Construction of recombinant pseudorabies virus vector expressing foreign protein and preparation method of recombinant pseudorabies virus

A pseudorabies virus, rabies virus technology, applied in virus/phage, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of time-consuming, labor-intensive success rate, etc.

Active Publication Date: 2020-12-15
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this cloning process is time-consuming, laborious and less successful for viruses with large genes, such as herpes viruses

Method used

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  • Construction of recombinant pseudorabies virus vector expressing foreign protein and preparation method of recombinant pseudorabies virus
  • Construction of recombinant pseudorabies virus vector expressing foreign protein and preparation method of recombinant pseudorabies virus
  • Construction of recombinant pseudorabies virus vector expressing foreign protein and preparation method of recombinant pseudorabies virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, the construction of the recombinant pseudorabies virus strain of pseudorabies virus thymidine kinase TK gene insertion mutation and expression of natural green fluorescent protein

[0078] In this example, the CRISPR / Cas9 gene editing system was used to specifically insert the TK gene of the pseudorabies virus Bartha-K61 strain and introduce the EGFP gene. Schematic diagram of gene editing of pseudorabies virus vector figure 1 , the specific operation is as follows:

[0079] 1. Construction and identification of TK gene knockout plasmid

[0080] 1.1 The amino acid sequence of the TK protein of the pseudorabies virus Bartha-K61 strain is shown in SEQ ID No.1 in the sequence listing. The gene sequence of the TK gene of the pseudorabies virus Bartha-K61 strain refers to the gene sequence of Bartha-K61 (Suidherpesvirus 1stain Bartha) in Genbank (Genbank Accession No. JF797217.1, 02-NOV-2011).

[0081] In order to knock out the TK gene, two segments in the T...

Embodiment 2

[0118] Embodiment 2, the construction of the recombinant pseudorabies virus strain of pseudorabies virus thymidine kinase TK gene insertion mutation and expression of foreign genes

[0119] This example uses the CRISPR / Cas9 gene editing system to specifically insert the EGFP gene of the monoclonal pseudorabies virus Bartha-K61 strain (Bartha-K61-ΔTK-EGFP) obtained in Example 1 for insertion mutation and expression of EGFP mutation. The specific operation is as follows:

[0120] 1. Construction and identification of EGFP gene knockout plasmid

[0121]1.1 In order to knock out the EGFP gene of the Bartha-K61-ΔTK-EGFP strain, two segments located in the TK gene sequence were selected in line with the 5'-N 20 -NGG-3' or 5'-CCN-N 20 -The 3' sequence regular fragment is the target sequence, N represents any one of A, G, C and T, N 20 Indicates 20 consecutive deoxyribonucleotides. The two target sequences are designated as target sequence 3 and target sequence 4, respectively. ...

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Abstract

The invention provides a method for constructing pseudorabies virus replication non-essential gene insertion mutation by utilizing a CRISPR/Cas9 gene editing system and obtaining a recombinant pseudorabies virus expressing a foreign gene. After the CRISPR/Cas9 gene editing system is introduced into a cell, a viral genome is identified through a pre-screened target sequence, and a pseudorabies virus gene infecting the cell is edited and recombined. The recombinant pseudorabies virus constructed by the method is inserted into a target foreign gene at a specific gene part without affecting replication of the virus, and the recombinant virus is screened forwards or backwards by utilizing a marker in the construction process, so that the obtaining efficiency of the recombinant virus is remarkably improved, and a foundation is laid for constructing a recombinant pseudorabies virus vaccine expressing the foreign gene. The method for constructing the recombinant pseudorabies virus based on theCRISPR/Cas9 gene editing system can be used for quickly constructing a recombinant virus live vector vaccine and has important application value.

Description

technical field [0001] The invention relates to a method for constructing a recombinant pseudorabies virus vector expressing foreign proteins and preparing the recombinant pseudorabies virus. Background technique [0002] Pseudorabies virus (PRV) belongs to the subfamily Alphaherpesvirinae of the family Herpesviridae. The viral genome is double-stranded DNA with a total length of about 150kb, encoding about 70 proteins. Thymidine kinase (TK) and other genes of pseudorabies virus are non-essential genes for the replication of pseudorabies virus, and they are also important virulence genes of the virus. Proliferation and immunogenicity. Pseudorabies virus has strong genetic stability, and the insertion of foreign antigen genes in non-essential genes will not affect the proliferation of the virus. It has a wide range of hosts and can infect various domestic and wild animals such as pigs, dogs, cattle, and sheep. As a neurotropic virus, pseudorabies virus preferentially infec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N7/01C12N15/869
CPCC12N15/85C12N7/00C12N15/86C07K14/43595C12N2710/16721C12N2710/16743C12Y207/01021C12N2800/107
Inventor 商营利刘振孔正杰
Owner SHANDONG AGRICULTURAL UNIVERSITY
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