Experimental Research On The Interference And Inhibitation Of The Repication And Expression Of Hepatitis Be Antigen

OBJECTIVE: To construct the lentiviral vector expressing shRNA that specifically targets the pre-core gene sequence of hepatitis B virus and discuss the controlling function of RNAi in Hep-G2 2.15 cells. In order to apply the RAN disturbance (RNA interference, RNAi) technical treatment second grade hepatitis (HBV) lays the rationale. And this can form the basis for treating hepatitis by using RNA disturbing methods.METHODS: The oligonucleotides expressing shRNA that could specifically target the pre-core gene sequence of hepatitis B virus and the vector expressing shRNA that had nothing to do with HBV synthesized chemically by gene engineering recombinated with the lentiviral vectors plentilox3.7expressing U6 promoter and the genic of GFP. The recombinant vectors was transformed E.coli DH5αand was identified by restriction enzyme digestion and by sequence analyses. Then the vector was transducted into 293T cells with the helper plasmid system to make up lentiviral particles and infect Hep-G2 2.15 cells. Check the expression of HBeAg with MEIA to show the expression decreasing greatly. Checking prec/c mRNA with RT-PCR and limited PCR also shows the expression decreasing greatly.RESULTS: The lentiviral vectors expressing shRNA that constructed by gene engineer were confirmed by restriction enzyme digestion and sequencing. Succeed in making up lentiviral particles by transfecting 293T cells with the helper plasmid system and infect Hep-G2 2.15 cells.Check HBeAg with MEIA to show the expression decreasing greatly. Checking HBV prec/c mRNA with RT-PCR and limited PCR also shows the expression decreasing greatly.CONCLUSION:.①The observed results show that Hep-G2 2.15 cell can successfully express HBV antigen; secrete and produce HBeAg; and transcribe HBeAg mRNA as well, so it can be used as the targeting cell in RNA interference study.②The successful construction of the shRNA expression vector Pls expressing U6 promoter and the genic of GFP has established the substantial base for the concerned RNAi studies in vivo and vitro.③Succeed in making up lentiviral particles by transfecting 293T cells with the helper plasmid system and infect HepG2 2.15 cells .Checked the expression of HBeAg to show the expression decreasing greatly. It was possible to use RNAi for inhibiting the replication of Hepatitis B Virus.④Confirmations may obviously suppress the HBeAg expression in the HepG-2.2.15 cell two pair of oligonucleotides sequence, for used in the RNA disturbance technology the thorough research which the hepatitis B treated providing the important guarantee.