BNIP3干扰质粒的构建及鉴定

Construction and identification of a RNA interference plasmid for rat BNIP3 gene

目的构建大鼠Bcl-2/E1B-19K-interacting protein 3(BNIP3)干扰质粒,研究其对BNIP3蛋白表达水平的影响。方法利用Invitrogen公司在线软件设计大鼠BNIP3(NM_053420)干扰片段,合成互补的单链寡核苷酸,蒋退火后形成的双链克隆入pcDNATM6.2-GW/EmGFP-miR表达载体,连接产物转化感受态细胞,增菌,质粒小量提取并测序。将构建的干扰质粒与大鼠BNIP3表达质粒共转染到HEK293细胞中,采用实时荧光定量PCR和Western blotting检测对目的基因的干扰情况。结果构建的2个干扰质粒经测序证实序列正确,与BNIP3表达质粒共转染后显著抑制外源性BNIP3的mRNA和蛋白表达。结论成功构建了大鼠BNIP3干扰质粒,为进一步研究BNIP3的功能打下了基础。

Objective To construct a RNA interfering plasmid targeting rat Bcl-2/E1B-19K-interacting protein 3（BNIP3） and assess its effect on exogenous BNIP3 expression in HEK293 cells.Methods The miRNA sequences were designed using Invitrogen BLOCK-iT RNAi Designer and synthesized into double-strand oligonucleotides,which were cloned into the pcDNATM6.2-GW/EmGFP-miR vector,followed by transformation of the product into competent Top10 E.coli cells.After expansion of the transformed bacteria,the plasmid was extracted and sequenced beforeits co-transfection with pEGFP-C3rBNIP3 plasmid into HEK293 cells.The interference effect of the constructed plasmid on BNIP3 mRNA and protein expression were detected by real-time PCR and Western blotting.Results The sequencing result indicated that the interfering plasmid targeting rat BNIP3 was constructed correctly.After transfection into HEK293 cells,the interfering plasmid significantly inhibited exogenous BNIP3 mRNA and protein expressions.Conclusion The RNA interfering plasmid targeting rat BNIP3 has been constructed successfully,which provides a useful tool for studying the function of BNIP3.