Expression,purification and bioactivity analysis of porcine interferon-gamma gene

A pair of primer were designed according to the sequence of porcine IFN-γ,then the gene was cloned into pET-30 a prokaryotic expression vector,recombinant expression vector pET-30 a-pIFN-γ was constructed.The recombinant plasmid was transformed into E.coli BL21(DE3) and then identified by PCR,double enzyme digesting and sequencing,and induced by IPTG.The soluble expressed product was purified through Nickel-affinity chromatography column,the protein was further purified by Sephadex-G100.The inclusion body was washed by DOC,then dissolved with SKL and subsequently renatured by dialysis,and the expressed protein was analyzed by SDS-PAGE and Western-blot.It was confirmed that the highly purified pET-30a-IFN-γ was harvested.Antiviral effect of the purified product was tested by cytopathogenic effect inhibition assay,the results indicated that the purified product had a higher activity of interferencing virus replication,the activity of IFN-γ against PRV was about 2.0×103 U·mg-1,the activity of IFN-γ against FMDV serotype O was about 2.56×105 U·mg-1.