红系分化相关基因相互作用蛋白质的分离与鉴定

Isolation and Identification of EDAG Interacting Proteins

目的制备红系分化相关基因(erythroid differentiation-associated gene,EDAG)蛋白的单克隆抗体,利用免疫沉淀联合质谱技术对EDAG相互作用蛋白质进行分离与鉴定。方法构建EDAG原核表达载体,通过诱导、表达、纯化获得EDAG融合蛋白,杂交瘤技术建立分泌EDAG单克隆抗体的杂交瘤细胞株,利用Western印迹筛选阳性杂交瘤细胞,免疫小鼠得腹水,最后用免疫共沉淀与质谱联合鉴定EDAG相互作用蛋白。结果纯化获得EDAG重组蛋白,筛选出4株分泌EDAG单克隆抗体的杂交瘤细胞株,并制备了腹水。该抗体均可用于内源EDAG蛋白的检测,其中两种抗体可用于免疫共沉淀实验,运用质谱技术筛选获得EDAG候选相互作用蛋白。结论利用EDAG单克隆抗体,筛选到EDAG候选相互作用蛋白质13种,涉及细胞增殖及转录等过程,为EDAG的功能研究及其分子机制的阐明提供了新的线索。

Objective Preparing the monoclonal antibody （mAb） against EDAG, separating and identifying EDAG interacting proteins. Methods The EDAG fusion protein was expressed by gene recombinant and purified with affinity chromatography. Hybridoma cell lines producing antiEDAG mAb were established by cell fusion and selected by Western blot. Co-immunoprecipitation combined with mass spectrum was used to identify EDAG interacting proteins. Results Recombinant EDAG protein was purified. Four hybridoma cell lines secreting anti-EDAG mAb were selected and relevant mouse ascites was prepared. All four variants of ascites were usable to detect the endogenous EDAG protein, two of which were applicable for immunoprecipitation. Furthermore, by mass spectrum technique, thirteen potential EDAG associating proteins were identified to be involved in cellular proliferation and transcription. Conclusions Our results may provide new clues to further elucidate cellular function and molecular mechanism of EDAG.