鼠-人嵌合抗粘蛋白1 IgG1 全抗体构建及其功能初步分析.docVIP

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，获得IgG全抗体表达质粒，瞬时转染293T哺乳动物细胞，直接免疫荧光法确定细胞内人源IgG抗体表达，收获转染细胞培养上清，经亲和层析纯化后，经SDS蛋白电泳、Western-blot、流式细胞仪检测和表面等离子共振技术等分析所制备IgG抗体分子特点及其与MUC1抗原的结合活性及动力学参数。结果 所构建鼠-人嵌合IgG表达质粒能够在哺乳动物细胞中有效表达并分泌到细胞上清，纯化后蛋白质电泳分析与预期IgG抗体分子大小一致，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，经表面等离子共振技术分析表明抗体的亲和力约为KD=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 Qingfeng1, Niu Zhaohe1,Liu Cuicui1, Wang Xiaofeng2, Wu Li1, Zhang Lei1 (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 mammalian cells were transiently transfected with the constructed chimeric IgG expression plasmid, intracellular IgG antibody expression was confirmed by immunofluorescence test and IgG was purified from the supernatant, which was characterized on SDS, the binding activity was analyzed on denatured and natural MUC1 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