Evaluation and comparison of Clostridium epsilon-alpha fusion gene expression using different commercial expression vectors

Document Type: Original Articles

Authors

1 Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran

2 Clostridia Specialized Research Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

3 Department of Avian Bacterial Diseases, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

Abstract

Clostridium perfringens and Clostridium septicum are gram positive, anaerobic, spore forming rods and are pathogen for human and livestock, which are widespread in nature, human and animal digestive system. C. perfringens produce 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; also toxins secreted from C. septicum include alpha, beta, delta and gamma. Epsilon and alpha toxins of C. perfringens and C. septicum are major causes of enterotoxemia and braxy respectively, in sheep and goat. The production of recombinant immunogenic proteins of these bacteria using suitable expression vectors and expression prokaryotic hosts can be convenient method to reduce the costs, and time of production of clostridial anaerobic vaccines. In the present study, we used recombinant E. coli strain TOP10 containing pJETεα for evaluation C. perfringens type D and C. septicum epsilon-alpha fusion protein into different commercial vectors. After extraction of pJETεα from recombinant cell it was digested by NdeI and XhoI restriction enzymes and was subcloned into pET22b (+), pET26b (+) and pGEM-B1 expression vectors in E. coli/Rosetta and E. coli/BL21 (DE3). The expression of recombinant fusion toxin was evaluated by SDS-page and western blotting in three different temperatures, various IPTG gradients and different times with using pGEMεα, pET22εα and pET26εα vectors in E. coli/Rosetta and E. coli/BL21 (DE3). According of the results, recombinant E. coli/ Rosetta/pET22εα at 37◦C and 6h after from induction by IPTG showed most expression.

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