinson L, et al. foot-and-mouse Disease Virus inhibition an Altered Tropism in the Presence of sensitivity immunology inhibitors, engineering production Infection and vaccination of Dendritic cells. J Virol.2011,85: 2212-2223). Therefore, the dendritic cells are taken as target cells, which is helpful for developing effective anti-foot-and-mouth disease virus vaccines. Although the molecular mechanism of the interaction of foot and mouth disease virus with dendritic cells is not well understood, Heparin Sulfate (HS) mediated endocytosis is most effective for the internalization of foot and mouth disease virus in dendritic cells that present antigens to lymphocytes that induce IgG responses specific to foot and mouth disease virus. non-Heparan sulfate-bound viruses and their infectious RNA also induce low levels of specific Immune responses to dendritic cell binding (Harwood LJ, et al. Dendritic cell interpretation of Foot-and-motion Disease Virus: infection of liver surface binding on Virus Uptake and infection of the Immune response. J Virus. 2008,82: 6379-.

The molecular basis of foot-and-mouth disease virus utilization of cell surface receptors is the characteristic amino acid sequence or domain in the outer capsid protein VP 1-3 located on the surface of the virion. Wild-type foot-and-Mouth Disease viruses recognize integrins using the RGD motif and infect sensitive tissue cells via a clathrin-mediated endocytosis pathway (O' Donnell V, et al. analysis of Foot-and-mouse Disease Virus intervention Events in harvested cells. JVirol.2005,79: 8506-. While cytotoxicity gradually can be adsorbed to the Cell Surface by Heparan Sulfate during adaptation (Bai X, et al. effects of Two Amino acids in the peptide proteins on the Interaction of Two Cell-adsorbed Panasia-1 Strains of Foot-and-Foot Disease Virus section O with a liver Disease receptor. virol J.2014,11:132), some Foot-and-Mouth Disease Virus Cell adapted Strains also gain the ability to utilize Heparan Sulfate as a receptor (Jackson T, et al. effective Infection of Cell in Type O Foot-and-Foot Disease Virus strain 5282 + Cell Surface Virus H. JVIROL. 1996). Heparin-sensitive cytotoxins accomplish the process of cellular internalization mediated by caveolin protein (O' Donnell V, et al, liver surface-Binding Foot-and-motion Disease Virus Enters Cells via vesicle A-mediated encystosis.J. Virol.2008,82: 9075-. The amino acid variations that lead to the conversion of the type of foot-and-Mouth Disease Virus receptor are mostly located near the pentameric, triatomic and pentameric (dyad) interface of the foot-and-Mouth Disease Virus particle (Sobrino F, et al. foot-and-motion Disease Virus: Current research and engineering trends. Norwalk: separator Academic Press.2017).

The most valuable vaccine strain OZK/93 for the O-type foot-and-mouth disease in China can infect host cells BHK-21 and CHO-K1 (Baixing Wen, molecular basis of biological phenotype difference of the O-type pan-Asia 1-series foot-and-mouth disease virus in China, Beijing, Chinese academy of agricultural sciences, 2012) by utilizing a heparan sulfate receptor path, but the vaccine strain can not achieve the complete cross-protection immune effect on the O-type foot-and-mouth disease virus of a few subtypes. Some foot and mouth disease virus cell adapted strains that utilize heparan sulfate receptors may also exhibit a attenuated virulence phenotype on the host cells (except OZK/93, etc.). The foot-and-mouth disease vaccine strain antigen spectrum can be expanded by performing site-directed mutagenesis on the B cell epitope of the foot-and-mouth disease virus, namely a plurality of amino acids in the antigen site by reverse genetic operation [ Liu is new, etc.. the foot-and-mouth disease vaccine strain antigen spectrum and the vaccine preparation method are expanded by reverse genetic operation ZL201010256781.22013.01.09 ]. However, the Genetically engineered Virus rHN and its vaccine candidate lost the ability to infect CHO-K1 cells (Li P, equivalent. evaluation of a genetic Modified Foot-and-mouse Disease Virus vaccine expressed by Reverse genetics. BMC Vet Res.2012,8:57), and some Amino Acid variation(s) in the Foot-and-Mouth Disease Virus T/B cell epitopes were strictly limited by the dual role of antibody reaction and cell adsorption (Mateu MG, et al. A Single Amino Acid catalysis inhibition of infection antigens Multiple overlay epitopes in the Major antibacterial Site of Foot-and-mouse Disease Virus of Virus C. J. virol.1990,71: 629), and further could significantly affect the neutralizing property of BHK production and the protecting ability of BHK-21 Virus in BHK production.

Disclosure of Invention

In view of the above, the present invention aims to provide an O-type foot-and-mouth disease virus mutant strain, and a preparation method and an application thereof, wherein amino acids mutated in the O-type foot-and-mouth disease virus mutant strain are located on a non-T/B cell epitope (such as VP 3G-H loop), and the O-type foot-and-mouth disease virus mutant strain has an ability to induce a neutralizing antibody with a strong cross-protection level.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an O-type foot-and-mouth disease virus mutant rHN^{D3173N+V3174E+N3179C}The rHN strain is used as a parent strain, and the following amino acid positions in the G-H loop of the VP3 protein are mutated:aspartic acid 173 is mutated to asparagine, valine 174 to glutamic acid and asparagine 179 to cysteine.

The invention provides the O-type foot-and-mouth disease virus mutant strain rHN^{D3173N+V3174E+N3179C}The preparation method comprises the following steps:

(1) carrying out PCR by using pOFS plasmid as a template and using OSEP3+ and NEC-primer pairs to obtain an A amplification product, and carrying out PCR by using NEC + and ONNP3' -primer pairs to obtain a B amplification product;

the nucleotide sequence of the OSEP3+ is shown as SEQ ID NO. 1in the sequence table;

the nucleotide sequence of the NEC-is shown as SEQ ID NO.2 in a sequence table;

the nucleotide sequence of the NEC + is shown as SEQ ID NO.3 in a sequence table;

the nucleotide sequence of the ONNP3' is shown as SEQ ID NO.4 in a sequence table;

(2) carrying out fusion PCR by using the amplification products A and B as templates and using primers OSEP3+ and ONNP3' -to obtain VP3^{D173N+V174Y+N179C}A DNA fragment;

(3) mixing the VP3^{D173N+V174Y+N179C}The DNA fragment and the pSK-HDV vector are subjected to double digestion by Spe I/Not I and are connected to obtain pSK-VP3^{D173N+V174E+N179C}A plasmid;

(4) cloning VP0 gene segment, VP1+ P2 gene segment, 5'UTR + L gene segment and P3+3' UTR gene segment into pSK-VP3^{D173N+V174E+N179C}In the plasmid, pOFS was obtained^{D3173N+V3174E+N3179C}A plasmid;

(5) subjecting said pOFS^{D3173N+V3174E+N3179C}Carrying out plasmid linearization treatment, and transfecting the obtained linear plasmid into BSR/T7-5 cells to obtain O-type foot-and-mouth disease virus mutant strain rHN^{D3173N+V3174E+N3179C}。

Preferably, the PCR system for the amplification product A or B in step (1) is 10 × LA Taq buffer 5. mu.l, 2.5mmol/L dNTP 5. mu.l, upstream primer 0.5. mu.l, downstream primer 0.5. mu.l, template 0.5. mu.l, LA Taq enzyme 0.5. mu.l, ddH₂O 38μl。

Preferably, the PCR program of the A amplification product or the B amplification product in the step (1) or the fusion PCR program in the step (2) is independently: 5min at 94 ℃; 30 cycles of 94 ℃ for 1min, 61 ℃ for 1min for 20s, and 72 ℃ for 3 min; 10min at 72 ℃.

Preferably, the reaction system for the Spe I/Not I double digestion in step (3) is 10 × Basal buffer 5. mu.l, template 25. mu.l, Not I5. mu.l, Spe I5. mu.l, ddH₂O10 mu l; the reaction condition of the Spe I/Not I double enzyme digestion is that the temperature bath is carried out for 2h at 37 ℃.

Preferably, the primer pair used for amplifying the VP0 gene fragment in the step (4) is OSBP4+ and OEP 2-; the nucleotide sequence of the OSBP4+ is shown as SEQ ID NO.5 in the sequence table; the nucleotide sequence of the OEP 2-is shown as SEQ ID NO.6 in the sequence table;

the primer pair for amplifying the VP1+ P2 gene fragment is OS2A +/OBN-; the nucleotide sequence of the OS2A + is shown as SEQ ID NO.7 in the sequence table; the nucleotide sequence of the OBN-is shown as SEQ ID NO.8 in the sequence table;

the primer pair used for amplifying the 5' UTR + L gene fragment is OST7 +/OBL-; the nucleotide sequence of the OST7+ is shown as SEQ ID NO.9 in the sequence table; the nucleotide sequence of the OBL-is shown as SEQ ID NO.10 in the sequence table.

