创伤弧菌溶细胞素在大肠杆菌中的表达及其对应激因子的调控  

Expression of fusion protein VVC in E.coli and it's regulatory effect on the stressors

作　　者：李桂军[1] 楼永良[1] 桂静[1] 肖美英[1] 

机构地区：[1]温州医学院检验医学院,硕士研究生浙江温州325000

出　　处：《检验医学教育》2008年第4期39-44,共6页

基　　金：浙江省自然科学基金(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SMMC772应激因子基因表达情况。方法:构建pET28a(+)-vvhA表达载体,对包涵体进行三步洗涤后,用金属亲和层析(6×His Tag)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、HSP90 mRNA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. coil, to detect the haemolysis activity, and to observe it＇s regulatory effect on the stressors of SMMC7721 ceils. Methods：The expression plasmid pET28a （＋）-vvhA was constructed, and inclusion body was washed by three-step washings. The recombinant protein, rVVC, was purified by Ni affinity chromatography and it＇s 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 ceils stimulated by rVVC.

关 键 词：创伤弧菌溶细胞素 大肠杆菌表达 溶血活性 应激因子 

分 类 号：R394.8[医药卫生—医学遗传学] R378.3[医药卫生—基础医学]