[Construction and application of subgenomic replicon vectors of Japanese encephalitis virus].
Based on the infectious clone of JEV vaccine SA14-14-2, the subgenomic replicons pCTCJEV, pCTMJEV with large deletions in the structural region were constructed. Then they were transfected into BHK-21 cell, the RNA replication of JEV subgenome can be monitored by RT-PCR and the non-structural protein can be found expressed in the cell by IFA. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive-strand and the negative-strand JEV RNA, we inserted an enhanced green fluorescence protein (EGFP) gene into the 3'-UTR of pCTCJEV, pCTMJEV under the control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA. After transfection, the EGFP fluorescence could be seen under the fluorescence microscope 1 day later,and maintained for more than a week with no apparent cytopathic effect. The constructed JEV replicons would provide valuable tools to provide a possible vector for a long-lasting RNA virus expression system.
Huang
Ying
Y
Lab. for Protein Engineering, Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
Shao
Wei
W
Jia
Li-Li
LL
Yu
Wei-Yuan
WY
Yu
Yong-Xin
YX
D000818:Animals; D002460:Cell Line; D004664:Encephalitis Virus, Japanese / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012093:Replicon / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D012367:RNA, Viral; D049452:Green Fluorescent Proteins