慢病毒介导的shRNA干扰人HepG2.2.15细胞Akt2基因表达的研究

摘要

Objective To construct the shRNA interference lentivirus vector forAkt2 and evaluate its effects on human HepG2.2.15 cell mediated by lentivirus.Methods RNAi sequences forAkt2 were designed using bioinformatics methods. Lentiviral vectors, expressed inE.coliand packaged by 293T cells, were used to construct theAkt2 shRNA vectors. Dilution method was applied to measure the transfection efifciency and titer according to the green lfuorescent protein (GFP) tracer. Real-time lfuorescence quantitative method were used to measure the interference effects of target sequences.ResultsThreeAkt2 targeting sequences were constructed and their corresponding shRNA lentiviral vectors were screened for efifciency. OneAkt2 shRNA lentiviral vector was screened by transient transfection (interference efficiency reached 85%) as the best efifciency and its working condition was established as well.ConclusionAkt2 shRNA lentiviral vectors was successfully constructed and screened, which can inhibit the expression ofAkt2 in human HepG2.2.15 cell effectively.%目的：针对Akt2基因构建shRNA慢病毒干扰载体并评价慢病毒介导的RNA干扰在人HepG2.2.15中的基因沉默效应。方法设计Akt2的RNAi寡聚核苷酸序列，利用慢病毒载体构建Akt2的shRNA载体，转染入大肠埃希菌并观察重组表达状况，利用293T细胞包装得到重组腺病毒，以绿色荧光蛋白（GFP）作为标记，逐孔稀释法确定转染效率及滴度，以实时荧光定量法比较各组对Akt2 mRNA的干扰效果。结果筛选了所构建的3个Akt2靶向序列，包装shRNA慢病毒后转染HepG2.2.15细胞，慢病毒转染后的沉默效率可达85%，比较得出沉默效率最高的靶序列和工作条件。结论本研究成功构建并筛选了针对Akt2的shRNA慢病毒载体，有效抑制HepG2.2.15细胞中Akt2 mRNA的表达。