탄저의 방어항원과 치사인자 정제 및 방어효과 분석

Purification of protective antigen and lethal factor of bacillus anthracis and analysis on protective effects

Anthrax is one of the most important zoonotic diseases in both clinically and economically in the worldwide. Bacillus anthracis, the causative agent of anthrax, possesses two known virulence determinants : a tripartite protein toxin and a poly-D-glutamic acid capsule. The tripartite toxin consists of protective antigen(PA), lethal factor(LF) and edema factor(EF) To investigate pathogenesis of anthrax, firstly, we developed purification techniques for PA and LF of tripartite toxin. We applied DEAE sepharose CL-6B, Mono-Q, Superose 12 and phenyl-superose column using FPLC system in order. We purified 2.88 mg of PA and 0.76 mg of LF from 5 liter of RM-medium and recovery rates of PA and LF were 0.107, 0.028% for total protein of culture supernatant. Molecular weights of PA and LF were 86,87 kDa by SDS-PAGE analysis. PA and LF were conformed by monoclonal antibody and mouse lethality. Mouse lethality could not be detected by single inoculation of PA and LF. However, in the combined inoculation of PA and LF, 50% mouse lethal dose was detected PA, 6.00㎍ and LF, 1.25㎍. Protective efficacy of the purified PA and LF was analyzed by challenge virulent strain mice immunized by single or combined inoculation of each components. The protection rates were 66.7% in the PA and LF combined inoculation group, 45.8% in the only PA inoculated group.