Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing Laboratory for Food Quality and Safety, Beijing, 100193, People's Republic of China.
Shenzhen Center for Disease Control and Prevention, Shenzhen, 518055, People's Republic of China.
J Mol Model. 2020 Feb 4;26(3):49. doi: 10.1007/s00894-020-4286-y.
A recombinant single-chain variable fragment (scFv) antibody was produced from a hybridoma cell strain secreting the monoclonal antibody for amantadine (AMD), and then its recognition mechanisms for AMD were studied using the molecular docking and molecular dynamics. Complex dockings revealed that three regions are involved in antibody recognition; framework 2 of the VL chain (LFR2) GLU40 and TYR42, complementarity-determining region of the VL chain (LCDR3) TYR116, and framework 2 of the VH chain (HFR2) HIS40 and TRP52 were the key amino acid residues. The results of molecular dynamics show that the most important amino acid residues in the interaction between AMD and scFv are HIS40 and TYR116. On the basis of the results of virtual mutation, the scFv antibody was evolved by directional mutagenesis of amino acid residue GLY107 to PHE. Indirect competitive ELISA (icELISA) results indicated that the scFv mutant had highly increased affinity for AMD with up to 3.9-fold improved sensitivity. Thus, the scFv antibody can be applied for mechanistic studies of intermolecular interactions, and our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-AMD antibody design and preparation in future.
从分泌金刚烷胺（AMD）单克隆抗体的杂交瘤细胞株中制备了一种重组单链可变片段（scFv）抗体，然后使用分子对接和分子动力学研究了其对 AMD 的识别机制。复合物对接表明有三个区域参与抗体识别；VL 链的框架 2（LFR2）GLU40 和 TYR42、VL 链的互补决定区（LCDR3）TYR116 和 VH 链的框架 2（HFR2）HIS40 和 TRP52 是关键的氨基酸残基。分子动力学的结果表明，AMD 和 scFv 相互作用中最重要的氨基酸残基是 HIS40 和 TYR116。基于虚拟突变的结果，通过将氨基酸残基 GLY107 定向突变为 PHE 对 scFv 抗体进行了进化。间接竞争 ELISA（icELISA）结果表明，scFv 突变体对 AMD 的亲和力大大提高，灵敏度提高了 3.9 倍。因此，scFv 抗体可用于研究分子间相互作用的机制，我们的工作通过定点突变提供了亲和力成熟的抗体，这有利于未来有价值的抗 AMD 抗体的设计和制备。
