Cell Death Analysis of Recombinant Mature Epsilon Toxin on the Kidney Cell Line

Background and objectives: epsilon toxin is the third hazardous bacterial toxin causing abs enterotoxaemia in domestic animal. in addition, epsilon toxin is known as a biological warfare agent. the aim of this study was to produce the recombinant mature epsilon toxin to evaluate cell death impact on the kidney cell line. materials and methods: for this purpose, the sequence of mature epsilon toxin (46-328 aa) in pet28a was cloned and expressed in escherichia coli bl21 (de3) and purified by nickel-nitrilotriacetic acid (ni-nta) column and confirmed by western blot analysis using hrp conjugated anti-his antibody. then, to assess the anti-proliferative effects of different concentrations of recombinant epsilon toxin, the mtt assay was done on the hek293 cell line. the annexin v/pi staining was done to investigate the apoptotic and necrotic cell populations after exposure to epsilon toxin. results: induction by 1 mm iptg for 4 h at 37°c was an optimized condition for expressing mature epsilon toxin in e. coli strain bl21 (de3). electrophoresis on sds-page 12% gel showed the desired band approximately at 38 kda. our results showed that recombinant epsilon toxin is mainly expressed as an inclusion body. furthermore, 100, 150, and 200 μg/ml of mature epsilon toxin are significantly reduced the cell viability (p≤0.05). the considerable increase of necrotic cell percentage was shown after exposing to 100, 150, and 200 μg/ml of mature epsilon toxin (p≤0.05). conclusion: the recombinant mature epsilon toxin had cytotoxic effects and could induce necrosis.