| In recent years, increasing attention has been given to the possible use of recombinant proteins such as antibacterial peptides or interferons to reduce the susceptibility of various teleost species to diseases. Administration of recombinant fish IFN is one of the most efficacious methods of maintaining a healthy fish population and is a promising alternative to the use of antibiotics and vaccines.In this study, firstly,semi-quantitative PCR assays were carried out to compare the effect of Poly I:C and recombinant IFN on expression of interferon system genes in grass carp (Ctenopharyngodon idellus). Recombinant grass carp IFN (rCiIFN) was expressed in E. coli and purified. Healthy grass carps were injected intraperitoneally with Poly I:C and rCiIFN, respectively. The expression patterns of several interferon system genes, including IFN, IRF-1, IRF-7, STAT-3, Mx and PKR were detected in grass carp tissues (kidney, spleen, gill, liver, intestine and heart) at various time points (6h,12h,24h,48h and72h). The results showed that a similar kinetics was observed after stimulation with either Poly I:C or rCilFN. IFN was expressed ubiquitously in all the tissues tested, and significantly increased at12h post-treatment, peaked at24h-48h and thereafter decreased at72h in most tissues (gill, spleen, kidney and liver). IRF7was also significantly enhanced at12h post-treatment in spleen, kidney, liver, gill and intestine, then displaying a significantly high level of expression until72h. STAT-3was up-regulated significantly at6h post-treatment in all tissues, and maintaining a significantly high level of expression until72h, with a relatively strong expression in spleen, kidney and gill. The expression of Mxl was up-regulated at6h post-treatment and significantly enhanced at12h, with a high level of expression till48h, thereafter decreased to a low level at72h. A relatively strong expression of Mxl was observed in kidney, spleen and gill, followed by intestine and heart. PKR displayed a significantly low level of constitutive expression. It was up-regulated significantly at6h post-treatment, and maintaining a relatively high level of expression until72h, with a relatively strong expression in spleen, kidney and gill. Compared with the expression of all genes above induced by Poly I:C, the results illustrated that recombinant IFN could up-regulate expression of interferon system genes in grass carp more quickly, strongly, and some interferon system genes induced by rCiIFN could maintain a significantly high level of expression. In addition, real-time quantitative PCR assays were carried out to compare the effect of Poly I:C and recombinant IFN on expression of interferon system genes at12h time point. The results showed that a similar kinetics was observed after stimulation with either Poly I:C or rCiIFN. The expression of the six genes in all the tested tissues increased significantly at12h post-treatment. Compared with gene expression levels observed in PBS-injected controls, the expression of the six genes in liver increased9.32-,9.00-,8.00-,10.3-,10.3-, and3.1-folds, respectively. In spleen they were up to6.29-,3.00-,25.18-,20.753-,0.934-, and10.934-folds, respectively. In kidney the figures were6.651-,9.00-,10.44-,8.65-,10.566-and5.460-folds, respectively. In gill the figures were4.56-,7.23-,13.0-,13.49-,1.985-and19.150-folds, respectively. Some of the six genes in intestine and heart were also up-regulated significantly, E.g., the figures were in intestine1.692-,3.00-,3.67-,4.58-,0.881,6.81-folds and in heart the figures were1.68-,6.00-,1.47-,1.180-,1.948-,5.890-fold, respectively.After feeding the fish with pellets mixed with rCiIFN for12h,24h,48h, and72h,Control fish were fed only the commercial pellets mixed with PBS. The expression patterns of IFN were detected in grass carp tissues (kidney, spleen, gill, liver, intestine and heart) at various time points (6h,12h,24h,48h and72h). semi-quantitative PCR assays showed that the expression level of IFN was significantly up-regulated and reached its peak level in tissues of those fish treated for1or2days. To compare the expression of IFN from the tested tissues,it was found that a relatively strong expression was in liver,spleen, kidney and gill. Additionally, After feeding the fish with pellets mixed with rCilFN for12h, real-time quantitative RT—PCR was performed to detect the expression of IFN from different tissues include kidney, spleen, gill, liver, intestine and heart at12h time point. The expression of IFN in all the tested tissues was observed,with a relatively strong expression in liver,spleen, kidney and gill.These results demonstrated that recombinant IFN could up-regulate the expression of interferon system genes,including IFN, IRF-1, IRF-7, STAT-3, Mx and PKR in grass carp (Ctenopharyngodon idellus). These data provide a base for promoting the actual use of recombinant IFN in the aquaculture. |
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