淘选
噬菌体展示
互补决定区
免疫球蛋白轻链
肽库
分子生物学
化学
克隆(Java方法)
单链可变片段
计算生物学
抗体
图书馆
单克隆抗体
肽序列
生物
遗传学
生物化学
基因
16S核糖体RNA
作者
Kuntalee Rangnoi,Kiattawee Choowongkomon,Richard O’Kennedy,Florian Rüker,Montarop Yamabhai
标识
DOI:10.1021/acs.jafc.8b01141
摘要
A human antiaflatoxin B1 (AFB1) scFv antibody (yAFB1-c3), selected from a naı̈ve human phage-displayed scFv library, was used as a template for improving and analysis of antibody–ligand interactions using the chain-shuffling technique. The variable-heavy and variable-light (VH/VL)-shuffled library was constructed from the VH of 25 preselected clones recombined with the VL of yAFB1-c3 and vice versa. Affinity selection from these libraries demonstrated that the VH domain played an important role in the binding of scFv to free AFB1. Therefore, in the next step, VH-shuffled scFv library was constructed from variable-heavy (VH) chain repertoires, amplified from the naı̈ve library, recombined with the variable-light (VL) chain of the clone yAFB1-c3. This library was then used to select a specific scFv antibody against soluble AFB1 by a standard biopanning method. Three clones that showed improved binding properties were isolated. Amino acid sequence analysis indicated that the improved clones have amino acid mutations in framework 1 (FR1) and the complementarity determining region (CDR1) of the VH chain. One clone, designated sAFH-3e3, showed 7.5-fold improvement in sensitivity over the original scFv clone and was selected for molecular binding studies with AFB1. Homology modeling and molecular docking were used to compare the binding of this and the original clones. The results confirmed that VH is more important than VL for AFB1 binding.
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