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A kind of attenuated mutant strain of foot-and-mouth disease virus and its preparation method and application

A technology of foot-and-mouth disease virus and mutant strain, which is applied in the field of biotechnology and biological products, and can solve the problem of no attenuated strain of foot-and-mouth disease.

Active Publication Date: 2022-04-15
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

So far, there is no Lb AUG modified foot-and-mouth disease virus available at home and abroad, and there are no reports of Lb AUG modified foot-and-mouth disease attenuated strains

Method used

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  • A kind of attenuated mutant strain of foot-and-mouth disease virus and its preparation method and application
  • A kind of attenuated mutant strain of foot-and-mouth disease virus and its preparation method and application
  • A kind of attenuated mutant strain of foot-and-mouth disease virus and its preparation method and application

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preparation example Construction

[0028] In the present invention, the preparation method of the attenuated mutant strain of foot-and-mouth disease virus preferably comprises the following steps, on the basis of the full-length infectious cloning plasmid of the foot-and-mouth disease virus strain OZK / 93-08, mutating LbAUG to obtain two kinds of different mutated recombinant plasmids, The virus was rescued by using the screened positive recombinant plasmid to obtain the mutant strain of foot-and-mouth disease virus.

[0029] In the present invention, the mutation is preferably performed by site-directed mutagenesis. The primers used for point mutations are listed in Table 1. At the same time, in order to verify whether the LbAUG site is mutated into any codon and whether it can affect the replication ability of the virus, the present invention also provides 2 kinds of contrast mutation schemes, LbAUG is mutated into TGG and CTG (see figure 1 ), the mutation method is the same as the above method, and the prime...

Embodiment 1

[0048] 1. Construction of mutant full-length plasmid

[0049] In order to obtain LbAUG mutated foot-and-mouth disease virus, on the basis of the full-length infectious cDNA clone plasmid pOZKF-Z1234 of the foot-and-mouth disease virus OZK / 93-08 strain by site-directed mutagenesis, LbAUG was point-mutated into AUC and AAA, and sequentially constructed Plasmids modified with different mutations of LbAUG. The primers used for point mutations are shown in Table 1, and the PCR amplification system is shown in Table 2. The amplification program was: 94°C for 5 min, 98°C for 20 min, 68°C for 1 min / 1kb, after 35 cycles, 72 ℃ 10min. Whether the resulting recombinant plasmid introduced the expected mutation was verified by sequence determination.

[0050] Table 1 Primers used for Lb AUG point mutation

[0051]

[0052] Table 2 PCR reaction system

[0053]

[0054] 2. Rescue of Mutant Viruses

[0055] The sequenced correct mutant plasmid was linearized with Not I, in Lipofecta...

Embodiment 2

[0063] Indirect immunofluorescence identification of rescued virus

[0064] The BHK-21 monolayer cells grew to about 70%, and the rescued parental virus (WT) and four strains of foot-and-mouth disease virus mutants (rV-O / TGG, rV-O / ATC, rV-O / CTG and rV-O / AAA), incubated at 37°C for 4h, fixed the virus-infected cells in 4% paraformaldehyde, washed 3 times with PBS buffer and permeabilized with 0.5% TritonX-100 (Sigma-Aldrich) for 15min , add 1% BSA and place in a 37°C incubator to block for 1h, add foot-and-mouth disease virus 3A monoclonal antibody 3A24 dropwise, incubate at 37°C for 1h, rinse with PBS for 3 times, add FITC-labeled goat anti-mouse IgG antibody (Sigma), 37°C Incubate for 1 h, rinse with PBS 3 times, add DAPI (Beyotime) to incubate for 15 min, rinse with PBS 3 times, and use a laser scanning confocal microscope (LSCM) to observe the fluorescence in virus-infected cells.

[0065] The identification results of the rescued virus were as follows: figure 2 As show...

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Abstract

The invention provides a foot-and-mouth disease virus attenuated mutant strain and its preparation method and application, belonging to the technical field of biotechnology and biological products. The attenuated mutant strain of foot-and-mouth disease virus is to mutate the Lb AUG in the parental foot-and-mouth disease virus leader protein gene to ATC or AAA. The level is reduced, the pathogenicity to suckling mice is significantly weakened, and this mutation type can be inherited stably. Based on the genome structure of the OZK / 93‑08 vaccine strain, the invention mutates the second start codon AUG in the lead protein gene to ATC or AAA, which can significantly attenuate the O-type foot-and-mouth disease virus. The method of the attenuated vaccine strain will provide a safer platform for the production of the FMD O-type inactivated vaccine antigen.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and biological products, and in particular relates to an attenuated mutant strain of foot-and-mouth disease virus and its preparation method and application. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious animal disease caused by foot-and-mouth disease virus (FMDV). , pigs, sheep and goats etc. The disease is one of the animal infectious diseases legally reported by the World Organization for Animal Health (OIE), and my country ranks it as the first class of animal diseases. Currently, the most effective control strategies for FMD are mainly achieved through inactivated vaccination and slaughter of infected and exposed animals. After decades of immunization with inactivated foot-and-mouth disease vaccines, some countries in the European Union and South America have effectively controlled foot-and-mouth disease, and are considered the benc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/04C12N7/00A61K39/135A61P31/14C12R1/93
CPCC12N7/00A61K39/12A61P31/14C12N2770/32121C12N2770/32134C12N2770/32162A61K2039/5254A61K2039/552
Inventor 卢曾军刘在新李平花袁红张杰白兴文孙普包慧芳李娜
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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