Patent ID: 6329156
Filing Date: 2001-12-11
Classification: C12Q

Abstract:
A method for screening inhibitors of the toxicity of Bacillus anthracis, which comprises the steps of:(a) generating the recombinant PA32 DNA fragment, SEQ ID NO. 7, from region 4 of protective antigen PA83 of Bacillus anthracis and ligating the PA32 DNA fragment to enhanced green fluorescent protein, EGFP, to form EGFP-PA32;(b) expressing the EGFP-PA32 to produce EGFP-PA32 protein;(c) contacting the EGFP-PA32 protein with individual cells in a first sample of mammalian cells, thereby generating a first sample of fluorescent cells;(d) measuring the fluorescence from individual cells in the first sample of fluorescent cells;(e) mixing EGFP-PA32 protein with a potential toxicity inhibitor of Bacillus anthracis;(f) contacting the mixture of the EGFP-PA32 protein and the potential toxicity inhibitor with individual cells in a second sample of mammalian cells, forming thereby a second sample of fluorescent cells;(g) measuring the fluorescence from individual cells in the second sample of fluorescent cells; and(h) comparing the fluorescence from individual cells in the first sample of fluorescent cells with the fluorescence from individual cells in the second sample of fluorescent cells, whereby the effectiveness of the toxicity inhibitor is determined from the decrease of the fluorescence from individual cells from the second sample of fluorescent cells relative to the fluorescence from individual cells in the first sample of fluorescent cells.