小鼠Hsp27基因siRNA重组腺病毒载体的构建与鉴定

Construction and Identification of Recombinant Adenovirus Vector of siRNA Targeting Hsp27

Hsp27是小分子量热休克蛋白亚家族的重要成员之一,主要参与细胞内蛋白质的折叠、抗凋亡、甾体激素合成、氧化应激及信号通路等生理过程。为进一步研究其生理与病理生理功能,本文构建了针对小鼠Hsp27基因的小分子干扰RNA(siRNA)重组腺病毒载体,并在AD-293细胞中与Hsp27超表达载体共同转染,观察其对Hsp27表达的影响。根据siRNA靶序列的设计要求,设计并合成针对小鼠Hsp27的siRNA靶DNA序列,克隆至穿梭载体pShuttle-H1中,与腺病毒骨架质粒pAdeasy-1在B J5183细菌中进行同源重组,转染AD-293细胞,包装得到含siHsp27的重组腺病毒,在体外与Hsp27超表达载体共转AD-293细胞。通过酶切鉴定、序列测定和W estern b lot鉴定,确定小鼠Hsp27 siRNA重组腺病毒载体构建成功,且该重组腺病毒能降低67%的Hsp27蛋白表达。本文构建的针对小鼠Hsp27基因的siRNA重组腺病毒载体,为进一步研究Hsp27基因的功能奠定了基础。

Hsp27 is a member of the heat shock protein family, which plays important roles in a variety of physiological processes including protein chaperoning, steroidogenesis and especially protection against apoptosis. To construct an adenovirus vector that expresses small interfering RNA （siRNA） against Hsp27 to shut down its gene expression, the Hsp27 template DNA sequence was designed by using online tools. The sense and antisense siRNA oligonucleotide templates were chemically synthesized, annealed and cloned into adenoviral shuttle vector pShuttle-H1. The recombinant adenovirus vector was obtained by homologous recombination with pAdeasy-1 in bacteria BJ5183. The recombined adenovirus was produced in 293 cells and subsequently infected AD-293 cells by co-transfect with adenovirus vector expressing Hsp27. The adenovirus vector expressing small interfering RNA for Hsp27 gene could specifically shutdown the 67% Hsp27 protein by western blot. The re- combinant adenoviruses expressing small interfering RNA forHsp27 gene was constructed successfully. The ade- novirus vector can effectively down-regulate Hsp27 expression by co-transfect with adenovirus vector expressing Hsp27 in the present study, which will facilitate further functional studies of Hsp27.