Immunoprotection Of Crucian Carp(Carassius Auratus) With Recombinant Interferon RCiIFN

Type I interferon(IFN) is a class of important cytokines in the innate immune response, with broad effectiveness in antiviral and anticancer therapy. Crucian carp(Carassius auratus) type I IFN shares high homology with that of grass carp(Ctenopharyngodon idella). In our previous study, the immunostimulatory effect and antiviral activity of grass carp recombinant IFN(rCiIFN) were proved in cells and tissues. In order to study the immune protection of rCiIFN for crucian carp and its mechanism, we have established a model about virus infected crucian carp using poly I:C induction. Then r CiIFN was used for immune protection experiment.So in this study, a recombinant plasmid pBV220-CiIFN was constructed and rCi IFN protein was expressed in Escherichia coli BL21(DE3) plys S. A large quantity of rCi IFN protein was obtained through prokaryotic expression at 42? for 4 h. In order to obtain higher purity protein, gel extraction method was used. Finally, as determined by SDS-PAGE, the r CiIFN protein is about 21 kD. The concentration of rCi IFN protein was 330 ? g/ml and was measured by protein Assay kit. Then the protein diluted to suitable concentration. To evaluate in vivo protective efficacy of rCi IFN against poly I:C, we administered it by oral feeding in our experiments.Crucian carp were fed with pellets mixed with rCiIFN at 1 ?g/g fish body weight once daily for 1, 2 and 3 days before intraperitoneal(ip) injection of poly I:C. The results indicated that oral rCi IFN had good protection effect for crucian carp compared with the control group, and that the relative percentage survival(RPS) from feeding for 3consecutive days(54.29 %) was higher than those from feeding for just 1 d or 2consecutive days(38.81 % and 47.83 % respectively).While the survival rate of the control group was much lower than that of the experimental group, the survival rates of feeding for 1d, 2d and 3d were 16.25%, 13.75% and 12.50% respectively.To study the immune protection of rCi IFN for crucian carp and its molecular mechanism, we analyzed the effect of rCiIFN on the expression levels of the PKR and IFN genes by real-time fluorescence quantitative PCR. Meanwhile, we extracted total RNA from liver, kidney, intestine, heart and gill tissues of crucian carp at 0, 24, 48 and 72 h after intraperitoneal injection of rCiIFN and that of control group intraperitoneal injection of PBS, then got the corresponding cDNA. The expression of IFN and PRK genes in all tested crucian carp tissues were detected by RT-PCR. By comparison with gene expression levels observed in PBS-injected controls, theexpression of IFN and PKR were found to be significantly up-regulated after stimulation with rCi IFN-injected in all tested tissues, and most of IFN and PKR reached the highest level within 48 h or 72 h post-treatment. These results demonstrated that r CiIFN had antiviral activity when crucian carp were infected with poly I:C and oral administration of rCi IFN was effective in protecting crucian carp against virus.