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为了提高抗VEGFR2单链抗体AK404R的亲和力,本研究采用亲水突变法将AK404R的重链CDR3区进行突变,建立次级突变单链抗体库。利用噬菌体展示技术从次级突变库中筛选具有抗VEGFR2特异性、高亲和力抗体,获得的抗体突变株经大肠杆菌HB2151分泌表达,镍亲和色谱柱纯化,并采用竞争性ELISA法、生物信息学方法分别对其亲和力和结构进行了分析。本研究最终建立了6.4×105的次级突变单链抗体库,其中两株突变株的亲和力有明显提高,两株突变株经分离纯化得到电泳纯的蛋白,竞争性ELISA结果显示突变体WZ01和WZ02的亲和力比亲本提高了3倍;生物信息学方法分析突变体与抗原的作用面增大、契合紧密,这可能是亲和力提高的原因之一。研究结果表明,在重链CDR3区引入亲水性氨基酸构建抗体突变库,可有效提高scFv的亲和力。
In order to improve the affinity of the anti-VEGFR2 single-chain antibody AK404R, we mutated the heavy chain CDR3 of AK404R using hydrophilic mutation method to establish a secondary mutant single-chain antibody library. The phage display technique was used to screen anti-VEGFR2-specific and high-affinity antibody from secondary mutant library. The obtained antibody was secreted by E. coli HB2151 and purified by nickel affinity chromatography. The competitive ELISA and bioinformatics Methods The affinity and structure were analyzed respectively. In the present study, a 6.4 × 105 secondary mutant single-chain antibody library was finally established, in which the affinity of the two mutants was significantly increased. The two mutants were purified by electrophoresis to obtain pure proteins. The competitive ELISA results showed that the mutants WZ01 and The affinity of WZ02 was three times higher than that of the parent. Bioinformatics analysis showed that the interaction between the mutant and the antigen increased, which was closely related to the antigen. This may be one of the reasons for the increased affinity. The results show that the introduction of hydrophilic amino acids in the heavy chain CDR3 region to construct antibody mutant library can effectively improve the scFv affinity.