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

A foot-and-mouth disease virus and mutant strain technology, which is applied in the field of vaccine candidate strains, can solve the problems of loss of CHO-K1 cells, impact on seed virus replication performance, virus titer, neutralizing antibody cross-protection ability, limitation, etc.

Active Publication Date: 2019-03-29
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the genetically engineered virus rHN and its vaccine candidate strains lost the ability to infect CHO-K1 cells (Li P, et al. Evaluation of a Genetically Modified Foot-and-Mouth Disease Virus Vaccine Candidate Generated by Reverse Genetics. BMC Vet Res. 2012,8:57), and some (one) key amino acid variations in the FMD virus T / B cell epitope are strictly limited to the dual effects of antibody response and cell adsorption (Mateu MG, et al.A Single Amino Acid Substitution Affects Multiple Overlapping Epitopes in the Major Antigenic Site of Foot-and-Mouth Disease Virus ofSerotype C.J Gen Virol.1990,71:629–637), which may significantly affect the replication performance of the seed virus in the BHK-21 cells used for the production of foot-and-mouth disease vaccines and virus titer and cross-protection ability of neutralizing antibodies

Method used

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  • Type O foot-and-mouth disease virus mutant and preparation method and application thereof
  • Type O foot-and-mouth disease virus mutant and preparation method and application thereof
  • Type O foot-and-mouth disease virus mutant and preparation method and application thereof

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preparation example Construction

[0046] The invention provides the O-type foot-and-mouth disease virus mutant strain rHN D3173N+V3174E+N3179C The preparation method comprises the following steps:

[0047] (1) Using the pOFS plasmid as a template, PCR was performed with OSEP3+ and NEC-primers to obtain the A amplification product, and NEC+ and ONNP3'-primers were used to perform PCR to obtain the B amplification product;

[0048] The nucleotide sequence of the OSEP3+ is shown in SEQ ID NO.1 in the sequence listing;

[0049] The nucleotide sequence of said NEC- is shown in SEQ ID NO.2 in the sequence listing;

[0050] The nucleotide sequence of said NEC+ is shown in SEQ ID NO.3 in the sequence listing;

[0051] The nucleotide sequence of the ONNP3'- is shown in SEQ ID NO.4 in the sequence listing;

[0052] (2) Using the A amplification product and the B amplification product as a template, carry out fusion PCR amplification with OSEP3+ and ONNP3'-primers to obtain VP3 D173N+V174Y+N179C DNA fragments;

[0...

Embodiment 1

[0124] Full-length cDNA clone of foot-and-mouth disease virus genome with site-directed mutation of VP3 (pOFS D3173N+V3174E+N3179C Plasmid) construction

[0125] The first step, first, use the pOFS plasmid as a template, and use OSEP3+ / NEC- and NEC+ / ONNP3'- to perform PCR amplification. The reaction system is (total volume 50 μl): 10×LA Taq buffer 5 μl, 2.5mM dNTP 5 μl, 0.5 μl upstream primer, 0.5 μl downstream primer, 0.5 μl template, 0.5 μl LA Taq enzyme, ddH 2 O 38 μl. The reaction procedures were all: 94°C for 5 minutes; 94°C for 1min, 61°C for 1min20s, 72°C for 3min, 30 cycles; 72°C for 10min. Two fragments A and B were amplified.

[0126] Then, the target product was obtained using the agarose gel recovery kit of Treasure Bioengineering (Dalian) Co., Ltd.

[0127] Table 1 The primers used to construct the full-length cDNA clone of foot-and-mouth disease virus VP3 site-directed mutation genome

[0128]

[0129] Using the recovered two fragments as templates (OSEP3...

Embodiment 2

[0144] The correct pOFS of the sequencing results in Example 1 and Comparative Example 1 D3173N+V3174E+N3179C Plasmids and pOFS V3174Y Using NotI to carry out enzyme digestion and fragment recovery, respectively, to obtain two linearized plasmids.

[0145] Press Lipofectamine TM According to the 2000 operating instructions, 2.5 μg of linearized plasmids were transfected into BSR / T7-5 cells, and the transfected cells and their suspensions were harvested within 72 hours. Freezing and thawing were repeated three times, and the BHK-21 cells were continuously passaged until the time when more than 90% of the cells showed typical cytopathic effect (CPE) tended to be stable. Select the 10th generation genetically engineered virus and follow the instructions of the RNeasy Mini Kit to extract total RNA. Using total RNA as a template, carry out RT-PCR amplification of the P1 gene [upstream primer 204: 5'-acctccgacgggtggtacgc-3' (SEQ ID No.15), downstream primer NK61: 5'-gacatgtcctcc...

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Abstract

The invention provides a type O foot-and-mouth disease virus mutant and a preparation method and application thereof, and belongs to the technical field of vaccine candidate strains. According to thetype O foot-and-mouth disease virus mutant disclosed by the invention, an rHN virus strain is used as a female parent virus strain, and the following amino acids in a G-H ring of VP3 protein are mutated: the 173rd aspartic acid is mutated into asparagine, the 174th valine is mutated into glutamic acid and the 179th asparagine is mutated into cysteine. The virus mutant has heredity stability and obtains the capacity of caveolin for performing mediated infestation on CHO-K1cells. The result of detecting the cross neutralization capacity of immune positive serum indicates that compared with a female parent virus strain, the virus mutant has the advantages that the cross protection capacity of the virus mutant for inducing organisms to produce foot-and-mouth disease virus neutralizing antibodies is notably improved, and the virus mutant shows excellent antigen broad spectrum properties. Vaccines prepared through inactivation of the virus mutant can be used for preventing infection with type O foot-and-mouth disease viruses.

Description

technical field [0001] The invention belongs to the technical field of vaccine candidate strains, and in particular relates to an O-type foot-and-mouth disease virus mutant strain and a preparation method and application thereof. Background technique [0002] Foot-and-mouth disease (foot-and-mouth disease, FMD) is an acute, febrile disease caused by foot-and-mouth disease virus (foot-and-mouth disease virus, FMDV) in pigs, cattle, sheep and other main domestic animals and other domesticated and wild cloven-hoofed animals. , highly contagious and highly contagious infectious diseases that can spread quickly and over long distances. The disease is one of the animal infectious diseases legally reported by the World Organization for Animal Health (office international desépizooties, OIE), and my country also lists it at the top of the list of first-class animal diseases [Ministry of Agriculture Announcement No. 1125, 2008]. Since the first relatively definite written record of ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85C07K16/10A61K39/135A61P31/14
CPCA61K39/12A61K2039/5252A61K2039/552A61P31/14C07K16/1009C12N7/00C12N15/85C12N2770/32121C12N2770/32134
Inventor 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
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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