论文部分内容阅读
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。
Objective To construct stable expression cell line of anti-human CD4 chimeric antibody and identify its expression product. Methods CHO-DHFR cells were stably transfected with chimeric antibody against human CD4 chimeric plasmid pHDC4 by lipofectamine. The cells were stably cultured in CHO-DHFR- Factory expansion training. The supernatant was collected and purified by protein A affinity chromatography. The expression of antibody gene was observed by laser scanning confocal microscopy. The expression of antibody gene in the cells was analyzed by mass spectrometry, protein N-terminal sequencing and equilibrium dissociation constant (KD) . Results The antibody expression levels of the anti-human CD4 chimeric antibody stably expressing cell line were 4.29-10.52μg / ml. The BCA antibody expression level was 12.68 mg / L. The results of confocal laser scanning microscopy showed that anti-CD4 The co-antibody stable expression cell strain can stably express the human constant region and the mouse variable region. The mass spectrometry analysis showed that it contained murine and human components. The N-terminal amino acid sequence of the protein showed that the light chain and the parent antibody N - The terminal amino acid sequence is exactly the same; its KD is 2.67 × 10-9M. Conclusion The anti-human CD4 chimeric antibody stably expressing cell line was successfully constructed. The expressed anti-human CD4 chimeric antibody retained the antigen-binding specificity and affinity of the parent antibody.