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针对由中国仓鼠卵巢细胞(CHO)表达的多聚亚基蛋白HBsAg在离子交换层析过程中容易因亚基解离而导致蛋白解聚和丧失生物活性的难题,实验中选择聚乙二醇(PEG)作为保护剂伴随式(Polyethylene Glycol-Accompanied)离子交换层析分离纯化HBsAg。实验表明,在流动相中加入1%PEG10000(WV)作为纯化伴侣,HBsAg的回收率由55%左右提高到80%以上,纯化倍数基本保持在12左右。对纯化产物进行SDS_PAGE分析表明,1%PEG10000的纯化伴侣伴随式离子交换层析能全部保留HBsAg的糖基化蛋白单体(27kD和30kD),高效液相色谱联用多角度激光散射(High Performance Size Exclusion Chromatography-Multiangle Laser Light Scattering,HPSEC-MALLS)进一步分析阐明了PEG能促使HBsAg颗粒尺寸分布更均一,结构更接近天然乙肝表面抗原。
In order to solve the problem that the poly-subunit HBsAg expressed by Chinese hamster ovary cells (CHO) is easy to dissociate and lose its biological activity due to dissociation of subunits during ion exchange chromatography, polyethylene glycol PEG) was isolated and purified as a Polyethylene Glycol-Accompanied ion exchange chromatography. Experiments show that adding 1% PEG10000 (WV) as the purification partner to the mobile phase, the recovery rate of HBsAg is raised from 55% to over 80%, and the purification fold is basically maintained at about 12. SDS-PAGE analysis of the purified product showed that the purified conjugate of 1% PEG10000 could completely retain the glycosylated protein monomers (27kD and 30kD) of HBsAg by ion exchange chromatography. High Performance Liquid Chromatography combined with High Performance Laser Size Exclusion Chromatography-Multiangle Laser Light Scattering, HPSEC-MALLS) further elucidated that PEG can promote the uniform distribution of HBsAg particle size and structure closer to the native hepatitis B surface antigen.