肾素(前体)受体miRNA干扰质粒的构建及鉴定

目的 设计及构建肾素(前体)受体微小干扰核糖核酸(miRNA)质粒,并鉴定出有效干扰质粒.方法 设计及构建4对肾素(前体)受体的pcDNATM6·2-GW/EmGFPmiR miRNA及1对阴性对照miRNA干扰质粒,通过测序鉴定.将干扰质粒用LipofectamineTM 2000转染大鼠血管平滑肌细胞,通过顺时转染获得细胞系,倒置荧光显微镜下观察绿色荧光及流式细胞术确定转染效率,Real-time PCR和Western blot检测4对干扰质粒、阴性对照质粒肾素(前体)受体的mRNA及蛋白表达水平.结果 测序显示肾素(前体)受体干扰序列及读框完全正确,倒置荧光显微镜及流式细胞术结果显示干扰质粒瞬时转染的大鼠血管平滑肌细胞系的转染效率均在60%以上.Real-time PCR和Western blot结果显示X2-2-3、X2-3-3干扰质粒对肾素(前体)受体 mRNA及蛋白有较好的抑制效果.结论 成功构建了肾素(前体)受体干扰真核表达载体,筛选出有效干扰质粒,为进一步研究肾素(前体)受体在大鼠血管平滑肌细胞中的作用提供参考.

Construction and Identification of miRNA Recombinant Eukaryotic Expression Vectors of (pro)Rennin Receptor

Aim To construct and identify the miRNA eukaryotic expression vectors of(pro)renin receptor((P)RR).Methods miRNA nucletides of(P)RR were chemically synthesized and inserted into pcDNATM6·2-GW/EmGFPmiR vector,which were confirmed by sequencing,then the recombinant miRNA vectors were transfected into A7r5 cell by LipofectamineTM 2000.The mRNA expression of(P)RR was detected by Real-time PCR and Western blot.Results Sequencing suggested that miRNA eukaryotic expression vectors targeting(P)RR possessed correct read frame and nucleotide sequence,and green fluorescene of the transient transfected A7r5 cell lines could be observed under inverted fluorescence microscope.Real-time PCR and Western blot results showed that the sequence of X2-2-3 and X2-3-3 could effectively knockdown the level of mRNA and protein of(P)RR.Conclusions miRNA eukaryotic expression vectors targeting(P)RR were successfully constructed and the effectively interference RNA were identified,which may be used for understanding the effect of(P)RR in the vascular smooth muscle cells.