Hefei National Laboratory for Physical Sciences at Microscale, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine , University of Science and Technology of China , Hefei 230027 China.
Healthy Processed Foods Research Unit, USDA-ARS , Western Regional Research Center , 800 Buchanan Street , Albany , California 94710 , United States.
J Agric Food Chem. 2019 Oct 9;67(40):11219-11229. doi: 10.1021/acs.jafc.9b02388. Epub 2019 Sep 25.
Peanut allergy is a major health problem worldwide. Detection of food allergens is a critical aspect of food safety. The VHH domain of single chain antibody from camelids, also known as nanobody (Nb), showed its advantages in the development of biosensors because of its high stability, small molecular size, and ease of production. However, no nanobody specific to peanut allergens has been developed. In this study, we constructed a library with random triplets (NNK) in its CDR regions of a camel nanobody backbone. We screened the library with peanut allergy Ara h 3 and obtained several candidate nanobodies. One of the promising nanobodies, Nb16 was further biochemical characterization by gel filtration, isothermal titration calorimetry (ITC), cocrystallization, and Western blot in terms of its interaction with Ara h 3. Nb16 specifically binds to peanut major allergen Ara h 3 with a dissociation constant of 400 nM. Furthermore, we obtained the Ara h 3-Nb16 complex crystals. Structure analysis shows the packing mode is completely different between the Ara h 3-Nb16 complex crystal and the native Ara h 3 crystal. Structural determination of Ara h 3-Nb16 will provide the necessary information to understand the allergenicity of this important peanut allergen. The nanobody Nb16 may have application in the development of biosensors for peanut allergen detection.
花生过敏是全球范围内的一个主要健康问题。检测食物过敏原是食品安全的一个关键方面。骆驼科单链抗体的 VHH 结构域，也称为纳米抗体 (Nb)，由于其高稳定性、小分子量和易于生产，在生物传感器的开发中显示出优势。然而，目前还没有针对花生过敏原的纳米抗体被开发出来。在本研究中，我们构建了一个含有随机三联体 (NNK) 的骆驼纳米抗体骨架 CDR 区文库。我们用花生过敏原 Ara h 3 对文库进行筛选，获得了几个候选纳米抗体。其中一个有前途的纳米抗体 Nb16，我们进一步通过凝胶过滤、等温滴定量热法 (ITC)、共结晶和 Western blot 对其与 Ara h 3 的相互作用进行了生化特性分析。Nb16 特异性地与花生主要过敏原 Ara h 3 结合，解离常数为 400 nM。此外，我们获得了 Ara h 3-Nb16 复合物的晶体。结构分析表明，Ara h 3-Nb16 复合物晶体与天然 Ara h 3 晶体的堆积方式完全不同。Ara h 3-Nb16 的结构测定将为理解这种重要的花生过敏原的致敏性提供必要的信息。纳米抗体 Nb16 可能在开发用于检测花生过敏原的生物传感器方面有应用。
