The Preliminary Study On Formulation Of Cationic Polymer/CpG Plasmid Nanoparticles And Their Immunostimulatory Activity

Unmethylated CpG oligonucleotides (CpG ODN) can activate innate and adaptive immunity, particularly in the form of PEI or chitosan complex. In this study, we first optimized the conditions for preparation of PEI and chitosan/plasmid DNA nanaoparticles, the quality of which was tested using electrophoresis, gel retardation, TEM and in vitro plasmid release test. Chicken peripheral blood mononuclear cells (PBMCs) were prepared using Ficoll gradient centrifugation for proliferation assay using mitogens, CpG-containing plasmids and the PEI or chitosan nanoparticles as the stimulants. MTT chromatometry assay showed that recombinant plasmid pcDNA-CpG60 containing 60 copies of CpG ODN could stimulate stronger proliferatiobn of PBMCs than other recombinant plasmids containing less copies of CpG ODN. The PEI nanoparticles, formulated in physiologic saline with N/P ratio of 6 have strongest immunostimulatory activity. Large amount of plasmid pcDNA-CpG60 was prepared with high density fermentation the recombinant E.coli and purified with our modified CTAB precipitation. The purified plasmid or its PEI nanoparticles was co-administered to 21-day healthy chickens with live vaccine against Newcastle disease. Serum HI antibody titers were tested at 7, 14, 21, 28, 35 days post inoculation, which showed that PEI/plasmid DNA nanoparticle can stimulate higher and more uniform antibody response than the naked plasmid.