Abstract:
The use of oral vaccination in aquaculture has lagged behind injectable vaccines for a long time in protective immunity. In this study, we constructed the DNA vaccine using plasmid vector (pEGFPN2) and ORF012R gene of rock bream iridovirus (pEGFPN2-ORF012R). Then it was encapsulated to chitosan nanoparticles (CNPs) according to a complex coacervation method and denoted as pEGFPN2-ORF012R-CNPs. The pEGFPN2-ORF012R-CNPs had diameter of 189.5 nm. Encapsulation efficiency and loading capacity were determined as 92.57% ± 0.87% and 9.32% ± 0.19%, respectively. Final encapsulated product (pEGFPN2-ORF012R-CNPs) had +12.11 mV zeta potential. In vitro vaccine release assay showed that the plasmid DNA was sustainably released from the pEGFPN2-ORF012R-CNPs, up to 84.26% ± 3.16% of the total amount. By in vitro cell culture experiment we confirmed that the cloned pEGFPN2-ORF012R-CNP was expressed in HEK-293 cells. Oral vaccination was carried out by feeding of pEGFPN2-ORF012R-CNPs (250 ng/zebrafish/day) for 14 days. Quantitative real time PCR results clearly showed the transcriptional upregulation of IFN and IFN-stimulatory genes (Mx) in kidney and gut of zebrafish upon oral vaccination of pEGFPN2-ORF012R-CNPs compared to control fed diet, suggesting that CNPs is potential DNA vaccine delivery agent.
