smad4 shRNA真核表达质粒构建及鉴定.docVIP

smad4 shRNA真核表达质粒构建及鉴定 　 作者：吴鹏，田媛，桂伶俐，陈刚，卢运萍，周剑锋，马丁 【摘要】 目的针对smad4基因的不同部位,构建不同smad4 shRNA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smad4 shRNA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smad4 shRNA 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、Western blotting均证实: 3种shRNA重组质粒中pGenesilsmad4shRNA2可明显降低细胞内smad4 mRNA丰度及smad4蛋白表达。结论成功构建了smad4 shRNA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。 【关键词】 宫颈癌; shRNA; smad4 　　Abstract：Objective To explore the feasibility of selective inhibiting smad4 expression using smad4 short hairpin RNA (shRNA) interference. MethodsThree 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. ResultsIt 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. ConclusionThe 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. 　　Key words：Cervical cancer; shRNA; smad4 　　0引言 　　TGFβ/smads 信号转导通路是由TGFβ超家族、TGFβ受体、smads蛋白家族及其核内转录调节因子组成的肿瘤抑制通路。通路中任何元件异常都可以引起TGFβ/smads 信号转导紊乱,从而导致肿瘤的发生。而smad4是调节转录和TGFβ抗增殖应答的核心分子,在肿瘤中的灭活表明它是抑癌基因，smad4突变或表达丢失与多种肿瘤的发生有关。因此，我们构建了针对smad4的特异性短发夹状RNA(shRNA)真核表达载体,转染宫颈癌细胞株HeLa,得到了基因表达抑制作用较强的永久干涉细胞克隆，为研究宫颈癌细胞的恶性生物学行为奠定基础。 　　1材料与方法 　　1.1材料真核表达质粒