【摘要】 目的: 构建并表达抗人CD3嵌合抗体, 以克服鼠源单克隆抗体(mAb)用于临床的局限性。方法: 采用基因工程技术, 将抗体VL、VH 基因分别克隆入嵌合抗体表达载体VLExpress及VHExpress中。共转染COS- 7细胞后, 用ELISA检测培养上清中嵌合抗体的表达水平; 采用ProteinA亲和层析法纯化抗体, 并进行Westernblot鉴定。用FACS检测嵌合抗体结合抗原的活性; 混合淋巴细胞培养检测抗体的功能。结果: 成功地构建了VH Express VH 及VLExpress VL表达载体并表达纯化。Westernblot的结果显示, 表达的抗人CD3抗体为人鼠嵌合抗体。FACS的结果显示, 该抗体具有良好的结合抗原活性; 混合淋巴细胞培养结果显示, 该抗体具有一定的免疫抑制功能。结论: 成功地构建、表达了抗人CD3嵌合抗体, 为进一步的研究打下了基础。更多还原

【Abstract】 AIM: To construct and express anti-human CD3 chimeric antibody. METHODS: The genes of variable regions of the light chain (V L) and heavy chain (V H) were cloned respectively into the expression vectors (V L Express, V H Express), and co-transfected into COS-7 cells. Expression level of the chimeric antibody in culture supernatant was detected by ELISA. The antibody was purified through protein A affinity chromatography and identified by Western blot. Binding activity of the chimeric antibody to the antigen was determined by FACS. Biological activity of the chimeric antibody was determined by mixed T-lymphocyte culture test. RESULTS: The expression vectors were constructed and the anti-human CD3 chimeric antibody was expressed and purified successfully. Western blot showed that the purified antibody was human-mouse chimeric antibody. FACS result showed that the chimeric antibody had antigen-binding activity. Mixed T-lymphocyte culture test showed that the chimeric antibody could suppress proliferation of T lymphocytes. CONCLUSION: The anti-human CD3 chimeric antibody has been constructed and expressed successfully, which lays the foundation for its further study.更多还原