实时定量检测NF-κB p65亚基核易位方法的建立

摘要

目的:建立可直观检测NF-κB p65亚基核易位的方法.方法:构建NF-κB p65的真核表达重组质粒pEG-FP-p65,转染HeLa细胞,加入肿瘤坏死因子α(TNF-α)后采用活细胞成像技术观察绿色荧光蛋白在HeLa细胞内的变化.结果:TNF-α处理后0～2 h,EGFP-p65融合蛋白主要聚集在胞浆处;TNF-α处理后4～12 h,随处理时间的延长,细胞核处的绿色荧光蛋白逐渐增强,但细胞内总荧光值基本保持稳定.结论:TNF-α可以促进EGFP-p65入核.活细胞成像技术具有操作简单、实时动态、直观、定量等优点,可用于探讨人类免疫缺陷病毒(HIV)潜伏感染再激活剂的作用机制.%Objective:To establish a feasible method that visualized EGFP-p65 fusion protein nuclear translocation.Methods:Recombinant pEGFP-p65 was constructed,then transfected into HeLa cells.Live cell imaging was adopted to observe the subcellular localization of green fluorescent protein.Results:EGFP-p65 fusion protein mainly exists in the cytoplasm from 0～2 h after TNF-α treatment.The signals of green fluorescence in the nucleus gradually increased from 4～12 h,while the total fluorescent intensity in cells remained unchanged.Conclusion:TNF-α can promote the nuclear translocation of EGFP-p65 fusion protein.The technique of live cell imaging has many advantages over other methods such as simple operation,real-time dynamic,intuitive and quantitative,which can be applied to probe the mechanism of HIV latency reactivators.