Objective Immunogenicity and subcellular localization of ORF 121R of large yellow croaker iridovirus (LYCIV) were analyzed to understand the pathogenesis role of the protein.
Method Sequence of LYCIV ORF 121R was compared with those of the homologous proteins, and subcellular localization predicted by Cell-PLoc 2.0. The gene sequence was cloned into pET32a and pEGFP-N1 vectors to construct recombinant plasmids, ORF 121R-His and ORF 121R-EGFP. ORF121R-His was expressed and purified to prepare polyclonal antibodies from mice. Titer and specificity of the antibody were measured using ELISA, western-blotting, and indirect immunofluorescence techniques. After transfecting EPC cells with the ORF 121R-EGFP plasmid, subcellular location of ORF 121R-EGFP was observed under a confocal microscope.
Result LYCIV ORF 121R was 79.96% homologous in sequence with the immunogenic ISKNV ORF 117R proteins and predicted to localize in the cytoplasm. Successfully constructed from the 1 377 bp orf 121R, the recombinant plasmids were further analyzed. The purified soluble ORF 121R-His had a molecular weight of 72.6 kDa with a polyclonal antibody specifically recognizing natural LYCIV viral protein with a titer of 1:128 000. Whereas ORF 121R-EGFP exhibited a cytoplasmic distribution in EPC cells with the fluorescence signal evenly enriched in the perinuclear region.
Conclusion LYCIV ORF 121R was mainly found in the cytoplasm. The immunogenicity and specificity of the protein as demonstrated in this study lent it a potential candidate for vaccine production and disease diagnosis on yellow croakers.
DownLoad: