抗肿瘤muc1单链抗体的构建表达及其功能初步分析 - 第三军医大学学报.doc

鼠-人嵌合抗粘蛋白1 IgG1全抗体的构建及其功能初步分析 陈庆峰1,牛兆河1,刘翠翠1,王笑峰2,吴俐1, 张磊1(266003 山东青岛，青岛大学医学院：附属医院乳腺外科1，微生物教研室2） [摘要] 目的 获得鼠-人嵌合抗MUC1 IgG全抗体，降低鼠源抗粘蛋白1(mucin1,MUC1)单克隆抗体（monoclonal antibody, mAb）的异种免疫原性，并在哺乳动物细胞中表达制备，初步分析其特性。方法 PCR扩增鼠mAb-6E3可变区编码基因，通过引入特异性限制性酶切位点克隆入含有人源IgG1抗体恒定区的哺乳动物细胞重组表达载体，转染293T细胞表达纯化IgG抗体后，分析其与MUC1抗原的结合活性及动力学参数。结果 获得了在哺乳动物细胞内表达的鼠-人嵌合抗MUC1 IgG全抗体，蛋白电泳分析、Western blot和流式细胞术分析表明该抗体能够与变性或天然状态下的T47D肿瘤细胞表面MUC1特异性结合，亲和力KD为2.8 × 10-7 M。结论 获得鼠-人嵌合抗MUC1 IgG抗体，具有与MUC1特异性结合的生物学活性。 [关键词] 乳腺肿瘤；粘蛋白类 [中图法分类号] R392 [文献标志码] A Construction and characterization of a mouse-human chimeric anti-mucin1 IgG1 antibody Chen Qingfeng, Niu Zhaohe,Liu Cuicui, Wang Xiaofeng, Wu Li, Zhang Lei (1Department of Breast Surgery, Affiliated Hospital, 2Department of Microbiology, Medical College, Qingdao University, Qingdao, Shandong Province, 266003, China） [Abstract] Objective o construct a mouse-chimeric anti-mucin1 IgG antibody with decreased immunogenicity of the mouse mAb-6E3 in human, and characterized the binding properties of mammalian cells produced chimeric IgG antibody. Methods The variable genes of a mouse mAb were PCR amplified and cloned into a human IgG mammalian expression vector through corresponding restriction sites. 293T cells were transiently transfected with the constructed chimeric IgG expression plasmid, and IgG was purified from the supernatant, which was characterized on SDS, denatured and natural antigen by Western blotting and flow cytometry, the binding affinity was determined via surface plasmon resonance technology. Results An anti-MUC1 mouse-human chimeric IgG antibody was obtained by the combination of the mouse variable genes with human IgG1 constant gene, which was produced and purified in 293T cells. Western blotting and FACS analysis showed that the obtained IgG could bind to nature and denatured MUC1 of T47D tumor cells specifically，and the binding affinity was 2.8 × 10-7 M. Conclusion A functional anti-MUC1 chimeric IgG is obta