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Enterotoxemia is one of the most important prevalent diseases in ruminants, specially sheep and goats. The epsilon toxin (ε-toxin), produce by Clostridium perfringens, is the main cause of enterotoxemia. The nontoxic epsilon mutants (genetic ε-toxoids) are applicable for different purposes, especially vaccine design. The aims of this study were to design and produce different nontoxic epsilon mutants and to investigate their properties. ε-I51C, ε-V56C, ε-H106P, ε-A114C, and ε-F118C mutants were synthesized by site-directed mutatgenesis (SDM) in the particular regions of ε-toxin gene with complementary mutagenic primers, based on the overlap-extension PCR method. Another mutant, sε, was also produced by gene manipulation and gene synthesis. These mutants were cloned in pET-26b(+) expression vector and the obtained recombinant plasmids (pET-εI51C, pET-εV56C, pET-εH106P, pET-εA114C, pET-εF118C, and pET-sε) transformed into E. coli (DE3). Positive clones of E.coli-I51C, E.coli-V56C, E.coli-H106P, E.coli-A114C, E.coli-F118C, and E.coli-sε were identified by plasmid extraction, enzymatic cut, cloning PCR, and sequencing. Protein expression of epsilon mutants was induced by IPTG and evaluated by SDS-PAGE, western blot and dot blot. The results showed that all of the mutants were synthesized and expressed successfully in E. coli, but according to ELISA results, the expression of sε and I51C mutants were better than the other mutants. Therefore, these mutants were identified and chosen as good candidates for the later in vitro and in vivo immunologic investigations.
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