The Study Of Using RNAi To Inhibit The Expression Of HBeAg In Vitro

AIM: To construct a vector expressing shRNA and green fluorescent protein in mammalian cells and a vector expressing HBeAg in eukaryotic cells, to observe the inhibition of RNAi on the expression of HBeAg in transfected Hela cells .Methods:The U6 promotor was amplified from the PTZU6+1 vector by PCR, the product of PCR was cloned into the pEGFP-C1, the recombinant vector was confirmed by restriction enzyme digestion,PCR and sequencing. Then vector was transducted into Hela cells to ensure the expression of green fluorescent protein. The prec/c gene was amplified from the PHBV1.5 vector by PCR, the product of PCR was cloned into the pCDNA3.1, the recombinant vector was confirmed by restriction enzyme digestion,PCR and sequencing. Then the vector was transfected into Hela cells to ensure the expression of HBeAg.Both of recombinant vetors were transfected into Hela cells and the inhibition was analyzed by semi-quantity PCR and MEIA.RESULTS: The recombinant vector was confirmed by restriction enzyme digestion, PCR and sequencing. Green fluorescent protein and HBeAg were expressed .The expression of HBeAg could be inhibited by psiHBV2. CONCLUSION:1.The recombinant vectors expressing shRNA or HBeAg were constructed successfully,these layed the foundation for using the vectors in further researches on RNAi.2. The expression of HBeAg was inhibited by psiHBV2 about 50 percent, It was possible to use RNAi for inhibiting the replication of Hepatitis B Virus.3. Only one of the three designed sequences surpressed the expression of HBeAg, It indicated that the inhibiting effect of RNAi was sequence dependent.