创伤弧菌天然溶血素的制备及其活性研究

Preparation and Activity Study of Natural Hemolysin from Vibrio Vulnificus

创伤弧菌溶血素(V.Vulnificus Hemolysin, VVH)是创伤弧菌重要的致病因子之一,但目前国内外重组表达的VVH多为包涵体形式.本研究建立了创伤弧菌培养上清中分泌型溶血素的纯化制备方法,并评价了胆固醇及雌二醇对天然VVH溶血活性的影响,为进一步深入研究VVH蛋白的生物学功能及致病机制奠定了基础.研究采用硫酸铵沉淀法结合脱盐柱、阴离子层析及疏水相互作用纯化制备出天然VVH,用溶血试验验证了蛋白活性,评价了胆固醇、雌二醇对创伤弧菌YJ016培养上清以及天然VVH溶血活性的影响.结果显示:从创伤弧菌培养上清中纯化制备的天然VVH纯度较高,其具有良好的溶血活性(2μg/HU),100μg/mL的胆固醇或雌二醇均能有效抑制VVH的溶血活性.本研究成功建立了制备天然VVH的有效方法,为创伤弧菌重症感染的致病机制、防控与治疗研究奠定了基础,同时对其他致病弧菌溶血素的制备也具有借鉴意义.

Vibrio Vulnificus Hemolysin(VVH)is one of the important pathogenic factors of Vibrio vulnificus,but currently,recombinantly expressed VVH in both domestic and international studies often exists in the form of inclusion bodies.This study established a purification preparation method for secreted hemolysin in Vibrio vulnificus culture supernatant,and evaluated the effects of cholesterol and estradiol on the hemolytic activity of natural VVH,which lays a foundation for further in-depth study of the biological functions and pathogenesis of VVH protein.The study used ammonium sulfate precipitation combined with desalting column,anion chromatography,and hydrophobic interaction to prepare natural VVH,and the protein activity was verified by hemolysis experiments.The effects of cholesterol and estradiol on the hemolytic activity of Vibrio vulnificus YJ016 culture supernatant and natural VVH were evaluated.The results show that the natural VVH purified from the Vibrio vulnificus culture supernatant is of high purity and has good hemolytic activity(2μg/HU).Cholesterol or estradiol at 100μg/mL can effectively inhibit the hemolytic activity of VVH.This study successfully established an effective method for preparing natural VVH,which lays a foundation for research on the pathogenesis,prevention,and treatment of severe infections of Vibrio vulnificus,and also has implications for the preparation of hemolysins from other pathogenic Vibrios.