Soluble Expression and Activity Analysis of Apoptin Fusion Protein

Objective:To construct the recombinant expression vector of pET-28a-SPA and to soluble expression highly in cytoplasmic fraction in E.coli BL21(DE3),examine the cytotoxicity of the purified products against the cancer cells.Methods:Recombinant expression plasmid pET-28a-SPA was constructed according to recombinant plasmid.The correct recombinant expression plasmid was transformed into the host strain BL21(DE3) induced by IPTG.The specific protein expressed was detected by SDS-PAGE and Western blot,the cytotoxic ability of the purified products were examined by MTT.Results: The specific protein expressed was detected by SDS-PAGE.The fusion protein was expressed at high level amounting to 20% of the total bacterial protein analyzed by computer software.The amount in supernatant is about 10%.After purified the supernatant protein,Western blot showed the monoclonal antibody raised against the recombinant Apoptin in murines could react to the protein expressed specifically,and exerted cytotoxic effect on A549 and Hela cancer cells.Conclusions: The fusion protein was expressed highly in cytoplasmic fraction in BL21(DE3),and as soluble form in E.coli BL21(DE3).Supporting that it would be a promising candidate for tumor targeted immunotherapy.