A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage
A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage
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DOI：
10.1016/j.vetimm.2007.02.001
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发表时间：
2007-05-15
影响因子：
1.8
通讯作者：
Yang, Yi-Yuan
中科院分区：
文献类型：
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作者：
Lee, Yu-Ching;Leu, Sy-Jye C.;Yang, Yi-Yuan
严重急性呼吸综合征(SARS)是一种新出现的人类疾病，需要快速诊断和有效治疗。在抗体来源中，免疫球蛋白Y(IgY)是鸡蛋中发现的主要抗体，可用作研究和免疫治疗中通常使用的哺乳动物抗体的替代品。本研究选用表达鸡源单链抗体的噬菌体，采用噬菌体展示抗体技术对其进行鉴定。将SARS冠状病毒刺突蛋白的截短片段克隆到pET-21载体中，并在BL-21大肠杆菌中表达。纯化后，用SDS-PAGE检测重组Spike蛋白的纯度，用抗His抗体和SARS-CoV恢复期患者血清进行Western印迹分析，验证其同源性。用这些细菌来源的蛋白免疫鸡，经Western印迹和免疫细胞化学染色分析，发现蛋黄和血清中的多克隆IgY抗体对免疫原具有较高的反应性。用5×10(7)克隆的免疫鸡脾B细胞，建立了噬菌体展示单链抗体文库。经过四次淘洗循环后，洗脱噬菌体滴度增加了10倍。在与鸡胚系基因的序列分析中，5个噬菌体克隆在互补决定区有较大差异，差异在31%~62%之间，其中一个优势噬菌体4S1与位于S蛋白456-650氨基酸残基之间的片段Se-e有较强的结合，该噬菌体与SARS冠状病毒感染的Vero E6细胞有显著的结合。根据这些结果，我们认为利用噬菌体展示系统可以成功地产生表达ScFv的特异性噬菌体结合子，该知识可以应用于临床或学术研究。(C)2007 Elsevier B.V.保留所有权利。
Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. In this study, phage-expressing chicken monoclonal scFv antibody was chosen and characterized with phage display antibody technology. Truncated fragments of SARS-CoV spike protein were cloned in pET-21 vector and expressed in BL-21 Escherichia coli (E. coli) cells. After purification, the purity of these recombinant spike proteins was examined on SDS-PAGE and their identity verified with Western blot analysis using anti-his antibodies and sera from convalescent stage SARS-CoV-infected patients. Using these bacteria-derived proteins to immunize chickens, it was found that polyclonal IgYantibodies in the egg yolk and sera were highly reactive to the immunogens, as shown by Western blot and immunocytochemical staining analysis. A phage displaying scFv library was also established from spleen B cells of immunized chicken with 5 x 10(7) clones. After four panning cycles, the eluted phage titer showed a 10-fold increase. In sequence analysis with chicken germline gene, five phage clones reacted, with large dissimilarities of between 31 and 62%, in the complementarity-determining regions, one dominant phage 4S 1 had strong binding to fragment Se-e, located between amino acid residues 456-650 of the spike protein and this particular phage had significantly strong binding to SARS-CoV-infected Vero E6 cells. Based on the results, we conclude that generating specific scFv-expressing phage binders with the phage display system can be successfully achieved and that this knowledge can be applied in clinical or academic research. (C) 2007 Elsevier B.V. All rights reserved.