Induced Expression of Alpha-Toxin Gene of Clostridium perfringens in Recombinant Lactobacillus casei and their Immunoprotective in mice

Abstract:

Abstract:[Objective] The study was aimed to prepare an oral vaccine by constructing recombinant Lactobacillus casei expressing alpha-toxin gene of Clostridium perfringens, for preventing poisoning by Clostridium perfringens. [Methods] The constructed cell-surface expression plasmid pPG1-ɑ/L.393 and secretion expression plasmid pPG2-ɑ/L.393, both with alpha-toxin gene, were electroporated into L.casei 393, generating recombinant bacteria pPG1-ɑ/L.casei393 and pPG2-ɑ/L.casei393. The recombinant strains were induced by 1% lactose in De Man, Rogosa and Sharp (MRS) broth, and the target protein was detected by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), Western blot and indirect immunofluorescence assay. BALB/C mouse were used as animal model immunized with recombinant strains by intragastric administration, and the immune efficacy was analyzed. Specific anti- alpha-toxin protein sIgA was detected by indirect enzyme linked immunosorbent assay (ELISA) in the feces, vaginal lavage, eye washing of mice after intragastric administration, and Specific IgG was detected by indirect ELISA in the serum of immunized mice. The resistance of immunized mice to alpha-toxin and the neutralization ability of antibodies to alpha-toxin were also tested. [Results] Mice immunized with pPG1-ɑ/L.casei393 and pPG2-ɑ/L.casei393 could produce remarkable anti-alpha-toxin antibodies, sIgA and circulating antibody IgG had completely neutralization ability against alpha-toxin. The test of alpha-toxin challenge in mice showed that the immunized mice could resist three times’ Minimum Lethal Dose(MLD). [Conclusion] All the results indicated that mice inmmunized by the recombinant Lactobacillus casei expressing alpha-toxin gene of Clostridium perfringens could elicit regional and systematic immunity response and neutralization ability.