BMI-1 shRNA载体的构建及其在Hela细胞中的表达

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psiRNA—hH1neo中，构建BMI-1shRNA表达载体BMI-1shRNA1、BMI-1shRNA2、BMI-1shRNA3和BMI-1shRNA4，用阳离子脂质体介导转染Hela细胞以筛选其有效序列。结果：①4个BMI-1shRNA表达载体BMI-1shRNA1、BMI-1shRNA2、BM-1shRNA3和BMI-1shRNA4经测序鉴定证明插入正确。②在Hela细胞中转染BMI-1shRNA2和BMI-1shRNA4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-1shRNA1、BMI-1shRNA2、BMI-1shRNA3和BMI-1shRNA4，其中BMI-1shRNA2和BMI-1shRNA4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。

Construction of eukaryotic vector BMI-1 shRNA and its inhibition in Hela cells

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 transfeered 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.