Clonagem e expressão do gene da toxina épsilon de clostridium perfringens tipo D e sua aplicação na imunização de animais

Abstract

The epsilon toxin is produced by Clostridium perfringens types B and D, causing enterotoxaemia in sheep, goats and calves. Enterotoxaemia is a disease with acute or super-acute profile, causing sudden death and huge economic loss. The therapeutic intervention may be difficult since the disease evolution is very rapid. However, this process can be prevented by immunizations with specific immunogenic vaccines. In the present work the etx gene encoding the epsilon toxin was cloned into pET-11a and the recombinant epsilon toxin was expressed as inclusion bodies. The protein concentration was determined and the lethal doses (LD) were estimated in mice, reaching 25 x 102 LD50/mL. Serum protection and cross-serum neutralization tests in mice were also used to characterize the recombinant toxin. For potency test, rabbits were immunized with 200, 100 and 50 µg of recombinant toxin, using aluminum hydroxide gel as adjuvant. Obtained titers of 40, 30 and 10 UI/mL, respectively, were higher than the minimum level of 5 UI/mL required by the European Pharmacopoeia (1998) and the minimum level of 2 UI/mL required by the Code of Federal Regulation/USA. The recombinant epsilon toxin produced emerges as a strong candidate for the production of a vaccine against enterotoxaemia caused by epsilon toxin of C. perfringens type D