| 慢病毒载体介导的人肝癌HepG2细胞BC047440基因沉默 |
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| 引用本文： | 钟扬,李靖,黄小兵,郑璐,杨彤翰,赵弘智,梁平. 慢病毒载体介导的人肝癌HepG2细胞BC047440基因沉默[J]. 第三军医大学学报, 2010, 32(8): 744-748 |
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| 作者姓名： | 钟扬 李靖 黄小兵 郑璐 杨彤翰 赵弘智 梁平 |
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| 作者单位： | 第三军医大学新桥医院肝胆外科,重庆,400037;第三军医大学新桥医院肝胆外科,重庆,400037;第三军医大学新桥医院肝胆外科,重庆,400037;第三军医大学新桥医院肝胆外科,重庆,400037;第三军医大学新桥医院肝胆外科,重庆,400037;第三军医大学新桥医院肝胆外科,重庆,400037;第三军医大学新桥医院肝胆外科,重庆,400037 |
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| 基金项目： | 国家自然科学基金(30570843)~~ |
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| 摘 要： | 目的构建BC047440基因RNAi慢病毒干扰载体,并检测其对人肝癌细胞系HepG2细胞中BC047440基因的沉默效果。方法针对已经筛选确定的BC047440基因RNAi有效靶序列,合成BC047440基因特异性shRNA序列,退火形成双链DNA,与经HpaⅠ和XhoⅠ双酶切后的pFU-GW-iRNA载体连接产生pFU-GW-shBC047440慢病毒载体,PCR、DNA测序鉴定阳性克隆。用脂质体转染法将pFU-GW-shBC047440、pHelper 1.0载体和pHelper 2.0载体共转染293T细胞,产生具备感染能力的慢病毒。以293T细胞中绿色荧光蛋白的表达水平测定病毒滴度,并测定其对人肝癌细胞株HepG2细胞最适感染复数(MOI)。以最适MOI感染HepG2细胞,分BC047440-shRNA组、control-shRNA组和HepG2组;RT-PCR和Western blot检测各组细胞BC047440 mRNA及蛋白的表达差异。结果经PCR鉴定,成功构建BC047440基因RNAi慢病毒载体。测定慢病毒滴度为5×108TU/ml,其在HepG2细胞中最适MOI为20;RT-PCR和Wes...
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| 关 键 词： | BC047440 RNA干扰 慢病毒载体 人肝癌细胞系HepG2 |
BC047440 silencing in HepG2 cells mediated by lentiviral vector |
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| Zhong Yang,Li Jing,Huang Xiaobing,Zheng Lu,Yang Tonghan,Zhao Hongzhi,Liang Ping. BC047440 silencing in HepG2 cells mediated by lentiviral vector[J]. Acta Academiae Medicinae Militaris Tertiae, 2010, 32(8): 744-748 |
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| Authors: | Zhong Yang Li Jing Huang Xiaobing Zheng Lu Yang Tonghan Zhao Hongzhi Liang Ping |
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| Affiliation: | Department of Hepatobiliary Surgery;Xinqiao Hospital;Third Military Medical University;Chongqing;400037;China |
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| Abstract: | Objective To construct a lentivirus vector for RNA interference(RNAi) of BC047440 gene,and detect the silence effect of the vector in human hepatocellular liver carcinoma cell line(HepG2).Methods The targeting sequence of BC047440 gene which can be effectively silenced in RNA inference was confirmed in our previous study.The cDNA encoding for sense and antisense Oligo DNA fragments of the targeting sequence was designed,synthesized and cloned into the pFU-GW-iRNA vector which had been double digested by the... |
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| Keywords: | BC047440 |
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