APE1和p53蛋白在A549细胞和HeLa细胞中的结合作用

发布时间：2018-02-28 13:54

本文关键词： APE蛋白 p蛋白相互作用　出处：《第三军医大学学报》2013年21期 　论文类型：期刊论文

【摘要】：目的探讨脱嘌呤/脱嘧啶核酸内切酶(apurinic/apyrimidinic endonuclease,APE1)和p53蛋白内源性和外源性相互结合作用。方法将重组质粒pET42a-hAPE1转染E.coli BL21(DE3),应用IPTG诱导GST-APE1蛋白表达,鉴定后经金属螯合层析技术纯化,通过Western blot鉴定纯化蛋白,得到GST-APE1融合蛋白。免疫共沉淀(Co-Immunoprecipitation,Co-IP)和GST蛋白沉降实验分别检测内源性和外源性APE1和p53蛋白结合作用,免疫荧光标记结合激光共聚焦显微镜技术确定APE1和p53蛋白亚细胞定位。结果转染质粒经IPTG诱导表达,应用SDS-PAGE发现GST-APE1在预期位置64×103处存在阳性条带,"Ni"柱纯化后得到GST-APE1融合蛋白,通过Western blot鉴定。免疫共沉淀和GST蛋白沉降实验证实APE1和p53存在内源性和外源性结合作用。激光共聚焦显微镜观察免疫荧光标记的APE1和p53蛋白均为核浆共表达蛋白,氧化应激处理后,蛋白逐渐由胞浆向胞核移位,二者共定位于核膜处较明显。结论 APE1和p53蛋白在A549和HeLa细胞中存在内源性和外源性结合作用,可能与其转录调控及肿瘤放化疗疗效有关。
[Abstract]:Objective to investigate the interaction between apurinic / aprimidinic endonuclease (APE1) and p53 protein. Methods Recombinant plasmid pET42a-hAPE1 was transfected into E. coli BL21NDE3, GST-APE1 protein was induced by IPTG and purified by metal chelation chromatography. GST-APE1 fusion protein, Co-immunoprecipitation Co-IP) and GST protein sedimentation assay were used to detect the binding effect of endogenous and exogenous APE1 and p53 protein, respectively. The subcellular localization of APE1 and p53 protein was determined by immunofluorescence labeling and laser confocal microscopy. Results the expression of the transfected plasmid was induced by IPTG. The positive bands of GST-APE1 were found at the expected position of 64 脳 10 ~ 3 by SDS-PAGE, and the fusion protein of GST-APE1 was obtained after purification by "Ni" column. The results of Western blot identification, immunoprecipitation and GST protein sedimentation showed that APE1 and p53 had both endogenous and exogenous binding effects. Laser confocal microscopy showed that both APE1 and p53 proteins labeled by immunofluorescence were nucleoplasmic coexpression proteins. After oxidative stress, the proteins were gradually translocated from cytoplasm to nucleus, and the two proteins were located in the nuclear membrane. Conclusion APE1 and p53 proteins have endogenous and exogenous binding effects in A549 and HeLa cells. It may be related to its transcriptional regulation and the curative effect of radiotherapy and chemotherapy.
【作者单位】：第三军医大学大坪医院野战外科研究所肿瘤中心;第三军医大学大坪医院野战外科研究所病理科;
【基金】：国家自然科学基金(81101783,81272499) 重庆市自然科学基金(CSTC2010BB5198)~~
【分类号】：R341

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