Arbabi-Ghahroudi Mehdi, Tanha Jamshid, MacKenzie Roger
Institute for Biological Sciences, National Research Council, The Antibody Engineering Group, Ottawa, Ontario, Canada.
Methods Mol Biol. 2009;502:341-64. doi: 10.1007/978-1-60327-565-1_20.
Techniques developed over the past 20 years for the display of foreign peptides and proteins on the surfaces of filamentous bacteriophages have been a major driving force in the rapid development of recombinant antibody technology in recent years. With phage display of antibodies as one of its key components, recombinant antibody technology has led to the development of an increasing number of therapeutic monoclonal antibodies. Antibody gene libraries are fused to a gene encoding a phage coat protein. Recombinant phage expressing the resulting antibody libraries in fusion with the coat protein are propagated in Escherichia coli. Phage displaying monoclonal antibodies with specificities for target antigens are isolated from the libraries by a process called panning. The genes encoding the desired antibodies selected from the libraries are packaged within the phage particles, linking genotype and phenotype. Here, we describe the application of this technology to the construction of a phage-displayed single-domain antibody (sdAb) library based on the heavy chain antibody repertoire of a llama, the panning of the library against a peptide antigen and the expression, purification, and characterization of sdAbs isolated by panning.
在过去20年里开发的用于在丝状噬菌体表面展示外源肽和蛋白质的技术，是近年来重组抗体技术快速发展的主要驱动力。重组抗体技术以噬菌体展示抗体作为其关键组成部分之一，已促使越来越多治疗性单克隆抗体的研发。抗体基因文库与编码噬菌体外壳蛋白的基因融合。表达与外壳蛋白融合的所得抗体文库的重组噬菌体在大肠杆菌中增殖。通过一种称为淘选的过程从文库中分离出对靶抗原具有特异性的展示单克隆抗体的噬菌体。从文库中选出的编码所需抗体的基因被包装在噬菌体颗粒内，从而将基因型与表型联系起来。在此，我们描述了该技术在构建基于羊驼重链抗体库的噬菌体展示单域抗体（sdAb）文库、针对肽抗原对文库进行淘选以及对通过淘选分离出的sdAb进行表达、纯化和表征方面的应用。
