Production and characterization of recombinant beta toxin of Clostridium perfringens

Abstract

newlineClostridium perfringens causes several forms of enteric disease in human and animals. Different toxinotypes of C. perfringens produce different combinations of lethal toxins. C. perfringens type C produces beta toxin which is a kind of lethal pore-forming toxin that is responsible for necrotic enteritis in animals. Although C. perfringens type C infects several livestock species, the juvenile pigs are most susceptible. In India, the north-eastern states have the highest pig population in the country but no indigenous vaccine against C. perfringens type C is currently available. Therefore, the present study was conducted with an aim to produce recombinant beta toxin protein in a heterologous host and to evaluate the immunogenicity of the recombinant toxin adjuvanted with calcium phosphate nanoparticles in the mice model. For this, the cpb gene of C. perfringens type C that encodes the beta toxin was cloned into a prokaryotic expression vector, pET28a(+). Then the recombinant clone was transformed into BL21-CodonPlus®(DE3)-RIL E. coli cells. Expression of the recombinant beta toxin was induced by 1 mM IPTG for 12 hours at 37and#61616;C. The recombinant beta toxin protein was present as inclusion bodies in the insoluble fraction of the cell lysate which was further purified by Ni-NTA column affinity chromatography and confirmed by SDS-PAGE analysis. The specificity and the reactivity of the recombinant beta toxin protein were confirmed by western blotting using anti-sheep C. perfringens beta toxin serum. In-vitro and in-vivo toxicity of the recombinant beta toxin protein was evaluated in MDCK cell line and mice, respectively. The recombinant beta toxin protein did not show cytotoxicity in the concentrations from 3500-6.89 ng/ml as well as failed to produce clinical signs or death in mice when administered intravenously at a 100and#956;g dose. The recombinant beta toxin protein was loaded into calcium phosphate nanoparticles (CaP-NPs) used as an adjuvant, and a calcium phosphate nanoparticles-recombinant beta toxin prot