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Porcine pseudorabies double gene deletion mutant virus strain and establishment method thereof

A porcine pseudorabies and mutant virus technology, applied in the field of viruses, can solve the problem of no high-efficiency and sensitive vaccines, and achieve good immunogenicity

Active Publication Date: 2019-09-20
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention proposes a kind of porcine pseudorabies double-gene deletion mutant virus strain and its construction method, which solves the problem that the existing PRV is prevalent and has no high-efficiency and sensitivity vaccine

Method used

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  • Porcine pseudorabies double gene deletion mutant virus strain and establishment method thereof
  • Porcine pseudorabies double gene deletion mutant virus strain and establishment method thereof
  • Porcine pseudorabies double gene deletion mutant virus strain and establishment method thereof

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: the construction of transfer plasmid pUC-gE / gILR, pUC-gE / gILRE and knockout carrier CRISPR / Cas9-gRNA-EGFP

[0037] 1. The microbial materials used in this application

[0038] The PRV / HN2012 strain was isolated, identified and preserved by Zhengzhou Key Laboratory of Swine Major Disease Prevention and Control; pig testis (ST) cells were purchased from China Veterinary Drug Control Institute and frozen by Zhengzhou Key Laboratory of Swine Major Disease Prevention and Control; Competent Cell DH5α was purchased from Kangwei Century Biotechnology Biotechnology Co., Ltd.; PRV universal transfer plasmid pG and CRISPR / Cas9 knockout vector Px459 / EZ were constructed and preserved by Zhengzhou Key Laboratory of Swine Major Disease Prevention and Control.

[0039] Second, the construction steps of the carrier

[0040] 2.1 Proliferation and culture of virus: Inoculate ST cells in good growth state on the bottom area of ​​25cm 2 Add an appropriate amount of DMEM cult...

Embodiment 2

[0084] Embodiment 2: recombinant porcine pseudorabies virus rPRV HN2012-gE - / gI - rescue and purification

[0085] 1. Microbial materials

[0086] rPRV HN2012-gE - / gI - -EGFP + The strains were isolated, identified and preserved by Zhengzhou Key Laboratory of Swine Major Disease Prevention and Control; pig testis (ST) cells were purchased from China Veterinary Drug Control Institute and frozen by Zhengzhou Key Laboratory of Swine Major Disease Prevention and Control; competent cells DH5α were purchased Zikangwei Century Biotechnology Biotechnology Co., Ltd.; CRISPR / Cas9-PX459-gRNA1-EGFP-EZ-gRNA2-EGFP, transfer plasmids pUC-gE / gILR and pUC-gE / gILRE are all controlled by the Zhengzhou Major Pig Disease Prevention and Control Center The lab builds and saves.

[0087] 2. rPRV HN2012-gE - / gI - Construction of Double Gene Deletion Strains

[0088] 2.1 Preparation of transfected cells: trypsinized ST cells with good shape and strong growth vigor were dispersed into single...

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Abstract

The invention relates to the field of viruses, in particular to a porcine pseudorabies double gene deletion mutant virus strain and an establishment method thereof. The virus strain is a gE, gI double gene deletion strain, the preservation number is CCTCC NO. V201749, preservation unit is: China's typical culture collection center, the preservation address is: Wuhan University, Wuhan, China, the postcode is 430072. rPRV HN2012-gE- / gI-immunized piglets established do not have any adverse reactions during the experiment, the neutralizing antibody level verifies that the deletion virus maintains good immunogenicity, the obtained rPRV HN2012-gE- / gI-double gene deletion virus is safe for the piglets, PRV (pseudorabies virus) specific antibody can be produced effectively and rapidly after immunization, and the porcine pseudorabies double gene deletion mutant virus strain is a vaccine strain which is expected to be used in the prevention and control of PRV epidemic.

Description

technical field [0001] The invention relates to the field of viruses, in particular to a porcine pseudorabies double-gene deletion mutant virus strain and a construction method thereof. Background technique [0002] Pseudorabies (PR), that is, Aujeszky’s disease, its pathogen is pseudorabies virus (Pseudorabies virus, PRV), cattle, sheep, pigs and other livestock and wild animals can suffer from the disease [i] . The main symptoms of PR are fever, severe itching (except for pigs), encephalomyelitis, nervous and reproductive system disorders, and it is an acute febrile highly contagious infectious disease [ii] . Pigs are the natural host and main source of infection of the disease. PRV can infect pigs of different ages, mainly causing abortion, stillbirth and mummification in pregnant sows; neurological symptoms, exhaustion and death in suckling piglets, with a mortality rate of up to 100%. So far, no drug that can successfully treat PR has been developed, so inoculation o...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/11C12N15/113C12N15/63C12N15/65C12N15/90C12R1/93
CPCC07K14/005C12N7/00C12N15/113C12N15/63C12N15/65C12N15/907C12N2310/10C12N2710/16721C12N2710/16722C12N2310/20
Inventor 陈红英郑兰兰赵宇张宇郑慧华刘芳
Owner HENAN AGRICULTURAL UNIVERSITY
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