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Clostridium perfringens is the causative agent of animal gastrointestinal diseases, especially ruminant enterotoxemia. It cause food poisoning, gas gangrene and necrotizing enteritis in humans. There isn’t type A component in Razi enterotoxemia vaccine. Nowadays, the nontoxic mutants of α-toxin are suggested as new candidates for vaccination. In this study, different nontoxic α mutants were design and produced, and their properties had been investigated. Mutants of α-H68G, α-H148L, and α-H148G were synthesized by site-directed mutatgenesis (SDM) in the particular region of α-toxin gene. Another mutant, sα (cpa247-370), was also produced by gene manipulation and gene synthesis. These α mutant genes were cloned in pET-26b (+) vector, and the obtained plasmids (pET-αH68G, pET-αH148L, pET-αH148G, and pET-sα) were transformed into E. coli (DE3) to express these genes. Positive clones of E.coli-H68G, E.coli-H148G, E.coli-H148L, and E.coli-sα were identified by plasmid extraction, restriction enzymatic cut, cloning PCR, and sequencing to confirm mutation that led to the conversion of histidine 68 (H68) to glycine and histidine 148 (H148) to glycine or lysine. Protein expression was optimized according to SDS-PAGE and evaluated by western blot and dot blot. The results showed the expression of these mutants in E.coli. Among them, α-H68G and sα mutants were chosen as the best for in vitro and in vivo analysis.
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