抗人肿瘤坏死因子-α人源单克隆抗体在CHO细胞中的表达及鉴定

Expression of human monoclonal antibody against human tumor necrosis factor-α in CHO cells and identification of expressed product

目的在CHO细胞中表达抗人肿瘤坏死因子(human tumor necrosis factor-alpha,h TNF-α)人源单克隆抗体,并对抗体纯化后进行鉴定。方法将含有编码抗h TNF-α人源单克隆抗体轻、重链可变区及恒定区基因的真核表达质粒p HL,利用脂质体Lipofectamine TM 2000转染CHODG44细胞,经氨甲喋呤(methotrexate,MTX)加压筛选和单克隆筛选,获得分泌表达抗h TNF-α人源单克隆抗体的CHO细胞株。应用Mab Select Su Re和Capto adhere两步层析纯化抗体后,分析抗体的纯度、N-末端序列、结合动力学平衡常数及中和活性。结果经两步层析后,抗h TNF-α人源单抗经SDS-PAGE分析,可见纯度较好的抗体重链、轻链和完整抗体条带,SEC-HPLC纯度为98.79%;抗体的轻、重链N-末端氨基酸序列与预期一致,其结合动力学平衡常数(KD)为1.34×10-10 M,能拮抗TNF-α对L929细胞的杀伤作用,对TNF-α细胞毒的中和率达90%左右。结论在CHO细胞中成功表达了具有较好中和活性的抗h TNF-α人源单克隆抗体。

Objective To express the human monoclonal antibody against human tumor necrosis factor（h TNF）-α in CHO cells,then purify and identify the expressed product. Methods Plasmid p HL containing the genes encoding the variable and constant regions of light and heavy chains of human monoclonal antibody against h TNF-α was transfected to CHODG44 cells in mediation of Lipofectamine^TM 2000,based on which the cell strain for secretory expression of monoclonal antibody against h TNF-α was screened by methotrexate（MTX）pressure screening and limiting dilution cloning.The target antibody was purified by Mab Select Su Re affinity chromatography and Capto adhere chromatography,then analyzed for purity,N-terminal amino acid sequence,binding dynamic equilibrium constant and neutralizing activity.Results After two steps of chromatography,the light and heavy chains as well as intact bands of human monoclonal antibody against h TNF-α were shown on SDS-PAGE profile,of which the SEC-HPLC purity was 98. 79%. The amino acid sequences at N-terminus of light and heavy chains of antibody were consistent with those expected,while the binding dynamic equilibrium constant（KD）was 1. 34 × 10^10 M. The antibody agonized the killing effect of TNF-α on L929 cells,with a neutralization rate of about 90%. Conclusion Human monoclonal antibody against h TNF-α with high neutralizing activity was expressed successfully in CHO cells.