Construction of Recombinant PA28γ Plasmid and Expression and Preparation of Polyclonal Antiserum

Objective To express PA28γ antigen as a GST-PA28γ fusion protein and prepared anti-PA28γ antiserum. Methods The PA28γ gene was cloned into the vector pGEX-4T-1. The recombinant plasmid was transferred into E.coli BL-21-STAR(DE3) and the GST-PA28γ fusion protein was expressed by inducing with IPTG. The fusion protein was purified by glutathione sepharose 4B affinity chromatography. BALB/c mice were immunized with recombinant PA28γ,and western blot was used to detect the expressed protein and polyclonal antiserum. Results DNA sequencing confirmed the PA28γ sequence was correct. Expressed fusion protein was mainly in soluble supernatant and accounted for about 15% of the total bacterial proteins. Western blot results demonstrated the recombinant GST-PA28γ was antigenic and could induce antibody production in mice. Conclusion The successful preparation of PA28γ protein and anti-PA28γ polyclonal antiserum facilitates further studies on the structure,functions and protein-protein interaction of PA28γ.