人STAT3基因siRNA真核表达质粒的构建及鉴定

Construction and identification of the eukaryotic vector expressing siRNA targeting human STAT3 gene

目的:构建针对人STAT3基因的siRNA真核表达质粒,检测其在细胞水平对STAT3基因表达的抑制效果。方法:用DNA重组技术将针对人STAT3基因mRNA序列不同位点设计的3个siRNA序列克隆到真核表达质粒pRNAT-U6.1/neo中构建重组体pRNAT-U6.1-siRNA,重组质粒经PCR检测及测序分析,用脂质体转染重组质粒至人食管癌Eca-109细胞,G418筛选获得阳性克隆,RT-PCR和Western blot检测STAT3基因mRNA和蛋白的表达,筛选最佳沉默效率的siRNA。结果:PCR检测及测序分析结果均提示重组质粒构建正确。RT-PCR和Western blot检测证实pRNAT-U6.1-siRNA3具有最佳的沉默效率。结论:成功构建人STAT3基因siRNA真核表达质粒,并证实其能够从mRNA和蛋白水平抑制STAT3基因的表达。

Objective：To construct siRNA eukaryotic expression vector targeting of human STAT3 gene,and identify its suppressive effect.Methods：Three specific siRNAs targeting of human STAT3 gene were synthesized and cloned into eukaryotic expression plasmid pRNAT-U6.1/neo to reconstruct recombinant plasmid pRNAT-U6.1-siRNA-STAT3,which was then identified by PCR test and DNA sequence analysis.The recombinant plasmid was transfected into human esophageal carcinoma cell line Eca-109,the positive cell clones were screened with G418,the suppression effect of STAT3 mRNA and protein was measured by RT-PCR and Western blot to select the optimal siRNA.Results：PCR test and DNA sequence analysis showed that the recombinant plasmid was constructed successfully.RT-PCR and Western blot analyses demonstrated that the optimal siRNA which could effectively silence the target gene pRNAT-U6.1-siRNA3.Conclusion：The siRNA eukaryotic expression vector targeting of human STAT3 gene was constructed successfully,which could inhibit the expression of STAT3 gene.