Cell death analysis of recombinant mature epsilon toxin on the kidney cell line

Abstract

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.

Keywords

• Cell death;
• Clostridium perfringens;
• Epsilon toxin;
• Necrosis;
• Recombinant expression