Sequence‐based design and construction of synthetic nanobody library

计算生物学 肽库 噬菌体展示 抗体库 基因组文库 剧目 图书馆 融合蛋白 牛血清白蛋白 抗原 生物 抗体 化学 分子生物学 遗传学 肽序列 生物化学 重组DNA 物理 基因 细菌 声学 16S核糖体RNA
作者
Chuanyong Liu,Yanping Li,Qinghua He,Jinheng Fu,Qingting Wei,Hao Lin,Ying Luo,Zhui Tu
出处
期刊:Biotechnology and Bioengineering [Wiley]
卷期号:121 (6): 1973-1985
标识
DOI:10.1002/bit.28707
摘要

Abstract 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 (IGHV3S65*01‐IGHJ4*01) and complementary determining regions of constrained diversity. The S‐Library containing 2.19 × 10 8 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.
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