作　　者： ; ; ; ;

机构地区： 暨南大学

出　　处： 《生物技术通报》 2013年第6期209-214,共6页

摘　　要： 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Primer 5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。Western blot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protein G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∶64 000;Western blotting证明抗血清能与副溶血弧菌28 kD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72 h,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。 It was to construct recombinant gene cloning and express the membrane surface protein outer the membrane protein K（ompK） of Vibrio parahaemolyticus,developing a method to detect Vibrio parahaemolyticus via immunomagnetic separation combined with color plate method.Using Primer 5 to design primers of ompK gene,the ompK gene was amplified by PCR subsequently and cloned into pET28a（＋） prokaryotic expression vector,optimally transferred into E.coli BL21 to express.The expression products were purified by Nickel column and immunized mouse to prepare polyclonal antibody.Western blotting analysis was applied to identify the recombinant protein and ELISA was used to analyze its immunogenicity.The titer and cross reactivity of the polyclonal antibody were determined using indirect ELISA,then the polyclonal antibodies we prepared were coupled with protein G immunomagnetic beads to develop a chromogenic plating detection of Vibrio parahaemolyticus.The sensitivity of immunomagnetic beads reached to 104CFU/mL.We managed to clone and express Vibrio parahaemolyticus outer membrane protein ompK,in addition,we prepared mouse anti-Vibrio parahaemolyticuspolyclonal antibody to create a new detection method using immunomagnetic beads,this method which combined with color plate method could save 72 hours comparing with traditional enrichment method,all process only need one-third of the time of traditional methods.When compared to the common immune detection methods,it was two magnitudes more sensitive.

关 键 词： 副溶血弧菌 多克隆抗体 免疫磁珠

领　　域： [农业科学] [农业科学] [农业科学]