RNA干扰ezrin基因真核生物表达载体的构建及鉴定

摘要

Objective To construct the expression vector of small hairpin RNA(shRNA) and to test its efficiency in silencing ezrin gene.Methods According to the ezrin cDNA sequence in GenBank,2 pair oligo nucleotides were designed and synthesized.After primer annealing,they were inserted into plasmid pGenSil-l to construct the shRNA eukaryotic expression vector.The recombinant plasmid was transformed into DH5a and the positive strain was identified by enzyme digestion and sequence analysis.The recombinant plasmid were transfected into 786-0 cells with Lipofectamine 2000.Then the expression level of ezrin gene was analyzed by the real-time fluorescent quantitative polymerase chain reaction (qRT-PCR)and Western bloting.Results The restriction enzyme analyses demonstrated that shRNA was inserted into vectors.Sequencing analysis demonstrated that shRNA was inserted into vectors and sequencing analyses demonstrated that the sequences were as same as the designed.The results of qRT-PCR and Western bloting showed that the expression of ezrin gene was reduced dramatically in mRNA and at protein level in shRNA-ezrinl.Conclusions We have successfully constructed shRNA eukaryotic expression vectors targeting at ezrin gene.shRNA-ezrinl can efficiently suppress ezrin expression in 786-0 cells,which is important for further research on ezrin gene function in 786-0 cell and the future RNAi experimental research in vivo and in vitro.%目的 构建用于RNA干扰(RNAi)的小发夹RNAshRNA表达载体并检测其对ezrin基因的沉默效果.方法 以ezrin为靶基因,以pGenesil-1质粒为载体,设计和构建重组体,根据GeneBank数据库提供的ezrin核苷酸序列,按照Tuschl设计原则,设计2条小发夹结构的DNA序列,经退火形成互补双链,克隆到空载体pGenesil-1中,转化DH5a菌株,提取质粒,予以酶切和测序鉴定；重组质粒转染786-0肾癌细胞株,运用实时荧光定量PCR和蛋白质印迹法进行筛选鉴定.结果 酶切及测序鉴定表明成功地构建重组质粒shRNA-ezrinl、shRNA-ezrin2；荧光实时定量PCR和蛋白质印迹法检测结果显示,肾癌细胞中shRNA-ezrinl、shRNA-ezrin2 mRNA相对表达量分别为(0.3376±0.0166)及(0.4661±0.0266),shRNA-ezrinl与shRNA-ezrin2相对表达量差异有统计学意义(P＜0.01),根据基相对表达量算出shRNA-ezrinl,shRNA-ezrin2 ezrin-mRNA的表达量抑制率分别为66.33％及53.29％.转染重组质粒后显著抑制786-0细胞中ezrin mRNA和蛋白表达,其中shRNA-ezrinl的抑制效率最高.结论 成功构建ezrin基因的shRNA表达载体,并筛选出抑制率较高的重组质粒载体shRNA-ezrinl,为进一步研究ezrin基因沉默对肾癌786-0细胞株生物学行为的影响奠定基础.