慢病毒介导shRNA干扰前列腺癌PC3细胞Keap1基因表达的研究

Interference of shRNA on Keap1 in prostate cancer PC3 cell mediated by lentivirus

目的:针对Keap1构建shRNA慢病毒干扰载体,并评价慢病毒介导的RNA干扰在人前列腺癌细胞PC3中的基因沉默效应。方法:利用生物信息学方法设计针对Keap1的RNAi寡聚核苷酸序列;采用慢病毒载体构建Keap1的shRNA载体,利用大肠杆菌进行重组表达,利用293T细胞包装得到重组腺病毒;依据绿色荧光蛋白(GFP)示踪,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靶序列的基因干扰效果。结果:筛选了所构建的4个Keap1靶向序列,以慢病毒载体构建完成对应Keap1的shRNA质粒。通过瞬时转染筛选得到效率最佳(干扰效率达到80%)的靶序列和工作条件。结论:本研究成功构建并筛选了针对Keap1的shRNA慢病毒载体,有效抑制PC3细胞中Keap1的表达。

AIM： To construct the shRNA in- terference lentivirus vector for Keapl and evalu- ate the interference effects in human prostate cancer PC3 cell mediated by lentivirus. METH- ODS： Bioinformatics methods were used to de- sign RNAi sequences for Keapl. A lentiviral vector was applied for construction of Keapl shRNA vectors, which was expressed in E. coli packaged by 293T cells. Transfection efficiency and titer were measured by dilution method ac- cording to the green fluorescent protein （GFP） tracer. Real-time fluorescence quantitative method was applied to compare interfere effects of target sequences. RESULTS： Four Keapl tar-geting sequences were constructed completely and the corresponding Keapl shRNA lentiviral vectors were screened for efficiency. One shR- NA lentiviral vector with best efficiency was screened by transient transfection （interference efficiency reached 80 %） and the working condi- tion was established as well. CONCLUSION. shRNA lentiviral vectors for Keapl was con- structed and screened successfully in PC3 cells, which effectively inhibit the expression of Keapl.