Smad2小干扰RNA载体的构建及其沉默效应的鉴定

目的构建smad2特异性小干扰RNA（siRNA）真核表达载体,并检测肿瘤细胞中其对smad2基因表达的干扰效果。方法根据smad2基因序列设计合理的smad2 siRNA,并将合成的寡核苷酸序列克隆到pSliencer 2.1-U6 neo载体中,转化大肠杆菌DH5α;挑取阳性菌落进行质粒酶切和序列分析;将构建正确的smad2 siRNA重组质粒转染,或与带Flag标签的smads真核表达载体共同转染293T细胞或HeLa细胞,收集细胞裂解物,Westem印迹检测siRNA的干扰效果。结果正确构建了smad2 siRNA重组质粒,对smad2表达的特异性干扰效果可达70%以上。结论成功构建了smad2 siRNA载体,为进一步探讨smad2在肿瘤发生发展中的作用奠定了基础。

Construction of siRNA Specific for Smad2 and Identification of its Efficiency in Carcinoma Cell Line

Objective To construct the small interfering RNA(siRNA) eukaryotic expression vector specific for smad2 and to observe its silencing effect on smad2 gene in carcinoma cells.Methods Oligonucleotides were designed specific for smad2 gene and cloned into siRNAs expression vector Psilencer2.1-U6 neo,then transformed into E.coli DH5α.The expression vectors were identified by enzyme digestion and sequence analysis.The smad2 siRNA vector was transfected or co-transfected with Flag-smads expression vectors into 293T cells and Hela cells.The expression of smad2 was analyzed by Western blot.Results The result of restriction enzyme digestion and nucleotide sequencing confirmed that the recombinant plasmids were correct.In transfected 293T cells and HeLa cells,Smad2 protein expression level was suppressed over 70%.Conclusion The Psilencer2.1-U6/siRNA specific for smad2 were successfully constructed,which laid foundation for further study of smad2 in base research of cancer.