Construction and identification of nuclear factor erythroid-2-related factor 2 gene RNA interference recombinant plasmid / 临床神经病学杂志

ABSTRACT

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 signidicantly inhibit the expression of Nrf2.These findings could provide an experimental basis for further study on Nrf2 signaling pathway in stroke field.