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副溶血弧菌外膜蛋白ompK免疫磁珠检测方法的建立

, PP. 209-214

Keywords: 副溶血弧菌,ompK,多克隆抗体,免疫磁珠

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Abstract:

构建重组基因克隆表达副溶血弧菌外膜蛋白ompK并制备多克隆抗体,建立副溶血弧菌的免疫磁珠富集,结合化学显色检测方法。利用生物软件Primer5设计副溶血弧菌ompK基因的引物,通过PCR扩增出ompK基因,并将其克隆至pET28a(+)原核表达载体,转化至大肠杆菌BL21进行优化表达。镍柱纯化表达产物,免疫小鼠,制备其多克隆抗体。Westernblot鉴定重组蛋白,ELISA分析其免疫原性。间接ELISA检测多抗效价及交叉性,并与proteinG磁珠偶联结合显色平板检测副溶血弧菌。结果显示,成功表达ompK蛋白;免疫小鼠获得特异性抗血清,效价为1:64000;Westernblotting证明抗血清能与副溶血弧菌28kD处条带特异性结合。制备的免疫磁珠敏感度最高能达到104CFU/mL。成功克隆表达副溶血弧菌外膜蛋白ompK并制备副溶血弧菌特异性鼠多克隆抗体,建立的免疫磁珠检测方法与显色平板法结合较传统的二次增菌法能够节省72h,整个过程只需要传统方法时间的1/3;相比于常用的免疫学检测方法检测灵敏度提高两个数量级。

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