ELK-1 rniRNA干扰质粒构建及鉴定

目的 设计及构建ELK-1微小干扰核糖核酸(miRNA)质粒,最终鉴定出有效干扰质粒.方法 设计及构建4对ELK-1的pcDNATM 6.2-GW/EmGFPmiR miRNA及1对无效对照miRNA干扰质粒,通过测序鉴定.将干扰质粒用脂质体法转染293T细胞,通过顺时转染获得细胞系.通过倒置荧光显微镜下观察绿色荧光确定转染效率,RT-PCR检测4对干扰质粒、阴性对照质粒ELK-1的mRNA的表达水平.结果 测序表明,ELK-1干扰序列及读框完全正确,干扰质粒瞬时转染的293T细胞系在倒置荧光显微镜下呈绿色荧光达90%以上.RT-PCR结果显示,MR-2、MR-3干扰质粒对ELK-1 mRNA有较好的抑制效果.结论 成功构建了ELK-1干扰真核表达载体,筛选出有效干扰质粒,为进一步研究ELK-1在肿瘤细胞中的作用提供参考.

Construction and identification of miRNA recombinant eukaryotic expression vectors of ELK-1

Objective To construct and identify the miRNA eukaryotic expression vectors of ELK-1. Methods Microinterference RNA(miRNA) nucletides of ELK-1 were chemically synthesized and inserted into pcDNA~(TM)6. 2-GW/EmGFPmiR vector, which were confirmed by sequencing, then the recombinant miRNA vectors were transfected into 293T cell by Lipofectamine~(Tm) 2000. The mRNA expression of ELK-1 was detected by RT-PCR. Results Sequencing suggested that miRNAi eukaryotic expression vectors targeting ELK-1 possesse correct read frame and nucleotide sequence, and green fluorescene of the transient transfected 293T cell lines could be observed under in verted fluorescence microscope. RT-PCR results showed that the sequence of MR-2 and MR-3 could effectively knowdown the level of mRNA of ELK-1. Conclusion miRNA eukaryotic expression vectors targeting ELK-1 were successfully constructed and the effectively interference RNA were identified, which may be used for understanding the effect of ELK-1 in the tumor cells.