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Attenuated strain freeze-dried vaccine of porcine pseudorabies virus

A technology of porcine pseudorabies virus and porcine pseudorabies, which is applied in the direction of vaccines, viruses, antiviral agents, etc., and can solve the problems of inability to effectively prevent and control infection

Pending Publication Date: 2021-05-11
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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  • Attenuated strain freeze-dried vaccine of porcine pseudorabies virus
  • Attenuated strain freeze-dried vaccine of porcine pseudorabies virus
  • Attenuated strain freeze-dried vaccine of porcine pseudorabies virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Screening and identification of porcine pseudorabies virus

[0022] 1. Screening virus strains

[0023] In November 2018, pigs who had been injected with porcine pseudorabies live 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:

[0024] 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.

[0025] 2. Vir...

Embodiment 2

[0047] Embodiment 2: Construction of porcine pseudorabies virus gE / gI double gene deletion strain

[0048] 1. Construction of TRZYT strain gE / gI double gene deletion strain

[0049] 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.

[0050] 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.

[0051] 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.

[0052] Table 2: Primer Sequence Information T...

Embodiment 3

[0083] Embodiment 3 prepares porcine pseudorabies virus attenuated strain vaccine

[0084] With Vero cells as cultured cells, according to the conventional method for culturing porcine pseudorabies virus, the cells were infected with PRV TRZYT / gE- / gI- strain and PRV QD / gE- / gI- strain respectively, when the cytopathic pathology (CPE) reached Harvest the virus at about 90%, freeze and thaw three times at -80°C, and measure the TCID of the virus 50 . According to the virulence of harvested virus liquid, carry out appropriate dilution, the volume ratio of diluted PRV TRZYT / gE- / gI- virus liquid, PRV QD / gE- / gI- strain virus liquid and protective agent is 1:1:1.5 , and mix thoroughly, 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 PRV TRZYT / gE- / gI- virus and PRV QD / gE- / gI- virus in the vaccine is not less than 10 7 TCID 50 / 0.1ml.

...

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Abstract

The invention provides an attenuated strain freeze-dried vaccine of porcine pseudorabies virus, which comprises antigens and a vaccine freeze-drying protective agent, the antigens are attenuated strain of TRZYT strain and attenuated strain of QD strain. The attenuated strain of TRZYT strain is prepared by carrying out virulence gene deletion on a porcine pseudorabies virus TRZYT strain with the preservation number of CCTCC (China Center For Type Culture Collection) NO.17403 and then carrying out rescue on cells; the attenuated strain of QD strain is prepared by carrying out virulence gene deletion on a porcine pseudorabies virus strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.10266 and then carrying out rescue on cells. The antigen used by the attenuated strain freeze-dried vaccine of porcine pseudorabies virus provided by the invention contains a gene-deleted attenuated strain of porcine pseudorabies virus, which is subjected to continuous passage in a mouse through horizontal transmission infection, the virulence reversion phenomenon is avoided, the heredity is stable, and the standard that the porcine pseudorabies virus with deleted vaccine strain has no virulence reversion is met; and the freeze-dried vaccine provided by the invention can be used to prevent porcine pseudorabies effectively.

Description

technical field [0001] The invention belongs to the technical field of vaccine attenuated strain preparation, in particular to a freeze-dried vaccine of attenuated porcine pseudorabies virus strain. Background technique [0002] Porcine pseudorabies virus (Pseudorabies virus, PRV) belongs to herpesviridae αherpesvirinae porcine herpesvirus type I taxonomically, mainly causing pseudorabies (Pseudorabies, PR) 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 p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/245A61K47/26A61K47/42A61P31/22
CPCA61K39/12A61K47/26A61K47/42A61P31/22A61K2039/525A61K2039/552A61K2039/5254C12N2710/16734A61K2300/00
Inventor 孙伟刘杉杉罗杰杨帆梁浩
Owner TONGREN POLYTECHNIC COLLEGE
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