Expression Of Canine Interferon ?/RTB Fusion Protein In Insect Baculovirus System And Preliminary Study On Antiviral Activity

Interferon?IFN?,a glycoprotein with strong antiviral activity,is one of the first lines of defense against invasion of pathogens by the host.Interferons participate in many immune interactions during viral infection and contribute to the induction and regulation of innate and adaptive antiviral mechanisms.However,viruses can"interfere"with the antiviral function of interferons and escape immune clearance,causing chronic viral infections.The development of recombinant interferon in genetic engineering makes interferon still attractive as an antiviral drug choice,however,due to the short half-life,frequent administration,neutralizing antibody production and species differences,the therapeutic effect of currently available recombinant canine interferon for canine viral disease has been greatly reduced.The ricin B chain itself is non-toxic and can better promote the adhesion and absorption of substances while keeping their respective biological activities unaffected as carrier molecules.In this study,canine interferon alfa?CaIFN??and ricin B chain?RTB?were co-expressed by an insect baculovirus expression system to prepare a recombinant CaIFN?/RTB fusion protein with interferon biological activity and desirable to obtain anti-viral biological agents that have specific biological activity on canine with highly efficient and inexpensive.This study includes the following two aspects:1.Preparation of Recombinant CaIFN?/RTB Fusion ProteinCaIFN?and RTB sequences were ligated in tandem by PCR and cloned into the pMD18T vector.The pFastBac^TM1-CaIFN?/RTB recombinant transfer vector was successfully constructed and transformed into DH10Bac competent cells for transposition,through antibiotics and blue-white screening,recombinant shuttle plasmid Bacmid-CaIFN?/RTB was obtained.A large amount of recombinant baculoviruses was obtained by electrotransfection into Sf9 insect cells and the virus titer of the P3 virus was 7.6×10⁷ IFU/mL.The optimal expression conditions for the virus infection were determined to be 300?L of virus fluid for 3 days.The virus collected was concentrated and purified and then the recombinant CaIFN?/RTB fusion protein was successfully obtained by SDS-PAGE and Western-blot identification.2.Detection of anti-virus activity of recombinant CaIFN?/RTB fusion protein in vitroThe cytotoxicity of the recombinant CaIFN?/RTB fusion protein was detected by MTT assay and the result was non-toxic.The VSV virus was amplified and the TCID₅₀ was measured at 10^-7.37/100?L.The MDCK-VSV system was used to demonstrate that the recombinant CaIFN?/RTB fusion protein had good antiviral activity,and the titer of the fusion protein was measured to be4.17×10⁶IU/mL?protein concentration was 1.40 mg/mL?stained by crystal violet.The above results indicate that the recombinant CaIFN?/RTB fusion protein was successfully obtained by using an insect baculovirus expression system,and the biological activity tested in vitro proved that it had high antiviral activity against MDCK cells,which provided a new basis for the development of antiviral biological agents for canine interferon.However,whether the body has the same biological activity still needs further exploration.