Construction and evaluation of human Dp71 shRNA vector / 中南大学学报(医学版)

ABSTRACT

Objective: To construct effective short hairpin RNA (shRNA) recombinant plasmids targeting humanDystrophin Dp71 gene, and evaluate their interference effciency. Methods: hTree pairs of siRNA sequences targeting human Dp71 gene and one pair of control siRNA sequence were designed, synthesized, and then inserted into the pRNAT-U6.1/Neo vector. hTe shRNA recombinant vectors were evaluated by enzyme digestion and sequencing. Dp71-shRNA and control shRNA plasmids were transfected into human normal gastric epithelial cells (GES-1) and humanbronchial epithelium (HBE). Western blot was used to evaluate its interfering effciency. Results: Restriction enzyme digestion and sequencing showed that the Dp71-shRNA vectors were successfully constructed. Western blot displayed that Dp71 protein expression was reduced to a signiifcant degree atfer transfection with the 3 Dp71-shRNA plasmids, and Dp71-shRNA2 plasmid inhibit the Dp71 expression most effciently. Conclusion: Dp71-shRNA vectors have been successfully constructed. The 3 Dp71-shRNA plasmids can inhibit Dp71 expression in GES-1 and HBEC, with Dp71-shRNA2 plasmid displaying the highest inhibition effciency.