en ميکروب شناسي Microbiology Research Article Research Article <div style="text-align: justify;"><span style="font-size:12px;"><span style="font-family:Times New Roman;"><b>Background:</b> Lactic acid bacteria (LAB) are promising platforms for mucosal vaccine development. <a name="_Hlk211428095"></a><a name="_Hlk211164959"><em>Staphylococcus aureus</em> </a>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 <em>Vibrio cholerae</em> 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 <em>Lactobacillus plantarum</em> as a dual-purpose vaccine candidate targeting <em>V. cholerae</em> and <em>S. aureus</em> by expressing CTB and SEB antigens.<br> <b>Methods:</b> A modified gene sequence encoding SEB (Lacking superantigenic activity) and CTB was successfully designed, synthesized, and cloned for secretory expression in <i>L. plantarum</i>. The resulting recombinant protein, tagged with His, was purified using Ni-NTA agarose ion-exchange chromatography and confirmed with Western blot analysis.<br> <b>Results:</b> 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.<br> <b>Conclusion</b>: The construct LP-pNZ7021&ndash;SP-seb-ctxB may be a promising candidate for recombinant vaccine development targeting <i>V. cholerae</i> (Cholera toxin-producing) and <i>S. aureus</i> (SEB-producing), providing dual protection against both pathogens.</span></span><br> &nbsp;</div> Staphylococcus aureus enterotoxin B, Lactobacillus Plantarum, Cloning, Organism, Vaccines, Cholera toxin subunit B 36 39 http://mlj.goums.ac.ir/browse.php?a_code=A-10-1038-1&slc_lang=en&sid=1 Fatemeh Maghsood Ahmadi maghsood_ahmady@yahoo.com 100319475328460031377 100319475328460031377 No Department of Microbiology NT.C., Islamic Azad University, Tehran, Iran Arash Mahboubi mahboubi@sbmu.ac.ir 100319475328460031378 100319475328460031378 Yes Department of Pharmaceutics, School of Pharmacy, Shahid Beheshti University of Medical Science, Tehran, Iran; Food Safety Research Center, Shahid Beheshti University of Medical Science, Tehran, Iran Farzaneh Hosseini farzaneh953@yahoo.com 100319475328460031379 100319475328460031379 No Department of Microbiology NT.C., Islamic Azad University, Tehran, Iran Davoud Esmaeili esm114@gmail.com 100319475328460031380 100319475328460031380 No Department of Microbiology, Baqiyatallah University of Medical Sciences, Tehran, Iran; Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran Bahareh Hajikhani hajikhani@sbmu.ac.ir 100319475328460031381 100319475328460031381 No Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran