创伤弧菌溶细胞素融合蛋白重组、表达与细胞毒活性鉴定

Recombination Expression and Cytolytic Bioactivity of vvhA Fusion Protein:Cytolysin of V.vulnificus

研究重组创伤弧菌溶细胞素融合蛋白(rVvhA)诱导人脐静脉内皮细胞(human umbili-cal vein endothelial cells,human ECV304)凋亡的作用。诱导含pET-28a(+)vvhA重组质粒的BL21大肠杆菌表达创伤弧菌溶细胞素融合蛋白,应用Ni2+亲和层析方法对rVvhA进行纯化;利用分步稀释加透析相结合的方法进行蛋白质复性;绵羊红细胞裂解试验对复性后的rVvhA进行溶血活性初步测定;MTT法检测rVvhA对人ECV304细胞的体外毒性作用;应用Hochest33342/PI活细胞荧光双染及流式细胞术分析rVvhA对人ECV304细胞诱导凋亡的影响。结果显示,用Ni2+-NTAHisBand亲和层析柱纯化rVvhA纯度达88.6%左右;复性后的rVvhA有一定的溶血活性,其溶血活性具有时间-剂量依赖性;MTT结果显示rVvhA具有降低人ECV304细胞的存活率活性;浓度为2.0HU/mlrVvhA作用人ECV30412h后,其诱导凋亡的活性高于对照组和浓度为0.5HU/mlrVvhA处理组,具有剂量依赖性;浓度为2.0HU/mlrVvhA处理组加用40μmol/Lcaspase全酶抑制剂(Z-VAD-FMK)后凋亡率较2.0HU/mlrVvhA处理组有一定程度降低。rVvhA对人ECV304细胞具有诱导凋亡的生物学活性,推测诱导调亡途径可能与caspase家族有关。

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 dosedependent 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.