Porcine pseudorabies variant virus strain and application thereof

A porcine pseudorabies virus strain technology, applied in the field of virus molecular biology and genetic engineering, can solve respiratory diseases, occasionally death, laboratory confirmed PRV wild virus infection, economic loss, breathing difficulties and other problems

Pending Publication Date: 2021-10-15
JIANGXI ZHENGBANG TECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PRV currently has only one serotype, but the biological characteristics and virulence of different strains are different. Since 2011, new PRV mutant strains have emerged in Northeast, East and South my country, causing serious damage to pig farms immunized with PRV classic vaccines. The economic loss is manifested as abortion, stillbirth and mummified fetuses in sows, central nervous symptoms, dyspnea, high mortality in piglets, respiratory diseases in growing and finishing pigs, and occasionally death, laboratory confirmed as wild PRV infection, epidemic The results of the scientific survey showed that the virulence genes gI and gE of the PRV mutant strains had multiple site insertions or mutations, and the main antigen genes gB, gC and gD also had multiple site insertions, deletions and point mutations, resulting in and antibody mutations; immune challenge protection test studies show that the traditional Bartha K61 strain vaccine can provide good immune protection against classic strains of PRV (such as Ea strain), but can only provide partial protection against mutant strains of popular viruses , which is consistent with the repeated occurrence of RPV infection in pigs immunized with Bartha K61 vaccine clinically

Method used

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  • Porcine pseudorabies variant virus strain and application thereof
  • Porcine pseudorabies variant virus strain and application thereof
  • Porcine pseudorabies variant virus strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Isolation and Identification of Pseudorabies Epidemic Variants and Virulent Strains

[0039] The aborted fetuses from a pig farm in Shandong were tested for porcine pseudorabies field virus antigen, and the result was positive for gE antigen. The supernatant of the positive disease material was inoculated into VERO cells for virus isolation, and a strain was isolated. Carry out gene identification and specific identification, it is found that the strain can be amplified to the corresponding PRV gene (NO.1~6), and the strain can be neutralized by pseudorabies virus-specific positive serum, identified as PRV virus, named It is PRV SD2020 strain.

[0040] PRV SD2020 strain induced VERO cell pathology such as figure 1 shown. figure 1 -a, figure 1 -b is the situation after 24h and 48h of inoculation with the PRV SD2020 strain, respectively, by figure 1 It can be seen that the PRV SD2020 strain specifically causes typical regular lesions (CPE) in infected cells...

Embodiment 2

[0045] Embodiment 2: Construction of porcine pseudorabies virus epidemic variant virulent strain genetically engineered strain (PRV SD2020ΔgI / gE strain)

[0046] Using CRISPR / CAS9 technology to knock out the gI / gE dual virulence gene on PRV, the specific process is outlined as follows:

[0047] 1. sgRNA sequence design and recombinant vector construction

[0048] Referring to the complete PRV genome sequence published in GenBank, the sgRNA sequences of gI and gE genes were designed and synthesized. The sgRNA sequences of gI and gE genes are shown in Table 2, and inserted into the BbsI restriction site of the psgRNA vector to construct the targeting plasmid.

[0049] Table 2 Design gI / gE sgRNA sequence

[0050] The PCR amplification product was identified by electrophoresis, and the electrophoresis results were as follows: figure 2 shown.

[0051] After the PRV SD2020ΔgI / gE strain was obtained, the gene sequence upstream and downstream of the gene deletion region was obtain...

Embodiment 3

[0054] Embodiment 3: Porcine pseudorabies virus epidemic variant virulent strain genetically engineered strain (PRV SD2020ΔgI / gE / TK strain) construction

[0055] Using CRISPR / CAS9 technology to knock out the gI / gE / TK three virulence genes on PRV, the specific process is outlined as follows:

[0056] Similarly, with reference to the process of Example 2, the TK gene target site was further designed (see Table 3 for the TK gene sgRNA sequence), the recombinant vector was constructed, transfected, and the plasmid and the viral genome were homologously recombined, and then purified by plaque to obtain The PRV SD2020ΔgI / gE / TK strain lacking gI, gE, and TK genes was identified.

[0057] Table 3 TK gene sgRNA sequence

[0058]

[0059] The results of PCR identification of the virus strains were as follows: Figure 5 As shown, the sequenced TK sequence is shown in SEQ ID NO.8, the electropherogram file

[0060]

[0061] 2. Transfection

[0062] The targeting plasmid was tran...

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Abstract

The invention provides a porcine pseudorabies variant virus strain and application thereof, and belongs to the technical field of virus molecular biology and genetic engineering. A virus separated from a PRV (Porcine Reproductive Virus) infected body in a pig farm is confirmed to belong to a PRV virulent virus through PCR (Polymerase Chain Reaction) identification, sequencing and virulence test, the virus is named as a PRV SD2020 strain, the strain is taken as a parent, a gI-gE gene and a TK gene of the strain are knocked out by utilizing a gene editing technology (CRISPR/CAS9 system), and a double-gene lost strain PRV SD2020 delta gI-gE and a three-gene lost strain PRV SD2020 delta gI-gE-TK are constructed. When the two gene deletion strains are used for preparing an inactivated vaccine and a live vaccine, an immune challenge protection test result shows that the prepared vaccine has good safety and immunogenicity, and shows that the two strains are suitable for preventing, controlling and purifying pseudorabies diseases.

Description

technical field [0001] The application belongs to the technical field of virus molecular biology and genetic engineering, and more specifically relates to a porcine pseudorabies mutant virus strain and its application. Background technique [0002] Pseudorabies is an important infectious disease of pigs caused by pseudorabies virus (hereinafter referred to as PRV), which is characterized by itching, fever and neurological symptoms of encephalomyelitis. It is currently one of the most serious infectious diseases that endanger the pig industry. It is widely popular in the world and causes huge economic losses to the world's pig industry every year. Because the virus can infect a variety of domestic animals (pigs, cattle, sheep, goats, dogs, rabbits, horses, etc.) and wild animals (foxes, wolves, rats, bats, deer, bears, wild boars, etc.) Natural foci of disease. Because pigs are the only natural carriers, some large pigs are latently infected for a long time and shed the vir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/38A61K39/245A61P31/22
CPCC12N7/00C07K14/005A61K39/12A61P31/22C12N2710/16721C12N2710/16722C12N2710/16734A61K2039/552
Inventor 陈义锋王闯林峰陈智韡杨宏富刘阳阳刘志昌
Owner JIANGXI ZHENGBANG TECHNOLOGY CO LTD
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