Construction and evaluation of new dual-promoter DNA vaccine expression plasmid pCMVnir with in vivo-inducible bacterial nirB and CMVie promoters

Objective: To obtain a new dual-promoter DNA vaccine expression plasmid pCMVnir with in vivo-inducible bacterial CMVie and nirB promoters. Methods: Native nirB promoter was derived from E.coli, and a modified version of nirB promoter was designed and synthesized by deleting the nir-response element, whose activation depends on low-level of oxygen tension. In order to study the activity of these promoters, two different reporter genes EGFP-N1 and Ds-Red were inserted into the pCMVnir. Both pCN-EGFP and pCN-DsRed reporter plasmids were transfected into attenuated Salmonella and E.coli DH5to naked eyes. When the bacteria were cultured on the LB agar containing ampicillin, the colonies became lightly red and green. The expression strength of the reporter genes were confirmed by the flow cytometer, and the data indicated that nirB was activated at normal condition. Conclusion: The pCMVnir promoter could express antigens in vivo, and it is suitable for developing intracellular bacteria-based DNA vaccine, so it can express the antigen genes in both intracellular bacteria and mammalian cells, and has the capacity to enhance the immunogenicity of DNA vaccine.