Fevre Cindy, Scheepmaker Lisette, Haas Pieter-Jan
Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, 3484 CX, Utrecht, The Netherlands.
Methods Mol Biol. 2017;1535:43-61. doi: 10.1007/978-1-4939-6673-8_4.
Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.
旨在鉴定免疫逃逸蛋白的方法主要依赖于对序列的计算机预测、与已知逃逸蛋白的结构同源性,或采用蛋白质组学驱动的方法。尽管这些方法已被证明是成功的,但它们受到效率低下和/或缺乏功能鉴定的限制。在这里,我们描述了一种高通量基因组策略,使用噬菌体展示技术从功能上鉴定细菌免疫逃逸蛋白。将细菌基因组DNA随机片段化并连接到噬菌体展示载体中,该载体用于创建一个表达细菌分泌蛋白和膜结合蛋白的噬菌体展示文库。该文库用于筛选与宿主免疫成分相互作用的展示细菌分泌蛋白组。