[Application of PCR-based tRNAval Pol III-shRNA expression cassettes in screening of efficient siRNA for inhibiting hepatitis B virus].
To screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA(val) Pol III-shRNA expression cassettes (SECs).



Based on core gene sequence of HBV, five target sites of siRNA were designed. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were co-transfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting (FACS); HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2. 2. 15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay (RIA). HBV pgRNA from cell total RNA was detected by semi-quantitative PCR.



Co-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration.



The expressed shRNA, which targets sites on HBV C mRNA in 492i, is to have having most efficient RNAi effect. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.
Pan
Xiu-cheng
XC
Institute of Infectious Disease, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Chen
Zhi
Z
Ni
Qin
Q
Yang
Zheng-gang
ZG
Xu
Ning
N
Jin
Han-ying
HY
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D049452:Green Fluorescent Proteins / Q000235:genetics; D006512:Hepatitis B Core Antigens / Q000096:biosynthesis / Q000235:genetics*; D006513:Hepatitis B e Antigens / Q000096:biosynthesis / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000166:cytology; D008113:Liver Neoplasms / Q000473:pathology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering; D012366:RNA, Transfer, Val / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D014162:Transfection
D006512:Hepatitis B Core Antigens; D006513:Hepatitis B e Antigens; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012366:RNA, Transfer, Val; D012367:RNA, Viral; D049452:Green Fluorescent Proteins