Cloning and expression of a non-toxic mutant of Clostridium perfringens epsilon toxin, ETX-I۵۱C

BACKGROUND AND OBJECTIVESEpsilon toxin (ETX), produce by Clostridium perfringens toxinotypes B and D, is the main cause of enterotoxemia. Enterotoxemia is one of the most important prevalent diseases in ruminants, specially sheep and goats. The non-toxic epsilon mutants are applicable for different purposes, especially enterotoxemia vaccine design. The aim of this study was to design and produce a non-toxic epsilon mutant, ETX-I۵۱C, and to investigate its properties.MATERIALS AND METHODSThe I۵۱C mutant of C. perfringens epsilon toxin was synthesized by site-directed mutatgenesis (SDM) in the particular region of ETX gene with complementary mutagenic primers, based on the overlap-extension PCR. The ETX-I۵۱C mutant was cloned in pET-۲۶b(+) vector and the obtained recombinant pET-ETX-I۵۱C plasmid was transformed into Escherichia coli (DE۳). Positive clones were identified by plasmid extraction, cloning PCR, and sequencing. Protein expression of this mutant was induced by ۰.۵mM of IPTG and evaluated by SDS-PAGE, blotting, and ELISA.RESULTS AND DISCUSSIONBased on the sequencing results, the mutation was correctly created in the desired I۵۱ position. The results showed that ETX-I۵۱C mutant was successfully synthesized and cloned and its derivative toxoid could be well expressed in E. coli. According to ELISA results, the expression of ETX-I۵۱C mutant was better than the control group and it had a better biological activity in the reaction with specific anti-ETX antibody.CONCLUSIONTherefore, the ETX-I۵۱C mutant of C. perfringens epsilon toxin are suggested as suitable candidate for the later in vitro and in vivo immunologic investigations. Taken together, the results suggest that ETX-I۵۱C is a potential vaccine candidate against enterotoxemia.

کلیدواژه ها:

Clostridium perfringens ، Enterotoxemia ، Epsilon toxin ، Site-directed mutatgenesis (SDM) ، ETX-I۵۱C ، Mutant