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.

[Isolation and characterization of streptomyces in composted Iranian soil, using chemotaxonomy]

The Actinomycetes are gram-positive organisms that tend to grow slowly as branching filaments. They were thought to be related to both bacteria and fungi, but in recent years it has been undoubtedly shown that they are prokaryotic organisms. Actinomycetes have proved to be causal agents of many human and animal infections. They also play as an important rule in producing new antibiotic agents. Fifty soil samples collected from different regions of the country were analyzed to determine the presence and types of antibiotic-producing Streptomycetes using dilution plating method. Starch casein agar and glucose yeast extract agar were used as the culture media. 88 selected organisms from 140 isolates were subjected to chemotaxonomy determination. The isomeric form of diaminopimelic acid [DAP] was determined by thin-layer chromatography [TLC] of hydrolyzed whole-organism preparations. A sample of the hydrolysated extract was applied as a 2[cm] band from the base of the TLC plates. A standard containing meso and LL-DAP was also run at both sides of the sheet for comparison with test mixtures. The plates were developed for about 3 [1/2] hours in a glass tank containing 40 ml methanol, 2 ml 6Mol HCl, 5ml Pyridine, and 13 ml water. DAP appears as green/brown spots, changing to yellowish with time. The LL-DAP, meso-DAP and hydroxy-DAP isomers have approximate R[f] values. Approximately 97% of test strains contained LL-DAP and the remaining 3%, contained meso-DAP. This study demonstrated a wide variety of Streptomycetes bacteria isolates which are capable to produce antibiotics and TLC is an appropriate method in determination of Them