Prokaryotic expression of IFN-γ gene from rabbit and preparation of the monoclonal antibodies against the recombinant rabbit IFN-γ

The IFN-γgene was amplified by PCR from rabbit and cloned into pET-28a(+)to generate the recombinant pET-28a-IFN-γplasmid.The recombinant plasmid was then transformed into Escherichia coli BL21(DE3).The recombinant IFN-γprotein was expressed by inducing with IPTG and purified with HisTrap affinity chromatography.With the fusion of the spleen cells from the recombinant protein immunized BALB/c mice and mouse myeloma cells(SP2/0),four hybridoma cell lines were prepared for secreting monoclonal antibodies(McAbs)against IFN-γ.The McAb was named as 3H8,4G4,4F10 and 6C12,and belongs to the subtype of IgG1-κ,IgG2b-κ,IgG1-κand IgG1-κ,respectively.The antibody titers of the ascites produced against McAbs were 10-4 to 10-5 by ELISA measurement.In the present study,the rabbit anti-IFN-γmonoclonal antibodies were prepared and thus provides an important means for the study and detection of IFN-γ.