Expression And Antiviral Activity Analysis Of Canine Interferon ?4,?2

Interferons(IFNs)are very important cytokines of human and other animals,which are generated by specific cells induced by inducers.IFNs are glycoprotein that possess antiviral activity,anti-tumor activity and immunoregulation function.IFNs include three types:I,? and ?.Among them,IFN-a belongs to type I interferon,and IFN-as are secreted by leukocyte,when they are stimulated by specific or aspecific antigen.The IFNs possess certain species-specific.As canine interferon alpha has wide antiviral activities and hight research value.So the topic modified the gene of CaIFN-a4 and CaIFN-a2,expressed them in two different systems and analyzed their bioactivity.Part ?:Expression of Canine Interferon Alpha4 in E.coli and Antiviral Activity AnalysisIn order to obtain canine interferon alpha4(CaIFN-?4)with activity and high quantity,The recombinant expression plasmid pET28a-CaIFN-?4 was constructed by cloning the CalFN-?4 gene into the pET-28a(+)expression vector.The recombinant expression plasmid was transformed into E.coli Rosetta.Recombiant CaIFN-a4 proteins about 23kDa was induced by IPTG,Interferon inclusion bodies were obtained through the ultrasonic broken,Inclusion bodies were denatured by urea,then renatured and purified with HisTrap affinity chromatography.Finally,we successfully harvested CaIFN-a4 purificated.The purificate CaIFN-a4 proteins had the antiviral activity agianst VSV,detected with MDCK and MDBK,and the antiviral activity were 1.19×105U/mL and 1.86×103U/mL,respectively.This study successfully expressed the canine interferon alpha4 with active,which laid the experimental foundations for the development of the production system of canine interferon alpha4 with high activity.Part ?:Stable Expression of Canine Interferon Alpha4 in CHO Cell and Antiviral Activity AnalysisIn order to obtain canine interferon alpha4(CaIFN-a4)with high activity,we prepared the fusion gene of canine interferons alpha4 and GFP,CaIFN-alpha4-GFP,connecting the canine interferons alpha4 and green fluorescent protein(GFP)with two GGGGS-five peptide(2G4S).The recombinant expression plasmid,pL-CaIFN-a4-GFP was constructed by cloning the fusion gene(CaIFN-a4-GFP)into the pL-eGFP eukaryotic expression vector.Recombiant lentivirus,vCaIFN-a4-GFP,was harvested following transfection of 293FT cells with the pL-CaIFN-a4-GFP eukaryotic expression plasmid and other three lentivirus plasmids.Finally,we successfully established CHO cell expressing CaIFN-a4-GFP through infecting the Chinese hamster ovary(CHO)with the harvested recombinant lentivirus,followed by screened with blast fungus element,flow cytometry sorting and limited dilution transfection cell cloning.Green fluorescence could be observed in the established CHO cell,demonstrating the expression of interferon.The CHO cell culture supernatant had the antiviral activity agianst VSV,detected with MDCK and MDBK,and the antiviral activity were 1.39×105U/mL and 2.19×104U/mL,respectively.This study successfully developed CHO cell expressing the canine interferon alpha4 with active,which laid the experimental foundations for the development of the production system of dogs interferon alpha4 with high activity.Part ?:Expression of Canine Interferon Alpha2 in E.coli and Antiviral Activity AnalysisIn order to obtain canine interferon alpha2(CaIFN-a2)with high activity and quantity,The recombinant expression plasmid pET28a-CaIFN-a2 was constructed by cloning the CaIFN-a2 gene into the pET-28a(+)expression vector.The recombinant expression plasmid was transformed into E.coli Rosetta.Recombiant CaIFN-a2 proteins about 23kDa was induced by IPTG,Interferon inclusions bodies were obtained through the ultrasonic broken,Inclusion bodies were denatured by urea,then renatured and purified with HisTrap affinity chromatography.Finally,we successfully harvested CaIFN-a2 purificated,The purificate CaIFN-a2 proteins had the antiviral activity agianst VSV,detected with MDCK and MDBK,and the antiviral activity were 1.70×106U/mL and 3.16×103U/mL respectively.This study successfully expressed the canine interferon alpha2 with active,which laid the experimental foundations for the development of the production system of dogs interferon alpha2 with high activity.