亲水突变法提高抗VEGFR2单链抗体的体外亲和力

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。

Affinity maturation of a single chain antibody against VEGFR2 by hydrophilic shuffling

This study is to improve the affinity of scFv-AK404R against VEGFR2.The secondary mutational library was constructed by hydrophilic shuffling in CDR3 region of the heavy chain.VEGFR2-specific screening was performed by phage display technology and the protein of mutants was expressed in periplasm of E.coli HB2151 and purified by affinity chromatography.The affinity constant of scFvs was measured by competitive ELISA,and the structure of scFvs was analyzed by bioinformatics.The result showed that a library with 6.4×105 scFv members was established by electro-transformation.Two mutated clones with high absorbance value were isolated after screening.After purification by affinity chromatography,electrophoretically pure scFv proteins were obtained.The competitive ELISA showed that the affinities of WZ01 and WZ02 were three times higher than that of the parental AK404R,and bioinformatics analysis showed that the enlarged contact surface and fitted closely with KDR3 surface may be the reasons for improved affinity.These results suggest that introducing hydrophilic amino acids to the heavy chain CDR3 region is an effective approach to improve the affinity of scFv.