生物工程技术制备人源抗-HBs Fab片段

The preparation of human anti-HBs Fab fragment by bioengineering technique

目的:用生物工程技术制备人源性抗-HBs F-ah。方法:将从抗体文库中筛选出的人源抗-HBs Fab基因克隆人pBAD/gⅢA载体,进而转化Top10大肠杆菌。对重组质粒菌发酵表达后,利用Ni-NTA-Agarose螯合层析柱纯化周质腔可溶性Fab 蛋白。对所得包涵体依次变性、溶解、纯化后,利用透析进行复性。用Western blot检测Fab蛋白的特异性,Dot blot测定其生物学活性。结果:经Ni-NTA-Agarose柱纯化的周质腔可溶性Fab蛋白,有较好的生物学活性,并且总量达到80mg/L。对所获包涵体进行透析复性后,也可得到少量有活性的蛋白,但比例很小。结论:用pBAD/gⅢA-Top10表达系统表达人源抗-HBs Fab片段,发酵培养后,经有效纯化可得到生物学活性较好的可溶性蛋白,为人源抗-HBs Fab片段的大量制备提供了有效手段。

AIM: To prepare human anti-HBs Fab by bioengineering technique. METHODS: The specific human anti-HBs Fab gene screened from combinatorial library was cloned into plasmid pBAD/g IIIA, then positive clone was transformed into ? coli Top10. After the fermentation and expression processes, the soluble Fab fragment in the periplasm were purified by Ni-NTA-agarose affinity chromatography, and the inclusion bodies were in turn denatured, solubilized, purified and renatured. The specificity of Fab protein was confirmed by Western blot, and binding activity to HBsAg was verified by Dot blot. RESULTS: The quantity of soluble Fab protein purified from periplasm with Ni-NTA-Agarose which possessed good specificity as well as excellent binding activity to antigens was up to 80 mg per liter, but the biologically active protein acquired after renaturation of the inclusion bodies was quite small. CONCLUSION: Using pBAD/g IIIA-Top10 expression system, the soluble Fab protein with biological activity could be produced from periplasm of the E.coli /Top10, and the strategy is available to prepare human anti-HBsAg Fab fragments in large quantity by gene engineering technique.