猪日本乙型脑炎病毒DNA复制子载体的构建及外源基因表达分析

doi:10.11843/j.issn.0366-6964.2015.01.014

Abstract

Abstract:

Swine Japanese encephalitis virus (JEV) is one of pathogens that causing sow abortion.In this experiment，the DNA replicon vector was constructed and exogenous gene was expressed.Based on the JEV SA14-14-2 sequences，viral structural protein encoding gene prM and parts of E gene sequences were removed by mutant PCR methods，which retained nonstructural protein genes and non-coding region sequences，subgenomic replicon pAC-JEV that inserted FMDV-2A and HDV ribozyme sequences was constructed.In order to identify its abilities of self-replication，expression of nonstructural protein (NS3) were detected by western blotting and indirect immunofluorescence assay.The results showed that NS3 protein level increased gradually with extension of transfection times.To explore JEV replicon ability about expressing foreign proteins，we inserted an enhanced green fluorescence protein (EGFP) gene，HA gene of swine influenza virus and GP5 gene of porcine reproductive and respiratory syndrome virus into the multiple cloning site of pAC-JEV(pAC-JEV-EGFP，pAC-JEV-HA，pAC-JEV-GP5) and mutation of NS5 gene was deleted (pAC-JEV-ΔNS5).The plasmids were transfected into BHK-21 cell，EGFP mRNA level was significantly increased at 48，72，96 h in pAC-JEV-EGFP by real-time PCR，the EGFP fluorescence was directly detected by the fluorescence microscope at 12 h，and EGFP fluorescence signal lasted for 8 days.HA of swine influenza virus and GP5 of porcine reproductive and respiratory syndrome virus were detected using mouse anti-HA and GP5 antibodies by Western blotting.The results show that HA and GP5 can be expressed in BHK-21 cells.This JEV subgenomic replicon pAC-JEV can be used to study JEV gene function and viral protein interaction with cellular proteins；it also is a valuable tool for DNA vaccine research.

CLC Number:

S852.659.6

WANG Xiao-du，ZHAO Fan-fan，DAI Bing，SHAO Dong-hua，JIANG Chun-ying，ZHOU Qi，MA Zhi-yong. Construction of DNA Replicon Vector of Swine Japanese Encephalitis Virus and Application of Exogenous Gene Expression Analysis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(1): 111-118.

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Construction of DNA Replicon Vector of Swine Japanese Encephalitis Virus and Application of Exogenous Gene Expression Analysis

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