核转录因子红细胞系相关因子-2干扰质粒构建及鉴定

Construction and identification of nuclear factor erythroid-2-related factor 2 gene RNA interference recombinant plasmid

目的设计及构建小鼠核转录因子红细胞系相关因子-2(Nrf2)基因的干扰质粒,并筛选出效果最好的干扰质粒。方法设计3组针对Nrf2基因的核糖核酸干扰(RNAi)序列,应用基因重组技术克隆入载体中构建短发夹RNA(shRNA),分别为shRNA1、shRNA2及shRNA3,通过基因测序鉴定,经Lipofectamine2000转染至BV2细胞,实时PCR检测Nrf2 mRNA的表达,Western Blot法检测Nrf2蛋白的表达。结果测序表明,克隆入载体中的Nrf2干扰序列及读码框完全正确,实时PCR及Western Blot显示shRNA3干扰效果最强。结论成功构建小鼠Nrf2的有效干扰质粒,为Nrf2信号通路在脑卒中领域的功能研究奠定了基础。

Objective To construct and identify the shRNA plasmid vector targeting nuclear factor erythroid-2- related factor 2 （Nrf2） , and to collect the strongest RNAi effect of Nrf2 shRNA sequence. Methods Nrf2 gene was targeted gene. Three shRNA sequences were designed by software and synthesized by chemical method： shRNA-1, shRNA-2 and shRNA-3. The double strand shRNA oligo was ligated to the vector. The construct was verified by sequencing analysis. BV2 cells were transfected with expressing shRNA plasmid vectors using Lipofectamine 2000. The expression of Nrf2 in the levels of mRNA was detected by real-time PCR, and Western Blot was adopted to abserve the expression of Nrf2 protein. Results Sequencing analysis suggested that the shRNA vectors targeting Nrf2 possessed correct nucleotide sequence and read frame. The result of Real-time PCR and Western Blot showed that the sequence of shRNAi-3 could more effectively knockdown the expression level of Nrf2 than the others. Conclusions The shRNA vectors targeting Nrf2 are successfully constructed and the shRNA can signidieantly inhibit the expression of Nrf2. These findings could provide an experimental basis for further study on Nrf2 signaling pathway in stroke field.