通过构建6种荧光融合蛋白质粒载体pECFP-IκBα、pECFP-IκBαM、pECFP-IκBα243N、pECFP-IκBα244C、pp53-DsRed和pp53/47C-DsRed，应用激光共聚焦显微镜，观测静止细胞内p53与IκBα及其各种突变体的定位特点及相互作用关系，直观地说明了活体细胞内的p53可与IκBα的C端的PEST区发生相互作用，p53的N端也参与了p53·IκBα复合体的形成. 同时，实时观测外界刺激下细胞内DsRed/CFP两荧光比值的变化，探讨p53·IκBα复合体形成与解离的动态平衡，细霉素B(leptomycin B，LMB) 作用下细胞内p53与IκBα的动力学分布、及核穿梭动态过程的意义. IκBα蛋白可能已经成为p53和NF-κB信号通路的一个交叉点，从而扩展了对于这两条重要信号通路调控的认识.
英文摘要：
Six fluorescent fusion protein plasmids were constructed, and the location and interaction of p53 and IκBα (IκBαM, IκBα243N and IκBα244C) in living cells were investigated with cyan (CFP) and red fluorescent protein(DsRed) fusion proteins by fluorescence resonance energy transfer. p53 interacts with the non-ankyrin C terminus PEST domain of IκBα, and the N terminus in p53 plays an important role in the formation of p53·IκBα complex. The change of DsRed/CFP in response to UV-mediated DNA damage was observed in real time and the balance between p53·IκBα complex formation and dissociation was discussed. In addition, the shuttling of p53 and IκBα between cytoplasm and nucleus under LMB stimulation suggested that the relationship among NF-κB, p53, IκBα, Mdm2, and WOX1 is complicated and remains to be determined. IκBα has been an important cross-talk protein between p53 and NF-κB, and this might expand the understanding of these two signaling pathways considerably.
