Design and Fabrication of a DNA Vaccine against Brucella Abortus based on recombinant Lactococcus Lactis that Expresses Lumazine Synthase Protein

Vaccination is an efficient and cost-effective way to control brucellosis. This study aims to generate recombinant Lactococcus lactis (L. lactis) with Brucella abortus (B. abortus) BLS cytoplasmic protein.

The target vector, gene, and signal peptide (pNZ8148-Usp45-BLS) were developed and made in this study. Cloning accuracy was verified by PCR, enzyme digestion, and sequencing. Top10 F Escherichia coli was transformed using the recombinant expression vector. The column plasmid extraction kit selected and eliminated chloramphenicol-effected bacteria from an agar plate. In electroporation, Lactococcus lactis bacteria received the recombinant vector. Both SDS-PAGE and RT-PCR confirmed the transition.

To confirm the correctness of cloning and to confirm the presence of the BLS gene in the pNZ8148 vector, PCR and enzymatic digestion were performed. Observation of the BLS gene fragment with a length of 477 bp and plasmid pNZ8148 - Usp45 without the BLS gene fragment with a length of 2997 bp, the cloning of the BLS gene fragment was confirmed. Also, in the study conducted by the nanodrop device, the concentration of the extracted plasmid was estimated at 848.9 ng/µl and the degree of purity was 2.07. The results of RT-PCR indicated the success of the BLS gene transformation of Brucella abortus in L. lactis bacteria. Also, a single protein band of 18 kDa was observed in transformed L. lactis.

The present study showed that the BLS gene of the probiotic L. lactis transfected with pNZ8148-Usp45-BLS is expressed by electroporation.