Study on VVC recombinant protein cytolytic activity mechanism of an extracellular cytolytic toxin of Vibrio vulnificus / 中华微生物学和免疫学杂志

ABSTRACT

Objective To investigate the cytolytic activity of extracellular cytolytic toxin rVVC of Vibrio vulnificus on the apoptosis of human ECV304 cells, and to analyze the activities of Caspase-3,-8 and -9. Methods The cytotoxic effect of refolded rVVC on the growth and apoptosis of ECV304 cells was identified by MTT, Hochest33342/PI fluorescent staining, flow cytometry and DNA agarose electrophoresis analysis, respectively. The activities of Caspase-3, -8 and -9 was measured using a colorimetric method. Results The viability of human ECV304 cells exposed to rVVC was inhibited by rVVC after 24 h. 2.0 HU/ml rVVC groups had a better cytotoxic effect to human ECV304 than that of 0.5 HU /ml rVVC groups. The apoptosis of human ECV304 cells in 2.0 HU/ml rVVC+40 μmol/L Z-VAD-FMK groups was relative reduced than that of 2.0 HU/ml of rVVC groups. After 0.5 h treatment with 2.0 HU/ml of rVVC, the Caspase-3 activity in human ECV304 cells increased gradually and reached the peak at 3 h (versus control groups, P<0.01). The activity of Caspase-8 and -9 remained unchanging. Conclusion The rVVC has cytotoxic effect on human ECV304 and the cytolysin is probably correlated with Caspase-3.