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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。
Objective To construct Smad3-specific RNA interference plasmid vector and lay a foundation for exploring the significance of inhibiting Smad3 expression in the treatment of liver fibrosis. Methods According to the nucleotide sequence of Smad3 gene provided by GenBank database, a DNA sequence encoding small hairpin RNAs (shRNA) was designed and ligated with plasmid pSilencer3.1-H1-hygro to construct eukaryotic expression vector , The use of restriction enzyme identification and sequencing to identify whether the positive clones. Results The pSilencer3.1-Smad3 vector was successfully constructed, and its inhibitory effect on Smad3 gene expression was further investigated by RNAi technique to investigate its role in the treatment of liver fibrosis. Conclusion Smad3-siRNA eukaryotic expression vector was successfully constructed.