Method for constructing pseudorabies virus gene-deleted attenuated strain and application of attenuated strain

A technology of pseudorabies virus and gene, which is applied in the field of constructing attenuated strains of pseudorabies virus gene deletion, which can solve the problems of inability to provide protection, weak neutralization ability of strains, etc.

Active Publication Date: 2020-09-08
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most effective way to prevent porcine pseudorabies is to vaccinate, but the antibodies produced by the classic Bartha-K61 attenuated vaccine immunization have weak neutralization ability to the current epidemic strains, and can no longer provide efficient protection

Method used

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  • Method for constructing pseudorabies virus gene-deleted attenuated strain and application of attenuated strain
  • Method for constructing pseudorabies virus gene-deleted attenuated strain and application of attenuated strain
  • Method for constructing pseudorabies virus gene-deleted attenuated strain and application of attenuated strain

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Experimental program
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Effect test

Embodiment 1

[0057] Embodiment 1, construct the pseudorabies virus strain (being the recombinant pseudorabies virus of UL13 gene deletion mutation) of UL13 gene deletion

[0058] In this example, CRISPR / Cas9 gene editing technology was used to specifically delete the UL13 gene of the pseudorabies virus Bartha-K61 strain. The specific operation is as follows:

[0059] 1. Construction and identification of UL13 gene knockout vector

[0060] 1.1 The amino acid sequence of the UL13 protein of the pseudorabies virus Bartha-K61 strain is shown in SEQ ID No.1 in the sequence listing. For the gene sequence of the UL13 gene of the pseudorabies virus Bartha-K61 strain, refer to the gene sequence of Bartha-K61 (Suidherpesvirus 1 stain Bartha) in Genbank (Genbank Accession No. JF797217.1, 02-NOV-2011).

[0061] In order to knock out the pseudorabies virus UL13 gene, two segments in the UL13 gene sequence were selected to match the 5'-N 20 -NGG-3' or 5'-CCN-N 20 -The 3' sequence regular fragment is...

Embodiment 2

[0088] Example 2. Effects of UL13 Gene Deletion on Host Natural Immune Response and Replication of Pseudorabies Virus

[0089] 1. The effect of UL13 gene deletion on IFN

[0090] Mouse fibroblasts (Mouse Embryonic Fibroblasts, MEF) at 3.5×10 5 Cells / well were plated in 12 plates at 37°C, 5% CO 2 Culture overnight in the incubator of embodiment 1, the recombinant pseudorabies virus of embodiment 1 UL13 gene knockout and pseudorabies virus Bartha-K61 strain (wild type) are respectively infected with MOI=1 MEF cell, after infection 0h, 12h, 24h RNA samples were collected, reverse-transcribed into cDNA, and the constitutively expressed GAPDH gene was used as an internal reference to normalize the cDNA concentration of the samples. Real-time quantitative PCR was used to detect the expression of type I IFN gene. Wherein, the upstream and downstream primer sequences of PCR detection type I IFN gene are respectively:

[0091] mIFN-F: 5'-ATGAGTGGTGGTTGCAGGC-3';

[0092] mIFN-R: 5'...

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Abstract

The invention provides a method for constructing a pseudorabies virus gene-deleted attenuated strain, a recombinant virus constructed by the method and application. According to the method, a pseudorabies virus gene-deleted attenuated strain is constructed by utilizing a CRISPR/Cas9 gene editing technology, and the pseudorabies virus gene-deleted attenuated strain constructed by the method is subjected to specific deletion mutation at a UL13 gene part. The expression quantity of the I-type IFN gene induced by the obtained pseudorabies virus gene-deleted attenuated strain is remarkably increased, a stronger natural immune reaction can be generated, and the replication of the virus is not remarkably different. Therefore, the UL13 gene deletion mutation can significantly reduce immunosuppression caused by the pseudorabies virus, and has important potential application value.

Description

technical field [0001] The invention relates to a method for constructing a gene-deleted attenuated strain of pseudorabies virus and its application. Background technique [0002] Pseudorabies is an acute infectious disease caused by pseudorabies virus (Pseudorabies virus, PRV) infection, including a variety of livestock and wild animals. Pigs are the natural host and reservoir of the virus. Pseudorabies virus infection can cause abortion, stillbirth in sows, infertility in boars, neurological disorders and death in piglets, and respiratory diseases in fattening pigs. The emergence of mutant strains makes adult pigs also show severe symptoms similar to piglets, which adds new difficulties to the prevention and control of the disease. The most effective way to prevent porcine pseudorabies is to vaccinate, but the antibodies produced by the classic Bartha-K61 attenuated vaccine immunization have weak neutralization ability to the current epidemic strains, and can no longer pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/90C12N15/85A61K39/245A61P31/22C12R1/93
CPCC12N7/00C12N15/907C12N15/85C12N9/12C12Y207/11A61K39/12A61P31/22C12N2710/16721C12N2710/16751C12N2710/16734A61K2039/552A61K2039/5254C12N2800/107Y02A50/30
Inventor 商营利孔正杰
Owner SHANDONG AGRICULTURAL UNIVERSITY
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