21.5kD MBP RNAi有效靶点的筛选及验证

目的:构建21.5 kD MBP基因干扰序列21.5 kD MBP-shRNA(21.5 kD MBP short hairpin RNA interference),筛选最佳沉默效果的21.5 kD MBP-shRNA真核表达载体,为21.5 kD MBP基因功能研究提供实验材料。方法:将21.5 kD MBP-shRNA阳性真核表达载体和阴性真核表达载体经脂质体介导分别转入人神经胶质瘤细胞株U251中,观察转染24 h后的转染效率;提取各组细胞总RNA,用实时荧光定量PCR技术检测各组21.5 kD MBP基因在mRNA水平的表达差异性。结果:21.5kD MBP-shRNA真核表达重组载体被成功转染入U251细胞,与阴性对照质粒(pGenesil-1-MBP-neg)转染组相比,沉默质粒转染组(pGenesil-1-MBP-1、pGenesil-1-MBP-2、pGenesil-1-MBP-3质粒分别转染)细胞中21.5 kD MBP mRNA表过水平均显著下降(P<0.05),其中pGenesil-1-MBP-3转染组细胞中21.5 kD MBP mRNA表达水平最低。结论:成功筛选出最佳沉默效果的21.5 kD MBP-shRNA真核表达载体,为21.5 kD MBP基因功能研究及基因治疗研究奠定了实验基础。

The screening and verification of the effective target for 21.5 kD MBP RNAi

Objective:To construct the short hairpin RNA(shRNA) interference expression plasmid vectors of 21.5 kD MBP gene and choose the shRNA eukaryotic expression vector with the best silencing effects on the 21.5 kD MBP gene expression so as to provide experimental materials for the 21.5 kD MBP gene function research.Methods:The 21.5 kD MBP shRNA positive and negative eukaryotic expression vectors were respectively transfected into human glioma cell line U251 by liposome-mediated.The transfection efficiency was observed 24 h after the cells were transfected,and then the total RNAs of each groups were extracted,finally the 21.5 kD MBP gene expression of each groups at mRNA level was detected by real-time quantitative PCR.Results:The 21.5 kD MBP shRNA eukaryotic expression recombinant vectors were successfully transfected into U251 cells.Compared with the negative control group(transfected by pGenesil-1-MBP-neg),the 21.5 kD MBP expressions at mRNA level in silence plasmid transfected groups(transfected by pGenesil-1-MBP-1,pGenesil-1-MBP-2 and pGenesil-1-MBP-3 respectively) were decreased significantly(P<0.05),especially for the pGenesil-1-MBP-3 transfected group.Conclusion: The best shRNA expression plasmid vector which has the strongest silencing effects on the 21.5 kD MBP gene expression at mRNA level is successfully obtained,which will establish a good experimental basis for the 21.5 kD MBP gene function and gene therapy research.