State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang, China.
College of Food Science and Technology, Nanchang University, Nanchang, China.
Biotechnol Bioeng. 2024 Jun;121(6):1973-1985. doi: 10.1002/bit.28707. Epub 2024 Mar 28.
Nanobody (Nb), the smallest antibody fragments known to bind antigens, is now widely applied to various studies, including protein structure analysis, bioassay, diagnosis, and biomedicine. The traditional approach to generating specific nanobodies involves animal immunization which is time-consuming and expensive. As the understanding of the antibody repertoire accumulation, the synthetic library, which is devoid of animals, has attracted attention widely in recent years. Here, we describe a synthetic phage display library (S-Library), designed based on the systematic analysis of the next-generation sequencing (NGS) of nanobody repertoire. The library consists of a single highly conserved scaffold (IGHV3S6501-IGHJ401) and complementary determining regions of constrained diversity. The S-Library containing 2.19 × 10 independent clones was constructed by the one-step assembly and rapid electro-transformation. The S-Library was screened against various targets (Nb G8, fusion protein of Nb G8 and green fluorescent protein, bovine serum albumin, ovalbumin, and acetylcholinesterase). In comparison, a naïve library (N-Library) from the source of 13 healthy animals was constructed and screened against the same targets as the S-Library. Binders were isolated from both S-Library and N-Library. The dynamic affinity was evaluated by the biolayer interferometry. The data confirms that the feature of the Nb repertoire is conducive to reducing the complexity of library design, thus allowing the S-Library to be built on conventional reagents and primers.
纳米抗体（Nb）是已知能够结合抗原的最小抗体片段，现在已广泛应用于各种研究，包括蛋白质结构分析、生物测定、诊断和生物医学。生成特异性纳米抗体的传统方法涉及动物免疫，这既耗时又昂贵。随着对抗体库积累的理解，近年来，不含动物的合成文库引起了广泛关注。在这里，我们描述了一种基于纳米抗体库的下一代测序（NGS）系统分析设计的合成噬菌体展示文库（S-Library）。该文库由一个高度保守的骨架（IGHV3S6501-IGHJ401）和受约束多样性的互补决定区组成。通过一步组装和快速电转化构建了包含 2.19×10 个独立克隆的 S-Library。S-Library 针对各种靶标（Nb G8、Nb G8 与绿色荧光蛋白融合蛋白、牛血清白蛋白、卵清蛋白和乙酰胆碱酯酶）进行了筛选。相比之下，从 13 只健康动物来源构建了一个原始文库（N-Library），并针对与 S-Library 相同的靶标进行了筛选。从 S-Library 和 N-Library 中分离出结合物。通过生物层干涉测量法评估了动态亲和力。数据证实，Nb 库的特征有助于降低文库设计的复杂性，从而允许在常规试剂和引物上构建 S-Library。
