Hurwitz Amy M, Huang Wanzhi, Estes Mary K, Atmar Robert L, Palzkill Timothy
Interdepartmental Program in Translational Biology & Molecular Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Department of Pharmacology, Baylor College of Medicine, Houston, TX, USA.
Protein Eng Des Sel. 2017 Feb;30(2):129-139. doi: 10.1093/protein/gzw074. Epub 2016 Dec 28.
Norovirus infections are the leading cause of non-bacterial gastroenteritis and result in about 21 million new cases and $2 billion in costs per year in the United States. Existing diagnostics have limited feasibility for point-of-care applications, so there is a clear need for more reliable, rapid, and simple-to-use diagnostic tools in order to contain outbreaks and prevent inappropriate treatments. In this study, a combination of phage display technology, deep sequencing and computational analysis was used to identify 12-mer peptides with specific binding to norovirus genotype GI.1 virus-like particles (VLPs). After biopanning, phage populations were sequenced and analyzed to identify a consensus peptide motif-YRSWXP. Two 12-mer peptides containing this sequence, NV-O-R5-3 and NV-O-R5-6, were further characterized to evaluate the motif's functional ability to detect VLPs and virus. Results indicated that these peptides effectively detect GI.1 VLPs in solid-phase peptide arrays, ELISAs and dot blots. Further, their specificity for the S-domain of the major capsid protein enables them to detect a wide range of GI and GII norovirus genotypes. Both peptides were able to detect virus in norovirus-positive clinical stool samples. Overall, the work reported here demonstrates the application of phage display coupled with next generation sequencing and computational analysis to uncover peptides with specific binding ability to a target protein for diagnostic applications. Further, the reagents characterized here can be integrated into existing diagnostic formats to detect clinically relevant genotypes of norovirus in stool.
诺如病毒感染是引起非细菌性肠胃炎的主要原因，在美国每年导致约2100万新发病例，造成20亿美元的损失。现有的诊断方法在即时检测应用中的可行性有限，因此显然需要更可靠、快速且易于使用的诊断工具，以控制疫情爆发并防止不恰当的治疗。在本研究中，结合噬菌体展示技术、深度测序和计算分析，鉴定出与诺如病毒GI.1基因型病毒样颗粒（VLP）具有特异性结合的12肽。淘选后，对噬菌体群体进行测序和分析，以确定共有肽基序——YRSWXP。对包含该序列的两个12肽NV-O-R5-3和NV-O-R5-6进行进一步表征，以评估该基序检测VLP和病毒的功能能力。结果表明，这些肽在固相肽阵列、酶联免疫吸附测定和斑点印迹中能有效检测GI.1 VLP。此外，它们对主要衣壳蛋白S结构域的特异性使它们能够检测多种GI和GII诺如病毒基因型。两种肽均能够在诺如病毒阳性临床粪便样本中检测到病毒。总体而言，本文报道的工作展示了噬菌体展示与下一代测序和计算分析相结合，用于发现与目标蛋白具有特异性结合能力的肽以用于诊断应用。此外，此处表征的试剂可整合到现有的诊断形式中，以检测粪便中临床相关的诺如病毒基因型。
