Effect of transient and stable transfection on inhibition of hepatitis B virus S gene expression by RNA interference

    LIU Jiaª²Yun1, LI Qingª²Xia2, HUANG Hongª²Yan2, MA Longª²Yang2, BAO Wei1, JIA Linª²Tao2, XUE Caiª²Fang3, LIU Jun3, WANG Chengª²Ji2, YANG Anª²Gang1,2

    1Department of Immunology, 2Department of Biochemistry and Molecular Biology, 3Department of Etiology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct pSUPER vectors expressing short interfering RNAs (siRNA) targeting hepatitis B virus (HBV) S gene sequence and to evaluate the effect of transient and stable transfection on the inhibition of hepatitis B virus gene expression in 2.2.15 cells by RNA interference. METHODS: Based on the sequence of HBV in 2.2.15 cells, two sequences targeting HBV S gene were designed and cloned into siRNAs expression vector pSUPER. The constructed plasmids were transiently and stably transfected into 2.2.15 cells respectively. The HBsAg and HBeAg in the supernatant of the transfected cells were assayed with Abbott MEIA Kits and HBV mRNA was measured by RTª²PCR. The effect of transient and stable transfection on the inhibition of hepatitis B virus gene expression by RNA interference was also compared. RESULTS: In single cell strains stably transfected with recombinant pSUPER plasmids and screened in Hygromycin media, the pSUPERª²S1 and pSUPERª²S2 siRNA expression vectors could effectively inhibit the secretion of HBV surface and e antigen by 83% and 78% respectively (P<0.01), and RTª²PCR results showed that the HBV mRNA decreased. However, the results at protein level and mRNA level in transiently transfected cells were not as good as those in stably transfected cells. CONCLUSION: siRNAs targeting HBV S gene sequence can specifically inhibit HBV gene expression with great potency in vitro. The transfection efficiency has obvious effect on RNA interfering target gene. ......