摘要
目的:以Smad4基因为靶标构建小干扰RNA(siRNA)真核表达载体。方法:根据GenBank公布的人Smad4核酸序列及SiRNA设计原则,用AmbionRNAi在线软件,筛选得到2个19bp片段为靶序列,合成两端带有BamHⅠ、HindⅢ酶切位点的发夹结构寡核苷酸序列,经过退火,将此序列克隆到真核表达质粒pSilencerTM3.1-H1neovector中,构建成重组质粒,酶切及测序验证。脂质体介导转染人原代增生性瘢痕成纤维细胞,经G418筛选细胞克隆,并用RT-PCR检测Smad4基因的表达。结果:成功构建了pSilencerTM3.1-H1neo-Smad4shRNA表达载体克隆,插入片段测序结果与合成的siRNA序列一致。RT-PCR结果显示转染shRNA1、shRNA2重组质粒的成纤维细胞内Smad4 mRNA水平均降低,其中以pSilencerTM3.1-H1-Smad4 shRNA1更为明显(P<0.01)。结论:构建p-Smad4 shRNA表达载体成功,为进一步研究Smad4基因的RNA干扰奠定了基础。
Objective： To construct a eukaryotic expression vector for small interfering RNA （siRNA） targeting Smad4 gene. Methods： According to Smad4 mRNA sequence in the Genbank, three pairs of 58nt oligonucleotides, each containing the sites of restriction endonuclease at both ends, were designed and synthesized. Oligonucleotides were annealed and ligated with linearized pSilencerTM 3.1-H1 neo by T4 DNA ligase. The recombinants were finally sequenced and identified by enzyme digestion and sequencing. The recombinant plasmids were transfected into fibroblasts respectively by lipofectamine reagent. The stable cell clones were screened with G418. The alteration of Smad4 expression was examined by RT-PCR. Results： pSilencer^TM 3.1-H1 neo -Smad4 expression vector were successfully constructed and identified by double endonuclease digestion. Sequence analysis revealed that inserted fragment was identical to the synthesized siRNA oligonucleotides. The result of RT-PCR showed that transfection of pSilencer^TM 3. 1-H1 neo -Smad4 shRNA 1 significantly down-regulated the Smad4 mRNA expression. Conclusions： The recombinant plasmid expressing the siRNA targeting Smad4 gene has been successfully constructed, laying a foundation for study on RNA interference of the Smad4 gene.
出处
《现代生物医学进展》
CAS
2009年第6期1082-1084,1094,共4页
Progress in Modern Biomedicine
基金
湖南省卫生厅中医药局科研基金(No:06103-22)
湖南省教育厅青年基金(No:08B065)
