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Recombination Expression and Cytolytic Bioactivity of vvhA Fusion Protein : Cytolysin of V.vulnificus
Jing Gui, Mei-Ying Xiao, Yong-Liang Lou*, Die Hu, Jie Yan1, Ye-Jing Zhu
Department of microecology, Key Research Laboratory for Medical Genetics of Zhejiang, Wenzhou Medical College, Wenzhou 325035, China; 1Department of Medical Microbiology and Parasitology, College of Medicine, Zhejiang University, Hangzhou 31003
Abstract: The aim of the study is to investigate cytolytic bioactivity of vvhA fusion protein (rVvhA): cytolysin of V.vulnificus on the apoptosis of human ECV304 cells. rVvhA was expressed highly in E.coli BL21(DE3) bearing recombinant plasmid pET-28a(+)vvhA induced by 0.5 mmol/L IPTG. The expressed product was denatured and then purified by metal affinity chromatography using Ni2+-NTA resin. Finally， rVvhA was refolded using dilution and step dialysis. The bioactivity of rVvhA was confirmed by cytolysis against sheep erythrocytes and evaluated by MTT， Hoechst33342/PI fluorescent staining and flow cytometry. The results showed that rVvhA was purified to a purity of 88.6%. The fusion protein had a cytotoxic effect against sheep erythrocytes in time- and dose- dependent manner and reduced the viability of human ECV304 cells in a dose-dependent manner; The caspase pan inhibitor， Z-VAD-FMK， can partially inhibit rVvhA induced apoptosis. rVvhA has cytotoxic effect on human ECV304 cells and this process is probably correlated with the activation of caspase.
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