Evaluation and Comparison of Clostridium Epsilon-Alpha Fusion Gene Expression Using Different Commercial Expression Vector
Publish place: Archives of Razi Institute journal، Vol: 76، Issue: 1
Publish Year: 1400
نوع سند: مقاله ژورنالی
زبان: English
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JR_ARCHRAZI-76-1_002
تاریخ نمایه سازی: 6 دی 1402
Abstract:
Clostridium perfringens and Clostridium septicum are gram-positive, anaerobic, spore-forming rods and pathogens for humans and livestock, which are widespread in nature as well as human and animal digestive systems. C. perfringens produces numerous different exoproteins, which are various systems of action. The major C. perfringens toxins include alpha, beta, epsilon, and iota. C. perfringens are classified into five groups (A-E) on the basis of the production of these lethal toxins. Furthermore, toxins secreted from C. septicum include alpha, beta, delta, and gamma. Epsilon and alpha toxins of C. perfringens and C. septicum are the major causes of enterotoxemia and braxy in sheep and goats, respectively. The production of recombinant immunogenic proteins of these bacteria using suitable expression vectors and expression prokaryotic hosts can be a convenient method for the reduction of the costs and production time of clostridial anaerobic vaccines. In the present study, recombinant Escherichia coli strain TOP۱۰ containing pJETεα was used for the evaluation of C. perfringens type D and C. septicum epsilon-alpha fusion protein using different commercial vectors. After the extraction of pJETεα from the recombinant cell, it was digested by NdeI and XhoI restriction enzymes and subcloned into pET۲۲b (+), pET۲۶b (+), and pGEM-B۱ expression vectors in E. coli/Rosetta and E. coli/BL۲۱ (DE۳). The expression of recombinant fusion toxin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting in three different temperatures, various isopropyl β-D-۱-thiogalactopyranoside (IPTG) gradients, and different times using pGEMεα, pET۲۲εα, and pET۲۶εα vectors in E. coli/Rosetta and E. coli/BL۲۱ (DE۳). According to the obtained results, recombinant E. coli/Rosetta/pET۲۲εα showed better expression at a temperature of ۳۷°C after ۶ h of induction by IPTG.
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Authors
H. R Sepehrifar
Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran
R Pilehchian Langroudi
Clostridia Specialized Research Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
S Ataei
Department of Avian Bacterial Diseases, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
A Haddadi
Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran
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