Cloning and expression of new apoptin fusion protein and its bioactivity.

Objective To clone and express apoptin-EC-SOD3 fusion protein and determine its bioactivity in vitro.Methods Apoptin gene was amplified by PCR and cloned into prokaryotic expression vector EC-SOD3-pET-28a.The constructed recombinant plasmid pET-28a-EC-SOD3-apoptin was transformed to E.coli BL21(DE3) for the expression under the induction of IPTG.The expressed protein was purified by Ni2+-NTA affinity chromatography and its activity was identified by MTT.Results PCR analysis proved that the recombinant plasmid pET-28a-EC-SOD3-apoptin was correctly constructed.The expressed soluble fusion protein was consistent with that expected.Its purity was over 85 % after being purified with Ni2+-NTA agarose and the purified protein showed bioactivity by MTT assay.Conclusion The recombinant plasmid for prokaryotic expression of pET-28a-EC-SOD3-apoptin can be constructed and the apoptin-EC-SOD3 fusion protein can be expressed successfully in E.coli BL21(DE3).MTT result indicates that HeLa cells are highly sensitive to the purified apoptin-EC-SOD3.