The Study Of Using RNAi To Inhibit The Expression Of Antigen Of HBV

OBJECTIVE: To construct the vector expressing shRNA, and to observe the effect of the RNA interference-mediated inhibition on HBV expression in transfected Hela cells and in mouse model of acute HBV infection. METHODS: The eukaryotic expression vector containing1.5 fold HBV ayw type whole genome was confirmed by restriction enzyme digestion and PCR. Then the vector was transfected into Hela cells and the expression of HBsAg and HBeAg was confirmed by MEIA,RT-PCR and IHC. The vectors expressing shRNA constructed by gene recombinant technology were confirmed by restriction enzyme digestion and sequencing. Both pHBV1.5 and shRNA vectors were transfected into Hela cells with different ratio. After 72h, the inhibition rate was analyzed by semi-quantity PCR and MEIA. Hydrodynamic injection of naked plasmid was used to construct a mouse model of acute hepatitis B virus infection. When the infection was confirmed by detection for HBV antigens, the animal model was established successfully. Then pHBV1.5 and shRNA vectors were injected into mice to study the inhibition of RNAi on the expression of HBV in vivo. The inhibition rate was analyzed by meauring the HBsAg and HBeAg in the serum of mice and detecting the expression of HBV C mRNA in the liver. RESULTS: The pHBV1.5 was confirmed by restriction enzyme digestion and PCR. Both HBsAg and HBeAg were expressed in Hela cells and mice after transfection. Also the shRNA vectors were confirmed by restriction enzyme digestion, PCR and sequencing. The...