Co-expression Of Porcine Interferon-α And Interferon-γ In Silkworm And Its Antiviral Activity Detection

Porcine interferon comprises Type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ). IFNs areimportant components of the host immune in response to the presence of pathogens or tumor cells. IFNsshare several common effects such as antivirus, antitumor and immunomodulatory activity. IFN-α areproduced by most cells, while IFN-γ is synthesized by activated NK cells and activated T-cells.Combination of type I and type II interferons has much greater activity than that expected from theseparate contribution of each type. Bombyx mori baculovirus expression system was used for suchmixture recombinant proteins production.Porcine interferon-α gene and interferon-γ gene were cloned into the baculovirus transfer vectorpVL1393to generate a recombinant plasmid pVL-α-IRES-γ. Both interferon genes are under the controlof pH promoter and IRES, respectively. The internal ribosome entry site (IRES) was derived fromCricket paralysis virus (CrPV). The pVL-α-IRES-γ was co-transfected with linearized Bm-BacPAK6DNA into Bm-N cells by liposome-mediated method to generate a recombinant baculovirus,rBm-α-IRES-γ. rBm-α-IRES-γ allows the simultaneous expression of porcine interferon-α and porcineinterferon-γ from a single messenger RNA. The translation of porcine interferon-γ is mediated by IRES.Early fifth-instar silkworms were infected with the recombinant virus and its haemolymph was collectedaround96-120h post-infection. The expressed recombinant proteins were successfully detected in silkhaemolymph by western-blot analysis. The bioactivity of recombinant PoIFNs in silkwormhaemolymph was investigated. The result show that the dilution of2×106of haemolymph can protectthe Vero cells from the cytopathic effect cause by100TCID50of VSV*GFP. That will allow us toexplore IFNs as a potential therapeutic agent for porcine diseases using silkworm-baculovirusexpression system.