Grihalde N D, Chen Y C, Golden A, Gubbins E, Mandecki W
Aging and Degenerative Disease Department, Abbott Laboratories, North Chicago, IL 60064, USA.
Gene. 1995 Dec 12;166(2):187-95. doi: 10.1016/0378-1119(95)00658-3.
A large filamentous phage library (1 x 10(9) clones) displaying random 30-amino-acid (aa) sequences on the N terminus of the pIII coat protein was constructed and characterized. Clones in the library were affinity selected for binding to monoclonal antibodies (mAb) against two viral antigens, the HIV gp120 protein and the HCV core protein. The obtained aa sequences precisely identified the epitopes recognized by the mAb. Binding of peptide-carrying phages to the Ab was demonstrated by ELISA, Western blot and the surface plasmon resonance (SPR) method. The mAb-specific peptides were transferred via genetic techniques onto the N terminus of Escherichia coli alkaline phosphatase (AP). When fused to the enzyme, the peptides maintained their ability to bind their respective mAb, indicating that the peptides contained the necessary contact residues for binding. The affinity of the peptides was estimated to be 100 nM by SPR. A comparison is presented of the relative affinities of phage-derived peptides to the native viral epitopes also displayed on the AP scaffold. The approach of transferring epitopes from phage to AP for further evaluation should be applicable to many other mAb or receptors.
构建并表征了一个大型丝状噬菌体文库（1×10⁹个克隆），该文库在pIII外壳蛋白的N端展示随机的30个氨基酸（aa）序列。对文库中的克隆进行亲和筛选，以使其与针对两种病毒抗原（HIV gp120蛋白和HCV核心蛋白）的单克隆抗体（mAb）结合。所获得的氨基酸序列精确鉴定了单克隆抗体识别的表位。通过酶联免疫吸附测定（ELISA）、蛋白质印迹法和表面等离子体共振（SPR）方法证实了携带肽的噬菌体与抗体的结合。通过基因技术将单克隆抗体特异性肽转移到大肠杆菌碱性磷酸酶（AP）的N端。当与该酶融合时，这些肽保持了结合其各自单克隆抗体的能力，表明这些肽包含结合所需的接触残基。通过SPR估计这些肽的亲和力为100 nM。还比较了噬菌体衍生肽与同样展示在AP支架上的天然病毒表位的相对亲和力。将表位从噬菌体转移到AP上进行进一步评估的方法应适用于许多其他单克隆抗体或受体。
