Construction and Screening of Bovine Bax Gene shRNA Interference Expression Vector

Abstract

Bax is a pro-apoptotic member of the Bcl-2 family genes which regulate programmed cell death. To decrease the apoptosis of bovine fibroblast cells, we construsted specific short hairpin (shRNA) expression vector encoding shRNA targeting Bax gene to screen the most effective vector. Four shRNAs sequences based on the sequence of bovine Bax mRNA in the GenBank were designed, and one scrambled shRNA sequence was regarded as negative control. The designed and synthesised single-stranded primer were annealed to double-stranded oligo sequences and cloned into linear pRI-GFP vector digested by enzymes Xho I and Bgl II. Screening positive cloning after transformed into DH5α competent cells and identified by PCR amplification and DNA sequencing. Named the correct vectors as pRI-GFP-Bax-190, pRI-GFP-Bax-206, pRI-GFP-Bax-215, pRI -GFP-Bax-389, pRI-GFP-Bax-NC (the negative control) and seleced them by quantitative PCR after transfected after 24 h and 48 h. The results showed that pRI-GFP-Bax-190 was the highest efficiency (95.47%), and significant difference (P>0. 01) after 48 h transfection. RNA interference (RNAi) mediated by shRNA expression vector could significantly down-regulate the expression of Bax gene in bovine fibroblast cells, which laid a foundation for further research.