【摘要】 目的:针对BMI-1基因的不同部位构建4种不同的BMI-1 shRNA真核表达载体,转染人宫颈癌细胞系Hela并筛选其有效序列。方法:应用DNA重组技术将针对人BMI-1基因的不同部位所设计的4对shRNA序列克隆到真核表达质粒psi RNA-hH1neo中,构建BMI-1 shRNA表达载体BMI-1 shRNA1、BMI-1 shRNA2、BMI-1 shRNA3和BMI-1 shRNA4,用阳离子脂质体介导转染Hela细胞以筛选其有效序列。结果:4个BMI-1 shRNA表达载体BMI-1 shRNA1、BMI-1 shRNA2、BM-1 shRNA3和BMI-1shRNA4经测序鉴定证明插入正确。②在Hela细胞中转染BMI-1 shRNA2和BMI-1 shRNA4能显著下调BMI-1mRNA表达,抑制率分别为59.9%和67.4%(P<0.01)。结论:成功构建了真核表达载体BMI-1 shRNA1、BMI-1 shRNA2、BMI-1 shRNA3和BMI-1 shRNA4,其中BMI-1 shRNA2和BMI-1 shRNA4能有效地抑制BMI-1在Hela细胞中的表达。本工作为今后研究BMI-1在肿瘤基因治疗中奠定基础。更多还原

【Abstract】 Objective:To construct eukaryotic vector expressing short hairpin RNA(shRNA)of BMI-1,and to inhibit BMI-1 mRNA expression in human cervix cancer cell line Hela.Method:Designing four different shRNA targeting the coding sequence of BMI-1,the BMI-1 shRNA1,BMI-1 shRNA2,BMI-1 shRNA3 and BMI-1 shRNA4 were constructed by inserting the designed shRNA to the eukaryotic expression vector psiRNA-hH1neo,and transfected into Hela cells with Lipofectamine 2000.Result:It was verified by partial nucleotide sequencing that the constructed eukaryotic vectors expressing shRNA of BMI-1 were correct.The BMI-1 expression of mRNA was significantly suppressed by 59.9% and 67.4% in BMI-1 shRNA 2 and 4 transfected Hela cells as compared with untransfected Hela cells,respectively.Conclusion:The vector of BMI-1 shRNA 1,BMI-1 shRNA 2,BMI-1 shRNA3 and BMI-1 shRNA4 has been successfully constructed.BMI-1 shRNA 2 and BMI-1 shRNA 4 could efficiently suppress BMI-1 expression in Hela cells.This work has laid foundations for further study on cancer gene therapy and potential application of BMI-1.更多还原