The primer pair for amplifying the gene segment of P3+3'UTR is 3A' +/DNS-; the nucleotide sequence of the 3A' + is shown as SEQ ID NO.11 in the sequence table; the nucleotide sequence of the DNS-is shown as SEQ ID NO.12 in the sequence table.

The invention provides the O-type foot-and-mouth disease virus mutant strain rHN^{D3173N+V3174E+N3179C}Or the O-type foot-and-mouth disease virus mutant strain rHN prepared by the preparation method^{D3173N+V3174E+N3179C}Use in improving cross-protection of neutralizing antibodies.

The invention provides the O-type foot-and-mouth disease virus mutant strain rHN^{D3173N+V3174E+N3179C}Or the O-type foot-and-mouth disease virus mutant strain rHN prepared by the preparation method^{D3173N+V3174E+N3179C}The application in infecting CHO series cell lines.

The invention provides the O-type foot-and-mouth disease virus mutant strain rHN^{D3173N+V3174E+N3179C}Or the O-type foot-and-mouth disease virus mutant strain rHN prepared by the preparation method^{D3173N+V3174E+N3179C}The application in antigen presentation of immune cells.

The invention provides a foot-and-mouth disease virus type O mutant rHN^{D3173N+V3174E+N3179C}Or the O-type foot-and-mouth disease virus mutant strain rHN prepared by the preparation method^{D3173N+V3174E+N3179C}An inactivated vaccine strain.

The invention provides an O-type foot-and-mouth disease virus mutant rHN^{D3173N+V3174E+N3179C}The rHN strain was used as the parent strain, and the following amino acids in the G-H loop of VP3 protein were mutated: aspartic acid 173 is mutated to asparagine, valine 174 to glutamic acid and asparagine 179 to cysteine. The invention successfully saves O-type foot-and-mouth disease virus mutant rHN by means of a foot-and-mouth disease virus reverse genetic operation technical platform^{D3173N+V3174E+N3179C}RT-PCR verifies that the virus mutant strain is transmitted to the 10 th generation to successfully amplify a target strip, and a sequencing result shows that the virus mutant strain has better genetic stability; the results of the one-step growth curve show: with maternal virus strain rHN and single site-directed foot and mouth disease virus mutant rHN^V3174YComparative example 1 comparative example rHN^{D3173N+V3174E+N3179C}The proliferation trends on the BHK-21 cells are similar, and the replication kinetics of the three cells do not show obvious difference, which indicates that the replication capacity of the virus in the BHK-21 cells is not changed by mutation of a specific site; by TCID₅₀The test result shows that the virus mutant strain does not obviously change the infectivity of the virus mutant strain on BHK-21 cells in an in vitro test; at the same time, using LD₅₀The test result shows that the pathogenicity of the virus mutant strain to the suckling mouse is obviously weakened in an in vivo test, and rHN^V174YIn contrast, rHN^{D3173N+V3174E+N3179C}The weakening effect is more obvious; plaque formation tests and indirect immunofluorescence and laser confocal microscope detection results show that the virus mutant strain obtains the capability of caveolin mediated infection of CHO series cell lines. The result of detecting the cross-neutralization capacity of the immune positive serum shows that the cross-protection capacity of the neutralizing antibody induced and generated by the virus mutant strain is obviously improved, which indicates that the virus mutant strain has excellent antigen broad-spectrum property.

Drawings

FIG. 1 is a schematic diagram of VP3 site-directed mutagenesis foot-and-mouth disease virus genome full-length cDNA clone construction

FIG. 2 is pOFS^V3174YAnd pOFS^{D3173N+V3174E+N3179C}Identification results of plasmid and virus mutant strains thereof; FIG. 2A, M, DNA molecular mass standard (1 Kb); 1 to 3 in sequence of pOFS and pOFS^V3174YAnd pOFS^{D3173N+V3174E+N3179C}Spe I/Not I cleavage product of plasmid; FIG. 2B, M, DNA molecular mass standard (DL 5000); 1 to 3, sequentially rHN and rHN^V3174YAnd rHN^D3173N ^{+V3174E+N3179C}Amplified RT-PCR products; FIG. 2C shows rHN and rHN from left to right^V3174YAnd rHN^{D3173N+V3174E+N3179C}A sequence peak map of VP3 encoding amino acids 171-181;

FIG. 3 shows rHN and rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}Plaque morphology on BHK-21 and CHO-K1 cells;

FIG. 4 shows rHN and rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}The one-step growth curve of (1);

FIG. 5 shows rHN and rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}Inoculating BHK-21 and CHO-K1 cells, and detecting whether the non-structural protein 3A of the foot-and-mouth disease virus expresses an indirect immunofluorescence detection result;

FIG. 6 shows rHN and rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}Detecting results of a laser confocal microscope infecting BHK-21 and CHO-K1 cells;

FIG. 7 is a graph of virus neutralization assay to determine the variance of r-values of neutralizing antibodies from sera collected on day 21 after immunization of pigs.

Detailed Description

The invention provides an O-type foot-and-mouth disease virus mutant rHN^{D3173N+V3174E+N3179C}The rHN strain is used as a parent strain, and the following amino acid positions in the G-H loop of the VP3 protein are mutated: aspartic acid 173 is mutated to asparagine, valine 174 to glutamic acid and asparagine 179 to cysteine.

In the invention, the rHN virus strain is O/HN/CHA/93 (namely OZK/93, classical vaccine strain) genetic engineering virus rescued by a foot-and-mouth disease virus reverse genetic operation technical platform. The rHN strain has been published, in particular in the article (Li P, et al. evaluation of a genetic Modified Foot-and-motion Disease Virus vaccine Candidate Generated by Reverse genetics. BMC Vet Res.2012,8: 57).

In the present invention, the design concept of the mutation is as follows:

the foot-and-mouth disease virus target site-specific modification is carried out on a potential target site in a relatively conservative non-T, B cell epitope region (a mutant segment is not a T cell epitope region of the foot-and-mouth disease virus nor a B cell epitope region of the foot-and-mouth disease virus) which influences the in-vivo and inter-space conformation stability of an antigen on an external capsid protein of the foot-and-mouth disease virus, wherein the modification is carried out on the spatial conformation, and the T cell epitope and/or the B cell epitope are/is generally selected to improve the cross neutralization capacity of the foot-and-mouth disease virus different from other people), the flexibility of the most main neutralizing antigen epitope region (VP 1G-H ring, namely antigen site 1-1) of the foot-and-mouth disease virus is optimized and the targeting presentation efficiency is improved in a mode of changing the space occupation (position) of the target amino acid and the interacting amino acid thereof, the carried positive and negative charges, the disulfide bond and other intermolecular forces characteristically; the non-T, B epitope region is specifically, but not limited to, the VP 3G-H loop, which is spatially close to antigenic sites 1-1, 1-2(VP 1C-terminal); site-directed modifications are preferably mutations, such as substitutions; the characteristic change is specifically the property change of amino acid chemical structure, polarity, acidity and alkalinity and the like caused by site-directed modification; the target presentation is that the foot-and-mouth disease virus recognizes antigen presenting cells and an endocytosis path which is dependent on receptor mediation; dendritic cells, etc., present antigens to lymphocytes, inducing specific humoral and cellular immune responses.

the nucleotide sequence of the NEC-is shown as SEQ ID NO.2 in a sequence table;

(2) taking the amplification product A and the amplification product B as templates, and carrying out fusion PCR amplification by using an OSEP3+ and ONNP3' -primer pair to obtain VP3^{D173N+V174Y+N179C}A DNA fragment;

(5) subjecting said pOFS^{D3173N+V3174E+N3179C}Carrying out plasmid linearization treatment, transfecting cells with the obtained linear plasmids to obtain O-type foot-and-mouth disease virus mutant strain rHN^{D3173N+V3174E+N3179C}。

The construction schematic diagram of the VP3 site-directed mutated foot-and-mouth disease virus genome full-length cDNA clone is shown in figure 1.

