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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18.11μg/L,低于野生型抗体的27.25μg/L,亲和解离常数Kd为4.06×10-8mol/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.
In order to obtain a higher affinity anti-carbofuran (scFv) antibody, an antibody model was derived from the anti-CBF scFv amino acid sequence by homology modeling to find the active pocket region in the antibody, and then the small molecule Drugs and antibodies for molecular docking and found that the hydrophobic interaction and hydrogen bonding affinity for the antibody plays an important role in the pocket further hydrophilic amino acid residues HArg40 and LHis38 simulated replacement, and then molecular docking analysis found that when leucine as a mutation Amino acid, the docking score was the highest.On the basis of this, the evolved scFv was identified by ELISA and the IC50 of evoscFv to CBF was 18.11μg / L, lower than that of wild-type antibody 27.25μg / L, the affinity dissociation constant Kd is 4.06 × 10-8mol / L, the relative affinity is 2.23 times higher than the wild type scFv, indicating that by molecular docking analysis and antibody activity in the pocket Amino acid residues to replace, to obtain a more affinity mutant antibody.