核转录因子TR3小干扰RNA载体的构建及其效果鉴定

Construction of Small Interfenrence RNA Vector Targeting Nuclear Factor TR3 and its Gene Silencing Function

目的:构建核孤儿受体TR3的小干扰RNA(siRNA)真核表达载体,并进行沉默效果测定。方法:根据TR3 mRNA序列和载体psiSTRIKE的粘性末端,设计合成TR3的干扰RNA序列,退火后克隆至psiSTRIKE,经测序鉴定后用阳离子脂质体将重组子和TR3高表达载体TR3-pcDNA共转染至HEK293细胞中,以Western blotting检测干扰后HEK293细胞内TR3表达的变化。结果:Western blotting检测结果证实构建的TR3 siRNA表达重组载体可显著抑制HEK293细胞内TR3的高表达。结论:构建了核孤儿受体TR3 siRNA真核表达载体。

Objective： To construct the small interfenrence RNA（siRNA） eukaryotic vector of inhibitory nuclear factor TR3 and to observe its interfering effect in HEK293 cell line. Methods： The specific siRNA sequence was designed targeting the TR3 mRNA according to the adhesive end of psiSTRIKE vector. Annealed the single-stranded oligos to generate a ds-oligo, cloned it into psiSTRIKE, and then sequence analysis was performed. The recombinant plasmid was transfected into HEK293 cell by liposome. TR3 expression was detected by Western blotting. Results： Western blotting analysis demonstrated that TR3 siRNA expression could suppress the high expression of TR3 in HEK293 cells. Conclusion： A siRNA eukaryotic vector containing TR3-siRNA sequence was successfully constructed.