[Construction of HCV-producing cell model based on self-cleaving ribozyme].
OBJECTIVE
To construct a stable HCV-producing cell model for anti-HCV drug research.


METHODS
The HCV-ribozyme recombinant plasmid pJFH1-Rbz was constructed to generate the exact 5' and 3' ends of HCV genomic RNA by placing two self-cleaving ribozymes at both ends of the HCV JFH-1 cDNA. The plasmid was then transfected into HepG2 cells and the resultant clones were screened with G418. Subsequently, immunofluorescence and Western blot were performed to detect the expression of HCV core protein, HCV RNA level was quantitated by TaqMan real-time PCR method and HCV particles was detected by electron microscopy.


RESULTS
HCV core protein was detected in the screened cell clone, and the level of HCV RNA was up to 1000,0000 copies/ml in the culture medium. Electron microscopy showed the viral particles in the culture suspension were approximately 55 nm in diameter. IFN-treating experiment demonstrated that the HCV RNA level decreased with the increasing concentration of IFN alpha.


CONCLUSIONS
We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.
Wang
Sheng
S
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.
An
Xiao-Ping
XP
Mi
Zhi-Qiang
ZQ
Liu
Da-Bin
DB
Zhang
Bao-Zhong
BZ
Lv
Jun
J
Zhou
Yu-Sen
YS
Tong
Yi-Gang
YG
D018076:DNA, Complementary; D016679:Genome, Viral; D056945:Hep G2 Cells; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D010957:Plasmids; D016337:RNA, Catalytic / Q000235:genetics*; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics*; D014771:Virion; D014779:Virus Replication
D018076:DNA, Complementary; D016337:RNA, Catalytic; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus