基于免疫共沉淀-液质联用技术的新型孕烷X受体配体检测方法的建立  

Identification of Pregnane X Receptor Ligands via an Immunoprecipitation/Liquid Chromatography Tandem Mass Spectrometry Method

作　　者：赵爽[1] 姜棋予 孙慧伟 柴燕涛 李晓娟 王志杰 侯俊[2] 赵敏[1] ZHAO Shuang;JIANG Qi-Yu;SUN Hui-Wei;CHAI Yan-Tao;LI Xiao-Juan;WANG Zhi-Jie;HOU Jun;ZHAO Min(The 302 Military Hospital of China-Peking University Teaching Hospital, Beijing 100039;Research Center for Clinical and Translational Medicine, 302nd Hospital of Chinese PLA, Beijing 100039 China)

机构地区：[1]北京大学三〇二临床医学院,北京100039 [2]解放军第三〇二医院临床研究管理中心,北京100039

出　　处：《生物技术通讯》2018年第3期397-403,共7页Letters in Biotechnology

基　　金：国家自然科学基金(81470097)

摘　　要：目的:建立基于免疫共沉淀-液质联用技术的新型孕烷X受体(PXR)配体检测方法。方法:在HEK293细胞中转染带有Flag标签的PXR表达载体,裂解细胞后用偶联Flag抗体的微珠(beads)结合并分离细胞中表达的FlagPXR蛋白;以PXR的已知最为公认的配体/激动剂利福平为模型药物,配制1μmol/L利福平溶液,与结合有Flag-PXR蛋白的微珠孵育形成微珠-蛋白-利福平复合物;将微珠从体系中分离出来,用蛋白印迹实验检测复合物中的蛋白质,用液相色谱-质谱联用技术(液质联用技术)检测复合物中的利福平。在此基础上对利福平的作用进行验证,在肝细胞癌细胞Hep G2中检测系列浓度梯度的利福平对PXR转录因子活性的影响。结果:用免疫共沉淀技术从HEK293细胞中分离鉴定得到Flag-PXR蛋白;用液质联用技术检测到蛋白与小分子复合物中的利福平;利福平能够剂量依赖地诱导PXR的转录因子活性。结论:建立了基于免疫共沉淀-液质联用技术的新型PXR配体检测方法。Objective： To Identify the potential ligands of pregnane X receptor（PXR） via an immunoprecipitation/liquid chromatography tandem mass spectrometry method. Methods： HEK293 cells were transfected with Flag-PXR expression vectors. Next, cells were harvested for immunoprecipitation assays. Flag-PXR was incubated and separated by Flag-beads, and identified by Western blot. The Rifampicin in complex was identified by liquid chromatography tandem mass spectrometry（LC-MS/MS）. To determine the specificity of Rifampicin＇s effect on PXR, the transcription factor activity of PXR was identified by luciferase experiments. Results： Flag-PXR fusion protein was expressed and separated from HEK293 cells. Rifampicin was identified by the transition of molecular weight m/z823 Da/792 Da during LC-MS/MS. Protein in complex was identified by immunoblotting, whereas Rifampicin in complex was identified by LC-MS/MS. Moreover Rifampicin enhanced the transcription factor activity. Conclusion：This work developed a novel methods to identified the interaction between PXR and Rifampicin.

关 键 词：孕烷X受体 免疫共沉淀 蛋白-小分子复合物 液质联用 

分 类 号：Q25[生物学—细胞生物学] Q78