【摘要】 目的:构建抗人CD25嵌合抗体稳定细胞株,并对表达产物进行相关鉴定。方法:构建CD25嵌合抗体稳定表达质粒VEC-VTacH和3.3-VTacL并稳定转染CHO-DHFR-细胞,经选择性培养、有限稀释法克隆和MTX加压选择后扩大培养构建的工程细胞,Protein A纯化回收目的抗体后分别进行质谱分析、蛋白质N端测序和平衡解离常数(Kd)的测定。结果:CD25嵌合抗体稳定细胞株的抗体表达水平约为3.74～7.07μg/ml,质谱分析结果表明其含有鼠源成分和人源成分,蛋白质N端测序表明其与亲本抗体N端序列完全一致,其Kd为2.35×10-10mol/L,而亲本抗体的Kd为1.88×10-10mol/L。结论:抗人CD25嵌合抗体保留了亲本抗体的抗原结合活性和亲和力。更多还原

【Abstract】 Objective:To construct a stable cell line expressing anti-CD25 chimeric antibody,which then can be characterized.Methods:The stable expression plasmids VEC-VTac and 3.3-VTacL were constructed and transfected into CHO-DHFR-cells.After selective culturing,limiting dilution cloning and step-wise MTX amplification,the engineered cells were expanded.The Protein A affinity chromatograph was employed to purify the antibody,which was characterized by mass spectrometry,N-terminal amino acid sequencing and determination of equilibrium dissociate constant(Kd).Results:An engineered cell line expressing anti-CD25 chimeric antibody ranging from 3.74 to 7.07 μg/ml was established.The mass spectrometry indicated the anti-CD25 chimeric antibody consists of mouse and human components.The N-terminal amino acid sequencing proved the consistent sequences between chimeric CD25 antibody and WuTac,whose Kd are 2.35×10-10 mol/L and 1.88×10-10 mol/L respectively.Conclusion:The chimeric antibody against CD25 maintains the antigen binding specificities and affinity of parental WuTac.更多还原