Research On Immunoproteomics Of Bacillus Anthracis A16R

Bacillus anthracis is a pathogenic bacterium which can cause a severe zoonosis. Due to the spores, which are spread by the aerosol route, easily large-quantity, and difficult to be killed, B.anthracis is considered to be the first biological warfare agents in some countries. Since the "911 "events happened in the USA, anthrax had been focused again all over the world. The vaccine is the best method to prevent the anthrax, and selecting the novel protective antigen plays a key role in the new vaccine research. In this work, we screened a lot of protective antigens from the vegetative proteins and spore proteins of A16R strain using the immunoproteomics. All of these could be helpful in the prophylaxis and therapy of anthrax. The results are as follow:The BA vaccine strain A16R and 200 mL Luria-Bertani solid medium were chose to culture BA spores. The spores were cultured in 37℃incubator for one day, then transferred to 30℃incubator for 10-15 days and collected using 4℃sterilized water at the 8000 r/min for 10 minutes. The purity of the spores was up to 98% by the fuchsin dyeing. At the end the spores were resuspensed by 2 mL 4℃sterilized water and the concentration was 5.5×109 CFU/mL by counting.Four New-Zealand rabbits were immunized by the collected B.anthracis spores. The first immune style was inguinal groove subcutaneous injection, and the followed two challenged was ear-vein, the last two challenged were subcutaneous for 7 days interval. The titer was up to 12800 by the indirectly ELISA. The serum can make reaction with the BA protein using western blotting. So the serum can make attribution to the next test followed.The B.anthracis vegetatve protein and spore protein were made by sonication, separated using the two-dimension electrophoresis gel, transferred onto PVDF and western blotted using the serum prepared in the section 2. After comparison between the result of western blotting and that of the Coomassie brilliant blue-dyed gel, the interested protein spots were chosen, digested by trypsin in gel and identified by MS. In conclusion, we have screened 86 immunogenic proteins in the vegetative cell protein sample, and 13 in the spores protein sample.19 immunogenic proteins had been reported before. All of these immunogenic proteins will benefit he development of anthrax vaccines.