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Recombinant porcine pseudorabies virus gE/gI double-gene-deleted strain and application thereof

A technology of porcine pseudorabies and virulence genes, which is applied in the field of livestock vaccine preparation, can solve the problems of economic losses in immunized pig farms and the inability of vaccines to completely protect PRV, and achieve good commercial development prospects, effective immune protection, and genetically stable effects

Active Publication Date: 2015-09-02
SHANDONG SINDER TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most widely used vaccine in the world is mainly the PRV Bartha-K61 vaccine. However, in recent years, the epidemic situation of porcine pseudorabies in my country is that the existing vaccine cannot completely protect against the attack of the new epidemic PRV, which has caused certain economic losses to the immunized pig farms. loss

Method used

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  • Recombinant porcine pseudorabies virus gE/gI double-gene-deleted strain and application thereof
  • Recombinant porcine pseudorabies virus gE/gI double-gene-deleted strain and application thereof

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Experimental program
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Effect test

Embodiment 1

[0011] Embodiment 1: Breeding of CGMCC No.10266 porcine pseudorabies virus strain

[0012] In recent years, pseudorabies has occurred in many pig farms in my country, most of which are breeding pig farms, and the infected pigs have been injected with pseudorabies vaccine before, and it is speculated that the infected virus has mutated; Screening for porcine pseudorabies virus.

[0013] Obtain samples of internal organs of sick pigs, including: heart, liver, lungs, spleen, tonsils and lymph nodes. Homogenate the visceral sample and PBS (0.1M, pH7.2) at V / V1:5, freeze-thaw 3 times, centrifuge at 3000r / min for 15min, add double antibody to the supernatant, incubate at 37°C for 1h, Sterilize by filtration through a 0.22 μm membrane filter. Take 1ml of the virus filtrate and inoculate the Vero cells grown into a single layer, pass three generations blindly, and observe the cytopathic changes (CPE). The cell culture medium with CPE was plaque-purified, and the purified virus was s...

Embodiment 2

[0016] Embodiment 2: Construction of recombinant porcine pseudorabies virus gE / gI double gene deletion strain

specific Embodiment approach

[0017] Extract the DNA of the isolated strain from the isolated porcine pseudorabies virus (CGMCC No.10266), adopt the method of genetic engineering to carry out the deletion of the virulence gene gE and gl gene to the isolated strain, and name it after the rescue on the cell PRV / gE - / gI - , Toxic, after adding a protective agent as a seed poison. The specific implementation is as follows:

[0018] 1 PCR primer

[0019] Referring to the complete PRV genome sequence (BK001744), a series of primers were synthesized by ourselves, which were used to amplify the left arms that can be used for homologous recombination on both sides of the US7 (gI) gene and US8 (gE) gene (including part of the gI and gE genes). Fragment (L) and right arm fragment (R). Wherein L includes part of the US6 gene and part of the gI gene, and R includes part of the gE gene, all of the US9 gene and part of the US2 gene. In addition, primers were designed to amplify EGFP and EGFP eukaryotic expression c...

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Abstract

The invention aims at providing a porcine pseudorabies virus vaccine. The porcine pseudorabies virus vaccine consists of an antigen and a protective agent, wherein the antigen comprises an attenuated virus strain which is prepared by deleting virulence genes gE and gI of a porcine pseudorabies virus strain with a collection number of CGMCC No.10266. The prepared vaccine provided by the invention can be used for effectively preventing porcine pseudorabies; moreover, porcine pseudorabies virus which is taken as the antigen is a gene-deleted strain, and after the porcine pseudorabies virus is subjected to continuous passage in a mouse body by virtue of horizontal transmission infection, no virulence enhancement phenomenon can be caused, and thus the gene-deleted strain is stable in heredity, and can meet the standard that a porcine pseudorabies virus deleted vaccine strain does not have virulence enhancement; and the prepared vaccine can be used for providing effective immune protection, and has very good commercialized development prospects.

Description

technical field [0001] The invention belongs to the technical field of livestock vaccine preparation, in particular to a recombinant porcine pseudorabies virus gE / gI double gene deletion strain and application thereof. Background technique [0002] Porcine pseudorabies virus PRV (Pseudorabies virus) belongs to herpesviridae a herpesvirinae vesiculovirus genus porcine herpesvirus type I, pigs are the only natural host of the virus, causing pseudorabies (Pseudorabies, PR) in pigs. The disease is mostly outbreaks in pig herds, mainly harming sows, causing abortion or vertical transmission to piglets, resulting in a large number of deaths of newborn piglets, which has brought huge economic losses to the pig industry in my country and even the world. At present, there is no effective drug for the treatment of PR, so vaccination has become the main measure to control the occurrence and prevalence of the disease. The most widely used vaccine in the world is mainly the PRV Bartha-K...

Claims

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Application Information

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IPC IPC(8): C12N7/04A61K35/763A61P31/22
Inventor 李明义刘杉杉孙伟刘阳李晓林赵航李彦凤葛栋李佳琪
Owner SHANDONG SINDER TECH
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