Preparation of recombinant adenovirus mediated siRNA targeting HBVx gene and its suppressive effect on HBVx gene expression

    QIN Weiª²Wei, LIU Jiaª²Yun, ZHANG Rui, WANG Tao, WANG Kai1, MENG Yanª²Ling, YANG Anª²Gang

    1Department of Biochemistry and Molecular Biology, 3Department of Immunology, School of Basic Medicine,2Department of Clinical Laboratory, Xijing Hospital, Fourth Military Medical Univerª²sity, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct the recombinant adenovirus vectors for siRNA targeting HBVx gene (HBx) and to evaluate the inhibitive effect on the expression of HBx gene in human hepatoma HepG2 cell line. METHODS: The shuttle vector pShuttleª²HBx was coª²transfected into E. coli BJ5183 with adenovirus backbone plasmid. The homologous recombinants were transfected into HEK293 packaging cells and adenovirus was packaged and amplified, followed by infection of the HepG2 cells transfected with HBx gene. The expression levels of HBx were determined by RTª²PCR and Western Blot after the total RNA and protein were collected. RESULTS: The recombinant adenoviral vectors conª²taining siRNA targeting HBx gene were successfully constructed, which were confirmed by restriction enzyme digestion. RTª²PCR results demonstrated that the HBx mRNA was markedly decreased and Western Blot results showed that the expression of HBx protein was also significantly reduced in HepG2 cell line. CONCLUSION: Adenovirusª²based siRNA targeting HBx gene can inhibit HBx expression with great potency and specificity in vitro. ......