Expression Of Duck IFN-α、IFN-γ、IFN-λ In Silkworm And Determination Of Their Antiviral Activity

In recent years,the poultry industry in China has developed rapidly.As an integral part of animal husbandry practices,rearing poultry has become the core source of income for farmers in rural areas.Trends in poultry farming have gradually changed from open range,rural farming to intensive and commercial-scale farming.The outbreak of avian viral diseases in this mass scale farming often causes huge monetary losses.For instance,circulating viruses account for 36%of the total causes of poultry mortality,and waterfowl are the main carriers to harbor these invading pathogens.Interferons(IFNs)are cytokine in nature with peculiar characteristics as antiviral,immunomodulatory and immune booster biological activities.They possess beneficial therapeutic activities and demonstrate a preventive effect on viral diseases.In the present study,the tools of genetic engineering were employed to express a cohort of interferon including duck interferon alpha(DuIFN-α)as type I IFN,duck interferon gamma(DuIFN-γ)as type II IFN and duck interferon lambda(DuIFN-λ)as type III IFN in ducks,aiming to achieve therapeutic efficiency for the prevention and control of viral diseases in duck(poultry).Briefly,the gene sequence of duck interferon alpha,gamma,and lambda were selected and optimized.All these genes were synthesized by using the approach of chemical synthesis.The duck IFN-α,IFN-γ,and IFN-λgenes were constructed into the polyclonal loci of baculovirus transfer vector pVL1393.The recombinant transfer vectors pVL1393-DuIFN-α,pVL1393-DuIFN-γand pVL1393-DuIFN-λwere constructed.The recombinant transfer vectors and inactivated rescue silkworm were introduced in Baculovirus shuttle plasmid reBmBac and co-transfected in silkworm(Bombyx mori)derived BmN cells and get rBmBac-DuIFN-α、rBmBac-DuIFN-γ、rBmBac-DuIFN-λ..The recombinant virus was collected and infected to the 5th instar silkworm larvae.At 5 days post-infection,the expression products were harvested.The expression efficiency of three interferons expressed in silkworm were thoroughly investigated and verified by quantification assays including PCR,RT-PCR.It was proved that the three interferon genes were replicated and efficiently transcribed in silkworm larvae along with the viral genomeThen the anti-viral activity of duck interferon alpha,gamma and lambda in silkworm haemolymph was detected by CEF/VSV-GFP system,The inhibition effect of duck interferon on the proliferation of VSV-GFP virus was detected on CEF cells by CPE method.The results showed that the recombinant duck interferon alpha,gamma and lambda expressed in the hemolymph of silkworm had strong antiviral activity.The antiviral titers of recombinant duck interferon alpha,gamma and lambda were 8.13 x 10~5 U/mL,3.0 x 10~5U/mL and 6.1 x 10~5 U/mL,respectively.Finally,by using plaque assay,co-transfected viruses were purified.The viral strains demonstrated higher expression efficiency for the three interferons with the biological titers of(1.9±0.25)×10~6 U/mL,(8.65±0.52)×10~5 U/mL and(1.56±0.1)×10~6 U/mL for DuIFN-α,DuIFN-γ,and DuIFN-λrespectively.Compared with the unpurified recombinant virus,the titer of IFN expressed by the purified strain with the highest expression level increased by 2-3 times.In this study,interferons alpha,gamma and lambda of duck were highly expressed by the expression system of silkworm baculovirus.The results showed that the three interferons had strong antiviral activity.It laid a foundation for the production of IFN for cheap ducks and poultry.It is expected that it will be helpful for the prevention and treatment of duck(poultry)viral diseases.