Cloning, expression, and purification of recombinant Enterotoxin B and cholera toxin B fusion protein in Lactobacillus plantarum

Background: Lactic acid bacteria (LAB) are promising platforms for mucosal vaccine development. Staphylococcus aureus enterotoxin B (SEB), a potent superantigen, is associated with food poisoning and toxic shock syndrome. Similarly, cholera toxin is the primary to widespread virulence factor in Vibrio cholerae infections, with its B subunit (CTB) serving as a well-established immune adjuvant that enhances antigen-specific responses in recombinant vaccines. This study aimed to engineer recombinant Lactobacillus plantarum as a dual-purpose vaccine candidate targeting V. cholerae and S. aureus by expressing CTB and SEB antigens. Methods: A modified gene sequence encoding SEB (Lacking superantigenic activity) and CTB was successfully designed, synthesized, and cloned for secretory expression in L. plantarum. The resulting recombinant protein, tagged with His, was purified using Ni-NTA agarose ion-exchange chromatography and confirmed with Western blot analysis. Results: Enzyme digestion and PCR analysis confirmed successful cloning of the SEB-CTB fusion gene into the pBlueScript II SK (+) and pNZ7021 expression vectors, as evidenced by the expected band on agarose gel. SDS-PAGE revealed a ~49 kDa protein band, indicating expression of the recombinant rSEB-CTB protein, which was further validated by Western blot using an anti-His tag antibody. Conclusion: The construct LP-pNZ7021–SP-seb-ctxB may be a promising candidate for recombinant vaccine development targeting V. cholerae (Cholera toxin-producing) and S. aureus (SEB-producing), providing dual protection against both pathogens.