基于核酶自剪切机制稳定分泌丙型肝炎病毒的细胞模型

Objective To construct a stable HCV-producing cell model for anti-HCV drug research. Methods The HCV-ribozyme recombinant plasmid pJFHl-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 die screened cell clone, and the level of HCV RNA was up to 1 ×107 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 α. Conclusions We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.

Construction of HCV-producing cell model based on self-cleaving ribozyme