重组创伤弧菌溶细胞素缬氨酸V201和V289突变对其活性的影响

将重组创伤弧菌溶细胞素A（recombinant Vibrio vulnificus hemolysin，rVvhA）第201位和第289位的缬氨酸定点突变为甘氨酸，并表达rVvhAval201gly,val289gly突变蛋白。检测突变蛋白与未突变蛋白的溶血活性、对胞内钙离子浓度、钾离子外流及对细胞损伤的影响。结果显示，与rVvhAval201gly,val289gly的溶血活性和细胞毒性均降低，诱导人类脐静脉内皮细胞（human umbilical veinendothelial cells，HUVEC）凋亡、胞内钙离子内流和钾离子外流等作用均受到抑制。实验结果表明，在创伤弧菌溶细胞素的活性发挥中，rVvhA的V201和V289两个氨基酸与该蛋白质损伤靶细胞时引起胞内外离子平衡失调有关，并能影响该蛋白质的溶血活性和细胞毒作用。

The Mutation of Valine201 and Valine289 Influences the Activity of Recombiant Vibrio vulnificus Hemolysin

To compare the activity between rVvhA and rVvhAval201gly,val289gly, Val201 and Va1289 were mutated to glycines and recombinant proteins were expressed after mutations. The hemolytic activity, cytotoxic, intracellular calcium influx and potassium ion release between rVvhA and rVvhAval201gly,val289gly were compared. The results showed that the hemolysis of rVvhAval201gly,val289gly was reduced. Cell death was partly rescued by blocking calcium influx and potassium ion release. In conclusion, the mutations of Val201 and Val289 are relevent with the cells ions balance, hemolysis and cytotoxicity of rVvhA.