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Attenuated pseudorabies virus QD strain by three-gene deletion

A porcine pseudorabies virus and gene deletion technology, which is applied in the direction of viruses, antiviral agents, and virus antigen components, can solve the problems of high threshold for artificial genome construction, unsuitable for rapid operation, etc., and achieve good commercial development prospects and effective The effect of immune protection

Active Publication Date: 2021-03-16
SHANDONG SINDER TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the rapid growth of bacteria, it is easy to screen and greatly increase the efficiency of later operations, but the construction threshold of artificial genomes is high and difficult to obtain, so it is not suitable for rapid operations

Method used

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  • Attenuated pseudorabies virus QD strain by three-gene deletion
  • Attenuated pseudorabies virus QD strain by three-gene deletion
  • Attenuated pseudorabies virus QD strain by three-gene deletion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Construction of three gene-deleted strains of gE, gI and 11K of PRV-QD

[0025] 1. Construction of recombinant vector

[0026] Taking the PRV HLJ8 strain (Genebank: KT824771) as a reference, the complete US7 (gI) gene, the complete US8 (gE) gene and the US9 (11K) gene were designed to delete the Unique Short (US) gene. Primers were designed to amplify the homology arms on both sides of the deleted gene, and the primers are shown in Table 1.

[0027] Table 1: Sequence Information Table of Primers

[0028]

[0029]

[0030] Use 5'arm F / 5'arm R and 3'arm F / 3'arm R primers to establish 50μL amplification system: 2×PrimeSTAR HS GC Buffer 25μL; dNTP (2.5mM each) 4μL; Forward primer 1μL; Reserveprimer 1μL ; PRV genomic DNA 1 μL; PrimeSTAR HS 0.5 μL; deionized water 17.5 μL. Reaction conditions: pre-denaturation at 98°C for 3 minutes; 10s at 98°C, 5s at 55°C, 1kb / min at 72°C, 30 cycles of amplification. The amplified products are the 5' arm fragment and the...

Embodiment 2

[0043] Example 2: Immunological activity of PRV-QD gE- / gI- / 11K-strain

[0044] 1. Determination of stability and growth characteristics of PRV-QD gE- / gI- / 11K-strains

[0045] PRV QD strain and PRV QD-gE- / gI- strain were respectively inoculated with monolayer PK-15 cells at the ratio of MOI 0.1, and placed in an incubator to continue culturing. The virus liquid was collected every 12 hours to measure TCID50, and the growth curve was drawn. The PRV QD-gE- / gI-strain was continuously passaged to 15 passages, and the recombined region was amplified using RetestF2 / RetestR primers and sequenced for identification.

[0046] 2. Half infection dose of PRV-QD gE- / gI- / 11K- mice

[0047] Sixty-five 6-week-old Balb / c female mice were divided into 13 groups randomly. PRV QD strain and PRV QD-gE- / gI- cell supernatant were diluted to 10-6 times with 10 times of normal saline. Mice were subcutaneously injected with 0.2 mL of virus solution of each dilution, and the negative control group wa...

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Abstract

The invention provides a porcine attenuated pseudorabies virus QD strain by gE- / gI- / 11K-three-gene deletion, which is prepared by deleting gE, gI and 11K genes of a porcine pseudorabies virus strain.The porcine pseudorabies virus strain is a porcine pseudorabies virus PRV-QD strain, and the preservation number is CGMCC No. 10266. The vaccine strain prepared by the invention can effectively prevent porcine pseudorabies, the porcine pseudorabies virus serving as an antigen is a gene deletion strain, when continuously passaged in mice through horizontal propagation infection, a virulence enhancement phenomenon is not observed, the gene deletion strain is stable in hereditary, and conforms to the non-virulence enhancement standard of the porcine pseudorabies virus deletion vaccine strain, andthe prepared vaccine can provide effective immune protection and has a good commercial development prospect.

Description

technical field [0001] The invention belongs to the technical field of vaccine preparation, in particular to a pseudorabies virus QD strain gE - / gI - / 11K - Deletion of three genes weakens the strain. Background technique [0002] Porcine pseudorabies is an acute infectious disease caused by porcine pseudorabies virus (Pseudorabies Virus, PRV). PRV belongs to the alpha herpesvirus subfamily of the family Herpesviridae. It mainly infects pigs and can also infect cattle, sheep, dogs and other domestic animals and some wild animals. Pigs are the main source of PRV infection. The main manifestations are reproductive disorders in sows and death of piglets. The mortality rate of piglets within 15 days of age is as high as 100%. The spread of PRV has brought huge losses to the swine industry. At present, there is no effective drug to treat pseudorabies, and vaccine is the main method for preventing this disease. The most widely used PRV vaccine strain today is Bartha-K61, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/245A61P31/22C12R1/93
CPCC12N7/00A61K39/12A61P31/22C12N2710/16721C12N2710/16734A61K2039/5254A61K2039/552
Inventor 于泽坤单学强只勇张伦李思菲段笑笑栾志舫杨海明杨彦超郝光恩马广斌赵炳光朱焕星
Owner SHANDONG SINDER TECH
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