Enterotoxemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens Type D. To combat the disease, a vaccine consisting of formalin-inactivated Type D toxin is available, but several concerns regarding its production have been raised. The development of efficacious recombinant subunit vaccines can provide a means whereby many of the production problems may be eliminated or minimized. However, the production of purified antigens is often associated with weakened antigen immunogenicity. New vaccine strategies therefore rely on the incorporation of effective adjuvants, which stimulate the innate immune response and, in turn, activate the adaptive immune response. An increasing number of studies have demonstrated that flagellin is a potent activator of a broad range of cell types involved in innate and adaptive immunity. Consequently, the aim of this investigation was to produce different recombinant vaccine candidates, inclusive of a flagellin-epsilon toxin fusion protein, for preventing Type D enterotoxemia.
Attempts at expressing the native gene sequence for the Type D epsilon toxin in Escherichia coli have been characterized by low levels of expression. This may be due to differences in the codon bias between the heterologous gene and E. coli. Therefore, in this study, a completely synthetic codon-optimized gene encoding the epsilon toxin was obtained. The full-length etx gene (etx0), an etx gene lacking the first nine amino acids of the signal peptide sequence (etx1), as well as a flagellin-toxin fusion gene composed of the etx1 gene and a truncated hag gene of Bacillus halodurans Alk36 (Δhag::etx1) were expressed in E. coli BL21-Gold(DE3) cells. Expression of the respective recombinant proteins, designated ETX0, ETX1 and NC-ETX1, respectively, were confirmed by Western blot analysis using anti-epsilon toxin antibodies. The ETX0 protein was expressed in a soluble form, whereas the ETX1 and NC-ETX1 proteins were insoluble and accumulated in inclusion bodies. In an attempt to optimize expression of the recombinant proteins, different cultivation media and concentrations of the promoter inducer were evaluated. Under optimized expression conditions, the yields of ETX0, ETX1 and NC-ETX1 were determined to be 828 mg/l, 395 mg/l and 525 mg/l, respectively.
Despite having expressed the recombinant proteins with an N-terminal hexahistidine tag, the recombinant ETX0 protein could not be purified with nickel (Ni2+) chelate affinity chromatography. This was due to removal of the affinity tag as a consequence of intracellular processing of the N-terminal signal peptide sequence. The recombinant ETX0 protein was consequently purified to near homogeneity by ion exchange chromatography. The recombinant ETX1 and NC-ETX1 proteins were purified from inclusion bodies by affinity chromatography under denaturing conditions and then refolded in TBS buffer. The yield of purified ETX0, ETX1 and NC-ETX1 were determined to be 430 mg/l, 400 mg/l and 340 mg/l, respectively. The cytotoxicity of these purified recombinant proteins was assayed in vitro against Madin-Darby canine kidney (MDCK) cells, the results of which indicated that NC-ETX1 was not toxic towards the cells and both ETX0 and ETX1 displayed moderate toxicity towards the cells.
Cumulatively, the results indicated that the respective proteins were expressed to high levels in E. coli and large amounts of purified proteins could be obtained. These recombinant proteins can in future be evaluated as vaccine candidates against C. perfringens Type D enterotoxemia.