The Bacterial Surface Expression of SARS Viral Epitope using Salmonella typhi Cytolysin A

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.

Screening of Anti-Atopic Dermatitis Material by Using NC/Nga Mouse Whole Blood System

BACKGROUND: Allergic inflammation was induced by activated Th2 lymphocytes, leading to IgE production and eosinophil activation. A Th2 disproportion was shown in atopic children soon after birth. During specific allergen stimulation, an increase of Th2 cells was observed in most cases. In this study, we prepared new screening "whole blood" system for searching the anti-atopic materials. Cytokine production and IgE secretion from whole blood system were assessed and we confirmed the results by using animal system. METHODS: Pathological features in NC/Nga mice are similar to those observed in human atopic dermatitis. Whole blood from NC/Nga mouse was stimulated by using TNCB (Th2 activator) or candidate materials of anti-atopic dermatitis, and the production of cytokines (IL-4, IL-12, and IFN-gamma) were measured by ELISA. In order to confirm the results of whole blood system, in vivo test was done by using NC/Nga mice. RESULTS: In whole blood system, LPS and extracts of green tea, hardy orange and onion induced the production of IL-12 and IFN-gamma while they reduced the production of IL-4. Also, LPS and extracts of onion reduced IgE production. Though atopic dermatitis was observed from a mouse stimulated with TNCB, it was not when a mouse was co-stimulated in LPS or extracts of onion. The results are same as those observed in whole blood system. CONCLUSION: Whole blood system was simple and speedy methods for searching a materials compared with the conventional high-cost animal system. And the results using whole blood system was proved to be reliable in our experiments for screening anti-atopic material. We expect that the system can be applied to other experiments for searching similar materials.