Cloning and prokaryotic expression of Guangxi swamp type buffalo interferon-γ gene

Interferon-γ(IFN-γ) gene of Guangxi swamp type buffalo was clone and prokaryotic expressing plasmid containing buffalo IFN-γ gene was constructed.Total RNA was extracted from peripheral blood mononuclear cells(PBMCs) of healthy swamp type buffalo stimulated with Concanavalin A(ConA) for 13 hours.The cDNA of buffalo IFN-γ gene was amplified by RT-PCR using a pair of primers designed based on the published bovine IFN-γ gene sequence.The PCR products were cloned into vector pMD18-T.A 457 bp fragment of RT-PCR products was revealed by agrose gel electrophoresis.Sequence coding for mature buffalo IFN-γ protein was subcloned into an expression vector pET32a+.Successful inserting of the 457 bp fragment into the vector pET32a+ was confirmed by PCR,endonuclease digestion and sequencing.Positive recombinant clones were identified by restriction enzyme digestion and sequencing.The recombinant buffalo IFN-γ gene was highly expressed in Escherichia coli(43.6% of total bacterial proteins) after induced by Isopropylthio-β-D-galactoside(IPTG) and a remarkable 35 ku recombinant fusion protein was revealed by SDS-PAGE.