作　　者： ; ; ; ; ; ;

机构地区： 深圳市疾病预防控制中心

出　　处： 《癌变．畸变．突变》 2012年第4期290-294,共5页

摘　　要： 目的:构建CYP2E1基因shRNA慢病毒表达载体,为研究CYP2E1在药物代谢和环境污染物代谢中的作用提供技术支持。方法:通过NCBI检索CYP2E1序列,设计合成3对shRNA片段,退火后连接到慢病毒载体PLKO.1-puro,将重组质粒转化到感受态JM107中。挑取筛选的单菌落,经PCR和测序鉴定后提取质粒。将质粒和包膜蛋白质粒共转染到293FT细胞,收集病毒上清,转导肝细胞L02,利用嘌呤霉素筛选得到CYP2E1缺陷型细胞,最后利用荧光定量PCR和Western blot鉴定干扰效果结果:PCR和测序结果证明双链shRNA已经正确插入慢病毒载体PLKO.1-puro,共转染293FT细胞得到高滴度病毒,转导L02细胞筛选出CYP2E1基因沉默细胞,荧光定量PCR检测shRNA3的最高抑制效率为86.9%,Western blot鉴定shRNA3基本无CYP2F1表达。结论:利用慢病毒介导RNAi技术成功构建了CYP2E1基因沉默细胞。 OBJECTIVE： To construct and identify the lentivirus vectors that interfere CYP2EI gene expression in human hepatocytes （L02 cells）, so that these vectors could be applied for further studies of CYP2E1 in metabolism of environmental pollutants and drugs. METHODS ： Three pairs of shRNA were designed and synthesized, then ligated to the lentivirus PLKO.1 vector. The lentivirus vectors with shRNA targeting human CYP2E1 mRNA were transfected into 293FT cells by Lipofectamine, then the lentivirus supernatant was obtained and used for infecting 1.02 cells. After one week of selection with puromycin, the CYP2E 1-silent cells were obtained, then the efficiency of gene knockdown were determined by Real-time PCR and western blot. RESULTS： PCR and western blot data showed that shRNA were successfully inserted into PLKO.lvector, restructured PLKO.lvector was transfected into 293FT cells and high-titer lentivirus was formed. The lentivirus was transducted into L02 cells, CYP2El-silent cells were obtained after selection. PCR data showed the highest inhibitory efficiency was approximately 86.9% for CYP2E1 expression, and western blot data indicated that no obvious band came out in L02 cells with shRNA3 interfering target. CONCLUSION： CYP2El-silent cells were successfully constructed by using lentivirus-mediated RNA interference technology.

关 键 词： 干扰 慢病毒 载体

领　　域： [生物学]