靶向MUC1的siRNA表达载体的构建及鉴定

Construction and identification of siRNA targeting MUC1 expression vector

目的构建和鉴定针对黏蛋白1(MUC1)特异性的siRNA表达载体。方法人工合成一对互补并编码相应短发夹状MUC1-siRNA的寡核苷酸链,将其插入pGCsilencerTMU6/Neo/RNAi质粒载体中,用双酶切、阳性克隆聚合酶链反应(PCR)鉴定及DNA测序鉴定所构建的重组载体是否正确;以脂质体法将构建的重组载体导入人胆管癌细胞QBC939中,采用逆转录聚合酶链反应(RT-PCR)和蛋白质印迹法(Western Blot)分别检测转染细胞中MUC1mRNA和蛋白的表达。结果双酶切、PCR鉴定及DNA测序证实MUC1-siRNA真核表达载体构建成功,转染该载体后的QBC939(干扰组)中MUC1mRNA及其蛋白质表达水平明显下调(P<0.05)。结论成功构建的特异性siRNA表达载体,转染QBC939后可特异性地封闭MUC1的表达,为进一步研究MUC1-siRNA载体提供了实验基础。

Objective To constuct and identify a specific siRNA targeting mucins （MUC1） expression vector. Methods A pair of oligonucleotide completing and coding hairpin MUC 1-siRNA synthesized were inserted into pGC silencerTM U6/Neo/RNAi vector. Double enzyme digestion, PCR test and DNA sequencing were used to identify the recombinant plasmid. Then, special MUCI-siRNA was transfeeted into cholangiocarcinoma cells with Lipofeetamine TM 2000. In vitro, MUCI mRNA and protein expression was tested by RT-PCR and Western Blot respectively. Results Double enzyme digestion, PCR test and DNA sequencing confirmed that the MUC1 specific siRNA expression vector was constructed successfully. After transfeetion, the expression of MUC1 mRNA and protein in QBC939 （experimental group） decreased significantly （P 〈 0.05 ）. Conclusions The results indicate that the MUC1-siRNA expression vector was successfully constructed, and the siRNA can significantly inhibit the expression level of MUC 1. This study can provide an experimental basis for further research of MUC 1-siRNA vector.