The invention takes pOFS plasmid as a template, and uses OSEP3+ and NEC-, NEC + and ONNP3' -primer pairs to carry out PCR respectively to obtain an A amplification product and a B amplification product;

the nucleotide sequence of the NEC-is shown as SEQ ID NO.2 in a sequence table;

in the present invention, the pOFS plasmid is a recombinant plasmid using pSK-HDV as a vector and containing rHN maternal viral strain (engineered virus of OZK/93) genome full-length cDNA. The pOFS plasmid is reported in the prior art (Li P, equivalent. evaluation of a genetic Modified Foot-and-mouse Disease Virus vaccine Generated by Reverse genetics. BMC Vet Res.2012,8: 57).

In the present invention, the PCR system for the amplification product A or B is preferably 10 × LA Taqbuffer 5. mu.l, 2.5mmol/L dNTP 5. mu.l, upstream primer 0.5. mu.l, downstream primer 0.5. mu.l, template 0.5. mu.l, LA Taq enzyme 0.5. mu.l, ddH₂O 38μl。

In the present invention, the PCR procedure for the A amplification product or the B amplification product is independently preferably: 5min at 94 ℃; 30 cycles of 94 ℃ for 1min, 61 ℃ for 1min for 20s, and 72 ℃ for 3 min; 10min at 72 ℃.

After obtaining the amplification product A and the amplification product B, the invention takes the amplification product A and the amplification product B as templates, and carries out fusion PCR by using OSEP3+ and ONNP3' -primer pair to obtain VP3^{D173N+V174Y+N179C}A DNA fragment.

In the invention, the fusion PCR amplification program is preferably selected from the group consisting of 94 ℃ for 5min, 94 ℃ for 1min, 61 ℃ for 1min for 20s, 72 ℃ for 3min, 30 cycles, and 72 ℃ for 10min, and the fusion PCR amplification system is preferably selected from the group consisting of 10 × LA Taq buffer 5. mu.l, 2.5mmol/L dNTP 5. mu.l, upstream primer 0.5. mu.l, downstream primer 0.5. mu.l, A amplification product 0.5. mu.l, B amplification product 0.5. mu.l, LA Taq enzyme 0.5. mu.l, and ddH₂O37.5. mu.l. In the present invention, the primers for fusion PCR include OSEP3+ and ONNP3' -.

VP3 is obtained^{D173N+V174Y+N179C}After DNA fragmentation, the present invention will use VP3 as described^{D173N+V174Y+N179C}The DNA fragment and the pSK-HDV carrier are subjected to Spe I/Not I double enzyme digestion, and double enzyme digestion products are recovered and connected to obtain pSK-VP3^{D173N+V174E+N179C}A plasmid.

In the present invention, the pSK-HDV vector has been reported in the prior art (Cao Weijun et al. rescue of foot-and-mouth disease virus O/HN/93 vaccine strain and identification of virus activity. North China agro-Proc., 2010,25(3): 32-37).

In the present invention, the SpeI/Not I double cleavage reaction system is preferably 10 × Basal buffer 5. mu.l, template 25. mu.l, Not I5. mu.l, Spe I5. mu.l, ddH₂O10. mu.l. The Spe I/NotI bisThe reaction conditions for enzyme digestion are preferably 37 ℃ for 2 h. The method of the present invention is not particularly limited, and the method may be a method using a known connection scheme.

Obtaining pSK-VP3^{D173N+V174E+N179C}After plasmid cloning VP0 gene segment, VP1+ P2 gene segment, 5'UTR + L gene segment and P3+3' UTR gene segment into pSK-VP3^{D173N+V174E+N179C}In the plasmid, pOFS was obtained^D3173N ^{+V3174E+N3179C}A plasmid.

In the invention, a template for amplifying a VP0 gene fragment is a pOFS plasmid, a primer pair used for amplifying a VP0 gene fragment is preferably OSBP4+ and OEP2-, the nucleotide sequence of the OSBP4+ is preferably shown as SEQ ID NO.5 in a sequence table, the nucleotide sequence of the OEP 2-is preferably shown as SEQ ID NO.6 in the sequence table, and a PCR system for amplifying a VP0 gene fragment is the same as that of an A amplification product, except that 10 × LA Taq buffer (5 mu l) is replaced by 5 × PrimeSTART^TMHS DNA polymerase Buffer (10. mu.l), LA Taq (0.5. mu.l) was replaced with PrimeSTART^TMHS DNApolymerase (0.5. mu.l); accordingly, ddH₂O was reduced to 33. mu.l. The PCR procedure for amplifying the VP0 gene fragment is the same as that for the A amplification product. The obtained VP0 gene fragment and pSK-VP3^{D173N+V174E+N179C}Carrying out double enzyme digestion on the plasmid, recovering and connecting double enzyme digestion products to obtain pSK-VP0+ VP3^{D173N+V174E+N179C}A plasmid. The reaction system of double enzyme digestion is the same as the step (3). The restriction enzyme in the reaction system is Spe I/EcoR I.

In the present invention, the template for amplifying the VP1+ P2 gene fragment is pOFS plasmid. The primer pair used for amplifying the gene fragment VP1+ P2 is preferably OS2A +/OBN-. The nucleotide sequence of the OS2A + is preferably shown as SEQ ID NO.7 in the sequence table; the nucleotide sequence of the OBN-is preferably shown as SEQ ID NO.8 in the sequence table. The PCR system for amplifying the VP1+ P2 gene fragment is the same as the PCR system for amplifying the VP0 gene fragment. The PCR program for amplifying the VP1+ P2 gene fragment is the same as the PCR program for amplifying the VP0 gene fragment. The obtained VP1+ P2 gene fragment and pSK-VP0+ VP3^{D173N+V174E+N179C}Carrying out double enzyme digestion on the plasmids respectively, recovering and connecting double enzyme digestion products to obtain pSK-VP0+ VP3^{D173N+V174E+N179C}+ VP1+ P2 plasmidAnd (4) granulating. The reaction system of double enzyme digestion is the same as the step (3). The restriction enzyme in the VP1+ P2 gene fragment double enzyme digestion reaction system is Nhe I/Not I. The pSK-VP0+ VP3^{D173N+V174E+N179C}The restriction enzyme in the plasmid double digestion reaction system is Spe I/Not I (it should be noted that Nhe I and Spe I are isocaudarner).

In the present invention, the template for amplifying the 5' UTR + L gene fragment is pOFS plasmid. The primer pair used for amplifying the 5' UTR + L gene fragment is preferably OST7 +/OBL-; the nucleotide sequence of the OST7+ is preferably shown as SEQ ID NO.9 in the sequence table; the nucleotide sequence of the OBL-is preferably shown as SEQ ID NO.10 in the sequence table. The PCR system for amplifying the 5' UTR + L gene segment is the same as the PCR system for amplifying the VP0 gene segment. The PCR program for amplifying the 5' UTR + L gene segment is the same as the PCR program for the VP0 gene segment. The obtained 5' UTR + L gene segment and pSK-VP0+ VP3^{D173N+V174E+N179C}Carrying out double enzyme digestion on the plasmid of + VP1+ P2, recovering and connecting double enzyme digestion products to obtain pSK-5' UTR + L + VP0+ VP3^{D173N+V174E+N179C}+ VP1+ P2 plasmid. The reaction system of double enzyme digestion is the same as the step (3). The restriction enzyme in the reaction system is Spe I/BamH I.

In the present invention, the template for amplifying the P3+3' UTR gene fragment is pOFS plasmid. The primer pair used for amplifying the P3+3'UTR gene segment is preferably 3A' +/DNS-; the nucleotide sequence of the 3A' + is preferably shown as SEQ ID NO.11 in the sequence table; the nucleotide sequence of the DNS-is preferably shown as SEQ ID NO.12 in the sequence table. The PCR system for amplifying the P3+3' UTR gene segment is the same as the PCR system for amplifying the VP0 gene segment. The PCR program for amplifying the P3+3' UTR gene segment is the same as the PCR program for the VP0 gene segment. The obtained P3+3'UTR gene segment and pSK-5' UTR + L + VP0+ VP3^{D173N+V174E+N179C}Carrying out double enzyme digestion on the plasmid of + VP1+ P2, recovering and connecting double enzyme digestion products to obtain pSK-5' UTR + L + VP0+ VP3^{D173N+V174E+N179C}+ VP1+ P2+ P3+3' UTR, i.e. pOFS^{D3173N+V3174E+N3179C}A plasmid. The reaction system of double enzyme digestion is the same as the step (3). The restriction enzyme in the reaction system is BglII/NotI.

