期刊论文

中文

现代生物医学进展

1673-6273

2009

9

6

1082-1084+1094

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）.

本校为第一机构

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19 bp片段为靶序列,合成两端带有Bam H Ⅰ、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-H1 neo vector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.H1 neo-Smad4 shRNA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...

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 fi...