全合成噬菌体展示抗体库淘筛策略与多样性分析

Bio-Panning Strategies and Diversity Analysis of Fully Synthetic Phage Display Antibody Library

大容量全合成噬菌体展示抗体库可用于针对特异靶点生产高亲和力抗体,是重要的单克隆抗体来源,其中亲和淘筛策略是影响抗体筛选效果的关键。亲和淘筛中的多样性损失会降低高亲和力抗体获得概率,为探究亲和淘筛多样性损失最小的筛选策略,分别以PCSK9,CD20为靶点,用全合成噬菌体展示抗体库淘筛,通过滴度测定与测序分析比较首轮淘筛中酸洗脱与宿主菌直接感染所得洗脱物的框架与多样性差异。同时采取直接液体扩增、刮板后液体扩增,以及30、37℃两种扩增温度探究不同扩增条件对次级库框架分布的影响。最佳淘筛方法效果通过PCSK9,CD20多轮淘筛后的单克隆phage-Elisa结果验证。研究结果表明合并酸洗脱与宿主菌洗脱噬菌体,30℃直接液体扩增可以使合成库首轮淘筛多样性损失最低,采用此法获得了靶向PCSK9与CD20的阳性序列。最终开发了一种保留全合成噬菌体抗体库多样性效果好、实验周期短的淘筛方法。可供全合成噬菌体抗体库进行高效的抗体筛选,有效推进了抗体药物早期发现进程。

Fully synthetic phage display antibody libraries,large collections of diverse antibodies,can be used to produce high-affinity antibodies against almost any specific targets.One of the crucial factors to successful in vitro antibody discovery is bio-panning strategy.The current bottleneck is how to minimize the nonspecific bindings while to maximize the enrichment of antibodies with high specificity and affinity.Traditional screening methods have been improved and some novel strategies have been established to meet the need of antibody development.Considering the loss of diversity during screening would reduce the probability of high-affinity antibody acquisition,this research was designed to explore a proper panning strategy to minimize the diversity loss.A fully synthetic phage display antibody library was screened,in which PCSK9 and CD20 were used as targets respectively.The differences between acid elution and direct infection of TG1 in the first-round panning were compared according to titer determination and sequence analysis.Direct liquid amplification,liquid amplification after scraping,and amplification temperatures of 30°C and 37°C were respectively evaluated to investigate the impacts of different amplification conditions on the framework distribution of sub-library.Finally,the best panning strategy was verified by monoclonal phage-Elisa after PCSK9 and CD20 multiple rounds of panning.The research results demonstrated that diversity loss of synthetic library in first-round screening could be minimized when combining acid elution and host bacteria eluting phage meanwhile direct liquid amplification at 30°C.Moreover,framework distribution of the sub-library was not significantly affected even if scraping step was omitted.Positive sequences targeting PCSK9 and CD20 could be obtained by this method respectively.In conclusion,a bio-panning strategy with shorter experiment period was developed for maintaining the diversity of the fully synthetic phage antibody library,which could be used for efficient antibody screening,and promoting the early development of antibody products.