Expression And Antiviral Activity Assay Of Canine Interferon A2

Interferon(IFN)is one of the members of the secretory protein family caused by viruses infecting cells.It is an important class of cytokines in animals and has a broad spectrum of biological functions such as antiviral,anti-tumor cell proliferation and immune regulation.They are classified based on the nucleotide sequence,chromosomal location,interaction with its specific receptor,structure and physicochemical properties.IFN-a is a member of type I interferon family and is a kind of glycoprotein secreted by leukocytes.It has high antiviral activity and has high clinical application value in experimental research and antiviral therapy.IFN also has shortcomings such as short half-life and frequent administration,which limits its clinical application.The serum albumin is a kind of macromolecular naturally presenting in animals.It has the characteristics of stable properties and long half-life,and has important physiological functions in maintaining colloidal osmotic pressure,transporting substances and scavenging free radical.It is very suitable for the research that IFN fuses expression with albumin to prolong the half-life of IFN.This experiment identified the antiviral activity of canine interferon α2 expressed by prokaryotic and insect/baculovirus expression system.Then,the fusion IFN which fused CaIFN-a2 and canine serum albumin was secreted in an insect/baculovirus expression system and detected its antiviral activity,which laid the foundation for the research and clinical application of canine interferon.Experiment I:Expression in E.coli and antiviral activity analysis of canine interferon alpha 2According to the CaIFN-a2 sequence(M28625.1)published in Genbank,the signal peptide was removed,and the mature encoding sequence of canine interferon alpha 2 was designed and artifically synthesized.Meanwhile,the enzyme sites of EcoR I and Hind III were introduced.The artificially synthesized CaIFN-a2 gene was cloned into a prokaryotic expression vector pET-28a(+)to construct a recombinant expression plasmid pET-28a-CaIFN-a2.The recombinant expression plasmid pET-28a-CaIFN-a2,which was identified by PCR and restriction enzyme digestion,was transformed into Rosetta and induced by IPTG.The recombinant protein with a molecular weight of approximately 23 kDa was identified by SDS-PAGE and Western blot analysis.The results showed that the recombinant protein mainly existed in a form of inclusion body and accounted for 52.5%of the total bacterial proteins.The recombinant CaIFN-a2 with the purity of 92%was obtained through denaturation,renaturation and purification.The antiviral activity of the recombinant CaIFN-a2 was determined by the canine kidney cell(MDCK)/vesicular stomatitis virus(VSV)system,which was 3.16×106 U/mL.The expression plasmid pET-28a-CaIFN-a2 was successfully constructed and expressed in E.coli.The expressed recombinant CaIFN-a2 had a good antiviral activity,which provided a basis for further research on novel canine interferon agent.Experiment Ⅱ:Expression and antiviral activity analysis of canine interferon alpha 2 via insect/baculovirus expression systemThe baculovirus expression system was used to express canine interferon alpha 2 with antiviral activity.The artificially synthesized mature sequence of CaIFN-a2 was inserted into the vector pFastBacHTA,and transformed into the DH10Bac competent cells.After three times of blue and white spot screening,the shuttle plasmid Bacmid-CaIFN-a2 was obtained.The recombinant shuttle plasmid was transfected into Sf 9 cells,and the recombinant baculovirus rBac-CaIFN-a2 was harvested.After the recombinant baculovirus were transmitted to the third passage,Sf 9 cells were infected with the recombinant baculovirus,which were harvested after 3 days of infection.SDS-PAGE,indirect immunofluorescence(IFA)and Western blot were used to identify the expression of CaIFN-a2.The IFA assay showed that CaIFN-α2 could be expressed in insect cells;SDS-PAGE and Western blot detected the expression product with a molecular weight of approximately 25 kDa and 28 kDa.The antiviral activity of recombinant CaIFN-α2 was detected by MDCK/VSV system,which could effectively inhibit the attack of VSV on MDCK cells.The antiviral activity of cell lysates was 5.58×105 U/mL.Experiment Ⅲ:Expression of canine long-acting interferon alpha in insect cellsThe recombinant canine fusion interferon alpha 2 with antiviral activity was expressed using a baculovirus expression system.The gene encoding canine serum albumin and the mature protein gene of canine interferon alpha 2 were linked with flexible peptide(GGGGS),and a honeybee signal peptide(HBM)recognized by insect cells was fused at the N-terminus of canine serum albumin gene.The artificially synthesized fusion gene sequence(HBM-Alb-CaIFN-a2)was inserted into the vector pFastBacI,and transformed into the DH10Bac competent cells.The shuttle plasmid Bacmid-HBM-Alb-CaIFN-a2 was obtained after three times of blue and white spots screening and purification.,Sf 9 cells were transfected with the recombinant shuttle plasmids which identified by PCR to obtain recombinant baculovirus rBac-HBM-Alb-CaIFN-a2.After the recombinant baculovirus was transmitted to the third passage,Sf 9 cells were infected with the recombinant baculovirus,which were harvested after 3 days of infection.SDS-PAGE,indirect immunofluorescence(IFA)and Western blot were used to identify the expression of recombinant canine fusion interferon a2.The IFA results showed that recombinant fusion interferon a2 could be expressed in insect cells.SDS-PAGE and Western blot analysis showed that there were about 90 kDa expression products in culture supernatant,which indicated that secretory expression was consistent with the expected results.The MDCK/VSV system was used to detect the antiviral activity of recombinant canine fusion interferon α2 which could inhibit the attack of VSV on MDCK cells,The antiviral activity was 1.78×104 U/mL.