Gat, O., Grosfeld, H., Ariel, N., Inbar, I., Zaide, G., Broder, Y., Zvi, A., Chitlaru, T., Altboum, Z., Stein, D., Cohen, S. and Shafferman, A.
Notes: After bioinformatics analysis of the Bacillus anthracis genome for potential vaccine targets, the 197 ORFs were selected for amplification. Using the genomic DNA of B. anthracis strain Vollum, all PCR products were generated using a 5’ primer incorporating the T7 promoter, the Kozak sequence, unique restriction enzyme sites and a start codon and a 3’ primer with a stop codon and three more unique restriction sites. The amplicons were then in vitro transcribed and translated using the TNT® T7 Quick for PCR DNA system and radiolabeled with [35S]methionine. The products were analyzed by SDS-PAGE and autoradiography. To assess the immunoreactivity of the translated products, the proteins were immunoprecipitated using anti-B. anthracis antisera. The seropositive candidate amplified ORFs were cloned into the pCI Mammalian Expression Vector using compatible RE sites. These vectors were introduced into ICR mice using a Helios gene gun system (0.5µg plasmid DNA on 1µm gold particles) and the mice immunized in 2 week intervals. (3515)