Porcine pseudorabies virus virulent strain, and gene deletion vaccine strain thereof and applications thereof

A gene deletion vaccine, porcine pseudorabies technology, applied in the direction of antiviral agents, viruses/phages, microorganism-based methods, etc., can solve the problem that the new virus cannot be completely resisted, the newly isolated virus cannot be effectively neutralized, and the immunized pig cannot effectively resist Virus and other problems, to achieve the effect of facilitating differential diagnosis, high protection efficiency and good safety

Active Publication Date: 2013-03-27
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the current situation is that the pig farms affected by PR have been immunized with the vaccine according to the routine procedures. Is there an antigenic variation in the new epidemic strain that makes the immunized pigs unable to effectively resist the virus? We used the serum neutralization test and sheep immunization test to confirm that the neutralizing antibody produced by Barthak61 immunized pigs could not effectively neutralize the newly isolated virus, and the sheep immunized with Barthak61 could not completely resist the attack of the new virus

Method used

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  • Porcine pseudorabies virus virulent strain, and gene deletion vaccine strain thereof and applications thereof
  • Porcine pseudorabies virus virulent strain, and gene deletion vaccine strain thereof and applications thereof
  • Porcine pseudorabies virus virulent strain, and gene deletion vaccine strain thereof and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Isolation and identification of porcine pseudorabies virus virulent strain (PRVHeN1 strain)

[0034] The brain tissue of diseased pigs was collected from a pig farm suspected of PRV infection in Henan Province, my country, and the tissue genomic DNA was extracted, and PCR identification was carried out using PRVgE-specific primers ( figure 1 ), the positive brain tissue homogenate was centrifuged, filtered and the filtrate was frozen for later use. After Vero cells were cultured at 37°C for 48 hours to form a monolayer, each bottle was inoculated with 1ml of the supernatant of the filtered disease material suspension, and after adsorption for 1 hour, the DMEM culture medium containing 2% fetal bovine serum was changed to continue culturing at 37°C. 3 to 4 days, whether there is cytopathy ( figure 2 ). When 60-70% of the cells have CPE changes, the virus is collected, and the virus strains with lesions are passed on for 2 to 3 generations, and then PCR ampli...

Embodiment 2

[0035] Example 2 There are antigenic differences between the PRV HeN1 strain and the vaccine strain Barthak61

[0036] 1) Microneutralization test and sheep immune challenge test were used to confirm the antigenic difference between the newly isolated strain (PRVHeN1 strain) and the vaccine strain Barthak61. Neutralization test operation method: 12 40-day-old SPF pigs, 5 of them intramuscularly injected 1mL containing 10 7.0 TCID 50 The inactivated PRVHeN1 strain, intramuscularly inject 1mL containing 10 7.0 TCID 50 The other 2 heads were injected with DMEM culture medium as a control, and blood was collected every week before and after inoculation to separate serum, and they were killed 5 weeks after inoculation. The neutralizing potency of the serum from pigs inoculated with Barthak61 and HeN1 against two strains of viruses (Barthak61 and HeN1) was determined by the fixed virus dilution serum method. The virus volume is 100TCID 50 / well, the serum was collected at diffe...

Embodiment 3

[0040] Example 3 Gene Deletion Vaccine Strain rPRV-gE - -EGFP + build

[0041] The transfer vector plasmid pUCUsAB-EGFP was constructed by gene cloning technology using part of the gI and Us2 genes of the PRVHeN1 strain as homology arms, and the green fluorescent protein gene (EGFP) as a screening marker (recombination sites such as Figure 5 As shown, the enzyme digestion of the transfer vector was identified as Image 6 ), the PRVHeN1 genome and the transfer vector were co-transfected into Vero cells, according to the instructions of the calcium phosphate transfection kit, the Vero cells were pre-cultured in a 60mm dish to form a monolayer, and the fresh culture medium was replaced 3 hours before transfection, Mix 4 μg viral genomic DNA and 10 μg transfer vector pUCUsAB-EGFP, then add 2MCaCl 2 , so that the final concentration of 0.24M, mixed evenly, this DNA-CaCl 2 The mixture was then added to a new tube containing an equal volume of 2XHBS, mixed well, and allowed to r...

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Abstract

The invention discloses a porcine pseudorabies virus virulent strain, and a gene deletion vaccine strain thereof and applications thereof. The porcine pseudorabies virus virulent strain is named as HeN1, the microbial preservation number of the porcine pseudorabies virus virulent strain is CGMCC NO.6656, the deleted gE gene obtaines the gene deletion vaccine strain rPRV-gE-EGFP+ on the basis of the virulent strain HeN1, and the microbial preservation number is CGMCC NO.6657. The virulent strain can be prepared into inactivated vaccine (single vaccine or combined vaccine), the gene deletion vaccine strain rPRV-gE-EGFP+ can be prepared into activated vaccine or inactivated vaccine (single vaccine or combined vaccine) and the like, so that porcine pseudorabies can be effectively prevented or cured, or the gene deletion vaccine strain rPRV-gE-EGFP+ can be prepared into a diagnosis reagent for diagnosing the porcine pseudorabies. The gene deletion vaccine strain rPRV-gE-EGFP+ has the advantages of being good in safety, high in protection efficiency, beneficial to differential diagnosis.

Description

technical field [0001] The present invention relates to a virulent virus strain and a gene-deleted vaccine strain obtained on the basis thereof, in particular to a virulent strain of porcine pseudorabies virus HeN1 and a gene-deleted vaccine strain rPRV obtained by deleting the gE gene of the virulent strain HeN1 -gE - -EGFP + , the present invention also relates to HeN1 and vaccine strain rPRV-gE - -EGFP + The application in the prevention or treatment of porcine pseudorabies belongs to the field of biomedicine. Background technique [0002] Pseudorabies (Pseudorabies, PR also known as Aujeszky'sdisease, AD) is an acute infectious disease caused by a variety of domestic animals, wild animals, companion animals and experimental animals caused by pseudorabies virus (Pseudorabies Virus, PRV). Under natural conditions, the disease is most common in pigs, cattle, sheep, dogs, cats and rodents. Pseudorabies virus has a lethal course in all susceptible animals except pigs. F...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/01A61K39/245A61P31/20G01N33/569C12R1/93
Inventor 田志军彭金美安同庆
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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