Abstract

DNA-mediated vaccination is a new weapon in the battle against infectious diseases. The parameters responsible for effective genetic immunization in channel catfish have not been studied. The objectives of this project are to demonstrate the activity of mammalian promoter sequences in the channel catfish, and to evaluate the parameters controlling the magnitude of expression. Channel catfish ovary (CCO) cells were transfected with eukaryotic expression plasmid constructs containing either no gene, or a b-galactosidase reporter gene, driven by different transcription promoters, in order to demonstrate the in vitro activity of mammalian promoters in catfish cells. Enzyme activity was only observed in cells transfected with the cytomegalovirus (CMV) promoter/β-galactosidase gene plasmid construct (Figure 1). Channel catfish (Ictalurus punctatus) were injected intramuscularly with 50 ug of the CMV expression plasmid constructs containing either no gene or a β-galactosidase reporter gene. At 7 days post injection, the muscle surrounding the injection site, anterior kidney, posterior kidney, liver, spleen, gill, and intestine were processed for immunohistochemical analysis. In addition, muscle flanking the injection site was collected, lysed, and assayed for β-galactosidase activity. Positive-staining muscle bundles were observed in fish injected with the β-galactosidase gene. No staining was observed in control fish. Moreover, enzyme activity was only observed in the muscle flanking the injection site of the fish inoculated with the β-galactosidase plasmid.

Figure 1.