实时定量检测NF-κB p65亚基核易位方法的建立

Establishment of Real-Time Quantitative Method Detecting the Nuclear Translocation of NF-κB p65

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p EGFP-p65,转染He La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He La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-p65fusion protein nuclear translocation.Methods:Recombinant pEGFP-p65was constructed,then transfected into HeLa cells.Live cell imaging wasadopted to observe the subcellular localization of green fluorescent protein.Results:EGFP-p65fusion protein mainly exists in the cytoplasm from0~2h after TNF-αtreatment.The signals of green fluorescence in the nucleusgradually increased from4~12h,while the total fluorescent intensity in cells remained unchanged.Conclusion:TNF-αcan promote the nuclear translocation of EGFP-p65fusion protein.The technique of live cell imaging hasmany advantages over other methods such as simple operation,real-time dynamic,intuitive and quantitative,whichcan be applied to probe the mechanism of HIV latency reactivators.