RESUMEN
Objective To determine the effect of Vibrio vulnificus cytolysin (VVC) inducing ap-optosis in human umbilical vein endothelial cell(HUVEC) and its possible mechanism. Methods The en-tire vvhA gene that encoding VVC from V. vulnificus strain GTC333 was amplified by PCR and sequenced af-ter T-A cloning. E. coli BL21DE3pET-42a-vvhA, a prokaryotic expression system of the vvhA gene, was then con-structed. Ni-NTA affinity chromatography was applied to purify the target recombinant protein rVVC, and SDS-PAGE plus Bio-Rad Agarose Image Analyzor were used to measure the output of rVVC and to determine the purity of rVVC extract. The activity of rVVC dissolving rabbit erythrocytes was detected by hemolysis test. DPNH chromotometry and TphBNa chromotometry were performed to examine the contents of LDH and K+ in the supernatants of rVVC-treated HUVEC cultures, respectively. The effect of rVVC inducing apepto-sis of HUVEC was detected by flow cytometry, rVVC was labeled with FITC and the location of FITC-labe-ling rVVC in HUVEC was observed by laser canfocal microscopy. Results The cloned whA gene had 96.09% and 98.26% similarities of nucleotide and amino acid sequences compared to the corresponding se-quences in GenBank. rVVC, with a dosage of 1 μg/ml, could dissolve rabbit erythrocytes (P<0.01). 10 μg/ml rVVC was able to promote the increases of K+ content (P<0.01) but no change of LDH content could be found in the cell supernatants. HUVEC was apoptotic after the cell was treated with 1～100 μg/ml of rVVC for 2 h. In the 5～240 min duration of co-incubation of FITC-labeling rVVC and HUVEC, the rV-VC gradually moved from surface to inner side of the membrane and then entered the cytoplasms. When FITC-labeling rVVC treated HUVEC for 30 min, most of the rVVC was found to be intracellular location. Conclusion rVVC has cytolytic activity. VVC has an ability to enter HUVEC and causes injury of HUVEC via inducing apoptosis, which may be the major pathogenic mechanism of VVC.