A kind of recombinant porcine pseudorabies virus ge/gi double gene deletion strain and its application

A porcine pseudorabies and virulence gene technology, applied in the field of livestock vaccine preparation, can solve the problems such as the inability of vaccines to fully protect PRV and the economic loss of immune pig farms, and achieve the effects of good commercial development prospects, effective immune protection, and stable heredity

Active Publication Date: 2018-01-02
SHANDONG SINDER TECH
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  • Abstract
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  • Claims
  • Application Information

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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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  • A kind of recombinant porcine pseudorabies virus ge/gi double gene deletion strain and its application
  • A kind of recombinant porcine pseudorabies virus ge/gi double gene deletion strain and its application

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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 and 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 w...

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 purpose of the present invention is to provide a porcine pseudorabies virus vaccine, which is composed of an antigen and a protective agent, wherein the antigen is made by deleting the virulence genes gE and gl from the porcine pseudorabies virus strain with the preservation number CGMCC No.10266 attenuated virus strains. The vaccine prepared by the invention can effectively prevent porcine pseudorabies, and the porcine pseudorabies virus as an antigen is a gene-deleted strain, which is continuously passed down in mice through horizontal transmission and infection, and there is no phenomenon of virulence returning to strength, and the heredity is stable. The porcine pseudorabies virus deletion vaccine strain has no standard of strong virulence, and the prepared vaccine can provide effective immune protection, and has a good prospect for commercial development.

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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Patent Type & Authority Patents(China)
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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