抗人CD4嵌合抗体的稳定表达及鉴定

目的构建抗人CD4嵌合抗体稳定表达细胞株,并对表达产物进行鉴定。方法采用脂质体法将抗人CD4嵌合抗体质粒pHDC4稳定转染CHO-DHFR-细胞,经选择性培养、有限稀释法克隆和MTX加压筛选抗人CD4嵌合抗体稳定表达株,经细胞工厂扩大培养。收集培养上清,经Protein A亲和层析纯化目的抗体,激光共聚焦显微镜观察抗体基因在细胞内的表达,并分别进行质谱分析、蛋白质N-末端测序和平衡解离常数(KD)的测定。结果构建的抗人CD4嵌合抗体稳定表达细胞株的抗体表达水平为4.29～10.52μg/ml;BCA测得纯化回收后的抗体表达水平为12.68 mg/L;激光共聚焦检测显示,抗CD4嵌合抗体稳定表达细胞株可稳定表达人的恒定区和鼠的可变区;质谱分析表明,其含有鼠源性和人源性成分;蛋白质N-末端氨基酸测序表明,其轻链与亲本抗体N-末端氨基酸序列完全一致;其KD为2.67×10-9M。结论成功构建了抗人CD4嵌合抗体稳定表达细胞株,其表达的抗人CD4嵌合抗体保留了亲本抗体的抗原结合特异性和亲和力。

Stable expression and characterization of chimeric antibody against human CD4

Objective To construct a cell strain for stable expression of chimeric antibody against human CD4 and characterize the expressed product.Methods Plasmid pHDC4 containing anti-CD4 chimeric antibody gene was transfected into CHO-DHFR-cells in mediation of liposome,based on which the cell strain for stable expression of anti-CD4 chimeric antibody was screened by selective culture,limiting dilution cloning and MTX pressure screening,then cultured in a large scale by cell factory.The culture supernatant was collected,from which the target antibody was purified by protein A affinity chromatography determined for expression by laser confocal microscopy,and characterized by mass spectrometry,N-terminal amino acid sequencing and determination of equilibrium dissociation constant(KD).Results The expression level of anti-CD4 chimeric antibody in constructed cell strain reached 4.29 ~ 10.52 μg / ml,which was 12.68 mg / L after large-scale culture as proved by BCA method.Laser confocal microscopy showed that both the constant region from human origin and the variable region from mouse origin were stably expressed in the constructed cell strain.Mass spectrometry indicated that the anti-CD4 chimeric antibody consisted of components from mouse and human origins.The N-terminal amino acid sequence of light chain was completely consistent with that of its parental antibody WuT4.The KD of the chimeric antibody was 2.67 × 10-9 M.Conclusion The cell strain for stable expression of chimeric antibody against human CD4 was successfully constructed,and the expressed chimeric antibody maintained the antigen binding specificity and affinity of parental antibody WuT4.