通過構(gòu)建6種熒光融合蛋白質(zhì)粒載體pECFP-IκBα,、pECFP-IκBαM,、pECFP-IκBα243N、pECFP-IκBα244C,、pp53-DsRed和pp53/47C-DsRed,，應(yīng)用激光共聚焦顯微鏡，觀測(cè)靜止細(xì)胞內(nèi)p53與IκBα及其各種突變體的定位特點(diǎn)及相互作用關(guān)系,，直觀地說明了活體細(xì)胞內(nèi)的p53可與IκBα的C端的PEST區(qū)發(fā)生相互作用,，p53的N端也參與了p53·IκBα復(fù)合體的形成. 同時(shí)，實(shí)時(shí)觀測(cè)外界刺激下細(xì)胞內(nèi)DsRed/CFP兩熒光比值的變化,，探討p53·IκBα復(fù)合體形成與解離的動(dòng)態(tài)平衡,，細(xì)霉素B(leptomycin B,，LMB) 作用下細(xì)胞內(nèi)p53與IκBα的動(dòng)力學(xué)分布,、及核穿梭動(dòng)態(tài)過程的意義. IκBα蛋白可能已經(jīng)成為p53和NF-κB信號(hào)通路的一個(gè)交叉點(diǎn)，從而擴(kuò)展了對(duì)于這兩條重要信號(hào)通路調(diào)控的認(rèn)識(shí).
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.
