重组质粒pSUPER-HBs RNAi的构建及筛选

Construction and screening of pSUPER-HBs-siRNA efficiently targeting HBs gene

目的:利用pSUPER-RNAi载体系统进行HBsRNAi序列的初步筛选和干扰效果鉴定。方法:设计并合成针对HBs基因区的3条siRNA(HBs-siRNA1、HBs-siRNA2、HBs-siRNA3),构建3种重组载体pSUPER-HBs-siRNA,与HBV质粒同时瞬转人胚肾293T细胞,72h后观察转染效果,实时定量PCR和ELISA法鉴定3种干扰序列对HBs的干扰效果,筛选最佳干扰序列。结果:成功构建3种干扰质粒pSUPER-HBs-siRNA,能高效转染人胚肾上皮细胞293T,转染效率在70%以上;实时定量PCR和ELISA法结果均显示HBs-siRNA2可有效抑制HBs的表达,抵制率可达80%,而HBs-siR-NA1、HBs-siRNA3干扰效果不显著,差异具有统计学意义(P<0.05)。结论:成功构建3种干扰质粒pSUPER-HBs-siR-NA,筛选出其中能有效抑制HBs基因表达的pSUPER-HBs-siRNA2序列,为后续研究奠定了基础。

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 HB-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 transfeetion 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.