Expression And Purification Of Recombinant Canine IFN-? With High Biological Activity By The Use Of An Insect Baculovirus System

Interferons(IFNs)are critical for eliciting competent immune responses especially antiviral immunity,which have been used as antiviral and antitumor drugs in human and veterinary clinic showing good therapeutic effect.IFNs do not act directly on viruses,but exert biological effects by binding to IFN receptors to activate downstream signaling pathways that lead to induction of a wide range of IFN-stimulated genes(ISGs)encoding key immune effector molecules in target cells.To date,three types of IFNs,including type I IFNs,type II IFNs and type III IFNs,have been identified based on their structural features and receptors.Among them,type III IFNs,also termed IFN-?s(IFN?1/2/3)or interleukin 29(IL-29)and interleukin 28A/B(IL-28A/B),are the most recently described antiviral cytokines which are evolutionarily distant to type I IFNs but exhibit similar antiviral activity to type I IFNs.In contrast to a broad expression spectrum of type I IFN receptors,expression of type III IFN receptors are restricted in certain cells such as epithelial cells and dendritic cells,indicating that functions of IFN-? may be tissue-specific with lower side effects.Therefore,efficient expression of IFN-? with high biological activity by genetic engineering expression systems in vitro is an attractive way for developing novel antiviral drugs for both human and veterinary clinic.Compared with the prokaryotic expression system,the eukaryotic expression systems,such as insect baculovirus expression system,are stable product quality,high purity,easy to control the quality,and ease of large scale production.In this study,we used the insect baculovirus expression system to express recombinant canine IFN-?(rcaIFN-?)in vitro,and further study the antiviral activity of the recombinant protein.To obtain a large amount of rcaIFN-? that are close to natural structure and function,a recombinant baculovirus expression plasmid containing the canine IFN-? cDNA was constructed and was used to generate recombinant baculoviruses in Sf9 cells.The P1 generation of recombinant baculoviruses(P1 rBAC-CaIFN-?)was used to infect Sf9 cells in an optimal titer for expression of canine IFN-? proteins.Successful expression of His-taggedrcaIFN-? n Sf9 cells was validated by immunoblotting analysis and immunofluorescence staining.After confirmation of the expression of rcaIFN-?,Sf9 cells infected by P3 generation of the recombinant baculoviruses for 4 days were collected and were lysed by multigelation.The rcaIFN-? was then purified by use of AKTA protein purification system with His protein purification column.To confirm the biological activity of the purified rcaIFN-?,MDCK cells were stimulated with rca IFN-? and expression of canonical interferon-stimulated genes(ISGs)was examined by quantitative real-time PCR.Upon rcaIFN-? stimulation,mRNA expression of ISGs such as ISG15,Mx1 and OASb was extensively increased in MDCK cells,indicating that the purified rcaIFN-? can active the IFN signaling.More importantly,rcaIFN-? treatment inhibited the replication of vesicular stomatitis virus(VSV)in MDCK cells,demonstrating that the purified rcaIFN-? has it antiviral activity.In summary,by use of the insect baculovirus expression system,we have successfully expressed recombinant canine IFN-? that shows efficient antiviral activity in vitro in multiple doses after purification.These results suggest that the insect baculovirus expression system is an effective way to get a large amo of rca IFN-? with high biological activity,making it possible to develop a novel antiviral drug for preventing viral diseases of pets.