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 TOP10 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 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 sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting in three different temperatures, various isopropyl β-D-1-thiogalactopyranoside (IPTG) gradients, and different times using pGEMεα, pET22εα, and pET26εα vectors in E. coli/Rosetta and E. coli/BL21 (DE3). According to the obtained results, recombinant E. coli/Rosetta/pET22εα showed better expression at a temperature of 37°C after 6 h of induction by IPTG.