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Porcine pseudorabies virus double-gene deletion strain

A technology of porcine pseudorabies virus and porcine pseudorabies, applied in the direction of viruses, virus peptides, antiviral agents, etc., can solve the problems of inability to effectively prevent and control infection, and achieve the effect of preventing porcine pseudorabies and hereditary stability

Pending Publication Date: 2021-08-13
TONGREN POLYTECHNIC COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

That is, the existing Bartha-K61 vaccine cannot effectively prevent and control the infection caused by the circulating strain of PRV

Method used

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  • Porcine pseudorabies virus double-gene deletion strain
  • Porcine pseudorabies virus double-gene deletion strain
  • Porcine pseudorabies virus double-gene deletion strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: Screening and identification of porcine pseudorabies virus

[0018] 1. Screening virus strains

[0019] In November 2018, pigs who had been injected with live porcine pseudorabies vaccine (Bartha-K61 strain vaccine) in a certain pig farm in Guizhou developed symptoms of porcine pseudorabies, and porcine pseudorabies virus was screened from the diseased pigs. The specific screening steps are as follows:

[0020] Take the liver and lymph node samples of diseased pigs, homogenize the samples with 0.1M PBS solution with pH 7.2, after repeated freezing and thawing, centrifuge at 5000r / min for 10min, add 10μL penicillin to the supernatant, and let it stand at 37℃. Set aside for 1h, filter through a 0.22μm filter to sterilize. Take 10 μL of the filtrate and add it to Vero cells that have just grown into a monolayer ( figure 1 ), after the lesions appeared, the poison was harvested, and it was placed at -80°C for repeated freeze-thaw detection.

[0021] 2. Vir...

Embodiment 2

[0041] Embodiment 2: Construction of recombinant porcine pseudorabies virus gE / gI double gene deletion strain

[0042] The DNA of the isolated strain is extracted from the isolated porcine pseudorabies virus TRZYT strain, and the virulence genes gE and gI genes of the isolated strain are deleted by means of genetic engineering.

[0043]A series of primers were synthesized with reference to the whole PRV genome sequence (BK001744) to amplify the left arm fragments ( 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.

[0044] 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 expected PCR product sizes are listed in Table 2.

[0045] Table 2: Primer Sequence Information Table

[0046]

[0047] 2. Construction of gE and gI gene tran...

Embodiment 3

[0070] Preparation and Application of Example 3 Porcine Pseudorabies Virus Gene Deletion Vaccine

[0071] Vero cells were cultured according to conventional methods, with PRV / gE - / gI - The virus strain infected the cells, and when the cytopathic pathology (CPE) reached about 90%, the virus was harvested, placed at -80°C for three times, and the TCID of the virus was measured 50 . According to the virulence of harvested virus liquid, carry out appropriate dilution, the volume ratio of the diluted virus liquid and protective agent is 1:1.5, fully mix, wherein the final concentration of sucrose is 20%, and the final concentration of gelatin is 4.8%. Each bottle is filled with 2.5mL and placed in a lyophilizer for lyophilization. The content of porcine pseudorabies virus in the vaccine is 10 6.5 TCID 50 / 0.1ml.

[0072] 1. Finished product inspection

[0073] 1) traits

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Abstract

The invention provides a recombinant porcine pseudorabies virus double-gene deletion strain, which is prepared by performing gE and gI gene deletion on a porcine pseudorabies virus TRZYT strain with the preservation number of CCTCC (China Center For Type Culture Collection) NO.17403. The invention also provides a porcine pseudorabies virus vaccine which is composed of an antigen and a protective agent, and the antigen is the porcine pseudorabies virus gene deletion strain prepared by the invention. The porcine pseudorabies virus gene deletion strain prepared by the invention is a low virulent strain, has no virulence reversion phenomenon after continuous passage in a mouse body through horizontal transmission infection, the heredity is stable, and can meet the standard of no virulence reversion of a porcine pseudorabies virus-deleted vaccine strain. Vaccines prepared by using the porcine pseudorabies virus double-gene deletion strain as an antigen can effectively prevent porcine pseudorabies.

Description

technical field [0001] The invention belongs to the technical field of preparation of attenuated vaccine strains, in particular to a double gene deletion strain of porcine pseudorabies virus. Background technique [0002] Porcine pseudorabies virus (Pseudorabies virus, PRV) belongs to herpesviridae αherpesvirus subfamily porcine herpesvirus type I in taxonomy, and mainly causes pseudorabies (PRV) in pigs. Pigs are the only host of PRV. PRV mainly causes miscarriage, stillbirth and mummification after infecting pregnant sows. Infecting gilts mainly causes infertility. Infecting piglets mainly causes neurological symptoms and death. The mortality rate of porcine pseudorabies is even as high as 100%, which seriously endangers the pig industry in my country. [0003] There is currently no effective drug to treat the disease, and it mainly relies on vaccination for prevention and control. In recent decades, pig farms have mainly relied on the Bartha-K61 vaccine for prevention a...

Claims

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Application Information

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IPC IPC(8): C12N7/01A61K39/245A61P31/22C12R1/93
CPCC12N7/00C07K14/005A61K39/12A61P31/22C12N2710/16721C12N2710/16722C12N2710/16734A61K2039/5254A61K2039/552
Inventor 孙伟刘杉杉高俊波朱锋钊段俊红
Owner TONGREN POLYTECHNIC COLLEGE
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