[Display of avian influenza virus nucleoprotein on Bacillus thuringiensis cell surface]

S-layer protein CTC surface display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying avian influenza virus nucleoprotein (NP) on Bt cell surface. Four recombinant plasmids were constructed by replacing 3'-terminal or central part below the surface anchor sequence slh of S-layer protein gene ctc with full length of np gene or part np gene (npp). The four resulting plasmids were pSNP (harboring fusion gene ctc-np), pCSA-SNP (harboring fusion gene csa-ctc-up, csa represents csaAB operon which is very important to the anchoring of S-layer protein on the bacterial cell surface), pCTC-NPP (harboring fusion gene ctc-npp) and pCSNPP (harboring fusion gene csa-ctc-npp). Five recombinant Bt strains were constructed by electro-transferring recombinant plasmids to Bt plasmid-free derivative strain BMB171. The resulting strains were BN (harboring pSNP), BCN (harboring pSNP as well as the plasmid pMIL-CSA which carried csaAB operon), C-S (harboring pCSA-SNP), BCCN (harboring pCTC-NPP and pMIL-CSA) and CN (harboring pCSNPP). The vegetative cells of five recombinant strains were used as agglutinogens of slide agglutination assay. Slide agglutination assay showed recombinant NP proteins were successfully displayed on the surface of five recombinant strains, respectively. After immunizing mice with vegetative cells of five recombinant strains respectively, five recombinant strains all elicited humoral respones to NP and exhibited immunogenicity as assayed by enzyme-linked immunosorbent assay (ELISA). Meanwhile these assays showed recombinant strain CN (harboring fusion gene csa-ctc-npp) exhibited the highest immunogenicity among five recombinant strains. That means the best way of constructing S-layer fusion gene is csa-ctc-* (* denotes heterologous antigen gene) which means the central part of S-layer protein gene ctc replaced by the heterologous antigen gene and csaAB operon located on the upstream of fusion gene. The strategy developed in this study gives a possibility to generate heat stable,oral, veterinary vaccine with Bt S-layer protein CTC surface display system.