Obtaining pOFS^{D3173N+V3174E+N3179C}After plasmid, the pOFS was added^{D3173N+V3174E+N3179C}Site of plasmid linearizationThen, the obtained linear plasmid is transfected into cells to obtain O type foot-and-mouth disease virus mutant rHN^{D3173N+V3174E+N3179C}。

In the present invention, the restriction enzyme for linearization is preferably NotI. The Not I enzyme digestion system is the same as the reaction system in the step (3), and is characterized in that: restriction enzyme is Not I (5. mu.l) only, and correspondingly, ddH₂The volume of O was increased to 15. mu.l. The NotI enzyme digestion conditions are the same as the enzyme digestion conditions in the step (3).

After obtaining the enzyme digestion product, preferably recovering the enzyme digestion product to obtain the linear plasmid. The recovery is preferably carried out using a kit. In the present examples, the kit was purchased from agarose gel recovery and/or DNA fragment purification kit of precious bioengineering (Dalian) Limited.

The present invention is not particularly limited to the linear plasmid transfected cells, and may be any cells using transfection methods well known in the art. The cells are preferably BSR/T7-5 cells. The BSR/T7-5 cell line stably expressing T7RNA polymerase was given as a gift by professor Karl-Klaus Conzelmann, Germany.

In order to determine whether the viral mutants prepared according to the invention are genetically stable, the pOFS of the invention is used^D3173N ^{+V3174E+N3179C}The plasmid is linearized and then transfected into BSR/T7-5 cells, and the transfected cells and suspension are collected in 72 h. The cells were repeatedly frozen and thawed three times, and were serially passaged on BHK-21 cells until the time when more than 90% of the cells showed typical cytopathic effect (CPE) became stable. Selecting 10 th generation gene engineering virus, extracting total RNA according to the RNeasy Mini Kit instruction, RT-PCR amplifying P1 gene [ upstream primer 204: 5'-acctccgacgggtggtacgc-3' (SEQ ID No.15), downstream primer NK 61: 5'-gacatgtcctcctgcatctg-3' (SEQ ID No.16)]Agarose gel electrophoresis, recovering target fragment, and sequencing by Beijing Liuhe Hua Dagenescience and technology Co. The result shows that the virus mutant strain prepared by the invention has no mutation in the passage process and has better genetic stability.

The invention also provides another scheme of specific amino acid mutation in VP 3G-H loop of O type foot-and-mouth disease virus, namely rHN^V3174YThe 174 th valine in the VP 3G-H loop was mutated to tyrosine using rHN as the parent strain. The rHN^V3174YThe plasmid is prepared by first constructing pOFS^V3174YA plasmid. The pOFS^V3174YThe construction method of the plasmid is substantially the same as that of pOFS^{D3173N+V3174E+N3179C}The construction method of the plasmid is characterized in that: amplification of VP3^V174YThe primer pair is OSEP3+/3174Y-, 3174Y +/ONNP3 '-and OSEP3+/ONNP3' -. The nucleotide sequence of 3174Y-is shown as SEQ ID No.13 in the sequence table; the 3174Y + nucleotide sequence is shown as SEQ ID No.14 in the sequence table. The PCR amplification system and the amplification program are used for constructing pOFS^{D3173N+V3174E+N3179C}Plasmid amplification system and amplification procedure. The rHN^V3174YrHN illustrating the protection of the invention as a positive control^{D3173N+V3174E+N3179C}The biological property of (a).

To identify the success of rescue of the viral mutant and its biological properties, the following tests were performed: plaque formation test, one-step growth curve drawing, TCID₅₀And LD₅₀And (3) detecting by using an indirect immunofluorescence and laser confocal microscope.

Wherein the plaque formation test can identify whether the receptor utilization phenotype of the genetically engineered virus obtained in the present invention is changed by mutation. The test results are as follows: rHN in comparison to rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}The ability to infect CHO-K1 cells was obtained.

The results of the one-step growth curve show: with maternal virus strain rHN and single site-directed foot and mouth disease virus mutant rHN^V3174YIn comparison, viral mutant rHN^{D3173N+V3174E+N3179C}The proliferation trends on BHK-21 cells are similar, and the replication kinetics of the three cells do not show obvious difference, which indicates that the replication capacity of the virus in the cells is not changed by mutation of a specific site.

TCID₅₀Measurement of (1), TCID₅₀Corresponding to in vitro test, aiming at identifying whether the pathogenic effect capability of the obtained genetic engineering virus on BHK-21 cells is changed; TCID₅₀The results of the assay showed that the maternal virus strain rHN was associated with the hoofEpidemic virus mutant rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}TCID of₅₀Between 10^-6.500～10^-7.125The difference between the three is not obvious, and the result further proves that the specific amino acid mutation in the VP 3G-H loop of the O-type foot-and-mouth disease virus does not obviously change the infectivity of the BHK-21 cells.

LD₅₀Measurement of (D), LD₅₀Corresponding to in vivo experiments, the aim is to identify whether the pathogenicity of the obtained genetically engineered virus on suckling mice is changed. LD₅₀The results of the measurement (2) show that rHN is comparable to rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}The pathogenicity to the suckling mouse is obviously weakened, and the latter is especially obvious.

Meanwhile, the indirect immunofluorescence detection result shows that: rHN, rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}The gene can be normally replicated and translated in BHK-21 cells, and the capability of infecting CHO-K1 cells by specific amino acid mutation in VP 3G-H loop of the O type foot-and-mouth disease virus is confirmed.

The laser confocal detection result shows that: rHN, rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}Infection of BHK-21 cells relies on two endocytosis pathways mediated by clathrin and caveolin; infection on CHO-K1 cells was only rHN indeed^V3174YAnd rHN^{D3173N+V3174E+N3179C}The gene is co-localized with the caveolin, which indicates that the infection of CHO-K1 cells by two foot and mouth disease virus site-specific mutants is dependent on the caveolin mediated endocytosis path.

In order to detect the broad-spectrum antigen of the virus mutant strain provided by the invention, the cross-neutralization capacity of the virus mutant strain inactivated vaccine immune positive serum is detected, and the result shows that the cross-protection capacity of the virus mutant strain provided by the invention for inducing a neutralizing antibody generated by an organism (pig) is obviously improved.

The invention provides the O-type foot-and-mouth disease virus mutant strain rHN^{D3173N+V3174E+N3179C}Or the O-type foot-and-mouth disease virus mutant strain rHN prepared by the preparation method^{D3173N+V3174E+N3179C}Use in increasing neutralizing cross-protection of antibodies.

The method of inactivation is not particularly limited in the present invention, and may be carried out using an inactivation protocol well known in the art. In the examples of the invention, Montanide was added to the mutant virus strain at a 1:1(V/V) ratio^TMISA201VG is emulsified for 20 hours, and the inactivated foot-and-mouth disease virus vaccine is prepared in a laboratory.

The foot-and-mouth disease virus inactivated vaccine is applied to prevention and treatment of O-type foot-and-mouth disease.

The following examples are provided to describe the foot-and-mouth disease virus type O mutant strain of the present invention and its preparation method and application in detail, but they should not be construed as limiting the scope of the present invention.

Introduction to sources of raw materials

1.1 sources of plasmids or cells

The BSR/T7-5 cell line stably expressing T7RNA polymerase was given as a gift by professor Karl-Klaus Conzelmann, Germany.

BHK-21 cells (Ins +, HS +) and CHO-K1 cells (Ins-, HS +) were introduced from the Chinese type culture Collection and the American Standard Biometrics Collection, respectively.

1.2 Primary reagents

LA Taq、PrimeSTART^TMHS DNA polymerase, restriction endonuclease, T4DNA ligase, DNA molecular mass standard, agarose gel recovery and DNA purification kitThe plasmid extraction kit is purchased from Bao bioengineering (Dalian) Co., Ltd.

Coli JM109 competent cells were purchased from Transgene, inc.

Opti-MEM I、Lipofectamine ^TM2000. GMEM, DMEM, 2 × MEM were all purchased from Invitrogen.

Ampicillin-streptomycin and 0.25% Trypsin-EDTA were purchased from Gibco.

Fetal Bovine Serum (FBS) was purchased from Hyclone.

Phosphate buffered saline (PBS; pH 7.4) was purchased from Biologic industries, Inc.

RNeasy Mini Kit was purchased from Qiagen.

Tragacanth gum was purchased from MP Biomedicals.

Type O hoof-and-mouth disease guinea pig antiserum, non-structural proteins (NSPs) 3A monoclonal antibody (McAb) was provided by the lanzhou veterinary institute of chinese academy of agricultural sciences.

