Prokaryotic Expression And Anti-IBDV Activity Of Chicken IL-18 And IFN-?

The healthy and rapid development of poultry industry was troubled by avian viral diseases such as Infectious Bursal Disease(IBD).Chemical drugs of inhibiting virus are not good to IBDV,and poultry is prone to drug resistance and drug residues.The development of new antiviral drugs seriously lagging behind;although most avian viral diseases can be immunized against vaccines,the effect is not ideal,and in the course of the use of the vaccine,some individuals with low immunity are likely to cause seriously illnesses and unequal immune response.Therefore,it is necessary to research a new cytokine.It can enhance antiviral activity of the immune system.It has been shown that chicken interleukin 18(ChIL-18)and chicken interferon gamma(ChIFN-?)have an inhibitory effect on a variety of viral infections.Therefore,it is necessary to study the recombinant protein ChIL-18 and ChIFN-? and the two mixed antiviral effect and its mechanism.In this study,the whole protein gene of ChIL-18 and ChIFN-? was analyzed.The ChIL-18 and ChIFN-? genes were obstained through RT-PCR method.And genes were inserted into pET28 a prokaryotic expression vector.The plasmids were transformed into E.coli BL21 compenent cells.Recombinant bacteria were induced by IPTG,and the ChIL-18 and ChIFN-?proteins were expressed and purified.To identify protein antiviral activity,200 ng / mL ChIL-18 and ChIFN-? were added to chicken embryonic fibroblasts,respectively or together.After 24 hours,2.5 MOI IBDV were inoculated with chicken embryonic fibroblasts.The results showed that the antiviral effect of the combination group was better than that of ChIL-18 or ChIFN-?group.200 ?g of ChIL-18 and ChIFN-? recombinant proteins were injected into 14-day-old chicks,respectively,with PBS as the negative control,24 hours after re-injection,the IBDV challenge of 7 × 104 PFU was performed on the chickens.The results showed that ChIL-18 and ChIFN-? could inhibit the proliferation of IBDV,and the effect of mixed group of ChIL-18 and ChIFN-? was significantly better than of ChIL-18 or ChIFN-? on the proliferation of IBDV,indicating that the mixed group has a better antiviral effect.After the protein injection of chicks,the proliferation of spleen lymphocyte and cytokinessecretion of lymphocyte and serum were detected using kits.The results showed that the protein of mixed group could promote the proliferation of spleen lymphocytes and secrete of IL-6,IL-12,and IL-17.The levels of IL-4 and IL-10 have not obvious change.Cytokines of serum were similar to those in lymphocytes.These indicated that level of the cellular immune of the mixed group was better than that in the ChIL-18 or ChIFN-? group.To analyze the relation of ChIL-18 and ChIFN-? and several Toll-like receptors(TLRs),were analyzed their activity.Downstream signaling pathways and NO in the peripheral blood mononuclear cells of chickens were detected,too.The results showed that the expression of TLR2,3,4 and MHC-II were increased in the macrophages,the MAPK and NF?B signaling pathways were activated and the secretion levels of NO were significantly increased.These results suggest that mixed groups have a better antiviral ability.In summary,ChIL-18 and ChIFN-?,and the both of them can induce the body to produce effective defense effect on the virus infection,and provide reference for the research of new cytokine preparation.