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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 human tumor necrosis factor-alpha (h TNF-α) human monoclonal antibody in CHO cells and identify the purified antibody. Methods The eukaryotic expression plasmid pHL containing the light chain, the heavy chain variable region and the constant region gene of anti-h TNF-α human monoclonal antibody was transfected into CHODG44 cells with lipofectamine TM 2000. The expression of pHL- MTX) screening and monoclonal screening to obtain CHO cell line secreting anti-h TNF-α human monoclonal antibody. After purification of the antibody by two-step chromatography using Mab Select Su Re and Capto adhere, the purity, N-terminal sequence, binding kinetic equilibrium constant and neutralizing activity of the antibody were analyzed. Results After two-step chromatography, the purity of the anti-h TNF-α human monoclonal antibody was analyzed by SDS-PAGE. The purity of the heavy chain, the light chain and the intact antibody were 98.79% The N-terminal amino acid sequence of light and heavy chains was as expected. The binding kinetic equilibrium constant (KD) was 1.34 × 10-10 M, which could antagonize the killing effect of TNF-α on L929 cells. And the rate of 90% or so. Conclusion The anti-h TNF-α human monoclonal antibody with good neutralizing activity was successfully expressed in CHO cells.