副溶血性弧菌直接溶血相关毒素蛋白的纯化及性质初探  

A Study on Expression and purification of TDH-related hemolysin gene of Vibrio parahaemolyticus

作　　者：苏建新[1] 聂军[2] 苏桂栋[3] 王珊珊[1] 黄佳亮[1] 曾年华[1] 肖红[1] 王志斌[1] 姜普林[1] 

机构地区：[1]广州军区军事医学研究所流行病研究室,广东广州510507 [2]第一军医大学流行病学教研室 [3]第一军医大学附属南方医院妇产科

出　　处：《华南预防医学》2004年第1期18-20,共3页South China Journal of Preventive Medicine

基　　金：广东省卫生厅医学科研课题 (A2 0 0 0 3 47)

摘　　要：目的　纯化副溶血性弧菌直接溶血相关毒素 (TRH)蛋白 ,鉴定表达产物的生物学活性和免疫原性 ,为构建副溶血性弧菌快速检测方法奠定物质基础。方法　构建trh基因的原核融合表达系统PRⅡ (trh/pGEX 3X/DE3) ,在 31℃条件下 0 6mmol/LIPTG诱导表达。用SDS PAGE分析蛋白表达 ,溶血试验检测表达蛋白的溶血活性。将表达产物皮下注射免疫兔 ,溶血抑制实验检测该抗体体外对TRH溶血活性的抑制作用。结果　PRⅡ在上述条件下诱导后 ,TRH呈可溶性、融合性表达。溶血试验表明 ,表达的蛋白具有溶血活性。用表达的蛋白免疫兔产生的相应抗体 ,在体外具有抑制TRH溶血活性的作用。结论　trh基因在原核融合表达系统pGEX 3X/DE3中获得成功表达 ,并得到纯度较高的TRH蛋白 ,为基因工程大规模制备高纯度TRH抗原、制备抗TRH的多克隆和 (或 )单克隆抗体、构建基因工程菌苗。Objective In order to obtain highly pure protein of thermost direct related hemolysin (TRH) in Vibrio parahaemolyticus and identify the bioactivity and immunogenicity of the product for sake of facilitating rapid test of Vibrio parahaemolyticus. Methods Recombinant bacterial PRⅡ(trh/pGEX 3X/DE3) was induced by 0 6 mmol/L IPTG at the temperature of 31℃. Expressed protein was analyzed with SDS PAGE. Hemolysin test was performed to determine the hemolysis activity. Rabbits were immunized with the protein and anti agglutination test was used to detect the polyclonal antibodies. The hemolytic suppression test was used to detect suppressive effect of the antibodies on hemolytic activity to TRH in vitro. Results Induced by PRⅡ, the TRH protein had hemolytic activity. The related antibody from immunized rabbits could suppress the hemolytic activity of TRH in vitro. Conclusion TRH gene had been successfully expressed and pure protein was obtained through pGEX 3X/DE3. This paid the preliminary foundation for preparing anti TRH poly/monoclonal antibodies for diagnosis, constructing the vaccine candidate and elucidating pathogenesity of Vibrio parahaemolyticus.

关 键 词：弧菌 副溶血陛 直接溶血相关毒素 

分 类 号：Q939.1[生物学—微生物学]