靶向MAG基因shRNA慢病毒载体的构建及其对MAG基因表达的沉默  

Construction of short hairpin RNA lentiviral vector targeting MAG gene and its silencing effect on MAG gene

作　　者：陈舒[1] 罗铭[2] 蔡望青[1] 李方成[1] 何志威[3] 刘安民[1] 

机构地区：[1]中山大学孙逸仙纪念医院神经外科,广东广州510120 [2]中山大学孙逸仙纪念医院肿瘤科,广东广州510120 [3]香港中文大学

出　　处：《中国病理生理杂志》2013年第9期1725-1728,共4页Chinese Journal of Pathophysiology

基　　金：广东省自然科学基金资助项目(No.S2011010002638);广东省科技社会发展项目(No.2012B031800356)

摘　　要：目的:设计以MAG基因为靶点的短发夹状RNA(shRNA),构建重组慢病毒载体并鉴定此RNA干扰体系对MAG基因表达的影响。方法:将3条靶向大鼠MAG基因的shRNA片段插入至慢病毒载体pWPI,与pCDNA3-MAG-FLAG质粒用Lipofectamine 2000共转染293T细胞,Western blotting法鉴定出最有效的shRNA。此重组质粒与pAX2和pMD2G经293T细胞包装后,产生的重组慢病毒感染少突胶质细胞,48 h后Western blotting法检测MAG蛋白的表达情况。结果:经双酶切后测序鉴定,构建了MAG shRNA慢病毒载体pWPI-MAG,鉴定出shRNA-2为最为有效的shRNA。重组慢病毒能明显抑制少突胶质细胞中MAG的表达。结论:慢病毒介导的shRNA干扰技术可特异性阻断MAG的表达,为进一步探讨MAG特异性shRNA治疗神经系统髓鞘损伤奠定了基础。AIM：To construct lentiviral vector-based short hairpin RNA （shRNA） targeting myelin-associated glycoprotein （MAG） gene and to evaluate its inhibitory effect on the expression of MAG gene. METHODS：Three shRNA fragments targeting MAG gene （shRNA1, shRNA2 and shRNA3） were designed and cloned into lentiviral vector pWPI. The three recombinant plasmids were identified by enzyme digestion and sequencing. Positive plasmids were co-transfected with pCDNA3-MAG-FLAG into 293T cells, and the most effective shRNA for knockdown of MAG gene was screened by Western blotting. Cells transfected with empty pWPI served as a control. Oligodendrocytes were infected with recombinant lentivirus that was produced by 293T packaging cells co-transfected with the most effective shRNA, pAX2 and pMD2G. After 48 h, the expression of MAG protein was measured by Western blotting. RESULTS：The MAG shRNA lentiviral vectors were confirmed by double enzyme digestion and sequencing, and shRNA2 showed the highest inhibitory efficacy among the three shRNA fragments. Recombinant lentivirus carrying shRNA-2 markedly decreased the expression of MAG protein in oligodendrocytes. CONCLUSION：Lentiviral vector-based shRNA targeting MAG gene specifically knocks down the gene expression, which provides a useful tool for investigating the role of MAG-specific shRNA in regulating myelination of central nerve system.

关 键 词：短发夹状RNA 髓鞘相关糖蛋白 慢病毒 

分 类 号：R737.25[医药卫生—肿瘤]