shRNA表达载体的构建及对HBV复制和表达的抑制

目的:构建shRNA的表达载体,检测针对乙型肝炎病毒(HBV)的shRNA的稳定表达质粒对HBV复制和表达的影响。方法:扩增U6启动子构建shRNA表达载体pU6。针对HBV基因组序列设计并化学合成双链核苷酸克隆到pU6载体得到质粒pU6B/HBVi,脂质体介导转染带有HBV基因组的2.2.15细胞,定量PCR和ELISA法检测pU6B/HBVi对HBV复制和表达的影响。结果:成功构建了shRNA的真核表达载体;针对HBV的pU6B/HBVi质粒对HBV的复制和表达有明显的抑制作用。结论:稳定表达shRNA的pU6B/HBVi质粒可以抑制HBV在2.2.15细胞中的复制和表达。

Construction of plasmid stably expressing shRNA and inhibition of HBV replication and expression

AIM: To construct the vector stably expressing small hair RNA(shRNA) and investigate the effect of shRNA expressed with DNA vector on HBV replication and expression. METHODS: The vector expressing shRNA designated pU6 was constructed by U6 promoter. The sequence of shRNA targeting on specific region of HBV genome against HBV was designed and synthesized, then cloned into pU6. The plasmid designated pU6B/HBVi was transfected into 2.2.15 cells using LipofectamineTM2000 reagent. The HBsAg and HBeAg in the supernatants of the transfected cells were measured by ELISA. The HBV copies in the transfected cells were measured by quantitative PCR. RESULTS: Compared with controls, not only concentration of HBsAg and HBeAg but also HBV copies in transfected 2.2.15 cells decreased significantly by the plasmid expressing shRNA against HBV. CONCLUSION: HBV replication and expression in 2.2.15 cells are inhibited by stably expressed shRNA.