Abstract
Lactic acid bacteria (LAB) are Gram-positive bacteria and generally regarded as safe (GRAS) organisms. LAB could be used for heterologous protein secretion and fused antigen to form fusion protein. They are good potential candidates as antigen delivery vehicles. We developed an efficient secretion system in the Lactococcus lactis model. Staphylococcal nuclease (Nuc protein) is a small, stable, and biochemically well-characterized enzyme secreted by Gram-positive bacteria. It was used as the reporter protein. Plasmid pNuc10 encoding LEISSTCDA-NucT is a L. lactis cloning vector. The secretion efficiency was reduced further to ~30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). A 9-residue synthetic propeptide, LEISSTCDA, was fused immediately after the signal peptide cleavage site. Secretion efficiency was increased to ~90% by LEISSTCDA insertion without altering the signal peptide cleavage site. Because the cloning vector, pNuc10, could not replicate in E.coli. We fused pBluescriptⅡSK(+) and pNuc10 to form the shutter vector pBN. We transformed DNA to E.coli by heat shock, and it would replicate in supercoil form for ColE1 origin of E.coli. Then the expression vector is transferred to the L. lactis MG1363 strain by electroporation. In this study, the gp25 glycoprotein of EB virus was fused with histidine tag as a heterologous protein, and was cloned between NucT and LEISSTCDA to form fusion protein. We hope that the L.lactis MG1363 transformed with this expression vector can be used as a live vaccine system.