鼠-人嵌合抗粘蛋白1 IgG1全抗体的构建及其功能初步分析

Construction and characterization of a mouse-human chimeric anti-mucin1 IgG antibody

目的获得鼠-人嵌合抗粘蛋白1(mucin1,MUC1)IgG全抗体,降低鼠源抗MUC1单克隆抗体(monoclonalantibody,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-PAGE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=2.8×10-7M。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。

Objective To construct a mouse-human chimeric anti-mucin1（MUC1） IgG antibody with decreased immunogenicity of mouse anti-MUC1 monoclonal antibody（mAb）-6E3 in human,and to characterize the binding property of mammalian cell-produced mouse-human chimeric anti-MUC1 IgG antibody.Methods The variable region genes of mouse mAb-6E3 were amplified by PCR and were inserted into a human IgG mammalian expression vector through corresponding restriction enzyme sites.293T mammalian cells were transiently transfected with the recombinant chimeric IgG expression plasmid,and the intracellular IgG antibody expression was confirmed by immunofluorescence test.The chimeric anti-MUC1 IgG antibody was purified from cell supernatants by affinity chromatography,and was characterized by SDS-PAGE electrophoresis.Its binding activity with denatured and natural MUC1 antigen was analyzed by Western blotting and flow cytometry,and the binding affinity was determined by surface plasmon resonance technology.Results A mouse-human chimeric anti-MUC1 IgG antibody was obtained by fusion of mouse monoclonal antibody variable region with human IgG constant region.The obtained chimeric IgG antibody could specifically bind to natural and denatured MUC1 on the surface of T47D tumor cells,with binding affinity of 2.8×10-7 M.Conclusion A functional chimeric anti-MUC1 IgG is obtained successfully,and it can bind to tumor-associated MUC1 specifically.