Prokaryotic expression and antiviral activity of recombinant canine IFN-α
The aim of this experiment was to establish a method for the prokaryotic expression and purification of canine interferon-α(CaIFN-α)and to research its antiviral activity.rCaIFN-α was ef-ficiently expressed and purified by constructing pET-28a/CaIFN-α expression vector.The cytotoxicity of rCaIFN-α was determined on canine kidney cells(MDCK)by CCK-8 assay,and its antiviral activity po-tency was determined in the MDCK/VSV system.RT-qPCR was used to detect the expression level of down-stream ISGs after rCaIFN-α acted on the MDCK cells.TCID50 and indirect immunofluorescence(IFA)were further utilized to determine the ability of rCaIFN-α to combat infection of canine parainfluenza virus(CPIV)and canine distemper virus(CDV).The results of electrophoresis and Western-blot showed that specific rCaIFN-α was successfully expressed.CCK-8 results showed that the recombinant protein was not obviously cytotoxic,and the potency of anti-VSV viral activity determined by cytopathic inhi-bition assay was 1.39×107 U/mL.rCaIFN-α was significantly up-regulated in the transcript levels of ISG15,Mxl,and other ISGs as determined by RT-qPCR.IFA and other results showed that rCaIFN-α had ex-cellent antiviral effects on CPIV and CDV.In conclusion that of the rCaIFN-α prepared in this experi-ment have excellent antiviral activity,which lays the foundation for further advancement of novel pet antiviral agents.
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