Efficient Prokaryotic Expression And Antiviral Activity Determination Of Recombinant Canine Interferon-?

Interferons(Interferons,IFNs)are glycoprotein cytokines secreted by host lymphocytes and monocytes with various biological activities such as antiviral,antitumor and regulating immune function.IFNs do not act directly on viruses,but exert their biological effects by binding to cognate receptors on target cells to activate downstream signaling pathways that lead to induction of a wide range of IFN-stimulated genes(ISGs)encoding key immune effector molecules.Type III IFNs are recently discovered family of IFNs which are evolutionarily distant to type I IFN but exhibit similar antiviral functions to type I IFNs.In contrast to a broad distribution of type I IFN receptors,the expression of type III IFN receptor is primary expression on epithelial cells and some dendritic cells,indicating that functions of type III IFN may be tissue-specific with lower toxicity effects.Therefore,type III IFN has great potential clinical applications and is expected to become a new genetic engineering drug in the future.However,there is no biological product of type III IFN for canines in pet clinical practice.Prokaryotic expression was successfully established with high expression efficiency of foreign proteins.Because it's a series of advantages,such as high yield,low cost,easy purification and can be expressed in large quantities through high-density fermentation.Therefore,Prokaryotic expression system has a good foundation for industrialization and is an important technique to realize the large-scale production of foreign proteins and promote their commercialization.To obtain a large amount of recombinant CaIFN-? protein,the gene sequence of CaIFN-? was optimized based on the codon preference of E.coli,and a prokaryotic expression vector containing CaIFN-? optimized gene sequence was constructed,and the vector was transformed into BL21 E.coli expression bacteria for induction and expression.By optimizing expression conditions,the recombinant CaIFN-? protein was expressed in large quantities.Because the recombinant CaIFN-? protein was expressed in the form of inclusion bodies,purified recombinant CaIFN-? protein was obtained by urea treatment and affinity chromatography,and further dialysis technology was used to realize the renaturation and concentration of the recombinant protein.To study the antiviral activity of the refolded CaIFN-? protein,MDCK cells were treatment with CaIFN-? and expression of canonical interferonstimulated genes(ISGs)was examined by quantitative real-time PCR.m RNA expression of ISGs such as MXI,ISG15 and 2'-5'OAS were extensively increased in CaIFN-? stimulated MDCK cells,indicating that the refolded CaIFN-? can active the IFN signaling.To further study the antiviral activity of the recombinant CaIFN-? protein,by the vesicular stomatitis virus(VSV-GFP)and influenza A virus as research models,we found that the recombinant CaIFN-? protein significantly inhibited the replicate of VSV-GFP and influenza virus in a dose dependent,indicating that the prepared recombinant CaIFN-? protein has high antiviral activity.In summary,our study constructed a prokaryotic expression vector for the efficient expression of recombinant CaIFN-? protein,realized the high-efficiency expression of recombinant CaIFN-? protein,and obtained recombinant CaIFN-? protein with high antiviral activity through purification and renaturation.This study provides foundation for new pet antiviral drug development.