【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。更多还原

【Abstract】 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.更多还原