宫颈癌细胞放射损伤后IER5蛋白表达及其相互作用蛋白的筛选和验证

Preliminary screening of novel IER5 interacting proteins after ionizing irradiation

的 了解IER5蛋白经放射损伤后的表达变化,并筛选IER5蛋白在放射损伤后可能参与DNA损伤修复通路中的相互作用蛋白.方法 HeLa细胞经4 Gyγ射线照射后在不同时间点收集细胞,提取总蛋白质和细胞核蛋白行Western blot检验;构建3×Flag标签融合表达的IER5蛋白表达载体,转染人肾上皮细胞293T并照射,收集细胞行免疫沉淀富集IER5的结合蛋白,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离免疫共沉淀复合物,考马斯亮蓝染色观察差异条带并切胶酶解后行质谱分析,对可能相互作用蛋白结果进行Western blot验证.结果 IER5蛋白照后4h表达逐渐增加,12 h至高峰,持续至48 h;细胞核内IER5蛋白表达也有增加趋势;质谱分析数据表明,照射组检测出374个蛋白,对照组检测出256个蛋白,两组比较差异蛋白41个,照射组中包括与DNA结合、代谢、损伤修复相关的10个蛋白;其中,与DNA损伤修复密切相关的1型聚ADP核糖基化聚合酶（PARP1）得到Western blot验证.结论 IER5是放射损伤相关蛋白,其可能参与DNA损伤修复通路.

 Objective To investigate the expression of radiation-induced IER5 protein and screen its potential interaction proteins that may participate in DNA repair process.Methods HeLa cells were irradiated with 4 Gy ionizing radiation.IER5 protein expression in whole cell lysate and in nuclear fraction were detected by Western blot at different timepoints after irradiation.3 × Flag-IER5 pCMV plasmid was constrcuted and the Flag tagged-IER5 expression was verified by Western blot.293T cells were transfected with 3 × Flag-IER5 pCMV plasmid.After irradiation the cells were collected and proteins were extracted.The IER5 interaction proteins were purified using immunoprecipitation and separated by 12% SDS-polyacrylamide gel electrophoresis.Then the binding proteins were cut from the gel and analyzed by Mass spectrometry.Results The expression of IER5 protein began to increase 4 hour post-irradiation and its peak level was observed at 12 hour post-irradiation,and it lasted until 48 hour after irradiation.The expression level of IER5 protein in whole cell lysate and nuclear fraction were both increased.With the mass spectrometry analysis,a total of 347 proteins and 256 proteins were identified in irradiated and nonirradiated groups,respectively.Fourty one differential proteins were obtained,where 10 proteins were associated with DNA metabolic process and DNA rapair in the irradiated group and the poly（ADP-ribose） polymerase 1 （PARP1） protein was further confirmed by Western blot.Conclusions IER5 protein is an DNA damage related protein,and it may participate in DNA repair process.