Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

Figure 9

Biological activity of PA antigen and its mice immunized sera on J774.1 macrophage cells. (a) Cytotoxicity of recombinant PA in comparison to native PA from Bacillus anthracis when incubated along with 0.125 μg mL⁻¹ LF. Cells containing LF alone were taken as negative control. Recombinant PA showed 50% cytotoxicity at 0.3 μg mL⁻¹ while native PA showed 0.25 μg mL⁻¹. (b) Toxin neutralization of PA immunized sera. J774.1 cells were treated with 0.3 μg mL⁻¹ of PA, 0.25 μg mL⁻¹ of LF, and serial twofold dilution of PA immunized mice sera starting from 1 : 100. After 5 hours of incubation, 20 μL of 5 mg mL⁻¹ MTT dye was added, and after 30 minutes, the formazan crystals developed were dissolved by acidified isopropanol. The toxin neutralization titer was defined as the highest sera dilution showing 50% death of macrophage. For PA sera, it was found to be 6.4 × 10³.

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