The inhibition of expression of SGIV ICP46-GFP in FHM cell by RNA interferencing

Abstract

Singapore grouper iridovirus(SGIV),as a causative agent of serious systemic disease,resulted in significant economic losses in grouper aquaculture.In this study,recombinant eukaryotic vector pEGFP-ICP46 which was inserted with SGIV ICP46(infected cell polypeptides 46) gene was transfected into fathead minnow(FHM) cells,and ICP46-GFP fusion protein was successfully expressed in the cytoplasm of FHM cells.Candidate siRNA targeting SGIV ICP46 gene(siRNA-ICP46) was designed and chemically synthesized.To investigate the inhibition effect of siRNA-ICP46,pEGFP-ICP46 and siRNA was co-transfected into FHM cells,and the green fluorescence was observed by fluorescence microscope at the indicated time points(24,36,48,60 and 72 h post transfection).During 24-48 h after transfection,the green fluorescence in FHM cells co-transfected with siRNA-ICP46 /pEGFP-ICP46 were similar to that of positive control(co-transfected with siRNA-GFP/pEGFP-ICP46),and both of them are about 70% weaker than that of negative control(co-transfected with siRNA-Negative/pEGFP-ICP46),which show the siRNA-ICP46 can effectively inhibit the extrinsic SGIV ICP46 gene in FHM cells during 24-48 h after transfection.