Establishment Of HCV Subgenomic Replicon QSG7701 Cell Line And The CTE-Ribozyme Inhibition Of HCV RNA Replication

Objective To establish a HCV Subgenomic Replicon cell line;To study the effect of ribozyme-CTE on intracellular HCV mRNA。Methods1.The plasmid pHCVBM4-5with HCV Subgenomic Replicon was translated into HCV Subgenomic Replicon RNA Using RiboMAX Express T7。2.The HCV Subgenomic Replicon RNA were transfected into QSG7701 cells Using Lipofectmine^TM2000,and then selected the cell clone with G418。The non-transfected QSG7701 cells as control。The stable cell line was verified by RT-PCR and Western Blotting。3.To observe the effect of pPHCV5-R1,pPHCVS-R2,pPHCV5-CR1 as well as pPHCV5-CR2 on HCV Subgenomic Replicon RNA replication。Result1.HCV Subgenomic Replicon QSG7701 stable cell line was stablished,which was verified by RT-PCR and Western Blotting。2.The plasmids that contain these classic ribozyme genes and new ribozyme genes were transfected into QSG7701 cells using Lipofectmine^TM2000。The following experiments were carried out after 48 hours of transfection.RT-PCR and WesternBlotting were used to detect HCV RNA and corresponding protein respectively o The results of RT-PCR suggested that those new rebozymes with CTE could cut target RNAs more effective than classic ribozymes without CTE。Those new ribozymes with CTE could cut target RNAs that classic ribozymes could not cut。Conclusion1.successful establishment of HCV Subgenomic Replicon QSG7701 cell line。2.CTE-ribozymes with was more effective on HCV RNA inhibition than rebozymes。...