Clathrin heavy chain McAb and rabbit caveolin polyclonal antibody (PcAb) were purchased from ThermoFisher.

Goat anti-guinea pig IgG H&L(

647) Purchased from Abcam.

FITC-labeled goat anti-mouse IgG (H + L) and goat anti-rabbit IgG (H + L) were purchased from Beijing kang, a century Biotech Co., Ltd.

DAPI was purchased from shanghai bi yunnan biotechnology limited.

Divinylimine and sodium thiosulfate, which are offered by Zhongnong very good Biotech, Inc.

Montanide^TMISA201VG is purchased from Seppic corporation (Shanghai Qingpu plant).

A liquid phase blocking ELISA antibody detection kit for O-type foot-and-mouth disease virus and a 3ABC monoclonal antibody blocking ELISA antibody detection kit for foot-and-mouth disease virus are provided by the diagnostic center of Lanzhou veterinary research institute of Chinese academy of agricultural sciences.

O/rV-1 strain and O type O/BY/CHA/2010 strain foot-and-mouth disease virus inactivated vaccine immune positive serum (see Table 4) are provided BY OIE/foot-and-mouth disease reference laboratory in China.

1.3 Experimental animals

A suckling mouse (Kunming series) of 1-3 days old is provided by Experimental animal center of Lanzhou veterinary research institute, Chinese academy of agricultural sciences.

18-22 g mice (Kunming series) were provided by the laboratory animal center of Lanzhou veterinary research institute, Chinese academy of agricultural sciences.

Guinea pigs (250-350 g) were provided by the laboratory animal center of Lanzhou veterinary research institute, Chinese academy of agricultural sciences.

Healthy susceptible pigs (25-40 kg) were purchased from a fixed-point pig farm in Gansu province.

Example 1

VP3 site-directed mutagenesis foot-and-mouth disease virus genome full-length cDNA clone (pOFS)^{D3173N+V3174E+N3179C}Plasmid) construction

First, using pOFS plasmid as template, using OSEP3 +/NEC-and NEC +/ONNP3' -to perform PCR amplification, wherein the reaction system (total volume 50 ul) is 10 × LA Taq buffer5 ul, 2.5mM dNTP 5 ul, upstream primer 0.5 ul, downstream primer 0.5 ul, template 0.5 ul, LA Taq enzyme 0.5 ul, ddH₂O38. mu.l. The reaction procedures are as follows: 5min at 94 ℃; 1min at 94 ℃, 1min at 61 ℃,20 s and 3min at 72 ℃ for 30 cycles; 10min at 72 ℃. Two fragments A and B are obtained by amplification.

Then, the target product was obtained using an agarose gel recovery kit from Bao bioengineering (Dalian) Co., Ltd.

TABLE 1 primers used for construction of the foot-and-mouth disease Virus VP3 site-directed mutagenesis genomic full-length cDNA clone

Using the two fragments obtained as templates (OSEP3 +/NEC-and NEC +/ONNP3 '-amplification products), and using a fusion PCR to amplify the target product by using OSEP3+/ONNP3' -as a primer pair to obtain VP3^{D173N+V174Y+N179C}A DNA fragment.

The second step is that: enzyme-cleaved ligation

The pSK-HDV vector and VP3 were digested with Spe I/Not I^{D173N+V174Y+N179C}The reaction systems of the DNA fragment enzyme digestion are 10 × Basal buffer5 mul, template 25 mul, restriction enzyme 15 mul, restriction enzyme 25 mul, ddH (total volume 50 mul)₂Carrying out warm bath for 2h at 37 ℃ under the condition of 10 mu l of O, obtaining two enzyme digestion products, respectively recovering and connecting the enzyme digestion products, wherein the connected reaction system (the total volume is 10 mu l) is 2 × ligase buffer5 mu l, the recovered pSK-HDV carrier is 1 mu l, and the recovered VP3 is^{D173N+V174Y+N179C}3. mu.l of DNA fragment and 1. mu.l of T4DNA ligase. 16 ℃ overnight. Transforming to obtain pSK-VP3^{D173N+V174E+N179C}A plasmid.

The third step is to amplify VP0 gene fragment by using pOFS plasmid as template and OSBP4+/OEP 2-as primer pair, and the difference of the reaction system is the same as the first step, namely, 10 × LA Taq buffer (5 mu l) is replaced by 5 × PrimeSTART^TMHS DNApolymerase Buffer (10. mu.l), LA Taq (0.5. mu.l) was replaced with PrimeSTART^TMHS DNA polymerase (0.5. mu.l); accordingly, ddH₂O was reduced to 33. mu.l. The reaction procedure was as above. The recovery conditions were as above.

Then, the amplified VP0 gene fragment, pSK-VP3, was digested with Spe I/EcoR I^{D173N+V174E+N179C}The plasmid reaction system and reaction conditions are the same as those of the second step, the recovery and the connection are carried out, the reaction system (the total volume is 10 mu l) is 2 × ligase buffer5 mu l, the VP0 gene fragment recovered by enzyme digestion is 3 mu l, and the pSK-VP3 recovered by enzyme digestion is pSK-VP3 ^{D173N+V174E+N179C}1. mu.l of T4DNA ligase 1. mu.l. 16 ℃ overnight. Transforming to obtain pSK-VP0+ VP3^{D173N+V174E+N179C}A plasmid.

The fourth step: the target product of VP1+ P2 is amplified by taking pOFS plasmid as a template and OS2A +/OBN-as a primer pair. The reaction system and the reaction procedure are the same as those in the third step. And recovering the VP1+ P2 gene fragment.

Then, the amplified VP1+ P2 gene fragment was double digested with Nhe I/Not I, and pSK-VP0+ VP3 was double digested with Spe I/Not I^{D173N+V174E+N179C}Plasmid (Nhe I and Spe I are isocaudarner). The reaction system and reaction conditions are the same as those in the third step. Recovering enzyme digestion products and connecting, wherein the connected reaction system is(total volume 10. mu.l) 2 × ligase buffer 5. mu.l, cleaved and recovered VP1+ P2 gene fragment 3. mu.l, cleaved and recovered pSK-VP0+ VP3 ^{D173N+V174E+N179C}1. mu.l of T4DNA ligase 1. mu.l. 16 ℃ overnight. Transforming to obtain pSK-VP0+ VP3^{D173N+V174E+N179C}+ VP1+ P2 plasmid.

The fifth step: the 5' UTR + L gene fragment was amplified using pOFS plasmid as template and OST7 +/OBL-as primer set. The reaction sequence of the reaction system is the same as that of the third step. Recovering the amplification product.

Then, the 5' UTR + L gene fragment amplified by Spe I/BamH I double digestion and pSK-VP0+ VP3^D173N ^+V174E+N179C+ VP1+ P2 plasmid, reaction system and reaction conditions are the same as the third step, enzyme digestion product is recovered, and the reaction system of connection (total volume 10 mul) is 2 × ligase buffer5 mul, the 5' UTR + L gene fragment recovered by enzyme digestion is 3 mul, the pSK-VP0+ VP3 recovered by enzyme digestion^{D173N+V174E+N179C}+ VP1+ P21. mu.l, T4DNA ligase 1. mu.l. 16 ℃ overnight. Transforming to obtain final product and pSK-5' UTR + L + VP0+ VP3^{D173N+V174E+N179C}+ VP1+ P2 plasmid.

And a sixth step: the P3+3'UTR gene fragment was amplified using pOFS plasmid as template and 3A' +/DNS-as primer set. The reaction system and the reaction procedure are the same as those in the third step. Recovering the amplification product.

Then, the amplified P3+3'UTR gene fragment and pSK-5' UTR + L + VP0+ VP3 were digested with Bgl II/Not I^{D173N+V174E+N179C}+ VP1+ P2 plasmid, reaction system and reaction conditions are the same as the third step, enzyme digestion product is recovered and connected, the connection reaction system (total volume 10 mul) is 2 × ligase buffer5 mul, the gene fragment of P3+3'UTR recovered by enzyme digestion is 3 mul, the pSK-5' UTR + L + VP0+ VP3 recovered by enzyme digestion^{D173N+V174E+N179C}+ VP1+ P21. mu.l, T4DNA ligase 1. mu.l. 16 ℃ overnight. Transforming to obtain pSK-5' UTR + L + VP0+ VP3^{D173N+V174E+N179C}+ VP1+ P2+ P3+3' UTR plasmid, pOFS^D3173N ^{+V3174E+N3179C}A plasmid.

