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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 a monovalent antibody lentiviral shuttle plasmid by using ch3E1D7 chimeric antibody against human mesenchymal transition factor (c-Met) blocking chimeric antibody and to express it in HEK293T cells by lentivirus expression system. The purified antibody The affinity and neutralizing activity were tested. Methods Anti-c-Met monovalent antibody was designed and named as mono3E1D7. The lentiviral vector containing three chains of monovalent antibody was constructed by gene engineering technique. HEK293T cells were transfected by calcium phosphate method. The expressed monovalent antibody was purified by protein A Sepharose 4B affinity chromatography Antibody integrity was detected by SDS-PAGE, and the biological activity of monovalent antibody blocking the binding of c-Met to its ligand HGF in vitro was detected by ELISA. Results After purification of HEK293T cells infected with monovalent antibody lentivirus, three bands of 55 ku heavy chain, 25 ku light chain and 30 ku clubbing protein chain were observed by SDS-PAGE. The purified monovalent antibody can bind to the c-Met antigen and also has the activity of blocking the binding of c-Met to HGF. Conclusion The anti-human c-Met blocking monovalent antibody was successfully obtained.