Codon changed immobilization antigen (iAg), a potent DNA vaccine in fish against Cryptocaryon irritans infection.

The immobilization antigen (iAg) DNA sequence from Chiayi isolate of Cryptocaryon irritans was computationally reviewed to replace the stop codons with suitable amino acids and its GC content was intensified. The plasmid construct comprising the codon changed iAg (optiAg/optimized iAg) was successfully expressed in the bacterial strain BL21 and also in grouper fin cells (GF-1). Results of immobilization assay, ELISA and western blot of C. irritans theront and recombinant iAg by grouper antiserum against optiAg DNA indicated that the codon changed iAg retains the native conformation. The DNA vaccine construct pcDNA3.1-optiAg was encapsulated in water-oil-water triple layer emulsions measuring 19 μm diameters and was used for the immunization experiment. In trial I experiment, grouper fish were immunized twice via intramuscular injection with the pcDNA3.1-optiAg and were challenged with C. irritans at 8-day post immunization (dpi), which resulted in 46% relative percent survival (RPS). In trial II, single immunization with pcDNA3.1-optiAg boosted with recombinant iAg protein, resulted in 40% RPS. The data from this study reveal that codon change in iAg not only accomplished the expression of iAg protein in both prokaryotic and eukaryotic cell systems, but also optiAg was proved as immunogenic due to the protection it confers to the immunized fish against C. irritans infection. Hence, it is concluded that iAg can be a potent DNA vaccine in fish against infection of the ciliated protozoan, C. irritans.