创伤弧菌溶细胞素vvhA基因在大肠杆菌中的表达及其对应激因子的调控  

Expression of fusion protein WC in E. coli and its regulatory effect on the stressors

作　　者：李桂军[1] 桂静[1] 肖美英[1] 楼永良[1] 

机构地区：[1]温州医学院检验医学院,325035

出　　处：《中华微生物学和免疫学杂志》2008年第1期24-28,共5页Chinese Journal of Microbiology and Immunology

基　　金：浙江省自然科学基金项目（X205004）

摘　　要：目的用大肠杆菌表达系统表达创伤弧菌溶细胞素，对其溶血活性进行评价。并观察其作用肝癌细胞SMMC7721后，调控肝癌细胞SMMC7721应激因子基因表达情况。方法构建pET28a（＋）-whA表达载体，对包涵体进行三步洗涤后，用金属亲和层析（6×HisTag）纯化重组创伤弧菌溶细胞素（rVVC），并用溶血试验验证重组蛋白活性。复性重组蛋白作用肝癌细胞SMMC7721后，RT-PCR方法检验SMMC7721细胞肿瘤坏死因子α（TNF-α）、热休克蛋白90（HSP90）基因分泌调节情况。结果用基因工程的方法成功获得高表达、高纯度（纯度≥96％）rVVC。兔红细胞溶血试验检测表明，rVVC具有溶血活性，其活性为0．2μg／HU（溶血单位）。RT-PCR结果显示，rVVC能明显诱导肝癌细胞SMMC7721的TNF-α、HSP90 mRNA表达能力，但具有时间、剂量依赖性。结论表达、纯化并复性的rVVC能明显诱导肝癌细胞SMMC7721的TNF-α、HSP90mRNA表达。提示rVVC在组织细胞损伤过程中发挥了应激作用。Objective To express Vibrio vulnificus cytolysin in E. coli, to detect the haemolysis activity, and to observe its regulatory effect on the stressors of SMMC7721 cells. Methods The expression plasmid pET28a（ ＋ ）-whA was constructed, and inclusion body was washed by three-step washings. The recombinant protein, rVVC, was purified by Ni affinity chromatography and its activity was verified by hemolysis test. Gene expression of tumor necrosis factor-α （ TNF-α ） and heat shock protein 90 （HSP90） from SMMC7721 cells stimulated by rVVC were tested by RT-PCR. Results rVVC was richly expressed and successfully purified with haemolysis activity. The rVVC can induce SMMC7721 cells to up-regulate the expression of TNF-α and HSP90 in dose- and time-dependent manner. Conclusion rVVC was expressed, purified and refolded successfully. It can induce SMMC7721 cells to up-regulate the expression of TNF-α and HSP90 mRNA. Stress effect aggravates the injury of SMMC7721 cells stimulated by rVVC.

关 键 词：创伤弧菌溶细胞素 E.coli表达 溶血活性 应激因子 

分 类 号：R686[医药卫生—骨科学]