Chromosomal Insertions in the Lactobacillus casei upp Gene That Are Useful for Vaccine Expression

Abstract

bTo develop a stable and marker-free Lactobacillus strain useful for the expression of vaccines, we developed a temperature-sensitive suicide plasmid with expression cassettes containing an HCE promoter, a PgsA anchor, the alpha-toxin gene, and an rrnB T1T2 terminator (PP␣T) that uses a 5-fluorouracil (5-FU) counterselectable marker for Lactobacillus casei. Three strains containing the correct PP␣T expression cassettes were produced via the selective pressure of 5-FU screening. We confirmed that the upp gene was deleted and that the PP␣T expression cassettes were inserted into the upp site of L. casei ATCC 393 by genomic PCR amplification and sequencing. 5-FU resistance in recombinant bacteria could be stably inherited for as long as 40 generations following insertion. However, bacteria containing the integrated DNA grew more slowly than wild-type L. casei. An indirect enzyme-linked immunosorbent assay (ELISA) analysis demonstrated that the alpha-toxin gene was expressed. Also, we visualized expression of the protein on the surface of L. casei cells using laser confocal microscopy. These results taken together demonstrate that these recombinant bacteria should provide a safe tool for effective vaccine production.

Oral immunization with aLactobacillus casei-based anti-porcine epidemic diarrhoea virus (PEDV) vaccine expressing microfold cell-targeting peptide Co1 fused with the COE antigen of PEDV

“…The constitutive expression plasmid pPG‐T7 g10‐PPT, containing an HCE promoter, T7g10 enhancer, PgsA anchor and the rrnBT1T2 terminator, was constructed in our laboratory (Song et al . ).…”

Oral recombinant Lactobacillus vaccine targeting the intestinal microfold cells and dendritic cells for delivering the core neutralizing epitope of porcine epidemic diarrhea virus

“…After that, the COE-Col-DCpep fusion genes were cloned into the expression plasmid pPG-T7g10-PPT, to generate pPG-T7g10-PPT-COE-Col-DCpep. To construct the recombinant lactobacillus strains, the recombinant plasmids were electrotransferred into L. casei 393 as described previously [25], giving rise to the recombinant strain pPG-COE-Col-DCpep/L393. In addition, pPG-T7g10-PPT-COE/L393, pPG-T7g10-PPT-COE-Col/L393 and pPG-T7g10-PPT-COE-DCpep/L393 were also generated and maintained in our laboratory.…”

Display of recombinant proteins at the surface of lactic acid bacteria: strategies and applications

“…One of the most exploited N-terminal transmembrane helix anchors is the PgsA protein from Bacillus subtilis, which is part of the poly-γ-glutamate synthetase complex. Several reports describe coupling of antigens to the C-terminus of PgsA, leading to successful surface display in L. casei and L. lactis [12,33,[49][50][51][52][53][54]. In these studies, surface-display was confirmed using fluorescence microscopy or flow cytometry.…”