smad4 shRNA 真核表达质粒的构建及鉴定

 目的 针对smad4基因的不同部位,构建不同smad4shRNA表达质粒载体,在转录后水平抑制smad4的表达,对其克隆进行鉴定并挑选出抑制效率最高的克隆。方法 用DNA重组技术将针对人smad4基因不同部位所设计的3对shRNA序列克隆到真核表达质粒pGenesil-1中,构建smad4shRNA表达载体pGenesil-smad4-shRNA1、2、3。脂质体介导转染人宫颈癌细胞株HeLa,经G418筛选抑制smad4表达的稳定细胞克隆。结果 3个smad4shRNA表达载体pGenesil-smad4-shRNA1、2、3经限制性酶切及序列分析证明基因插入正确。RT-PCR、Westernblotting均证实：3种shRNA重组质粒中pGenesil-smad4-shRNA2可明显降低细胞内smad4mRNA丰度及smad4蛋白表达。结论 成功构建了smad4shRNA表达载体pGenesil-smad4-shRNA1、2、3,并筛选出特异而高效地阻断smad4表达的克隆。此实验结果为进一步研究smad4蛋白分子的生物学功能及应用奠定了基础。

Construction of Eukaryotic Vector Expressing shRNA of smad4 Gene

Objective To explore the feasibility of selective inhibiting smad4 expression using smad4 short hairpin RNA (shRNA) interference. Methods Three 19bp reverse repeated motifs targeting of smad4 gene were synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pGenesil-shRNA1, 2, 3 and pGenesil-con were transfected into HeLa cells respectively by lipofectamine reagent. The alteration of smad4 expression was examined by RT-PCR and Western blot. Results It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic vector expressing shRNA of smad4 was correct. shRNA2 in pGenesil-smad4 cells knocked down the expression of smad4 mRNA and protein dramatically compared with untransfected and control cells. Conclusion The shRNA can efficiently suppress smad4 expression in HeLa cells. The results of the study lay the foundation for further studying on biological functions and potential application of smad4.