Display of H5N1 Avian influenza virus haemagglutinin HA1 on Bacillus thuringiensis cell surface and its immunogenicity for mice

S-layer protein CTC surface display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying H5N1 Avian influenza virus haemagglutinin HA1 on B. thuringiensis cell surface. Two recombinant plasmids were constructed by replacing the central part below the surface anchor sequence slh of S-layer protein gene ctc with part ha1 gene (halp ). The two resulting plasmids were pCTC-HA1P (harboring fusion gene ctc-halp ) and pCSHA1P (harboring fusion gene csa-ctc-halp, csa represents csaAB operon which was very important to the anchoring of S-layer protein on the bacterial cell surface). Two recombinant B. thuringiensis strains were constructed by electro-transferring recombinant plasmids to B. thuringiensis plasmid-free derivative strain BMB171. The resulting strains were CH (harboring pCSHA1P) and BCCH (harboring pCTC-HA1P as well as the plasmid pMIL-CSA which carried csaAB operon). The vegetative cells of CH and BCCH were used as antigens of haemagglutination (HA) assay and haemagglutination inhibition (HI) assay. HA assay showed recombinant HA1 proteins were successfully displayed on the cell surface of CH and BCCH, respectively. HI assay showed recombinant HA1 proteins displayed on the cell surface were specific to standard positive HI (haemagglutination inhibition test) serum of subtype H5 AIV. After immunizing mice with vegetative cells of CH and BCCH respectively, CH and BCCH all elicited a humoral response to HA1 and exhibited immunogenicity as assayed by enzyme-linked immunosorbent assay (ELISA). ELISA also showed CH exhibited a higher immunogenicity than BCCH. The strategy developed in this study gives a possibility to generate heat-stable, oral, veterinary vaccine against AIV with B. thuringiensis S-layer protein CTC surface display system.