Expression of porcine interferon-γ and preparation of rabbit antisera against porcine interforn-γ

The total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of a healthy piglet.And porcine interferon-γ (IFN-γ) gene cDNA was amplified by RT-PCR using a pair of primers designed according to the published porcine IFN-γ gene sequence.The PCR product was cloned into pMD18-T vector,subcloned into an expression vector pET-30a.The sequencing results showed the IFN-γ sequence was almost the same as the reported porcine IFN-γ sequence in GenBank.The recombinant plasmid was transformed into E.coli,and then induced by IPTG at 37 ℃.The recombinant porcine IFN-γ was expressed efficiently in form of inclusion body with the yield accounting for 39 % of total bacterial proteins.SDS-PAGE and Western blotee analyses showed that the recombinant fusion protein had a molecular weight of approximately 25 ku.The inclusion body was denatured by 8mol/L urea,purified by ProBond~(TM) Purification Systerm,and then refolded by dialysis against water.Antisera against porcine IFN-γ was generated by immunizing rabbits with the purified protein.The expression and purification of the recombinant porcine IFN-γ fusion protein and preparation of the antisera against porcine IFN-γ will lay a foundation for the future application of porcine IFN-γ.