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Double gene-deleted strain of pseudorabies virus variant, construction method and application thereof

A pseudorabies virus, double-gene technology, applied to pseudorabies virus mutant double-gene deletion strains and its construction and application fields, can solve the problems of inability to provide immune protection, enhanced pathogenicity of PRV mutant strains, and antigenic variation , to achieve the effect of improving screening and purification efficiency and improving purification efficiency

Active Publication Date: 2014-09-24
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Preliminary research results show that compared with the previous strains, the antigenicity of the new circulating strains has mutated, and the pathogenicity of the PRV mutant strains is significantly enhanced. immune protection

Method used

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  • Double gene-deleted strain of pseudorabies virus variant, construction method and application thereof
  • Double gene-deleted strain of pseudorabies virus variant, construction method and application thereof
  • Double gene-deleted strain of pseudorabies virus variant, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The construction of embodiment 1 deletion virus

[0033] 1. Experimental method

[0034] 1.1 Construction of transfer vector

[0035] Two pairs of primers P1S / P1R and P2S / P2R were designed according to the US region gene sequence of PRV Kaplan strain (GenBank accession number JQ809328.1) and the deletion site of PRV Bartha-K61 strain (Table 1). Then take the PRV variant strain TJ strain virus DNA as a template, and use primers P1S / P1R (SEQ ID No.1 and SEQ ID No.2) and P2S / P2R (SEQ ID No.3 and SEQ ID No.4) to amplify respectively Left and right homologous recombination arms (L and R), and then L and R were cloned into the pOK12 vector through EcoRI and XbaI restriction sites to obtain the transfer vector pOK-LR. Using the pEGFP-N1 plasmid as a template, primers P3S / P3R (SEQ ID No.5 and SEQ ID No.6) (Table 1) were used to amplify the complete expression cassette containing EGFP and Neo genes. Then this fragment was cloned between the L and R fragments of pOK-LR through...

experiment example 1

[0046] Identification of experimental example 1 deletion virus strain rPRVTJ-delgE

[0047] 1. Experimental method

[0048] 1.1 Indirect immunofluorescence assay (Immunofluorescence assay, IFA)

[0049] Infect PK-15 cells with rPRVTJ-delgE-EGFP-Neo, rPRV-delgE strain and parental virus PRV TJ strain at 1MOI (multiplicity of infection, MOI)), and after 24h, the transitional virus rPRVTJ-delgE-EGFP-Neo inoculates the cells and places them directly Observed under a fluorescence microscope, the cells inoculated with rPRVTJ-delgE and the parental virus PRV TJ strain were fixed with cold absolute ethanol, and PRV gB (IDEXX, USA, batch number DJ358) and gE monoclonal antibody (IDEXX, USA, Batch number CJ291), after 2 hours at 37°C, wash the cells with PBS 5 times, add 1:80 diluted FITC-labeled goat anti-mouse IgG (Sigma Company), place in a wet box for 45 minutes at 37°C, wash 5 times with PBS , observed under an inverted fluorescence microscope.

[0050] 1.2 One-step growth curve...

experiment example 2

[0061] Experimental Example 2 Detection of the safety and immunogenicity of the deletion virus strain rPRVTJ-delgE in pigs

[0062] 1. Experimental method

[0063] 1.1 Immunity and attack

[0064] Twenty-one 35-day-old healthy piglets negative for both PRV antibody and antigen were selected and randomly divided into 5 groups. Among them, Group A and Group E each had 3 heads, and Groups B, C, and D each had 5 heads. Group A was inoculated with commercial pseudorabies Bartha-K61 strain attenuated vaccine (purchased from Harbin Veken Biotechnology Co., Ltd., production batch number 2012002), and the inoculation dose was 10 5 TCID 50 per head; groups B, C and D were inoculated with rPRVTJ-delgE strain, and the inoculation dose was 10 5 TCID 50 / head, 10 4 TCID 50 / head and 10 3 TCID 50 per head; Group E was inoculated with PBS at a dose of 1 mL / head. The inoculation site for all pigs was the left neck muscle. One week after immunization, all pigs were challenged with P...

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Abstract

The invention discloses a double gene-deleted strain of a pseudorabies virus variant, a construction method and an application thereof. The invention provides the pseudorabies virus strain with gI and gE genes being deleted. The pseudorabies virus variant is assigned the access number CGMCC. NO.8786. The invention further provides the construction method for the double gene-deleted strain. The construction method includes: (1) constructing a pseudorabies virus TJ strain transfer vector containing complete expression cassettes of EGFP and Neo; (2) transfecting the transfer vector to a cell which is inoculated with a pseudorabies virus TJ strain to obtain a transitional virus; and (3) performing an enzyme digestion process to a transitional viral genome, co-transfecting the enzyme digested transitional viral genome with the transfer vector and performing a plague screening process to obtain the double gene-deleted strain. The double gene-deleted strain is completely attenuated, is free of any clinical reaction after immunization of pigs, can rapidly induce generation of a specific antibody of pseudorabies virus, is high in neutralizing antibody titer and can provides a complete immune protection which aims at an attack of a presently-epidemic pseudorabies virus variant.

Description

technical field [0001] The present invention relates to a pseudorabies virus variant strain, in particular to a pseudorabies virus variant strain double-gene deletion strain and its construction method, and the present invention also relates to a pseudorabies virus variant strain double-gene deletion strain in the preparation and prevention of pseudorabies virus mutation The invention belongs to the field of construction and application of a pseudorabies virus mutant double-gene deletion strain. Background technique [0002] Pseudorabies (PR) is caused by pseudorabies virus (PRV) and is caused by pigs, sheep, cattle and other domestic and wild animals. Acute infectious disease characterized by neurological and nervous system disorders. PRV is a member of the Varicellavirus genus of the Alphaherpesvirinae subfamily of the Herpesviridae family. Its genome is a linear double-stranded DNA with a length of about 145kb and a G+C content of up to 73%. The entire genome consists o...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/63A61K39/245A61P31/22C12R1/93
Inventor 仇华吉孙元罗玉子李素李永锋
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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