基于定点突变的抗克百威单链抗体的亲和力成熟

摘要

To obtain higher affinity anti-carbofuran ( CBF) single-chainfragmen! ( scFv) antibody, the scFv model by homology modeling was constructed according to the arnino acids sequence and the active pocket region were determined. Through molecular docking model of CBF and scFv, it was found that hydrophobic interaction and hydrogen bonding played an important role of antibody affinity. Then the hydrophilic amino acids HArg40 and LHis38 were changed to hydrophobic amino acids. The molecular docking of CBF and mutant antibody indicated that higher docking scores could be obtained when the leucine was taken as the mutation ami-no acid. Furthermore, mutation scFv gene was constructed, soluble reeombinant protein was expressed and the affinity of evolved scFv (evoscFv) was identified by ELISA. The results showed that the 50% inhibition of binding of evoscFv against CBF was 18.11 μg/L which was lower than the wild type scFv antibody, and the affinity was also improved 2. 23-fold. All these indicated that directed affinity maturation anti-CBF evoscFv was obtained through the analysis of molecule docking and replacement of target amino acid residues.%为获得亲和力更高的抗克百威(CBF)单链抗体(scFv),从抗CBF scFv氨基酸序列出发,通过同源模建获得抗体模型,找出抗体中的活性口袋区域,进而将小分子药物与抗体进行分子对接,发现疏水作用和氢键对于抗体亲和力具有重要作用.进一步对口袋内亲水氨基酸残基HArg40和LHis38进行模拟替换,再进行分子对接分析,发现当以亮氨酸为突变氨基酸时,对接评分最高.在此基础上,通过构建突变scFv基因及可溶性表达,采用ELISA法对进化后的单链抗体(evoscFv)进行了鉴定.结果表明,evoscFv对CBF的IC50值为18.11 μg/L,低于野生型抗体的27.25 μg/L,亲和解离常数Kd为4.06×10-8 mol/L,相对亲和力比野生型scFv提高了2.23倍,说明通过分子对接分析及对抗体活性口袋中氨基酸残基进行替换,获得了一个亲和力更高的突变体抗体.