Evaluation and Comparison of Clostridium Epsilon-Alpha Fusion Gene Expression Using Different Commercial Expression...

Area of Science:

• Veterinary Microbiology
• Molecular Biology
• Vaccine Development

Background:

• Clostridium perfringens and Clostridium septicum are pathogenic bacteria causing diseases in humans and livestock.
• Key toxins include alpha and epsilon, responsible for enterotoxemia and braxy in sheep and goats.
• Developing recombinant vaccines offers a cost-effective alternative to traditional methods.

Purpose of the Study:

• To evaluate the expression of a recombinant epsilon-alpha fusion protein from C. perfringens type D and C. septicum.
• To optimize the production of this fusion protein using various expression vectors and hosts.
• To facilitate the development of novel clostridial anaerobic vaccines.

Main Methods:

• Construction of a recombinant Escherichia coli strain (TOP10) with pJETεα plasmid.
• Subcloning the epsilon-alpha fusion gene into pET22b(+), pET26b(+), and pGEM-B1 vectors.
• Expression analysis in E. coli/Rosetta and E. coli/BL21 (DE3) using SDS-PAGE and Western blotting under varying conditions (temperature, IPTG concentration, time).

Main Results:

• Recombinant E. coli/Rosetta/pET22εα demonstrated superior expression of the epsilon-alpha fusion protein.
• Optimal expression was achieved at 37°C after 6 hours of induction with isopropyl β-D-1-thiogalactopyranoside (IPTG).
• Different expression vectors and host strains influenced the protein yield.

Conclusions:

• The study successfully produced a recombinant epsilon-alpha fusion protein from C. perfringens and C. septicum.
• Optimized expression conditions in E. coli/Rosetta using the pET22εα vector are identified.
• This work provides a foundation for developing more efficient and economical clostridial vaccines.