关键词: 产气荚膜梭菌; α毒素; 克隆表达; 抗血清

Abstract: One pair of primers had been designed and synthesized based on the α-toxin gene of Clostridium perfringens.The complete α-toxin gene fragment was amplified by polymerase chain reaction (PCR), and then was cloned into pGEM-T Easy vector to construct pGEM-T-α.Digested with EcoRⅠ and Hind Ⅲ, a fragment of 1125 bp was cloned into the expression plasmid vector pET-28a(+).The recombinant plasmid was transformed into the BL21(DE3)plys and induced by 1.0 mmol/L IPTG at 37 ℃.The expression product was found to be 46.1 ku as expected one identified by SDS-PAGE, and confirmed by Western blotting with Clostridium perfringens type A antisera, indicating similar reactivity with native α-toxin.Recombinant α-toxin protein was simultaneously found in culture supernatant, postsonic supertanant and inclusion bodies, most protein was expressed in inclusion bodies, which indicated recombinant α-toxin protein was expressed in the extracellular, periplasm and cytoplasm.Recombinant α-toxin protein in postsonic supertanant could not make mice die, indicating its non-toxicity.Toxin-antitoxin neutralization test showed that antisera of recombinant α-toxin protein were specific to α-toxin.Upon immunization of rabbit with the recombinant α-toxin protein, antisera with high antibody titer neutralizing 100 MLD toxin per 1 mL were prepared.

Key words: Clostridium perfringens; α-toxin; cloning and expression; antisera