Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity.

Abstract

In order to develop a secure and competent technique to express the human immune gene for fighting infections, we cloned and expressed the BD2/3 using VR1020 (a eukaryotic expression plasmid). BD2/3 contains human β-defensin 2 (BD2) and human BD3. To explore safe and effective DNA delivery molecules in vitro and in vivo, the fusion genes of BD2/3 were used as an immune-labelled gene to verify transfection effectivness of modified chitosan (CS). Plasmid of VR1020-BD2/3 was packed with biomaterials: CS, average molecular weight: 25000D; polyethylene glycol-O-chitosan-polyethylenimine (PEG-O-CS-PEI); liposomes (LP); polyamine cationic liposomes (PCL); polyamine cationic liposomes of protamine (PCL-protamine) by ionotropic gelation. We observed that BD2/3 fusion gene showed high bioactivity in vitro and in vivo. The BD2/3 fusion protein inhibited the proliferation of bacteria (S. aureus, S. pneumoniae, P. aeruginosa and E. coli). The Kunming mice were immune to these nanoparticles and we analyzed their delivery efficiency and gene expression effect. BD2/3 results in multiple changes of innate and required immune system of mice. BD2/3 increases expression of IgG, IgG1, IgG2a, IL-2, IL-6, IFN-γ, as well as of lymphocytes and monocytes. Following challenge with virulent E. coli, CD4+ and CD8+ positive T-cell counts were highly elevated in the BD2/3 immunized mice, resulting in higher survival rates of mice. These results indicate that nanoparticles containing modified CS and BD2/3 are potentially safe and effective drugs in vivo to improve the immunity against bacterial infection and enhance innate immunity and adaptive immunity against infectious diseases.