利用酵母展示系统确定空间构象性抗原表位的方法研究

A New Strategy for Epitope Mapping by Yeast Surface Display System

酵母展示系统是研究可溶性蛋白间相互作用的有效系统.利用酵母展示系统可以简单快速地研究抗原与抗体相互作用.充分利用此系统的优势,在确定空间构象性的抗原决定簇方面发展出新的应用.利用酵母同源重组把巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)序列中的不同肽段以及不同点突变体展示在酵母表面,并用3个抗MIF单克隆抗体10C3,2A12和4E10分别标记,流式细胞仪检测抗体与抗原突变体的结合.用酵母展示方法确定了MIF上与3个单克隆抗体结合的关键氨基酸序列,从而建立起一个简便可靠的确定空间构象性抗原决定簇的方法.

As an effective method of studying soluble protein-protein interactions, yeast display system is now widely used for affinity maturation of single-chain antibodies. Due to the strong homology recombination machinery of yeast and the high-throughput nature of FACS detection, a rapid scan for interaction between antigen-antibody pairs could be easily achieved. Based on this system, a novel and reliable method for determining conformational epitopes was developed. Different fragments of macrophage migration inhibitory factor （MIF） and several point mutations of MIF were displayed on yeast cell surface using homologous recombination technology. Three MIF monoclonal antibodies, 10C3, 2A12 and 4El0, were screened for their binding affinity to each displayed peptide. Utilizing this technology, the key amino acids of MIF that bind to the MIF monoclonal antibodies were easily identified.