抗间质上皮转化因子(c-Met)单价抗体的制备及生物学活性检测

Preparation and biological activity of anti-human c-mesenchymal epithelial transition factor(c-Met) monovalent antibody

目的用抗人间质上皮转化因子(c-Met)阻断型嵌合抗体ch3E1D7表达质粒构建单价抗体慢病毒穿梭质粒,利用慢病毒表达系统实现其在HEK293T细胞中快速表达,并对纯化后抗体的亲和力及中和活性进行检测。方法设计抗c-Met的单价抗体,命名为mono3E1D7,利用基因工程技术构建单价抗体三条链的慢病毒表达载体,磷酸钙法转染HEK293T细胞,表达的单价抗体经蛋白A琼脂糖4B亲和层析柱纯化,SDS-PAGE检测抗体完整性,ELISA检测单价抗体在体外阻断c-Met与其配体HGF结合的生物学活性。结果感染单价抗体慢病毒的HEK293T细胞上清纯化后,SDS-PAGE分析可见55 ku的重链、25 ku的轻链以及30 ku的杵状结构链(Knob)三条带。且纯化后的单价抗体能与c-Met抗原结合,同时还具有阻断c-Met与HGF结合的中和活性。结论成功获得抗人c-Met阻断型单价抗体。

Objective To construct lentiviral vectors for the expression of monovalent antibody against human c-mesenchymal epithelial transition factor（c-Met） using anti-c-Met chimeric antibody ch3E1D7 plasmid,and test the affinity and neutralizing ability of the purified monovalent antibody in transfected HEK293 T cells.Methods The anti-c-Met monovalent antibody was designed,namely mono3E1D7.Three different lentiviral expression vectors of the monovalent antibody were then constructed using genetic engineering technology.The three expression vectors were co-transfected in HEK293 T cells to express the monovalent antibody,which was later purified by protein A-sepharose 4B affinity chromatography.The antibody structural integrity was identified by SDS-PAGE.Ability of the monovalent antibody to bind and neutralize hepatocyte growth factor（HGF） was tested by ELISA.Results Heavy,light and Knob chains of the mono3E1D7,with molecular masses of about55,25 and 30 k D,respectively,were observed on reduced 10% SDS-PAGE.ELISA showed that the expressed protein could bind to c-Met specifically and neutralize c-Met/HGF binding.Conclusion Monovalent antibody targeting c-Met has been successfully constructed,expressed and identified,which could help to study the important role of monovalent antibody targeting to c-Met in following experiments.