Finally, the pOFS with correct result identified by Spe I/Not I double enzyme digestion^{D3173N+V3174E+N3179C}The plasmid is sent to Beijing Liuhe Huada Gene science and technology Co., Ltd for sequence determination.

Comparative example 1

And respectively taking OSEP3 +/3174Y-and 3174+/ONNP3' -as two pairs of primer pairs and taking pOFS plasmid as a template, and carrying out PCR to obtain a C amplified fragment and a D amplified fragment, wherein the amplification system and the amplification procedure are the same as the scheme of the first step in the example 1. Fusion PCR was performed according to the protocol of the first step in example 1 to obtain VP3^V174YThe procedure of the second to sixth steps in example 1 was followed to obtain pOFS^V3174YA plasmid.

Example 2

pOFS with correct sequencing result in example 1 and comparative example 1^{D3173N+V3174E+N3179C}Plasmid and pOFS^V3174YAnd respectively carrying out enzyme digestion and fragment recovery by using NotI to obtain two linearized plasmids.

By Lipofectamine ^TM2000 instructions, 2.5. mu.g of linearized plasmid was transfected into BSR/T7-5 cells, respectively, and transfected cells and suspension were harvested in 72 h. The cells were repeatedly frozen and thawed three times, and were serially passaged on BHK-21 cells until the time when more than 90% of the cells showed typical cytopathic effect (CPE) became stable. Selecting 10 th generation gene engineering virus, and extracting total RNA according to the RNeasy Mini Kit instruction. The total RNA was used as a template to perform RT-PCR amplification of the P1 gene [ upstream primer 204: 5'-acctccgacgggtggtacgc-3' (SEQ ID No.15), downstream primer NK 61: 5'-gacatgtcctcctgcatctg-3' (SEQ ID No.16)]The amplified product is subjected to agarose gel electrophoresis, and a target fragment is recovered and sent to Beijing Liuhe Hua Dageno science and technology Co.

pOFS plasmid was used as positive control for the constructed pOFS^V3174YAnd pOFS^{D3173N+V3174E+N3179C}SpeI/Not I double enzyme digestion identification of the plasmid is carried out respectively, and as a result, two target bands which are consistent with the expected sizes are obtained (FIG. 2A). The sequencing result fully proves that the full-length cDNA clone construction of the foot-and-mouth disease virus genome of two VP3 site-directed mutations is successful.

pOFS^V3174YAnd pOFS^{D3173N+V3174E+N3179C}Significant CPE was observed within 48h after Not I linearization of the plasmids and transfection of BSR/T7-5 cells, respectively. The harvested transfected samples were serially passaged on BHK-21 cells, both of which resulted in the appearance of more than 90% of the cells starting from passage 4The CPE tends to be stable in time (about 8-10 h). The 10 th generation genetic engineering virus inoculated to BHK-21 cells was selected and subjected to RT-PCR identification, so that the band of about 2474bp (base pairs) was obtained, and the sequencing result of the gel recovered product was consistent with the original plasmid sequence (FIG. 2B, FIG. 2C). The two virus mutants are respectively named as rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}。

Example 3

Plaque formation test

rHN and rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}The virus mutants were serially diluted 10 fold and inoculated into monolayers of BHK-21 and CHO-K1 cells (200. mu.l/well) cultured in 6-well plates, respectively, gently shaken 1 time at 37 ℃ for 1 hour with 10-15 min intervals to keep the cell surface infiltrated, then the inoculum was aspirated and covered with 2ml of MEM medium (1ml of 2 × MEM containing 2% FBS +1ml of 1.2% tragacanth solution) per well and cultured for 48 hours (BHK-21) or 72 hours (CHO-K1). finally, the medium was aspirated and fixed (50% methanol + 50% acetone), 0.2% crystal violet stained, and Plaque Forming Units (PFU) were observed and calculated.

The plaque formation test results show that, compared with the maternal virus strain rHN, a single foot-and-mouth disease virus site-directed mutant strain rHN is generated on BHK-21 cells^V3174YThe plaque form of (A) is slightly enlarged and the replication level is slightly improved, while the virus mutant rHN^D3173N ^{+V3174E+N3179C}The plaque morphology of (a) tends to be smaller and less replication competent, but the statistically analyzed differences were not significant (fig. 3, table 2). rHN failed to form plaques on CHO-K1 cells, whereas rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}Plaques were formed on CHO-K1 cells (FIG. 3, Table 2).

TABLE 2 plaque formation test results

Example 4

Drawing of one-step growth curve

Will be at 1.0MOI (multiplicity of infection)rHN and rHN^V3174YAnd rHN^D3173N ^{+V3174E+N3179C}Respectively inoculating the virus mutant strains to BHK-21 cells, adsorbing at 4 ℃ for 1h, then absorbing the inoculated solution, adding DMEM medium without FBS again, and incubating at 37 ℃ until the viruses are harvested at the 2 nd, 4 th, 6 th, 8 th, 9 th, 10 th, 12 th, 16 th, 20 th and 24 th hours. After repeated freeze-thawing 3 times, PFU was measured according to item 1.6, and a one-step growth curve was plotted.

The results are shown in FIG. 4: rHN, rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}The one-step growth curve of (1). As shown in FIG. 4, rHN and rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}The growth curve of one step is substantially consistent, which shows rHN^V3174YAnd rHN^D3173N ^{+V3174E+N3179C}Does not affect the self-replicating capacity of the strain.

Example 5

1、TCID₅₀Measurement of (2)

BHK-21 cells were made into 2 × 10⁶Suspension in ml, with 10-fold serial dilutions of rHN, rHN^V3174YAnd rHN^D3173N ^{+V3174E+N3179C}50. mu.l of each was added to a 96-well plate (8 wells/dilution), and 10 wells were selected^-4～10^-6Three gradients, then 37 ℃ CO₂Incubate for 72 h. Absorbing and removing the culture medium, fixing with 10% formalin solution for 30min, staining with 0.05% methylene blue solution for 30min, washing, naturally drying, counting the number of lesion holes, and processing according to

Method of calculating TCID₅₀。

2、LD₅₀Measurement of (2)

Each get 10^-4～10^-8Inoculating diluted rHN and two foot and mouth disease virus mutant strains to 1-3 days old suckling mice (200 mul/mouse, 5 mice/dilution degree) subcutaneously, observing the morbidity of the suckling mice, counting the death number of the suckling mice by 7 days, and calculating LD according to Reed-Muench method₅₀。

Determination of TCID₅₀Is to know whether the pathogenic effect capability of the obtained genetic engineering virus on BHK-21 cells is changed, which is equivalent to an in vitro testAnd (6) testing. Determination of LD₅₀It is to know whether the pathogenicity of the obtained genetically engineered virus to the suckling mouse is changed, and the method is equivalent to an in vivo test.

TCID₅₀The results of the assay show that the parental virus strain rHN and the two virus mutants rHN^V3174YAnd rHN^D3173N ^{+V3174E+N3179C}TCID of₅₀Between 10^-6.500～10^-7.125The difference between the three is not obvious, and the result further proves that the specific amino acid mutation in the VP 3G-H loop of the type O foot-and-mouth disease virus does not obviously change the infectivity of the BHK-21 cells (Table 2). LD₅₀The results of the measurement (2) show that rHN is comparable to rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}The pathogenicity of the compound is obviously weakened, and the pathogenicity of the compound is especially obvious (the pathogenicity is reduced by more than 10 times, see table 2).

Example 6

Indirect immunofluorescence assay

rHN and rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}Respectively inoculating to BHK-21 or CHO-K1 cells, placing at 37 deg.C and CO₂An incubator. After 4-8 h, the inoculation liquid is aspirated and discarded, 4% paraformaldehyde is fixed for 30min, 0.2% TritonX-100 is permeated for 15min, 5% BSA is used for blocking for 1h, foot-and-mouth disease virus 3AMcAb (3A24, 1:400) is added, the mixture is incubated for 1h at 37 ℃, and FITC labeled goat anti-mouse IgG (1:50) is added for reaction for 1 h. DAPI (1:10000) was added to stain nuclei for 5 min. After each step is finished, washing the substrate with PBS for 3-5 times. Finally, the plate was observed under a fluorescent inverted microscope (EVOS FL).

The indirect immunofluorescence detection result shows that after the female parent virus strain and the virus mutant strain are inoculated to BHK-21 cells for 4 hours, green fluorescence generated by the reaction of 3A24(McAb) recognizing foot-and-mouth disease virus 3A and FITC-labeled host specific IgG can be observed, which indicates rHN and rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}It was normally replicated and translated in BHK-21 cells (FIG. 5). However, rHN and rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}After CHO-K1 cells are inoculated for 4-8H, only the CHO-K1 cells can not detect the expression of the foot-and-mouth disease virus 3A protein, thereby confirming that the O type foot-and-mouth disease virus VP 3G-H loop obtains infection by specific amino acid mutationCapacity of CHO-K1 cells (FIG. 5).

