OBJECTIVETo express and purify Hap protein of nontypeable Haemophilus influenzae (NTHi) in prokaryotic system, and study its immunogenicity and adhesive activity.

METHODHap protein was expressed in E.coli BL21 with pET32a (+)-Hap and purified by affinity chromatography. The adhesive activity of the recombinant Hap protein was observed in competitive adhesion assay using scanning electron microscope and bacterial counting. BALB/C mice were immunized intranasally with the purified recombinant Hap protein and cholera toxin B subunit (CT-B), and anti-Hap IgA and IgG were detected by enzyme-linked immunosorbent assay.

RESULTSSDS-PAGE analysis showed a single band of the target protein, whose purity reached 85% according to the result of Gel analysis software. The concentration of the protein was 3.2 g/L after ultrafiltration and condensation. Competitive adhesion assay showed that compared with control group, the recombinant Hap protein significantly inhibited the adhesion of NTHi to ECM (P<0.01). Compared with Hap immunization alone, immunization with Hap combined with CT-B resulted in significantly higher titers of anti-Hap IgG and IgA in mice (P<0.05).

CONCLUSIONHighly purified recombinant Hap protein has been obtained in a prokaryotic system and shows good immunogenic and adhesive activities. These results will establish the basis for further study of NTHi vaccine.