Construction And Functional Study Of Chicken α Interferon And Salmonella FliC Fusion Protein

Interferon is a kind of cytokine that can be widely expressed in various monocytes such as macrophages,dendritic cells and fibroblasts,and has various functions such as antiviral,antitumor and immune regulation.Since the first chicken α interferon was cloned in 1994,the function of chicken α interferon antiviral activity has received great attention in the field of veterinary medicine.Salmonella typhimurium flagellin FliC has a unique structure and function,can stimulate the body to produce certain inflammatory factors,and plays an important role as an effective activator in natural and adaptive immunity.The flagellin FliC is soluble in the prokaryotic system.As a basic vaccine adjuvant,it is widely used in the development of pathogen vaccines such as bacteria,viruses and parasites,and has good effects in veterinary clinics.The prokaryotic expression cost and high protein expression yield are effective methods for obtaining a large amount of protein of interest.However,chicken α interferon exists in the form of inclusion bodies in prokaryotic expression systems,which hinders widespread use in clinical practice.The aim of this study is to construct a fusion protein for the soluble expression of chicken α interferon by prokaryotic expression system.This protein not only has the function of anti-viral activity of chicken α interferon,but also has the function of immunomodulation of Salmonella typhimurium flagellin FliC.Therefore,it provides certain technical support for the clinical application of chicken α interferon.In this study,we constructed chicken α interferon expression plasmid pET28a-Chifn and Salmonella typhimurium flagellin gene fliC expression plasmid pET28a-FliC,and a ligated the gene Chifn and fliC to construct a fusion protein plasmid pET28a-EcBoc.Three plasmids were transformed into BL21(DE3)respectively,IPTG induced expression,and three expression proteins were analyzed by SDS-PAGE.After analysis,the fusion protein EcBoc with an expression product size of 72 KDa was obtained,the protein was soluble and expressed mainly in the supernatant;Under the same expression conditions,the chicken α interferon protein with an expression product size of about 18 KDa was obtained,mainly in the form of insoluble inclusion bodies;The FliC protein of Salmonella typhimurium with expression product size of about 54 KDa was obtained,which was mainly soluble.After purification of the expressed product by Ni column chromatography resin,the fusion protein EcBoc with higher purity was obtained.To verify the function of the fusion protein EcBoc,firstly,the dual luciferase plasmid pGL3 basic Mxp containing Mx-promoter was constructed to detect whether the protein can activate the Mx-promoter through the dual luciferase reporter gene system.In addition,the plasmid pGL3 basic Mxp-EGFP expressing green fluorescent protein was constructed,and was transfected into DF1 cells.After incubating the protein EcBoc,the green fluorescent luminescence was observed more directly by fluorescence microscopy to judge whether the protein can activate the Mx-promoter.On the other hand,the plasmid pGL3 basic Mxp-EGFP was transfected into DF1 cells,and the fusion protein EcBoc was incubated after transfection.Flow cytometry analysis of the proportion of DF1 fluorescent cells,and quantitative analysis of the activation of Mx-promoter by this protein.Finally,In order to analyze whether the protein has the activity and function of Salmonella typhimurium flagellin FliC,we detected the fusion protein EcBoc to stimulate the production of TNF-α by ELISA.Results showed that the fusion protein EcBoc can activate the Mx-promoter by the dual luciferase reporter gene system,and has the biological activity of chicken α interferon pro tein;Results observed by fluorescence microscopy that the DF1 cells transfected with the plasmid had obvious luminescence after incubation of the fusion protein;Flow cytometry analysis showed that the proportion of DF1 fluorescent cells reached 24.30% in the fusion protein EcBoc experimental group,and the proportion of DF1 fluorescent cells was 29.68% in the chicken interferon group.It was compared with the blank control group,the difference was significant(P < 0.01).In short,these methods verified that the fusion protein EcBoc has chicken α interferon function.Finally,the fusion protein EcBoc was detected by ELISA to stimulate the production of TNF-α in PBMC cells,and the concentration reached 54.68 pg/m L.It was compared with the control group,the difference was significant(P < 0.01).This confirmed that the fusion protein EcBoc has the activity and function of the Salmonella typhimurium flagellin FliC protein.In summary,this study successfully fused chicken α interferon with Salmonella typhimurium flagellin FliC to construct chicken α interferon fusion protein soluble in prokaryotic system,which has both chicken α interferon resistance’s viral activity and Salmonella typhimurium flagellin immunoregulatory function.