Example 7

Confocal laser microscopy

rHN and its two virus mutants were inoculated at 100MOI into BHK-21 or CHO-K1 cells, respectively. After incubation at 37 ℃ for 15min, the inoculum was aspirated and washed 3 times with PBS. Adding O type foot-and-mouth disease dolphin antiserum (1:500), Clathrin heavy chain McAb (1:500) or rabbit derived caveolin PcAb (1:200) in sequence,

647 labeled goat anti-Guinea pig IgG H&L (1:400), FITC-labeled goat anti-mouse or anti-rabbit IgG (1:50), at 37 ℃ for 1h each. And (3) staining nuclei with DAPI for 5min, washing with PBS for 5-8 times, sealing with 50% glycerol, and observing under a laser confocal microscope (TCS SP 8).

Laser confocal detection results show that after the maternal virus strain and the two virus mutant strains are inoculated to BHK-21 cells for 15min, the co-localization of foot-and-mouth disease virus, clatharin and caveolin can be respectively observed, which indicates rHN and rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}Infection of BHK-21 cells relied on clathrin and caveolin mediated two endocytosis pathways (FIG. 6). However, only rHN cells were found in CHO-K1 cells^V3174YAnd rHN^{D3173N+V3174E+N3179C}Co-localization with caveolin suggests that infection of CHO-K1 cells by both viral mutants should be dependent on caveolin-mediated endocytosis pathways.

Example 8

Representative strain of foot-and-mouth disease virus

Mya98 pedigree: O/BY/CHA/2010 (also one of the current representative species for the production of type O Foot-and-Mouth disease vaccines), GenBank accession JN998095(Zheng H, et al. genetic Characterization of a New Pan rendering A topic type string of section O Foot-and-motion DisaseVirus Isolated in China drug 2010.Virus genes.2012,44(1): 80-88).

Panasia-1 lineage representative strain: O/CHA/7/2011, GenBank accession number JF 837375.

Representative strain of Cathay topology: O/SCGH/CHA/2016, GenBank accession number KX 161429.

Branch representative strain IND2001 d: O/XJ/CHA/2017, GenBank accession number: MF461724(Zhu Z, actual. first Detection of Foot-and-mouse Disease Virus O/ME-SA/Ind2001inChina. transbound emery Dis.2018May 9.doi: 10.1111/tbed.12895).

The O/rV-1 strain is a foot-and-mouth disease genetic engineering virus related to a national invention patent and a new veterinary drug registration certificate which are newly applied by an applicant and are disclosed in the form of the national invention patent and the new veterinary drug registration certificate. The grant number of the national invention patent is ZL 201210035986.7. The name of the new veterinary drug is porcine foot-and-mouth disease O-type virus 3A3B epitope deletion inactivated vaccine (O/rV-1 strain), the certificate number is (2017) New veterinary drug certificate number 50, the research unit is Lanzhou veterinary institute of Chinese academy of agricultural sciences, Zhongxiang practical company, Zhongnong witt biotechnology company, and the certificate issue date is as follows: year 2017, month 10 and day 16.

The representative foot-and-mouth disease virus strain and rHN of 10 th generation^V3174YAnd rHN^{D3173N+V3174E+N3179C}(total 8 strains) As a research material, TCID of each virus strain was measured by the method of example 5₅₀The results are shown in Table 4.

1) Preparation and safety inspection of foot-and-mouth disease virus antigen

The 10 th generation rHN and rHN^V3174YAnd rHN^{D3173N+V3174E+N3179C}Inactivating, respectively adding diethylene imine to a final concentration of 3mmol/L, and reacting at 30 deg.C for 48h while stirring or shaking. Adding sodium thiosulfate to the final concentration of 1.2-2.0%, and fully stirring to block excessive inactivator.

Subsequently, the above 3 types of foot-and-mouth disease virus type O antigens were sampled for safety test (the remaining samples were rapidly cooled to below 4 ℃ and stored): 5 mice were used each, and 0.5ml of inactivated antigen was injected subcutaneously per mouse; 2 guinea pigs were used each, and 2ml of the inactivated antigen was injected subcutaneously per guinea pig. After 7 days of continuous observation, no typical clinical symptoms caused by foot-and-mouth disease virus infection appear.

2) Preparation of foot-and-mouth disease virus inactivated vaccine

After the safety of the inactivated antigen is qualified, Montanide is added according to the proportion of 1:1(V/V)^TMAnd emulsifying the mixture for 20 hours in ISA201VG to obtain 3O type foot-and-mouth disease virus inactivated vaccines.

3) Immune positive serum

Respectively immunizing 2 heads of pigs with the 3O type foot-and-mouth disease virus inactivated vaccines (the O type foot-and-mouth disease virus liquid blocking ELISA antibody titer is not higher than 1:8 and 3ABC antibody detection is negative), respectively collecting blood 7, 14 and 21 days after immunization, standing at 37 ℃ for 1h, and standing at 4 ℃ overnight. Centrifuging at 8000rpm for 15min, and collecting serum.

4) Liquid phase blocking ELISA antibody detection for O-type foot-and-mouth disease virus

According to the specification of the O-type foot-and-mouth disease virus liquid phase blocking ELISA antibody detection kit, the following operations are carried out:

each 100. mu.l of the serum of the pig to be tested was serially diluted 2 × with PBST (the same procedure was carried out for the negative and positive control sera).

Adding the serum to be detected in a ratio of 1: 4-1: 512 into a 96-well plate according to a row, wherein each 50 mu l of the serum is added into each well; and (3) setting a negative control and a positive control (negative control serum, 1: 1-1: 8; positive control serum, dilution of 1: 2-1: 256) and a virus antigen control hole (4 holes).

Viral antigens were diluted to working concentration (1:25) with PBST (containing Tween-20) and added to serum dilution wells (50. mu.l/well) and viral antigen control wells (100. mu.l/well); seal plate, shake, stand overnight at 4 deg.C or incubate for 90min at 37 deg.C.

Transferring the antigen-antibody complex from a 96-well plate to an ELISA plate coated with O-type foot-and-mouth disease virus rabbit antibody (body) in sequence, wherein each ELISA plate is 50 mu l; incubate on plate at 37 ℃ for 60 min.

Continuously washing the plate for 3-5 times by PBST, and patting to dry; adding a guinea pig antibody working solution of O type foot-and-mouth disease virus at 50 mul/well; incubate on plate for 30min at 37 ℃.

Continuously washing the plate for 3-5 times by PBST, and patting to dry; adding rabbit anti-guinea pig IgG-HRP working solution, 50 mul/hole; incubate on plate for 30min at 37 ℃.

Continuously washing the plate for 3-5 times by PBST, and patting to dry; mixing the TMB substrate A solution and the B solution (1:1, V/V), shaking, adding the mixed substrate solution according to the amount of 50 mul/hole, and incubating at 37 ℃ for 15min (color reaction).

After the color reaction is finished, adding a stop solution (50 mu l/hole) to stop the reaction; OD450nm values were read on a microplate reader.

OD of wells controlled with viral antigen₄₅₀And (5) determining the antibody titer of the serum to be detected by taking the nm value as a critical value.

The ELISA antibody titer results of the pig serum to be detected are shown in Table 3.

TABLE 3 liquid-phase blocking ELISA antibody detection results for type O foot-and-mouth disease viruses

On the 7 th day of the animals immunized by the 3O type foot-and-mouth disease virus inactivated vaccines, the O type foot-and-mouth disease virus specific ELISA antibodies are generated. By day 14, the level of the O-type foot-and-mouth disease virus specific ELISA antibody of the swine immunized by the foot-and-mouth disease vaccine is increased; after 21 days, the antibody levels of all immunized pigs were 1:90 or more (Table 3).

5) Virus Neutralization Test (VNT)

And (3) selecting immune positive serum with the O-type foot-and-mouth disease virus liquid phase blocking ELISA antibody titer being more than or equal to 1:128, and performing a virus neutralization experiment to judge the cross neutralization capacity of the immune positive serum to the O-type foot-and-mouth disease viruses with different genotypes. The specific steps are as follows (repeated three times):

test strains were separately diluted to 1000TCID₅₀、100TCID₅₀、10TCID₅₀And 0.1TCID₅₀(ii) a Selecting 100TCID₅₀The dilutions were added to columns 1 to 10 of a 96-well plate (50. mu.l/well), and columns 11 and 12 were added to 1000TCID wells with 4 wells₅₀、100TCID₅₀、10TCID₅₀And 0.1TCID₅₀The virus solution of (1).

