重组质粒pSUPER-HBs RNAi的构建及筛选

Objective: To develop a system to screen for the effective siRNA sequence targeting HBs gene and to identify the interference efficiency. Methods: Three HBs-targeting siRNA segments (HBs-siRNA1, HBs-siRNA2, and HBs-siRNA3) were designed, synthesized and cloned into pSUPER vector to construct three recombinant plasmids pSUPER-HBs-siRNA, which were then transfected into human embryonic kidney 293T cells together with HBV plasmid. The transfection efficiency was observed 72 h later, the interference efficacies of the 3 segments were identified by real-time PCR and ELISA analysis, and the best one was identified. Results: Three recombinant plasmids of pSUPER-HBs-siRNA were constructed successfully and effectively transfected into 293T cells to induce RNAi, with a transfection rate higher than 70%. The results of real-time PCR and ELISA analysis showed that HBs-siRNA2 silenced the HBs gene expression by more than 80%. Compared with HBs-siRNA2, HBs-siRNA1 and HBs-siRNA3 did not demonstrate obvious interfering effect (P＜0.05). Conclusion: We have successfully constructed 3 siRNA sequences targeting HBs, and pSUPER-HBs-siRNA2 can effectively silence HBs genes, which paves a way for future study.