Cloning and Expression of a Fusion Protein Containing Highly Epitopic Regions of Clostridium perfringens Epsilon Toxin and Clostridium novyi Alpha Toxin

Abstract

Clostridium perfringens and novyi species are two important toxin-producing pathogens which pose a risk to the livestock health. Epsilon and alpha toxins are major toxins of these two pathogens, respectively. Advances in current vaccine industrialization lead to the utilization of toxin epitopes instead of the whole pathogen/toxoids to produce novel vaccines. In the present study, bioinformatics approaches were applied to design a fused protein containing both toxin fragments of interest with the highest antigenicity score for B-cells. To do so different specialized algorithms including I-TASSER, IEDB, ElliPro, PyDock and CLC Main Workbench were applied. The chimeric protein was successfully cloned, expressed, and purified using an immobilized-metal affinity chromatography for His-tagged proteins. During in vivo experiments on rabbits, the levels of immunization provided by the recombinant protein or native alpha and epsilon toxins were compared based on serological studies. Results indicated that the designed protein was able to stimulate effective immune responses against both alpha and epsilon toxins. This can be used as a proper strategy to design novel peptide-based subunit vaccines. Clostridium perfringens and novyi species are two important toxin-producing pathogens which pose a risk to the livestock health. Epsilon and alpha toxins are major toxins of these two pathogens, respectively. Advances in current vaccine industrialization lead to the utilization of toxin epitopes instead of the whole pathogen/toxoids to produce novel vaccines. In the present study, bioinformatics approaches were applied to design a fused protein containing both toxin fragments of interest with the highest antigenicity score for B-cells. To do so different specialized algorithms including I-TASSER, IEDB, ElliPro, PyDock and CLC Main Workbench were applied. The chimeric protein was successfully cloned, expressed, and purified using an immobilized-metal affinity chromatography for His-tagged proteins. During in vivo experiments on rabbits, the levels of immunization provided by the recombinant protein or native alpha and epsilon toxins were compared based on serological studies. Results indicated that the designed protein was able to stimulate effective immune responses against both alpha and epsilon toxins. This can be used as a proper strategy to design novel peptide-based subunit vaccines.

Keywords

• clostridium
• alpha toxin
• epsilon-toxin
• fusion protein
• elisa
• immunization