Subjecting selected immune positive serum of foot-and-mouth disease vaccine to action at 56 deg.C for 30min, serial diluting with DMEM basic culture solution 2 ×, adding into one row of 96-well plate (1: 2-1: 1024, 50 μ l/well), adding into DMEM basic culture solution (50 μ l/well) in the 11 th and 12 th rows, and adding CO₂The reaction is carried out for 1h at 37 ℃ in a cell culture box.

BHK-21 cell suspension (2 × 10)⁶Individual cells/ml, about 10ml) were added to the above 96-well plate50 μ l/well, put at 37 ℃ in CO₂And (5) incubating for 72h in a cell incubator.

The liquid in the 96-well plate was aspirated, fixed, stained, and the result was judged (Reed-Muench method).

The results are shown in Table 4.

TABLE 4 results of antibody titer of the sera of pigs to be tested

The statistics of the VNT results of the 5 types of immune positive sera to the 8 types of foot-and-mouth disease viruses shows that the immune positive sera of the O/rV-1 strain for producing the virus strain for the swine foot-and-mouth disease virus O-type 3A3B epitope deletion inactivated vaccine has better neutralizing capacity (r value) to O-type Catay topological type, Mya98, Panasia-1 pedigree and IND2001d branched foot-and-mouth disease viruses>0.3), the neutralizing capacity of the O/BY/CHA/2010 immune positive serum to other genotype foot-and-mouth disease viruses is stable between 0.31 and 0.85 (r value); however, the rHN immune positive serum has poor neutralizing capacity (r value is between 0.08 and 0.14) to the foot-and-mouth disease virus of the Panasia-1 lineage, and the V174Y on the VP3 causes the neutralizing capacity to be respectively reduced and increased to the foot-and-mouth disease virus of the Mya98 lineage as well as the Panasia-1 lineage and the IND2001d branch; of note, rHN^{D3173N+V3174E+N3179C}The neutralizing capacity of the immune positive serum to the O-type four-genotype foot-and-mouth disease virus is obviously improved, and the r values of the immune positive serum are all more than 0.50 (Table 4).

According to the recommendation of OIE terrestrial animal diagnostic test and vaccine handbook, the ideal foot-and-mouth disease vaccine for preparing seed virus not only has good production performance (short lesion time on BHK-21 cells, stable heredity and the like), but also meets the requirement that the r value is not less than 0.3 for the neutralization level and the immunity efficacy of different genotype foot-and-mouth disease viruses of the same serotype in vitro (OIE, 2012). Analysis of variance of VNT results shows that D173N + V174E + N179C on rHN VP3 can remarkably improve cross-neutralization capacity of immune positive serum of foot-and-mouth disease virus, and the fact that rHN is involved^{D3173N+V3174E+N3179C}The genetically stable strain may have good cross-challenge protection against O-type Cathay topology, Mya98 and Panasia-1 lineage as well as the IND2001d branched foot-and-mouth disease virus (FIG. 7).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

1. O-type foot-and-mouth disease virus mutant rHN^{D3173N+V3174E+N3179C}The vaccine is characterized in that the vaccine is obtained by taking rHN virus strain as a mother virus strain and mutating the following amino acid positions in a G-H loop of VP3 protein: aspartic acid 173 is mutated to asparagine, valine 174 to glutamic acid and asparagine 179 to cysteine.

2. The foot and mouth disease virus type O mutant rHN of claim 1^{D3173N+V3174E+N3179C}The preparation method is characterized by comprising the following steps:

the PCR program of the A amplification product or the B amplification product is as follows: 5min at 94 ℃; 30 cycles of 94 ℃ for 1min, 61 ℃ for 1min for 20s, and 72 ℃ for 3 min; 10min at 72 ℃;

the PCR system of the amplification product A or B is as follows, 10 × LA Taq buffer5 mul, 2.5mmol/LdNTP 5 mul, upstream primer 0.5 mul, downstream primer 0.5 mul, template 0.5 mul, LATaq enzyme 0.5 mul, ddH₂O 38μl；

the nucleotide sequence of the NEC-is shown as SEQ ID NO.2 in a sequence table;

(3) mixing the VP3^{D173N+V174Y+N179C}DNA fragment and pSK-HDV vector were eachUsing Spe I/Not I double enzyme to cut and connect to obtain pSK-VP3^{D173N+V174E+N179C}A plasmid;

the template for amplifying the VP0 gene fragment is pOFS plasmid, the primer pair for amplifying the VP0 gene fragment is OSBP4+ and OEP2-, the nucleotide sequence of the OSBP4+ is shown as SEQ ID NO.5 in a sequence table, the nucleotide sequence of the OEP 2-is shown as SEQ ID NO.6 in the sequence table, and the difference between the PCR system for amplifying the VP0 gene fragment and the PCR system for amplifying the A amplification product is that 5 mul 10 × LA Taq buffer is replaced by 10 mul 5 × PrimeSTART^TMHSDNA polymeraseBuffer, 0.5. mu.l LA Taq exchange to 0.5. mu.l PrimeSTART^TMHSDNA polymerase; accordingly, ddH₂O is reduced to 33 μ l; the PCR program for amplifying the VP0 gene fragment is the same as the PCR program for amplifying the product A;

the template for amplifying the VP1+ P2 gene fragment is pOFS plasmid; the primer pair used for amplifying the gene segment of VP1+ P2 is OS2A +/OBN-; the nucleotide sequence of the OS2A + is shown as SEQ ID NO.7 in the sequence table; the nucleotide sequence of the OBN-is shown as SEQ ID NO.8 in the sequence table; the PCR system for amplifying the VP1+ P2 gene fragment is the same as the PCR system for amplifying the VP0 gene fragment; the PCR program for amplifying the VP1+ P2 gene fragment is the same as the PCR program for amplifying the VP0 gene fragment;

the template for amplifying 5' UTR + L gene segment is pOFS plasmid; the primer pair used for amplifying the 5' UTR + L gene fragment is OST7 +/OBL-; the nucleotide sequence of the OST7+ is shown as SEQ ID NO.9 in the sequence table; the nucleotide sequence of the OBL-is shown as SEQ ID NO.10 in the sequence table; the PCR system for amplifying the 5' UTR + L gene segment is the same as the PCR system for amplifying the VP0 gene segment; the PCR program for amplifying the 5' UTR + L gene segment is the same as the PCR program for amplifying the VP0 gene segment;

the template for amplifying the P3+3' UTR gene segment is pOFS plasmid; the primer pair for amplifying the gene segment of P3+3'UTR is 3A' +/DNS-; the nucleotide sequence of the 3A' + is shown as SEQ ID NO.11 in the sequence table; the nucleotide sequence of the DNS-is shown as SEQ ID NO.12 in the sequence table; the PCR system for amplifying the P3+3' UTR gene segment is the same as the PCR system for amplifying the VP0 gene segment; the PCR program for amplifying the P3+3' UTR gene segment is the same as the PCR program for amplifying the VP0 gene segment;

3. The method according to claim 2, wherein the fusion PCR process in step (2) is: 5min at 94 ℃; 30 cycles of 94 ℃ for 1min, 61 ℃ for 1min for 20s, and 72 ℃ for 3 min; 10min at 72 ℃.

4. The method according to claim 2, wherein the SpeI/Not I double cleavage reaction in step (3) is carried out in the reaction system of 10 × Basal buffer 5. mu.l, template 25. mu.l, NotI 5. mu.l, SpeI 5. mu.l, ddH₂O10 mu l; the reaction condition of the SpeI/NotI double enzyme digestion is that the temperature bath is carried out for 2h at 37 ℃.

5. The foot and mouth disease virus type O mutant rHN of claim 1^{D3173N+V3174E+N3179C}Or the O-type foot-and-mouth disease virus mutant rHN prepared by the preparation method of any one of claims 2 to 4^{D3173N+V3174E+N3179C}The application in the infection of CHO cell line.

6. The type-O foot-and-mouth disease virus mutant rHN according to claim 1^{D3173N+V3174E+N3179C}Or the O-type foot-and-mouth disease virus mutant rHN prepared by the preparation method of any one of claims 2 to 4^{D3173N+V3174E+N3179C}The inactivated vaccine strain of (1).