Development of a genetically engineered multivalent subunit vaccine against “struck”, “braxy”, lamb dysentery, and enterotoxemia in sheep

The development of genetically engineered multivalent subunit vaccines is needed to provide protection against infections caused by Clostridium septicum and multiple types of Clostridium perfringens. In this study, a pET-32a(+) plasmid encoding the C. septicum α-toxin gene and the C. perfringens β-toxin gene was constructed; a positive clone was designated pET-32a-α-β1. This plasmid, along with pET-32a-α-β2-ε-previously engineered to express a C. perfringens α-β2-ε trivalent recombinant chimeric fusion protein-was transformed into Escherichia coli DE3 for recombinant protein expression after isopropyl-β-D-thiogalactopyranoside induction. Recombinant proteins α-β1 and α-β2-ε were purified, mixed at a protein concentration ratio of 1:1, and subsequently freeze-dried. The freeze-dried proteins were reconstituted with an aluminum hydroxide adjuvant and administered to sheep at various doses. Based on antibody titers and serum neutralization titers in immunized sheep, 0.2 mg of mixed protein was identified as the minimum immunization dose for the subunit vaccine. Toxin neutralization tests performed 21 days post-vaccination showed that sera from immunized sheep neutralized 10, 10, 27, and nine times the mouse lethal doses of C. perfringens types B, C, and D, and C. septicum toxins, respectively. All immunized sheep were protected against C. perfringens types B, C, and D, and C. septicum toxins, with a survival rate of 100%; the negative control group showed a survival rate of 0%. These encouraging findings provide a foundation for the development of novel subunit vaccines.