Cdh1小干扰RNA载体的构建及鉴定

Construction and identification of Cdh1 small interfering RNA eukaryotic vector

目的:构建Cdh1小干扰RNA载体,并将其转染至Hela细胞进行鉴定.方法:根据pENTRTM/H1/TO中间载体要求,设计Cdh1小干扰RNA载体的干扰序列及无干扰作用的对照序列,合成相应的DNA单链,退火后连接到pENTRTM/H1/TO线性载体,形成完整载体,进行测序鉴定.分别将测序鉴定成功的Cdh1小干扰RNA载体及对照载体采用脂质体法转染Hela细胞,转染后48h提取细胞总RNA及细胞总蛋白,采用实时定量PCR和WesternBlot检测Cdh1的表达.结果:经测序鉴定成功构建Cdh1小干扰RNA载体和对照载体,分别命名为pENTR/shCdh1和pENTR/shcontrol.与未转染及转染pENTR/shcontrol的Hela细胞相比,转染pENTR/shCdh1的Hela细胞Cdh1表达降低(P<0.05).结论:成功构建Cdh1小干扰RNA载体,并能下调Hela细胞Cdh1的表达.

AIM： To construct Cdhl small interfering RNA （siRNA） eukaryofic expression vector and evaluate its expression in Hela cells. METHODS： The interfering sequence and control sequence for Cdhl small hairpin RNA （shRNA） were designed based on pENTR^TM/H1/TO vector and the oligonucleotides were synthesized. These oligonucleotides were annealed and ligated into linearized pENTR^TM/H1/TO vector respectively. After confirmation by DNA sequencing, positive recombinant plasmids were transfected into Hela cells respectively by the lipesome method. Forty-eight hours later, total cellular RNA and total protein were extracted, then the expression of Cdhl was analyzed by Real Time-PCR and Western Blot. RESULTS： DNA sequencing confirmed the successful construction of Cdhl siRNA eukaryotic expression vector and control vector, named as pENTR/shCdhl and pENTR/shcontrol respectively. Compared with the Hela cells transfected with pENTR/shcontrol or untransfected, the transfected pENTR/shCdhl cells showed significant suppression in the expression of Cdhl （P 〈 0. 05 ）. CONCLUSION： The Cdhl siRNA eukaryotic expression vector is successfully constructed and it can knockdown the expression of Cdhl in Hela cells.