Sp1基因RNA干扰载体的构建及鉴定

Construction and Identification of the RNA Interference Vector Targeting Sp1 Gene

目的:构建干扰载体pSilencer3.1-Sp1,并初步研究其对Sp1基因的干扰作用。方法:根据Sp1cDNA编码序列,设计并合成针对Sp1基因的特异性RNA干扰片段,并将其克隆入pSilencer3.1-H1neo干扰载体中,构建Sp1基因小干扰RNA(siRNA)真核表达载体pSilencer3.1-Sp1;分别将阴性对照载体pSilencer3.1与重组载体pSilencer3.1-Sp1经脂质体LipofectAMINE2000介导转染HeLa细胞,采用RT-PCR、Western blot方法分别检测Sp1基因的转录与表达水平。结果:构建了Sp1基因siRNA真核表达载体pSilencer3.1-Sp1,经酶切、测序鉴定证实克隆正确,并在mRNA水平和蛋白水平证实了载体的干扰效果。结论:特异性siRNA能明显抑制Sp1基因在HeLa细胞中的表达,为进一步研究Sp1的生物学功能和作用机制奠定了实验基础。

Objective：To construct the RNA interference（RNAi） vector targeting Sp1 gene.Methods：According to Sp1 cDNA coding sequence,the specific RNAi fragment targeting Sp1 gene was designed and synthesized,which was cloned into pSilencer 3.1-H1 neo plasmid vector,and the small interfering RNA（siRNA） eukaryotic expression vector pSilencer 3.1-Sp1 targeting Sp1 gene was constructed.The pSilencer 3.1 negtive vector and pSilencer 3.1-Sp1 vector were transfected respectively into HeLa cells by LipofectAMINE2000.RT-PCR and Western blot methods were carried out for the identification of integration of RNAi vectors on mRNA and protein levels.Results：The specific siRNA eukaryotic expression vector pSilencer 3.1-Sp1 targeting Sp1 gene was constructed successfully,and the knockdown effects on mRNA and protein levels were confirmed.Conclusion：Sp1 gene expression can be suppressed markedly by specific siRNA in HeLa cells.Based on the results,the further studying on the biological functions and mechanisms of Sp1 will be carried out.