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Pseudorabies virus JS-2012 infectious clone plasmid and construction method and application

A pseudorabies virus, infectious cloning technology, applied in the field of bioengineering, can solve the problems that mutant strains cannot be completely protected, and the virulence of pseudorabies virus mutant strains is enhanced.

Active Publication Date: 2017-07-11
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the classic PRV strain SC, the virulence of the variant strain of pseudorabies virus is enhanced, and Bartha vaccine cannot completely protect against the variant strain

Method used

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  • Pseudorabies virus JS-2012 infectious clone plasmid and construction method and application
  • Pseudorabies virus JS-2012 infectious clone plasmid and construction method and application
  • Pseudorabies virus JS-2012 infectious clone plasmid and construction method and application

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Experimental program
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Effect test

Embodiment 1

[0059] The construction of embodiment 1 recombinant virus rJS2012-gG / loxp

[0060] In order to construct an infectious clone of pseudorabies virus JS-2012 strain, in this example, the loxp sequence and the marker gene EGFP were inserted into the JS-2012 genome by homologous recombination to obtain recombinant virus rJS2012-gG / EGFP. Then, the EGFP marker gene in rJS2012-gG / EGFP was deleted by Cre enzyme, and the recombinant virus rJS2012-gG / loxp carrying loxp was obtained.

[0061] The specific implementation process is as follows:

[0062] 1.1 Construction of transfer vector pTEGGF

[0063] First, pEGFP-C3 was digested with XhoI and BamHI, filled in with T4DNA polymerase, and the large fragment was recovered from the gel and ligated with T4DNA Ligase to obtain the plasmid pPEGFP-C3D that deleted the sequence of the multiple cloning site between XhoI and BamHI. Using the PRV JS-2012 genome as a template, primers PgGF / PgGR (Table 1) and PDgGF / PDgGR (Table 1) were used to ampli...

Embodiment 2

[0071] Embodiment 2 Construction of recombinant virus rJS2012-BAC

[0072] In this example, the loxp-Cre enzyme system and the loxp site in rJS2012-gG / loxp were used to recombine the artificial bacterial chromosome vector carrying the marker gene EGFP into rJS2012-gG / loxp to obtain the recombinant virus rJS2012-BAC. The specific implementation process is as follows:

[0073] 2.1 Construction of transfer vector pBeloBAC11-EGFP

[0074] Using pEGFP-C3D as template, use primers PEGFPF / PEGFPR (Table 1) to amplify the EGFP expression cassette, PCR reaction system: 94°C for 5min, 94°C for 30s, 57°C for 45s, 72°C for 2min, a total of 30 cycles; 72°C 10min. The results of electrophoresis of PCR products were as follows: Figure 6 ( Figure 6 Middle, 1: DNA Marker DL2000, 2: PCR amplified fragment), the target band of about 1600bp was amplified. The target DNA band and pBeloBAC11 were respectively digested with BamHI and HindIII, recovered by gel, and the EGFP expression cassette ...

Embodiment 3

[0077] The construction of embodiment 3PRV infectious bacteria artificial chromosome

[0078] In this example, the infectious plasmid pBAC-JS2012 was obtained by electrotransforming the rJS2012-BAC genome into Escherichia coli DH10B. The specific implementation process is as follows:

[0079] Infect vero cells with rJS2012-BAC, 16-21 hours after infection, use the Hirt method to extract the circular genome during rJS2012-BAC replication in the cells, and electroporate Escherichia coli DH10B. The electroporation conditions are as follows: 1mm electroporation cuvette, 1750V, 25μF , 200Ω, electric shock once. Add 0.8mL S.O.C culture solution immediately after electric shock, transfer the electrotransformed bacteria into a 2.0mL centrifuge tube, incubate at 37°C for 1h, centrifuge at 5000rmp for 2min, spread the precipitated bacteria on the LB plate containing chloramphenicol (25μg / mL), 37 Cultivate at ℃ for 24h. Bacterial colonies on the plate were picked, placed in LB liquid ...

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Abstract

The invention provides a pseudorabies virus JS-2012 infectious clone plasmid and a construction method and application; the JS-2012 infectious clone plasmid is constructed on the basis of newly appeared PRV variant JS-2012 in China by insertion of a pBeloBAC11 vector sequence in the downstream of gG gene termination codon of JS-2012 by use of a loxP-Cre enzyme system, a galK positive and negative screening system is used for genetic sequence operation of the JS-2012 infectious clone plasmid to establish a variant JS-2012 reverse genetic operating system, and a technical platform is provided for deep research of PRV variant genetic variation mechanisms, virulence enhancement and virus latent infection mechanisms, vaccine development and the like.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a pseudorabies virus infectious cloning plasmid and a preparation method and application thereof. Background technique [0002] Pseudorabies is an important animal infectious disease caused by pseudorabies virus (PRV), characterized by fever, itching, and encephalomyelitis. PRV can infect a variety of hosts, but pigs are the main natural host, reservoir and disseminator of the virus. Pigs of different ages can be infected, mainly causing abortion, stillbirth, mummified fetuses in pregnant sows, high mortality in suckling piglets, and infertility in breeding pigs. PRV belongs to the herpesviridae herpesvirus subfamily A, its genome is a linear double-stranded DNA, about 150kb in length, composed of a long unique region (UL), a short unique region (US) and repeat sequences on both sides of the US, encoding More than 70 kinds of proteins. Some important viral genes, such a...

Claims

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Application Information

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IPC IPC(8): C12N15/869C12N7/01C12N7/04C12N15/38C12Q1/70C12Q1/68A61K39/245A61P31/22C12R1/93
CPCA61K39/12A61K2039/5254C12N7/00C12N15/86C12N2710/16721C12N2710/16734C12N2710/16743C12N2710/16762C12Q1/705
Inventor 童光志郑浩王涛童武李国新高飞
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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