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Recombinant porcine pseudorabies virus TK/gE/gI three-gene-deleted vaccine

A porcine pseudorabies and virulence gene technology, 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: 2015-08-12
SHANDONG SINDER TECH
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  • Abstract
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  • 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 TK/gE/gI three-gene-deleted vaccine
  • Recombinant porcine pseudorabies virus TK/gE/gI three-gene-deleted vaccine

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Experimental program
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Effect test

Embodiment 1

[0011] Embodiment 1: Breeding of 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 subpackaged and ...

Embodiment 2

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

specific Embodiment approach

[0017] The DNA of the isolated strain was extracted from the isolated porcine pseudorabies virus (CGMCC No.10266), and the virulence genes TK, gE and gI genes of the isolated strain were deleted by genetic engineering, and rescued on the cells Designated as PRV / TK - / 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 and right homology arms of the TK gene, and the homology arms located on both sides of the US7 (gI) gene and US8 (gE) gene (including part of the gI and gE genes). Left arm fragment (L) and right arm fragment (R) available for homologous recombination. In addition, primers were designed to amplify EGFP and EGFP eukaryotic expression cassettes, as well as primers for identifying gene deletions. The primer sequences and expect...

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Abstract

The invention aims at providing a porcine pseudorabies virus vaccine. The porcine pseudorabies virus vaccine comprises an antigen and a protective agent, wherein the antigen contains an attenuated virus strain which is prepared after deleting virulence genes, namely TK, gE and gI, by a porcine pseudorabies virus strain with the collection number of CGMCC No. 10266. The prepared vaccine can effectively prevent porcine pseudorabies; furthermore, because the porcine pseudorabies virus as the antigen is a gene-deleted strain, by continuous passage of horizontally transmitted infections in mouse bodies, no virulence reversion occurs, and the genetic stability is realized, thereby being in line with the standard of having no virulence reversion in the porcine pseudorabies virus deleted vaccine strain; and the prepared vaccine can provide effective immune protection, and has great commercialization development prospects.

Description

technical field [0001] The invention belongs to the technical field of livestock vaccine preparation, in particular to a porcine pseudorabies virus vaccine. 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-K61 vaccine. However, in recent years, the epidemic situation o...

Claims

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Application Information

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Patent Type & Authority Applications(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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