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Attenuated pseudorabies virus with deletion gene and application of attenuated pseudorabies virus as vaccine

A pseudorabies virus and gene deletion technology, which is applied to viruses, vaccines, antiviral agents, etc., can solve problems such as low immunity, unsatisfactory use effects, and unclear safety, and achieve good safety effects

Active Publication Date: 2020-01-17
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the fact that the existing pseudorabies virus vaccine has low immunity to fur animals such as dogs, minks, foxes and raccoons, the safety is not clear, and the use effect is not ideal. On the basis of the biological characteristics of the strain, it was found that the deletion of the Us3 gene in the Us region of the PRV virus genome resulted in the attenuation of the virulent virus and 100% of the parental PRV virulent virus attack on fur animals such as dogs, minks, foxes and raccoons induction of protection

Method used

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  • Attenuated pseudorabies virus with deletion gene and application of attenuated pseudorabies virus as vaccine
  • Attenuated pseudorabies virus with deletion gene and application of attenuated pseudorabies virus as vaccine
  • Attenuated pseudorabies virus with deletion gene and application of attenuated pseudorabies virus as vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Design of PRV-specific sgRNA

[0048] Design sgRNAs targeting gE, gl, TK and US3 gene sequences (see Table 1 for design).

[0049] Table 1 CRISPR / Cas9 sgRNA sequences targeting the PRV-GL genome

[0050]

Embodiment 2

[0051] Example 2: Construction of CRISPR / Cas9 vector

[0052] Heat the 2 primers of the 6 groups of sgRNA-gE designed in Example 1 at 95°C for 5 minutes, and cool naturally to anneal to form a double double-stranded structure. The annealed product is cloned into the PX330 vector after digestion with BbsI, and transformed into competent DH5α . The plasmids were extracted from the expanded culture of the correctly sequenced bacterial solution for later use.

Embodiment 3

[0053] Embodiment 3: the acquisition of recombinant virus

[0054] 3 μg of PRV-GL genome and 0.5 μg each of pX330-gE / gI sgRNA1 / 2 plasmids were co-transfected into monolayer VERO cells (6-well plate). Typical cytopathic changes (CPE) can be seen 48 hours after transfection. After 72 hours, the supernatant of the transfected cells was harvested, inoculated with PK-15 cells, and subjected to plaque purification and screening; plaques were picked under a fluorescent microscope, and after 10 rounds of purification , and the obtained virus was named rPRV-delgE / gI. Similarly, 3 μg of the rPRV-delgE / gI genome and each 0.5 μg of the pX330-TKsgRNA1 / 2 plasmid were co-transfected into monolayer VERO cells (6-well plate), and plaque screening was performed again to obtain rPRV-delgE / gI / TK ( triple gene deletion recombinant virus). Finally, rPRV-delgE / gI / TK was co-transfected with pX330-US3 sgRNA1 / 2 to obtain rPRV-delgE / gI / TK / US3 (four-gene deletion recombinant virus).

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Abstract

The invention relates to an attenuated pseudorabies virus with a deletion gene, as a vaccine, a vaccine and a construction method, and particularly provides an attenuated pseudorabies virus (PRV), wherein all or part of a Us3 gene of a Us region is deleted. Experiment proves that the virus strain has 100% protein induction for attack of parent PRV virulent viruses on skin and hair animals of dogs,minks, foxes, racoon dogs and the like. The attenuated PRV virus strain can be used as a vaccine for safely and effectively preventing and controlling epidemic situation of pseudorabies (PR) of the skin and hair animals, and has great social value.

Description

technical field [0001] The present invention relates to an attenuated pseudorabies virus, and the use of such virus for treating and / or preventing acute infectious diseases of livestock and wild animals, and also relates to an attenuated pseudorabies virus vaccine. Background technique [0002] Pseudorabies (PR, Pseudorabies) is an acute, highly pathogenic infectious disease caused by pseudorabies virus (PRV, Pseudorabies virus), characterized by fever, itching, encephalomyelitis, respiratory and nervous system disorders. In addition to the natural host pigs, cattle, sheep, dogs, mink, rabbits, badgers, raccoon dogs, polar bears and other domestic and wild animals are susceptible to the pathogen. Since 2010, the PR epidemic has spread to many regions and countries such as Europe, the United States, and my country, and has shown a spreading trend. From 2011 to 2015, it has spread to 22 provinces and cities in my country. The virus may become more virulent or worse. Variations...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/85C12N15/90A61K39/245A61P31/22C12R1/93
CPCC12N7/00C12N15/85C12N15/907A61K39/12A61P31/22C12N2710/16721C12N2710/16762C12N2710/16734C12N2800/107A61K2039/5254A61K2039/552Y02A50/30
Inventor 田进李志杰曲连东
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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