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Construction of pseudorabies virus double fluorescent marker 5 gene deletion strain

A pseudorabies virus and virus strain technology, applied in the fields of biotechnology and animal virology, can solve the problems of purulent reactive tissue, pneumonia, secondary bacterial infection, etc.

Active Publication Date: 2019-01-25
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, in the herpes virus genome, the envelope protein gN encoded by UL49.5 can inhibit the TAP-mediated polypeptide transport to the endoplasmic reticulum, block the assembly of MHC class I complex in virus-infected cells, and down-regulate MHC class I The expression of molecules on the cell surface makes it possible for viruses to evade host CD8 + Recognition of T cells and replication in the epithelial cells of the host nasal cavity and upper respiratory tract, causing purulent reactions or tissue ulceration, leading to bacterial secondary infection and severe pneumonia

Method used

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  • Construction of pseudorabies virus double fluorescent marker 5 gene deletion strain
  • Construction of pseudorabies virus double fluorescent marker 5 gene deletion strain
  • Construction of pseudorabies virus double fluorescent marker 5 gene deletion strain

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Embodiment Construction

[0022] 1. Carrier

[0023] The pMD-18T vector, used for cloning, sequencing and construction of transfer vectors, was purchased from Bao Biological Engineering (Dalian) Co., Ltd. The vector pU-EGFP containing the GFP gene and the vector pU-ERFP containing the RFP gene are both kept by the laboratory of the patent applicant.

[0024] 2. Primer Design

[0025] The following primers were designed and synthesized according to the PRV sequence published on GenBank:

[0026] (1) Amplify the left homology arm of gI gene:

[0027] US6F: 5'-GACTTAAGGATCTCCGACCCGCAGGTGG-3' (SEQ ID NO: 1)

[0028] US6R: 5'-GACTTAAGGGAGCCGGGTCACGTCGCGCG-3' (SEQ ID NO: 2)

[0029] (2) Right homology arm of US9 gene:

[0030] US2F: 5'-GACTAGTATGGGGGTGACGGCCATCAC-3' (SEQ ID NO: 3)

[0031] US2R: 5'-GACTAGTCGGAGAGATCCTGCCGTCTAGG-3' (SEQ ID NO: 4)

[0032] (3) GFP gene:

[0033] EGFP-F: 5'-GGTATACGGATCCAAGGAGATATAACA-3' (SEQ ID NO: 5)

[0034] EGFP-R: 5'-GACTAGTGAGCTCTTAAAGCTCATCAT-3' (SEQ ID NO: 6) ...

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Abstract

The invention relates to establishment of a pseudorabies virus double fluorescence labeled 5 gene deletion strain. The virus strain is characterized in that a self separated pseudorabies virus strain (SX strain) serves as a female parent, through a molecular biology means, a gE gene, a gI gene, a US9 gene, a UL49.5 partial gene and a TK gene of the pseudorabies virus are deleted, and a green fluorescent protein (GFP) and a red fluorescence protein (RFP) serve as labeled proteins to replace the deleted genes, and a double fluorescence labeled 5 gene deletion virus strain (rPRV / gE- / gI- / US9- / delta UL49.5 / TK- / GFP+ / RFP+) is obtained. The invention establishes a pseudorabies virus carrier system; the double fluorescence protein labels are convenient for future screening and authenticating of gene engineering; multiple exogenous genes can be inserted at the same time, and greater efficiency is obtained compared with a common live carrier system. The double fluorescence labeled deleted virus is successfully established as a live carrier, and lays a foundation for research on recombined pseudorabies virus gene engineering vaccine.

Description

technical field [0001] The invention relates to the construction of a strain of pseudorabies virus (or called pseudorabies virus) lacking 5 genes (gE, gl, US9, UL49.5, TK) with double fluorescent markers, and belongs to the fields of biotechnology and animal virology. Background technique [0002] Pseudorabies (PR; also known as Aujeszky’s disease, AD) is an acute infectious disease caused by pseudorabies virus (PRV), which can infect a variety of domestic animals, companion animals and wild animals. Pigs are the only species that can survive infection and are the virus reservoir. The main manifestations of infected pigs are neurological symptoms, salivation, dyspnea, reproductive failure of sows, high mortality of piglets, etc., causing huge economic losses to the pig industry. [0003] PRV is a member of the Herpesviridae, Alphaherpesvirinae, and Varicellavirus genus. The PRV genome is a linear double-stranded DNA, about 145Kb, composed of a long unique region (UL) and a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/38A61K39/245A61P31/22C12N15/869
Inventor 李俊平杨承槐陈晓春李慧姣孙莹张广川李启红曹明慧李岭
Owner CHINA INST OF VETERINARY DRUG CONTROL