猪胸膜肺炎放线杆菌毒素蛋白的表达、纯化及其间接ELISA检测方法的建立与应用  

Optimal Expression Condition and Purification of APP ApxⅡ Protein and Development of an App-ELISA

作　　者：梁望旺[1] 何启盖[1] 刘正飞[1] 陈焕春[1] 吴斌[1] 徐晓娟[1] 李涛[1] 

机构地区：[1]华中农业大学畜牧兽医学院,湖北武汉430070

出　　处：《中国兽医学报》2005年第2期145-147,共3页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30200011);技攻关项目(2001AA201B02);湖北省自然科学基金资助项目

摘　　要：将含有猪胸膜肺炎放线杆菌毒素 基因的质粒 p ET- Apx 转化到大肠杆菌 BL2 1 (DE3)。挑选出表达量最高的克隆子 ,于 37℃经 IPTG诱导表达。对表达条件进行优化 ,并对包涵体进行了提纯和复性。用复性后的蛋白作抗原 ,建立了检测猪传染性胸膜肺炎放线杆菌抗体的间接 EL ISA。试验的最佳反应条件为 :最佳抗原稀释度为 1∶ 1 6 0 ,抗原包被液用 Tris- HCl(p H8.0 ) ,封闭液用含 0 .4 %BSA的 PBST,血清稀释度为 1∶ 4 0 ,二抗稀释度为 1∶ 6 0 0 0 ,稀释液用含0 .4 %BSA的 PBST,血清反应时间为 30 min,二抗反应时间为 4 5 min,底物反应时间为 2 0 m in。与间接血凝 (IHA)检测结果比较 ,建立的 EL ISA诊断方法具有良好的稳定性和可重复性 ,并具有较高的特异性和敏感性。用建立的间接EL ISA对 2 5 0 3份临床送检血清进行了血清流行病学调查 ,结果表明 ,胸膜肺炎放线杆菌抗体的阳性率为 6 3%。The expression plasmid pET-ApxⅡ were tansformed into E.coli BL21(DE3).Clones of higher expression were selected,then grown in the presence of IPTG at 37℃ to induce expression.The conditions of expression were optimized and the inclusion bodies were purified and renatured.Using the renatured protein as antigen we developed an indirect enzyme-linked immunosorbent assay(ELISA) for detecting Actinobacillus pleuropneumoniae antibody.The test conditions were optimized by reagent titration utilizing known positive and negative sera.A positive/negative(P/N) value larger than 2.1 was determined as comprehensive positive standard for the ELISA.The performance of the ELISA was assessed for reproducibility and specificity.The results showed that the ELISA had good reliability,speciality and sensitivity for clinic serum samples.

关 键 词：猪 胸膜肺炎放线杆菌 毒素 基因表达 基因纯化 间接ELISA 

分 类 号：S858.28[农业科学—临床兽医学] S852.619[农业科学—兽医学]