【摘要】 目的 :构建抗CD71人 鼠嵌合抗体的真核细胞表达载体并在真核细胞中表达嵌合抗体。方法 :将鼠源性抗CD71单抗 (75 79)重、轻链可变区基因 (VH 和VL)分别克隆至含有人IgG轻、重链恒定区的表达载体pγ Expr和pκ Expr中 ;将嵌合表达载体 (Chi75 79 pγ Expr和Chi75 79 pκ Expr)经电转染技术共转染至真核细胞 (SP2 0 )中。结果 :经ELISA法检验阳性转染瘤细胞培养上清 ,证明其表达蛋白产物中同时具有人IgG重链恒定区 (Cγ)和轻链恒定区 (Cκ)蛋白 ;通过间接免疫荧光流式细胞术 (FCM)和间接免疫荧光显微技术 ,证明该表达产物能够特异性识别和结合表达于靶细胞表面的CD71分子。结论 :抗CD71人 鼠嵌合抗体 (D2C)在体外真核细胞表达成功。更多还原

【Abstract】 Objective:To construct the expression vectors of VH and VL from an anti -CD71 monoclonal antibody (line7579) , and express an anti-CD71 mouse/human chimeric antibody (D2C) in vitro.Methods:The variable regions of heavy and light chains from an anti -CD71 monoclonal antibody (line7579) cloned in the pGEM-T vectors were constructed respectively into the expression vectors,pγ-Expr(including human IgG1 constant region) and pκ-Expr(including human Igκ constant region).The expression plasmid vectors (Chi7579-pγ-Expr and Chi7579- pκ-Expr) ,confirmed by a clone-PCR and a further sequence analysis, were co-transfected by electroporation into the non-Ig-producing murine hybridomas SP2/0.Results:Screening for transfectomas secreting chimeric antibody was done by cross sandwich ELISA, which identified the transfectomas to secrete a chimeric antibody (D2C) with the human heavy constant region(Cγ) and light constant region(Cκ). FCM analysis of culture supernatants of the transfectomas demonstrated that the binding capacity of the chimeric Ab (D2C) to the antigen (CD71) was retained.Conclusion:The construction of the V H and V L expression vectors and the expression of an anti-CD71 mouse/human chimeric antibody (D2C) were accomplished successfully.更多还原