建立了离子交换层析介质一步纯化鸡白痢沙门氏菌兔抗血清获得多抗免疫球蛋白G (IgG)的方法，利用IgG与杂蛋白等电点的差异，指导阴阳离子交换层析介质筛选和操作条件优化；针对多抗IgG易失活的难题，采用差示扫描荧光法为纯化后的IgG筛选稳定剂。结果表明，通过毛细管等电聚焦测得兔抗血清多抗IgG的等电点为6.04?7.08，阳离子交换介质CM Sepharose Fast Flow层析纯化的IgG最高电泳纯度为63.5%，高效液相尺寸排阻色谱测得IgG回收率为15.5%；阴离子交换层析纯化效果更佳，pH 5.5条件下Q Sepharose XL(Q-XL)介质层析获得的IgG最高纯度达99.3%，回收率达67.5%；200 g/L山梨醇对IgG具有最佳稳定作用，IgG的2个热变性温度分别提高了5.52和8.84℃，70℃时山梨醇的稳定作用更明显；以200 g/L山梨醇为稳定剂、用阴离子交换层析介质Q Sepharose XL一步纯化所得IgG具有高纯度、高收率及高稳定性，且制备工艺简单。
A single step separation protocol was developed for purification of anti-Salmonella pullorum polyclonal immunoglobulins G (IgG) from rabbit serum. According to the difference of isoelectric point (pI) between IgG and the major impurities, the types of ion exchange media were systematically screened by comparison of their purification results. In order to prevent the IgG from denaturation, different stabilizers were screened by differential scanning fluorimetry (DSF). The results showed that pI of the IgG was 6.04?7.08 determined by capillary iso-electric focusing. After cation exchange chromatography with packing materials of CM Sepharose Fast Flow (CM), the purity analyzed by SDS?PAGE was 63.5% and IgG recovery rate measured by high performance size-exclusion chromatography was 15.5%, respectively. In contrast, the purity of IgG was 99.3% and recovery rate was 67.5% after anion exchange chromatography with packing materials of Q Sepharose XL (Q-XL). 200 g/L sorbitol was found to possess the best protection effect. The two thermal denaturation temperatures of IgG were increased by 5.52 and 8.84℃, respectively, and the stability at 70℃ was significantly improved. The results demonstrated that the one step anion exchange chromatography together with 200 g/L sorbitol protection provided a high purity, high recovery rate, and high stability of the IgG. The whole process is facile and efficient.
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