Development of a cell culture system based on recombinant hepatitis C virus expressing enhanced green fluorescent protein
10.3760/cma.j.issn.0254-5101.2012.12.008
- VernacularTitle:增强型绿色荧光蛋白标记的重组丙型肝炎病毒体外细胞培养系统的建立
- Author:
Hongtao XU
;
Li XIAO
;
Jianchun XIAN
;
Yabao CHEN
- Publication Type:Journal Article
- Keywords:
HCV;
EGFP;
Reporter gene;
Cell culture
- From:
Chinese Journal of Microbiology and Immunology
2012;(12):1034-1038
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a time saving and sensitive cell culture system based on hepatitis C virus chimera expressing enhanced green fluorescent protein(EGFP) and to facilitate the study on HCV pathogenesis and screening of anti-HCV drugs.Methods Enhanced green fluorescent protein reporter gene and a mutation V2440L that can yield higher virus titers were introduced into the C-terminus of non-structural protein 5A (NS5A) of the JFH1 viral genome by using recombinant PCR.The viral RNA was transfected into Huh7.5 cells.Viral RNA in supernatant of HCV RNA-transfected cells was determined after transfection by RT-PCR.HCV replication and infection were determined by immunofluorescence assay.IFN-α was used to evaluate the feasibility of this system for anti-HCV drugs screening.Results The viral RNA replicated efficiently in transfected cells.These cells can produce HCV-EGFP reporter virus.Viral RNA levels in supernatant were 3.06× 105 copies/ml and 7.96×106 copies/ml at 72 h and 9 d after transfection,respectively.The virus titer reached to 104 FFU/ml 9 d after transfection.The expression of EGFP was inhibited by IFN-α in a dose dependent manner in Huh7.5 cells infected by HCV-EGFP reporter virus.Conclusion The recombinant HCV JFH1-EGFP reporter gene system is a time saving,cost effective and sensitive method for studying viral replication cycles and screening of anti-HCV drugs.
