【摘要】 目的构建Smad3特异的RNA干扰质粒载体,为探讨抑制Smad3表达在肝纤维化治疗中的意义奠定基础。方法根据GenBank数据库提供的Smad3基因核苷酸序列,选择设计能转录小发夹结构RNA(small hairpinRNAs,shRNA)的DNA序列,并与pSilencer3.1-H1-hygro质粒载体连接,构建真核表达载体,使用限制性内切酶鉴定及测序鉴定是否为阳性克隆。结果成功构建pSilencer3.1-Smad3载体,拟进一步采用RNAi技术观察其对Smad3基因表达的抑制情况,从而研究其在肝纤维化治疗中的作用。结论 Smad3-siRNA真核表达载体被成功构建。更多还原

【Abstract】 Objective To construct eukaryotic expression vector of RNA interference specific for Smad3,and to lay a foundation for exploring the significance of Smad3 inhibition in treatment of hepatic fibrosis. Methods Genome sequences of Smad3 gene were retrieved from Genbank and cDNA was designed to code expression of shRNA(small hairpin RNAs) for Smad3 gene.The cDNA was synthesized and inserted into pSilencer3.1-H1-hygro which was eukaryotic expression vector and identified by the restriction map and sequencing. Results The recombinant plasmid of RNA interference specific for Smad3 identified by the restriction map and sequencing completely coincided with the designs. Conclusions The siRNA eukaryotic expression vector against Smad3 mRNA has been successfully constructed.更多还原