Notch1 miRNA干扰质粒构建及功能鉴定

Construction and function identification of Notch1 miRNA recombinant eukaryotic expression vectors

目的设计及构建大鼠Notch1微小干扰核糖核酸(miRNA)干扰质粒,最终筛选出效果最好的干扰质粒。方法针对大鼠Notch1基因分别设计3对pre-miRNA序列,通过T4连接酶克隆至pcDNATM6.2-GW/EmGFP-miRNA表达载体构建干扰质粒,基因测序鉴定,经Lipofectamine 2000转染至H9c2心肌样细胞,荧光显微镜下观察绿色荧光确定转染效率,Western blot检测3对干扰质粒、阴性对照质粒对Notch1胞内结构域(N1ICD)表达的影响。结果测序表明,Notch1干扰序列及读码框完全正确,荧光显微镜观察H9c2心肌样细胞miRNA瞬时转染效率在80%左右。Western blot显示miRNA6637对Notch1干扰效果最强。结论成功构建大鼠Notch1 miRNA的有效干扰质粒,为Notch1信号通路在心血管领域的功能研究奠定了基础。

Objective To construct and identify the microRNA（miRNA） eukaryotic expression vectors for Notch1,and to screen the ideal miRNA eukaryotic expression vectors with most interference effect.Methods Three pairs of pre-miRNA sequences for rat Notch1 were synthesized and inserted into pcDNATM 6.2-GW/ EmGFP-miR vector by T4 ligase for the construction of miRNA expression plasmid,which were confirmed by sequencing,then the recombinant miRNA vectors were transfected into H9c2 cells by Lipofectamine 2000.The transfection efficiency was observed under the inverted fluorescence microscope.The interference effect of miRNA on Notch1 was evaluated by Western blot for N1ICD（Notch1 intracellular domain）.Results Sequencing suggested that miRNA eukaryotic expression vectors targeting Notch1 possessed correct nucleotide sequence and read frame,and the express of the green fluorescent protein was over 80% when the transient transfected H9c2 cells observed under the inverted fluorescence microscope.The results of Western blot showed that the sequence of miRNA 6637 could more effectively knockdown the expression level of Notch1 than the others.Conclusion miRNA eukaryotic expression vectors targeting Notch1 are successfully constructed and the effectively interference RNA is identified,which lay a foundation of the function study of Notch1 signaling pathway in the cardiovascular field.