葡萄球菌A型肠毒素检测竞争ELISA方法的建立  

An Indirect competitive ELISA method for quantitative analysis of Staphylococcal Enterotoxin A

作　　者：徐丹丹[1] 黄金海[1] 刘莹[1] 孙跃辉[1] 周凤娟[1] 

机构地区：[1]天津大学农业与生物工程学院,天津300072

出　　处：《食品工业科技》2010年第10期380-382,385,共4页Science and Technology of Food Industry

基　　金：天津市应用基础及前沿技术研究计划项目(08JCZDJC22600)

摘　　要：为建立快速、灵敏、特异的检测食品中金黄色葡萄球菌A型肠毒素的方法,应用构建的pGEX-6P-SEA、pET28α-SEA重组质粒表达带有不同融合肽的葡萄球菌A型肠毒素,分别通过谷胱甘肽亲和纯化、镍螯合纯化获得SEA的重组融合表达蛋白HIS-SEA、GST-SEA,以HIS-SEA为包被抗原,以GST-SEA、天然SEA肠毒素为竞争抗原,确立了SEA型肠毒素ELISA检测的工作条件:包被浓度为2.5μg/mL,阳性血清稀释度为1∶50,辣根过氧化物酶标记的羊抗鸡免疫球蛋白工作浓度为1∶2000。建立了以重组肠毒素GST-SEA作为竞争抗原检测葡萄球菌肠毒素SEA的间接竞争ELISA(ciELISA)方法,竞争肠毒素GST-SEA蛋白浓度x与P/N值y的关系式:x(GST-SEA)=1.99-y/0.34,R2(GST-SEA)=0.9964,线性检测范围均为1～62.5ng/mL,最低检测量为1ng/mL,此方法丰富了食品中A型肠毒素的检测手段。In order to establish a （SEA）, the recombinant prote rapid, sensitive and specific detection method of Staphylococcal enterotoxins A n GST-SEA and HIS- SEA were expressed in Escherichia coll. Using two recombinant plasmids（ pET28α-sea, pEGX-6p-sea）and purled by affinity purification or metal immobilization affinity chromatography. Indirect competitive ELISA method was established and the optimal coating antigen concentration of HIS-SEA was 2μg/mL,the blocking solution was 5% skim milk,the dilution of serum sample was 1：50.The relationship between the concentration of competition antigen X（GST-SEA） and P/N y was XGST-SEA = 1.99-y/ 0.34,R^2（GST-SEA） =0.9964.The detection concentration range of 1-62.Sng/mL and the detection limit was 1 ng/mL The method riched the detection means of SEA.

关 键 词：葡萄球菌A型肠毒素(SEA) 重组蛋白 间接竞争酶联免疫吸附反应(ciELISA) 

分 类 号：Q814.9[生物学—生物工程]