慢病毒载体介导的人肝癌HepG2细胞BC047440基因沉默  

BC047440 silencing in HepG2 cells mediated by lentiviral vector

作　　者：钟扬 李靖[1] 黄小兵[1] 郑璐[1] 杨彤翰[1] 赵弘智[1] 梁平[1] 

机构地区：[1]第三军医大学新桥医院肝胆外科,重庆400037

出　　处：《第三军医大学学报》2010年第8期744-748,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(30570843)~~

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Western blot检测结果分别显示BC047440-shRNA组中BC047440 mRNA及BC047440蛋白表达较control-shRNA组和HepG2组明显降低,control-shRNA组与HepG2组间无明显差异。结论成功构建BC047440特异性RNAi慢病毒载体,感染人肝癌细胞系HepG2细胞后,实现了对HepG2细胞中BC047440基因的有效沉默。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 HpaⅠ and XhoⅠ,and vector then the results confirmed by PCR and DNA sequencing.293T cells were cotransfected with lentiviral vector pFU-GW-shBC047440,pHelper 1.0 and pHelper 2.0 by Lipofectamine 2000,to produce lentiviral vector which can infect other cell lines.The virus titer and the fittest multiplicity of infection（MOI） in HepG2 were measured by the expression level of green fluorescent protein（GFP）.The infected HepG2 was divided into BC047440-shRNA group,control-shRNA group and HepG2 group.The changes of BC047440 mRNA and protein level were detected by RT-PCR and Western blot analysis respectively.Results The data of PCR demonstrated that the lentivirus RNAi vector of BC047440 was constructed successfully.The titer of virus was 5×108 TU/ml,and the fittest MOI in HepG2 was 20.BC047440 and BC047440 mRNA expression of BC047440-shRNA group was obvious lower than the control-shRNA group and HepG2 group,but there was no visible different between the control-shRNA group and HepG2 group.Conclusion The lentivirus RNAi vector of BC047440 is constructed successfully.After this lentivirus vector infects HepG2 cells,the target of silencing the expression on BC047440 is achieved.

关 键 词：BC047440 RNA干扰 慢病毒载体 人肝癌细胞系HEPG2 

分 类 号：R394.33[医药卫生—医学遗传学] R73-354[医药卫生—基础医学]