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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。
OBJECTIVE: To prepare human anti-HBs F-ah using bioengineering technique. Methods: The human anti-HBs Fab gene screened from the antibody library was cloned into pBAD / gⅢA vector and transformed into E.coli Top10. After the recombinant plasmids were fermented, the periplasmic-cavity soluble Fab protein was purified by Ni-NTA-Agarose chelate chromatography. The resulting inclusion body in turn denaturation, dissolution, purification, the use of dialysis refolding. The specificity of Fab protein was detected by Western blot and its biological activity was determined by Dot blot. Results: The periplasmic soluble Fab protein purified by Ni-NTA-Agarose column had good biological activity and the total amount reached 80mg / L. After the obtained inclusion body dialysis refolding, but also a small amount of active protein, but a small proportion. Conclusion: The human anti-HBs Fab fragment was expressed by pBAD / gⅢA-Top10 expression system. After fermentation, the soluble protein with good biological activity was obtained after purification, which provided an effective method for mass production of human anti-HBs Fab fragment means.