Construction of a dual-promoter plasmid pCMVnir with HCMVie and in vivo-inducible bacterial nirB promoters and its advantages in genetic immunization

Objective To develop a plasmid matching the bacteria based DNA vaccine inoculation. Methods Dual promoter DNA vaccine with eukaryotic HCMVie and prokaryotic promoter nirB were designed to improve antigen expression in vivo by replacing the T7lac and P10 promoters of pTriEx 4 and keeping the CMVie promoter of pTriEx 4. The nirB promoter was derived from E.coli , a modified version of nirB promoter without nitrogen response element; its activation depends only on low level of oxygen tension. We constructed dual promoter expression plasmid pCN 16L1E7 and the other two single promoter vectors pCMV 16L1E7 and pNir 16L1E7, and detected the mucosal immune reactions induced by these vector transformed Salmonella strains. Results The pCMVnir plasmid is successfully developed. In addition, the recombinant Salmonella could replicate in vivo up to 6 weeks, especially in mucosa associated lymph organs and antigen processing cells. The nirB promoter could be activated by aerobic and anaerobic condition, high expression of antigen did not interfere the stability of the recombinant bacteria. Immune reaction induced by pCN EGFP is higher than that of pCMV EGFP. Dual promoter pCN 16L1E7 could induce stronger immune reaction than the other ones. Conclusion pCMVnir is a good candidate vector for developing intracellular bacteria based DNA vaccines which is more potential for APC Ag presentation and is more immunogenic.