Wang Rongzhi, Xiang Shuangshuang, Zhang Yonghui, Chen Qiuyu, Zhong Yanfang, Wang Shihua
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China.
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China
Appl Environ Microbiol. 2014 Jul;80(14):4126-37. doi: 10.1128/AEM.00936-14. Epub 2014 May 2.
Single-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy.
单链可变片段（scFv）抗体作为大多数人类疾病的诊断和治疗剂或生物传感器被广泛应用。然而，目前scFv抗体在稳定性、溶解性和亲和力方面的局限性，使得通过传统抗体筛选和表达形式来生产具有挑战性。我们在此描述一种创建基于绿色荧光蛋白（GFP）的抗体的可行策略。将保留抗原结合活性的互补决定区3（CDR3）引入超级折叠GFP的结构环中，结果表明插入CDR3的GFP显示出与野生型GFP几乎相同的荧光强度，并且插入CDR3的纯化蛋白显示出与相应scFv相似的抗原结合活性。在所有的互补决定区中，CDR3负责抗原识别，并且只有CDR3a插入是产生与特定抗原结合的基于GFP的抗体的最佳形式。通过将其他scFv的CDR3引入GFP的环9中，进一步验证了该系统的广泛通用性。我们开发了一种通过将CDR3插入GFP环中来快速有效地生产高亲和力基于GFP的抗体的可行方法。此外，亲和力可以通过特定氨基酸扫描和定点诱变来增强。值得注意的是，该方法在使用GFP作为支架创建针对各种抗原的抗体方面具有更好的通用性，这表明具有高亲和力和特异性的基于GFP的抗体可能对疾病诊断和治疗有用。
