Preparation And Biological Activity Of β,γ And β-γ Interferon Proteins From Panthera Tigris Altaica

Interferon(IFN)has broad-spectrum antiviral,immune regulation and anti-tumor biological functions,and it can be seen everywhere in the prevention and treatment of viral diseases.Different interferons have different antigenic properties and different molecular structures.In order to facilitate the generalization and application,the International Interferon Nomenclature Committee has classified them.Among them,Interferon-β(IFN-β)is an important member of type Ⅰ interferon,and its main role is to participate in the body’s resistance to viruses.Interferon-γ(IFN-γ)is the only type Ⅱ interferon.Its anti-virus effect is slightly weaker than that of type Ⅰ.It can induce the virus-infected cells to express the corresponding antigen of the virus,thereby prompting the immune system to respond to the infected cells.Successfully identify and kill,play a protective role on the body.At present,there are few studies on Panthera tigris altaica interferon,and the functional studies of IFN-β and IFN-γ are still unknown.In addition,after IFN-β and IFN-γ are connected and fused,their combined antiviral effect is still unknown.In this study,a single interferon was used to fuse the Panthera tigris altaica IFN-β and IFN-γ(PtIFN-β,PtIFN-γ)genes by using overlap extension PCR(gene splicing by overlap extension,SOE-PCR)technology.Prokaryotic expression is then carried out,and finally the cell line is used for in vitro biological activity detection,and the strength is compared with a single PtIFN-β and PtIFN-γ recombinant protein.It provides theoretical and material support for obtaining interferon preparations that are easy to produce,have strong activity and have superimposed antiviral activity in the future.It is divided into the following parts:1.Query the published PtIFN-β and PtIFN-γ gene sequences from GenBank,refer to and design primers,use the cDNA extracted from the peripheral blood DNA of Panthera tigris altaica and its lymphocytes as the template,and successfully amplify the signal peptide-free PtIFN-β,PtIFN-γ mature peptide genes.The two parts of PtIFN-β and PtIFN-γ were fused by SOE-PCR method.The three genes were respectively connected to the pET32a(+)vector to construct prokaryotic expression vectors of PtIFN-β,PtIFN-γ and PtIFNβ-γ genes.2.Utilize the Escherichia coli(E.coli)prokaryotic expression system to perform prokaryotic expression of each recombinant plasmid.Nickel affinity chromatography was used for protein purification.In addition,the protein expressed in the inclusion body needs to be renatured,and the gradient dialysis method is used.The final identification uses SDS-PAGE analysis and Western blot analysis.A series of results show that this study successfully induced the expression of the target protein of the corresponding size.3.Use the purified target protein to immunize rabbits by back injection.After three immunizations,the heart draws blood and separates the serum.The indirect ELISA method is used to detect the titers of the three antibodies.The results showed that the antibody titer of PtIFN-β,PtIFN-γ and PtIFNβ-γ in the serum reached 1:102400.4.Detect the antiviral activity of PtIFN-β,PtIFN-γ and PtIFNβ-γ on the VSV/F81,AIV/F81 and CDV/F81 systems using the microcytopathic inhibition method.The results showed that:PtIFN-β,PtIFN-γ and PtIFNβ-γ proteins all have significant anti-VSV,AIV and CDV activities,reaching 103-105 U/mg,and the order from high to low is:PtIFNβ-γ>PtIFN-β>PtIFN-γ.RT-PCR was used to detect the transcription of key genes in JAK-STAT under the effect of recombinant interferon proteins.The expression of antiviral proteins were detected by Western blot.With the use of JAK inhibitors,it was further confirmed that PtIFN-β,PtIFN-γ and PtIFNβ-γ proteins exert antiviral effects through the classical pathway.This study used E.coli to successfully express PtIFN-β,PtIFN-γ and PtIFNβ-γfor the first time,and found that they can inhibit the replication of VSV,AIV and CDV through the JAK-STAT pathway,and PtIFNβ-γ has the strongest effect on inhibiting virus replication.This provides better conditions and methods for the prevention and treatment of Panthera tigris altaica viral diseases,and lays the foundation for the industrial production of Panthera tigris altaica interferon.