论文部分内容阅读
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 construct and identify siRNA expression vector targeting mucin 1 (MUC1). METHODS: A pair of complementary oligonucleotides encoding the short hairpin MUC1-siRNA was synthesized and inserted into the pGCsilencerTMU6 / Neo / RNAi plasmid vector. The positive clones were identified by double enzyme digestion and PCR (positive cloned polymerase chain reaction) And DNA sequencing. The constructed recombinant vector was introduced into human cholangiocarcinoma cell line QBC939 by lipofectamine. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the recombinant vector. The expression of MUC1 mRNA and protein in transfected cells were detected respectively. Results MUC1-siRNA eukaryotic expression vector was successfully constructed by double enzyme digestion, PCR and DNA sequencing. The expression of MUC1 mRNA and protein in QBC939 (interference group) transfected with this vector was significantly down-regulated (P <0.05). Conclusion The specific siRNA expression vector successfully constructed can specifically block the expression of MUC1 after transfection with QBC939, which provides experimental basis for further study of MUC1-siRNA vector.