The cytolysin A (ClyA) is a 34 kDa pore-forming cytotoxic protein and expressed by some enteric bacteria including Salmonella typhi. This toxin is transported on the bacterial surface and secreted without posttranslational modification. Using the surface display of ClyA, the expression vectors for 193-aa immunogenic antigen of spike protein (termed S1E) from severe acute respiratory syndrome coronavirus (SARS-CoV) were constructed. The vectors carried a gene encoding S. typhi ClyA conjugated to S1E at the C terminus (termed ClyA-S1E) and asd gene in pGEM-T and pBR322, named pGApLCS1E and pBApLCS1E, respectively. An asd-mutated E. coli transformed with these vectors could grow without diaminopimelic acid (DAP), indicating that they were stably maintained in such mutants. ClyA-S1E recombinant proteins from these vectors were expressed on the surface of the attenuated S. typhimurium deficient of global virulence gene regulator, ppGpp. However, they did not show the hemolytic activity on the blood agar plate and cytotoxicity against HeLa cells. To examine whether bacteria expressing ClyA-S1E induced the immune response against S1E, S. typhimurium deficient of ppGpp and Asd was transformed with these vectors and orally immunized in mice. In the western blotting against GST-conjugated S1E using the immunized mouse sera, it was shown that the significant band was detected in the mouse serum by the bacteria transformed with pGApLCS1E but not with pBApLCS1E. It indicates that the immune response producing antibody was dependent on the expression level of ClyA-S1E. Therefore, ClyA delivery system can be used for SARS vaccine development.
Agar ; Animals ; Bacteria ; Blotting, Western ; Coronavirus ; Diaminopimelic Acid ; Enterobacteriaceae ; Genes, vif ; HeLa Cells ; Humans ; Mice ; Perforin ; Protein Processing, Post-Translational ; Recombinant Proteins ; Salmonella ; Salmonella typhi ; Severe Acute Respiratory Syndrome