以2-巯基-1-甲基咪唑(MMI)为混合模式配基,人血免疫球蛋白G(IgG)为模型蛋白,探讨混合模式配基双水相系统分离抗体的新方法,主要包括MMI-聚乙二醇(PEG)的制备和IgG分配研究。首先优化了PEG4000的环氧氯丙烷活化反应条件,环氧氯丙烷和PEG比例为3.5:1,三氟化硼乙醚溶液加量为0.05 mL g 1PEG,开环反应时间为8 h;40%氢氧化钠溶液加量为0.15 mL g-1PEG,闭环反应时间为4 h,环氧基团接入量可达到460μmol g-1PEG,偶联配基MMI,得到MMI-PEG混合模式配基。进一步考察了IgG在PEG4000/(NH4)2SO4双水相系统中的分配,发现添加MMI-PEG配基有效提高了IgG分配系数,加入适量NaCl,分配系数达到了17.1。结果表明,混合模式配基双水相萃取可以实现IgG的上相富集,为抗体分离提供了一个新途径。
Three hydrophobic charge-induction adsorbents with functional ligands of 4-mercapto-ethyl-pyridine, 2-mercapto-methyl-imidazole or 2-mercapto-benzimidazole were evaluated in the purification of porcine immunoglobulin from porcine blood. Adsorption isotherms were studied under different pH conditions. The adsorbent with 2-mercapto-methyl-imidazole as the ligand showed reasonable adsorption capacity(43.60 mg·g^(-1)gel)with great selectivity and it also showed the best elution performance in chromatographic studies. A multi-pH step elution process was proposed for the 2-mercapto-methyl-imidazole adsorbent, and the results showed that high immunoglobulin purity(94.3%) and a yield of 9.8 mg·(ml plasma)^(-1) could be achieved under the optimal condition of loading(pH 5.0)–pre-elution(pH 7.0)–elution(pH 3.8). Moreover, molecular simulation was employed to help in analyzing the binding mechanism between the ligands and immunoglobulin, and the results showed that both 2-mercapto-benzimidazole and 2-mercapto-methyl-imidazole ligands were docked on the same pocket(around TYR319 and LEU309) of the Fc fragment of immunoglobulin, with 2-mercaptobenzimidazole showing stronger binding interactions.
Qilei ZhangTiantian ZhuangHongfei TongHongyin WangDongqiang LinShanjing Yao
