Long-lasting specific antibodies against P277 induced by mucosal administration of P277 repeat sequences carried by Hsp65 in the absence of adjuvants.

To improve the weak immunogenicity of peptide P277, the recombinant expression plasmid pET28-Hsp65-6 x P277 was constructed by inserting 5 x P277 which was amplified by PCR into the vector pET28-Hsp65-P277. It was transformed into Escherichia coli BL21 (DE3) and the fusion protein (Hsp65-6 x P277) was expressed effectively as soluble protein after inducing by lactose. The fusion protein was purified by means of cell disruption, ammonium sulfate precipitation, double-distilled H2O dialysis, DEAE52-cellulose column chromatography, and then used to immunize female NOD mice with three i.n. inoculations in the absence of adjuvants. Serum samples from the immunized mice were collected at 3 weeks interval. Antibodies against P277 and HSP65 were detected in immunized mice sera by enzyme-linked immunosorbent assay (ELISA) and Western blot. Specific antibodies were successfully induced and lasted for more than 20 weeks in animals immunized with the fusion protein via intranasal route even in the absence of adjuvants.