| 摘要: |
| [目的]分离纯化基因工程重组蛋白金黄色葡萄球菌溶血素(α -hemolysin,α -HL)并检测其生物学活性,为疫苗研发奠定基础.[方法]对含pET32a+-α-HL质粒的BL21(DE3)进行诱导表达,诱导成功后,将重组菌BL21(DE3)培养物用超声波裂解,并用凝胶过滤层析法对该基因工程蛋白进行分离纯化,用Bradford法及兔红细胞分别测定纯化产物的蛋白含量和溶血比活.[结果]纯化产物在SDS-PAGE电泳中53 ku处呈现出单一清晰带,达电泳级纯度.纯化产物含量约为0.127 7 mg/mL,溶血比活为8 192 HU/mg.[结论]成功获得了金黄色葡萄球菌α-HL,且所获的α-HL具有良好的溶血活性. |
| 关键词: α-溶血素 金黄色葡萄球菌 溶血活性 基因工程蛋白 原核表达 |
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| 基金项目:山东省农业科学院高技术自主创新基金，山东省农业科学院重大成果培育基金 |
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| Purification and hemolytyic activity detection of Staphylococcus aureus α-hemolysin from gene expression product |
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| Abstract: |
| 【Objective】 The study was to purify the Staphylococcus aureus α-hemolysin（α-HL）which was expressed in the host strain BL21(DE3) as a genetic engineering recombinant protein,and then its biologic activity was analyzed to establish foundation for the vaccine research prospect.【Method】 The BL21(DE3) containing the plasmid pET32a+α-HL with IPTG was induced to made sure that the interest protein was expressin.Then the BL21(DE3) culture solution under ultrasonic was exposed,and the purification of α-hemolysin was achieved by gel filtration chromatography(GFC).The purified product was analyzed with SDS polyacrylamide gel electropheresis,and then subjected to the analysis with hemolytic tested on rabbit erythrocyte to detect the median hemolytic dose potency(HD50).Protein concentrations were determined by the method of Bradford.【Result】 There was an expected protein band with molecular mass of 53 ku by SDS PAGE.The protein level was 0.127 7 mg/mL,and hemolysis specific activity was 8 192 HU/mg. 【Conclusion】 The Staphylococcus aureus α-HL was obtained successfully,and it shows good hemolytic activity. |
| Key words: α-hemolysin Staphylococcus aureus hemolytic activity engineered protein prokaryotic expression |
