肝脏特异性表达载体的构建及Western blotting检测  

The Construction and Western Blotting Ananlysis of Liver-specific siRNA

作　　者：张利涛[1] 刘慧琳[1] 曹以诚[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《中国畜牧兽医》2011年第1期100-104,共5页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金重大研究计划项目(90412015)

摘　　要：研究靶向于肝细胞的组织特异性siRNA,实现siRNA基因治疗的组织特异性,可用于特异性治疗乙肝,突破RNA干扰技术在临床应用的一大障碍。用靶向于EGFP的siRNA(以下简称siEGFP)替代靶向于乙肝病毒保守区的siRNA,构建可以在肝细胞中特异性表达的载体,用来表达siRNA。将目标载体分别转染肝癌细胞HepG2、乳腺癌细胞MDB-MB-231、人胚肾细胞293,在蛋白质水平上检验siRNA抑制的组织特异性。Western blotting结果证实,siEGFP在肝组织来源的细胞系HepG2中抑制效率明显高于其他两组细胞。构建的载体可以在肝癌细胞系中特异性的表达siRNA,而在其他组织细胞中不表达,实现了组织特异性。进一步将siEGFP替换为抑制HBV表达的siRNA,同样可以实现肝脏特异性的表达。In order to achieve tissue-specific siRNA therapy,the liver-specific siRNA in vivo expression plasmid had been investigated.It could be used for liver-specific hepatitis B treatment,overcoming a barrier about the siRNA in the clinical application.We used anti-EGFP siRNA instead of the anti-HBV siRNA in this study.The vector was expressed specifically in hepatocyte through construction can express siRNA.The constructed vector had been transiently transfected HepG2,MDA-MB-231,MDA-MB-293 cells respectively.Then the inhibition of siEGFP in the different cell lines had been evaluated and compared by Western blotting ananlysis.siEGFP was proved by Western blotting result to be more efficient to inhibit in the hepatic tissue origin clone HepG2 than other two groups.Tissue specificity was achieved through constructed carrier expressing siRNA specifically in hepatoma cell line,but not in other tissue cell.If we replaced the siEGFP with anti-HBV siRNA in this modified plasmid,it was highly possible to develop a liver-specifically expressing anti-HBV siRNA therapy.

关 键 词：RNAI 组织特异性siRNA 肝脏特异性 EGFP WESTERN BLOTTING 

分 类 号：Q75[生物学—分子生物学]