Expression Of Recombinant Porcine Interferon-γ And Its Activity Against Senecavirua A

Interferon-gamma(IFN-γ)is a cytokine that is capable of multiple roles in adaptive and innate immune regulation and has also been developed as an immunomodulatory drug for clinical use because of its antiviral,antitumor,antiparasitic,and anticancer biological activities.It has been shown that recombinant porcine interferon(rPoIFN-γ)can inhibit the replication of viruses in animals significantly.As a major porcine breeding country in China,Senecavirus A(SVA)can cause blistering disease in acute death syndrome and sows in newborn piglets,with mortality rates as high as 70%-80%after infection,which has threatened the pig breeding industry development seriously.Therefore,it is important to use genetic engineering to develop immune-enhancing drugs and anti-viral with high activity and low cost.In this study,the CHO cell expression system was used to produce rPoIFN-γwith high bioactivity,and SVA was used as a model virus to analyze its ex vivo antiviral activity,providing materials and laying the foundation for the functional study of rPoIFN-γand the development of swine viral disease control agents.In this study,according to the sequence of the porcine IFN-γgene in Gen Bank,His tag and the signal peptide sequence were inserted at both ends of the gene’s open reading frame,CHO codon optimization was performed,and the target gene fragment was sent to the company for synthesis;the pc DNA3.1 vector plasmid and synthesized target fragment were double that was digested and ligated respectively,and the recombinant plasmid was identified by double digestion and sequencing.The recombinant plasmid pc DNA3.1-Po IFN-γwas transfected into suspension-cultured CHO cells,and the supernatant was collected and purified by a nickel ion affinity chromatography column to determine protein expression using SDS-PAGE and Western blot detection techniques.The results showed that the recombinant plasmid pc DNA3.1-Po IFN-γwas successfully constructed by Western blot and SDS-PAGE,and the specific protein bands appeared around 27 k Da.To detect the antiviral activity of purified rPoIFN-γ,the toxic effect of rPoIFN-γon IBRS-2cells was detected by CCK-8 assay.The antiviral potency of rPoIFN-γagainst vesicular stomatitis virus(VSV)was determined by the VSV/PK-15 system using cytopathic inhibition assay(CPEI)to investigate the antiviral effect of rPoIFN-γon SVA.The antiviral effect of rPoIFN-γon SVA was investigated by q PCR,Western-blot and viral titer assay.The rPoIFN-γinduced cytokine expression and ISGs in IBRS-2 cells by q PCR.The results showed that rPoIFN-γhad no toxic effect on IBRS-2 cells.The rPoIFN-γhad an antiviral potency of 5.59×10~7U/mg against VSV on PK-15 cells and an anti-SVA activity potency of 6.87×10~6U/mg.The rPoIFN-γinhibited SVA’s replication significantly in IBRS-2 cells(P<0.01).The expression levels of cytokines and 17ISGs were significantly increased by rPoIFN-γtreatment in IBRS-2 cells(P<0.01).In this study,the rPoIFN-γwas successfully prepared and demonstrated to have low cytotoxicity and high potency of antiviral activity,which provided the functional analysis of rPoIFN-γand the study of antiviral agents research basis and experimental materials.