Figure 6

Figure 6

(A) Vector map of pYA3493 (pBR ori), which is a periplasmic secretion AsdA⁺ vector. A DNA fragment encoding the β-lactamase signal sequence (bla SS) and 12 amino acid residues of the N-terminus of mature-lactamase was positioned under the control of the P_trc promoter. Map of pYA3493 shows the unique restriction enzyme sites in the multiple cloning site. (B) Vector map of recombinant plasmid pG8R8029. Codon-optimized gene of IAG52B non-cytoplasmic domain of 1269 bp was PCR amplified and cloned into the pYA3493 at EcoRI-BamHI sites. The IAG52B gene was fused into the same reading frame of bla SS and under the control of the P_trc promoter. (C) Synthesis of IAG52B antigen in E. piscicida, E. ictaluri and E. coli asdA mutant strains. pYA3493 (vector control) or pG8R8029 (encoding IAG52B) were electroporated into E. piscicida (χ16000) or E. ictaluri (J111) or E. coli (χ6212). IAG52B antigen synthesis was analyzed in these cells by western blotting using an anti-IAG52B antibody. (D) Subcellular location of synthesized IAG52B in E. piscicida and E. coli. Western blot showing IAG52B synthesis in whole cell lysate, periplasmic, cytoplasmic, outer membrane and supernatant fraction of χ16000 and χ6212 harboring pG8R8029. (E) Analysis of IAG52B antigen synthesis in E. piscicida vaccine strain χ16022. pYA3493 (vector control) or pG8R8029 (encoding IAG52B) was electroporated into χ16022, IAG52B antigen synthesis was analyzed by western blotting by using anti-IAG52B antibody.