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

To prepare human anti-HBs Fab by bioengineering technique.

The specific human anti-HBs Fab gene screened from combinatorial library was cloned into plasmid pBAD/g IIIA, then positive clone was transformed into E.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.

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.

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.