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A Pseudorabies virus qd strain weakened by three gene deletions

A porcine pseudorabies virus and gene deletion technology, which is applied in the directions of viruses, antiviral agents, viruses/phages, etc., can solve the problems of unsuitability for rapid operation, high threshold for artificial genome construction and difficulty in obtaining, etc., and achieves good commercial development prospects and effective The effect of immune protection

Active Publication Date: 2022-05-24
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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  • A Pseudorabies virus qd strain weakened by three gene deletions
  • A Pseudorabies virus qd strain weakened by three gene deletions
  • A Pseudorabies virus qd strain weakened by three gene deletions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0025] 1. Construction of recombinant vector

[0026] Taking PRV HLJ8 strain (Genebank: KT824771) as a reference, the complete US7(gI) gene, complete US8(gE) gene and US9(11K) gene were designed to delete the short unique region (Unique Short, US). The 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 for primers

[0028]

[0029]

[0030] Use 5'arm F / 5'arm R and 3'arm F / 3'arm R primers to establish a 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 min; 98°C for 10s, 55°C for 5s, 72°C for 1 kb / min, amplification for 30 cycles. The amplified products were the 5' end arm fr...

Embodiment 2

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

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

[0045] PRV QD strain and PRV QD-gE- / gI- strain were respectively inoculated with monolayer PK-15 cells at an MOI ratio of 0.1, and placed in an incubator to continue culturing. The virus solution was collected every 12h to measure TCID50, and the growth curve was drawn. The PRV QD-gE- / gI- strain was serially passaged to 15 passages, and the recombination region was amplified using RetestF2 / RetestR primers and identified by sequencing.

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

[0047] Sixty-five 6-week-old Balb / c female mice were divided into groups of 5 and randomly assigned to 13 groups. PRV QD strain and PRV QD-gE- / gI- cell supernatant were diluted to 10-6 times by 10-fold gradient dilution with normal saline. The mice were subcutaneously injected with 0.2 mL of each dilution virus soluti...

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Abstract

The invention provides a kind of porcine pseudorabies virus QD strain gE ‑ / gI ‑ / 11K ‑ The three-gene deletion weakened strain is produced by deleting the gE, gI and 11K genes of the porcine pseudorabies virus strain. The porcine pseudorabies virus strain is the porcine pseudorabies virus strain PRV-QD strain, and its preservation number is CGMCC No.10266. The vaccine strain prepared by the invention can effectively prevent porcine pseudorabies, and the porcine pseudorabies virus as an antigen is a gene-deleted strain, which can be continuously passed down in mice through horizontal transmission and infection, and there is no phenomenon of virulence returning to strength, and the heredity is stable. The vaccine conforms to the standard of no virulence reversion of the porcine pseudorabies virus deletion vaccine strain, and the 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, and in particular relates to a pseudorabies virus QD strain gE - / gI - / 11K - Three gene deletion attenuated strains. Background technique [0002] Porcine pseudorabies is an acute infectious disease caused by porcine pseudorabies virus (Pseudorabies Virus, PRV). PRV is a subfamily of alphaherpesviruses of the family Herpesviridae. The main infecting objects are pigs, and they can also infect cattle, sheep, dogs and other livestock and some wild animals. Pigs are the main source of PRV infection, mainly manifested as reproductive failure of sows and piglet mortality. 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 pig industry. Currently, there is no specific drug to treat pseudorabies, and vaccines are the main method to prevent the disease. The most widely used PRV vaccine strain today is Bartha-K61, and ...

Claims

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Application Information

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Patent Type & Authority Patents(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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