In order to induce stringent regulation of ghost cassette, a new ghost system was designed so that E gene is regulated by cI857 of a temperature sensitive regulator and antisense RNA under arabinose system at transcriptional and translational levels, respectively. The constructed system under stringent regulation owing to both regulatory factors was designed as pMMP184 which carries pBR replication origin. A ghost cell obtained from S. typhimurium harboring pMMP184 was examined for immune response after injection via intramuscular (IM) route against BALB/c mouse. The immune responses IgG, IgG1, IgG2a and vaginal IgA of the mice against S. typhimurium ghost cells were increased suddenly at 4 weeks post injection and maintained until 10 weeks. Additionally, pathogenic E. coli antigens, such as FedA, FedF, F41 and intimin, carried in the ghost cells elicited similar immune responses to those of S. typhimurium lipopolysaccharide. Proliferations of T and B cells, indicated by elevations of CD3e and CD23, respectively, were observed using FACS analysis.  The challenge assays against sublethal dose of virulent S. typhimurium were presented by protections of 25~50% more than that of control. Our results suggest that the S. typhimurium ghost cell produced from the new ghost system is a potential possibility as a ghost vaccine.

Key words: ara operon, asd Ghost vaccine, Salmonella typhimurium, Temperature-sensitive