重组人抗HBsAg Fab抗体的纯化方法比较研究

目的研究重组人抗HBsAg Fab抗体的纯化条件。方法采用羊抗人Fab抗体亲和层析柱、14F7单克隆抗体亲和层析柱、离子交换-分子筛层析柱，分别纯化由酵母工程菌(Gs115／Fab)发酵的重组人抗HBsAg Fab抗体，并对3种纯化方法所得Fab抗体的纯度、收率、与HBsAg的结合活性进行比较。结果3种纯化方法中，14W单克隆抗体柱纯化的Fab抗体的纯度达98％左右，Fab抗体柱纯化的Fab抗体的纯度为95％，但这两种亲和柱的目的蛋白收率都不高，分别为35％、55％。而离子交换柱纯化的Fab抗体的纯度为93．8％，经分子筛柱进一步纯化后，可达98％以上，Fab抗体蛋白收率可达80％以上。经ELISA分析，3种方法纯化的Fab抗体均具有较高的HBsAg抗原结合力和特异性。结论通过对3种纯化方法的比较得出，离子交换一分子筛层析法是重组人抗HBsAg Fab抗体的最佳纯化方法，这为抗HBsAg Fab抗体的产业化生产和临床研究打下良好基础。

Study on the purification of recombinant human anti-HBsAg Fab fragment

Objective The aim of the study was to develop a purification procedure on Pichia pastoris GS115/Fab expressing human anti-HBsAg Fab fragment. Methods Purity and yield ratio and conjugated activity of purified Fab fragment were analyzed with three purified ways of goat anti human Fab affinity chromatography and 14F7 monoclonal antibody affinity column, as well as Ion exchange Size exclusion column. Results 98% purity was reached through 14F7 monoclonal antibody column, and 95% purity was gained after goat anti human Fab fragment column. But yield ratio of the two affinity columns was low, being 35% and 55%, respectively. ForIon exchange Size exclusion column, purity and yield ratio of Fab fragment were very good, being 93.8% and 80% or more, respectively. Results of ELISA analysis showed that purified Fab fragment through three columns could bind to HBsAg specifically. Conclusion The purification process of recombinant anti-HBsAg Fab fragment was established. It lays a foundation for industrialization and clinical research of human anti-HBsAg Fab fragment.