针对乙肝病毒的串联序列amiRNA表达载体的构建

Construction of the expression plasmids of tandem sequence amiRNA against HBV

目的为优化RNA干扰研究方法,构建了针对乙肝病毒的串联序列amiRNA(artificial microRNA)质粒表达载体。方法设计针对乙型肝炎病毒S区的靶干扰序列,构建单一序列amiRNA质粒表达载体;将其中干扰效率好的两个序列串联起来,构建串联序列amiRNA质粒表达载体。结果经酶切及测序鉴定,插入序列与靶序列一致,载体构建正确。结论成功构建了新型针对乙肝病毒的串联序列amiRNA质粒表达载体,为进一步体外及体内实验奠定了基础。

Objective To optimize the design of RNA interference and construct the tandem sequence amiRNA expression plasmids against I-IBV. Method Aiming at the conservation sites, singular-sequence vectors amiRNA-HBV1, amiRNA-HBV2, amiRNA-HBV3 and amiRNA-HBV4 were constructed. These vectors were transfected into HepG2.2.15 transiently. The two sequences with high efficiency were chosen, and then the tandem-sequence vector amiRNA HBV3- HBV4 was built. Result Four constructed miRNA expression vectors and the tandem one were verified correctly by DNA sequencing and enzyme cut appraisal. Conclusion The tandem sequence ptasmid amiRNA-HBV 3-4 was successfully constructed. In this study we combined some advantages of amiRNAs reported previously with the aim to construct a more practical amiRNA with high inhibition effects on HBV, which paved the way for further studies in vitro and in vivo.