Expression Of Chicken IFN-?3 And Sheep IFN-?3 In Silkworm And Determination Of Their Antiviral Activity

Interferon is a kind of cytokine with the functions including broad-spectrum antiviral activity,immunoregulation and anti-tumor proliferation while working on the same kind of cells.According to the structure of interferon and their receptors on the surface of cells,interferon can be divided into three groups,type I,type II and type III interferon.Type I interferon has a very strong antiviral activity,but some viruses,such as PRRSV,produced a mechanism to escape from the host immune system in a long period of evolution,which makes the Type I interferon of the host do not have immune activity to these viruses in vivo.Type III interferon(IFN-?s)had been discovered in the recent years which include IFN-?1,IFN-?2,IFN-?3 and IFN-?4.It is similar to type I interferon in function of protein and its structure of genes is similar to IL-10.Fortunately,some researches show that type III interferon works on some of these viruses escaping from the host immune system.The aim of this experiment was to use the silkworm baculovirus expression system to express chicken interferon lambda 3(ChIFN-?3)and sheep interferon lambda 3(OvIFN-?3)with high antiviral activity,which set the stage for the interaction of type III interferon with a variety of common livestock and poultry viruses and combined usage of interferons.In this experiment,the amino acid sequences of ChIFN-?3 and OvIFN-?3 were determined by aligning the amino acid sequence related to the ChIFN-?3 and OvIFN-?3 on the NCBI.The sequence of target genes was optimized according to the preference of silkworm codon.After optimization,the GC content of ChIFN-?3 was adjusted to 48.38%and CAI value was 0.93;the GC content of OvIFN-?3 was adjusted to 52.44%,and the CAI value was 0.94.BamH I and EcoR I sites were added at both ends of the optimized sequence,and the Kozak sequence was added before the starting codon.The genes were synthesized by the chemical method.The synthesized ChIFN-?3 and OvIFN-?3 were inserted to the multiple cloning site of the baculovirus vector pVL1393.The recombinant transfer vector co-transfected BmN cells with inactivated rescue baculovirus reBmBac to obtain recombinant baculovirus.The recombinant baculovirus was purified by spotting and then injected into the 5 instar silkworm larvae to collect hemolymph of the infected silkworm.The genome PCR showed that ChIFN-?3 and OvIFN-?3 were successfully integrated into the baculovirus genome,and the recombinant baculovirus proliferated and expressed the target protein in the silkworm larvae.The antiviral activity of recombinant interferons was detected on the VSV-GFP system by cytopathic effect inhibition method,and their potency was calculated according to the Reed-Muench method.The results suggested that the potency of ChIFN-?3 can reach 5.5±0.8×10~6U/mL hemolymph,and that of OvIFN-?3 is3.1±0.6×10~6U/mL hemolymph.