人IgE分子Fc段受体结合区的表达、纯化及功能鉴定  

Molecular cloning,expression,purification,and characterization of Fc fragment of human IgE with CHO expression system

作　　者：李晶[1] 郭怀祖[1] 杨扬[1] 钱卫珠[1] 张大鹏[1] 薛静亚[1] 朱磊[1] 王皓[1] 

机构地区：[1]第二军医大学肿瘤研究所,上海200433

出　　处：《现代免疫学》2011年第6期445-449,共5页Current Immunology

摘　　要：利用哺乳动物细胞表达、纯化具有生物学功能的人IgE Fc段与受体结合的功能区(Fcε2-4)。以CRL8033细胞株为模板,PCR扩增人IgE Fcε2-4的cDNA并构建到真核表达载体pcDNA3.1(+)中,脂质体转染CHO-K1细胞并以G418加压筛选。利用有限稀释法、Dot-blot及FACS筛选出高表达目的蛋白的稳定细胞株并进行扩增。CHO-K1培养上清经偶联抗IgE抗体(Omalizumab)的亲和层析柱分离纯化后,聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定纯度,并采用表面等离子共振(SPR)法对该蛋白与其抗体的亲和力进行检测。结果表明,SDS-PAGE显示最终获得了纯度高于95%的人IgE Fcε2-4蛋白,呈二聚体状态,分子量约78kD,SPR检测结果显示,该蛋白与Omalizumab抗体特异性高亲和力结合(kD值约3.8nmol/L)。提示,本实验获得了结构及糖基化修饰正确的人IgE Fcε2-4结构域,为进一步研究其功能及探索新的抗体药物奠定了基础。In the present study,gene cloning,plasmid construction,CHO expression,purification,and biological activity of Fc fragment of human IgE were reported,in which the cDNA was constructed from the total RNA of CRL8033 cells extracted,and the expression plasmid IgE Fcε2-4/pcDNA3.1（＋） was constructed by inserting the sequence of coding region of the IgE Fcε2-4 into the eukaryotic expression vector pcDNA 3.1（＋） and then transfected into CHO-K1 cells.The high-expression strain was screened by the condition media containing 2 mg/ml G418 and cloned through the limited dilution method.IgE Fcε2-4 was purified by a chromatography using coupled anti-IgE antibody（Omalizumab） affinity resins.The bioactivity was confirmed by Dot-blot and FACS analysis.It was shown by SDS-PAGE analysis that the molecular weight of 7.8 kD and high purity of over 95% of the target protein could be demonstrated.By SPR testing,the affinity of Omalizumab and IgE Fcε2-4 was found to be about 3.8 nmol/L.This study can provide an foundation to provide active protein for the future structural and functional studies and to find out new antibody drugs.

关 键 词：IGE Fcε2-4 克隆表达 CHO 偶联抗IgE抗体 

分 类 号：R329.8[医药卫生—人体解剖和组织胚胎学]