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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pcDNATM6.2-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-1mRNA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。
Objective To design and construct the ELK-1 small interfering RNA (miRNA) plasmid and finally identify the effective plasmid. Methods Four pairs of pcDNATM6.2-GW / EmGFPmiR miRNAs targeting ELK-1 and one pair of null control miRNA plasmids were designed and constructed and sequenced. The interference plasmids were transfected into 293T cells by lipofectamine and the cell lines were obtained by transfection in time. The transfection efficiency was determined by observing the green fluorescence under an inverted fluorescence microscope. The mRNA expression of ELK-1 in 4 pairs of interfering and negative control plasmids was detected by RT-PCR. The sequencing results showed that ELK-1 interference sequence and the reading frame were correct. The 293T cell line transiently transfected with interference plasmids showed a green fluorescence of more than 90% under an inverted fluorescence microscope. RT-PCR results showed that MR-2, MR-3 interference plasmids had a good inhibitory effect on ELK-1 mRNA. Conclusion The recombinant eukaryotic expression vector ELK-1 was successfully constructed and the effective plasmid was screened out to provide a reference for further study on the role of ELK-1 in tumor cells.