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Iranian Journal of Microbiology، جلد ۱۳، شماره ۶، صفحات ۸۳۲-۸۳۸
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عنوان انگلیسی Cell death analysis of recombinant mature epsilon toxin on the kidney cell line
چکیده انگلیسی مقاله 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.
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نویسندگان مقاله | Roza Chehreara
Department of Genetics, Faculty of Basic Sciences, Islamic Azad University, Research Branch, Tehran, Iran


| Shohreh Zare Karizi
Department of Genetics and Biotechnology, School of Biological Science, Varamin-Pishva, Branch Islamic Azad University, Varamin, Iran


| Hamideh Mahmoodzadeh Hosseini
Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran


| Seyed Ali Mirhosseini
Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran


| Mohammad Shafiei
Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran


| Jafar Amani
Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran


| Rouhollah Kazemi
Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
نشانی اینترنتی https://ijm.tums.ac.ir/index.php/ijm/article/view/2913
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