突变型Myocilin真核表达载体的构建及体外表达  

Construction of Mutant Myocilin Plasmid and Its Expression in Human Trabecular Meshwork Cells

作　　者：王莉娜[1] 葛坚[1] 刘炳乾[1] 宋革[1] 侯飞[1] 

机构地区：[1]眼科学教育部重点实验室中山大学中山眼科中心,广州510060

出　　处：《眼科学报》2006年第2期125-128,共4页Eye Science

基　　金：国家自然科学基金(30271386;30300380);国家自然科学基金创新研究群体基金(30321004);卫生部临床重点学科项目(3030902005);广东省自然科学基金(36649;021823);全国百优博士论文作者专项基金(200363)

摘　　要：目的:构建P370L突变型Myocilin真核表达载体及体外转染体系,为Myocilin功能研究提供实验基础。方法:应用定点诱变技术进行Myocilin基因第1131位氨基酸残基位点的定点突变,利用阳离子脂质体介导体外转染技术瞬时转染人小梁细胞,RT-PCR和WesternBlot检测Myocilin表达.结果:经克隆、测序证实获得突变基因,突变载体转染后人小梁细胞MyocilinmRNA和Myocilin蛋白质表达显著增加。结论:P370L突变型Myocilin真核表达载体成功构建,阳离子脂质体是人小梁细胞有效的体外转染体系。Purpose： To establish the mutant Myocilin gene plasmid in order to study the function of Myocilin. Methods： The mutant site was induced by site-directed mutagenesis. This plasmid were transfected into human trabecular meshwork cells （ HTM cells） by cationic liposomes and the expression of Myocilin was examined by RT-PCR and Western blot analysis. Results ： The mutant plasmid were correctly constructed. Myocilin was efficiently expressed in HTM cells transfected with this plasmid. Conclusion： The constructed eukaryote expression plasmid pcDNA-MYOC-P370L could express Myocilin in HTM cells in vitro.

关 键 词：定点突变 脂质体 转染 MYOCILIN 

分 类 号：R346[医药卫生—基础医学] R775