Deep sequencing of phage-displayed peptide libraries reveals sequence motif that detects norovirus
Deep sequencing of phage-displayed peptide libraries reveals sequence motif that detects norovirus
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DOI：
10.1093/protein/gzw074
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发表时间：
2017-02-01
影响因子：
2.4
通讯作者：
Palzkill, Timothy
中科院分区：
文献类型：
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作者：
Hurwitz, Amy M.;Huang, Wanzhi;Palzkill, Timothy
诺如病毒感染是非细菌性胃肠炎的主要原因，在美国每年导致约2100万新病例和20亿美元的成本。现有的诊断方法对于床旁应用的可行性有限，因此显然需要更可靠、快速和简单易用的诊断工具，以遏制疫情爆发并防止不适当的治疗。在这项研究中，噬菌体展示技术，深度测序和计算分析的组合用于鉴定与诺如病毒基因型GI特异性结合的12聚体肽。1个病毒样颗粒（VLP）。在生物淘选后，对噬菌体群体进行测序和分析以鉴定共有肽基序-YRSWXP。进一步表征了含有该序列的两个12-mer肽，NV-O-R5-3和NV-O-R5-6，以评估基序检测VLP和病毒的功能能力。结果表明，这些肽有效地检测GI。1固相肽阵列中的VLP，ELISA和斑点印迹。此外，它们对主要衣壳蛋白的S结构域的特异性使它们能够检测广泛的GI和GII诺如病毒基因型。两种肽都能够检测诺如病毒阳性临床粪便样品中的病毒。总体而言，这里报道的工作证明了噬菌体展示与下一代测序和计算分析相结合的应用，以揭示具有特异性结合能力的肽与靶蛋白的诊断应用。此外，本文表征的试剂可以整合到现有的诊断形式中，以检测粪便中诺如病毒的临床相关基因型。
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 solidphase 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.