丝状噬菌体pⅧ展示系统表达赭曲霉毒素A模拟表位及应用研究  

Expression and application research of ochratoxin A mimotope by filamentous phage pⅧ display system

作　　者：陈兴龙[1] 徐玲[1] 刘仁荣[1] 裘雪梅[1] 朱立鑫[1] 

机构地区：[1]江西科技师范大学生命科学院,南昌330013

出　　处：《卫生研究》2012年第5期814-818,共5页Journal of Hygiene Research

基　　金：国家自然科学基金项目(No.30860240)

摘　　要：目的传统赭曲霉毒素A(OTA)竞争抗原毒性强、成本高、制备难度大。将OTA模拟表位展示于丝状噬菌体pⅧ表面,获得无毒、易于制备的OTA竞争抗原替代物。方法构建带有OTA模拟表位的重组噬菌粒pC89-COTA,为了提高OTA模拟表位与抗体的结合率,在OTA模拟表位序列5'端引入了肠激酶酶切位点。噬菌粒转化进大肠杆菌XL1-blue后再经野生KM13噬菌体感染,获得带有OTA模拟表位的重组噬菌体。用该噬菌体代替竞争抗原建立OTA的无毒ELISA分析方法。结果成功表达出带有OTA模拟表位的重组噬菌体,经肠激酶酶切的OTA模拟表位噬菌体与OTA抗体的结合效率显著高于未酶切的噬菌体。利用酶切后的噬菌体建立OTA的无毒ELISA分析方法,其最低检出限为100pg/ml,线性范围为250pg/ml～1000pg/ml。进行加标回收实验,回收率为99.8%～112.3%,相对标准偏差为8.19%～11.64%,测试16份市售样品,阳性率为31.25%。结论成功表达了OTA模拟表位噬菌体,建立了OTA的无毒ELISA检测方法。Objective Traditional ochratoxin A （OTA） competitive antigen are high toxicity, high price and difficult preparation. Non-toxic and easy prepared OTA competitive antigen substitutes were expressed by recombinant filamentous phage which have OTA mimotope displayed on its p VII surface. Methods Recombinant phagemid pC89-COTA which contain OTA mimotope nucleotide sequence was constructed. Moreover, to increase the binding rate of OTA mimotope with antibody, Enterokinase cleavage sits were led into 5＇ terminal of OTA mimotope nucleotide sequence. The pC89-COTA was transformed into E. coli XLl-blue. The E. coli XLl-blue were infected by wild KM13 phage and recombinant phage with OTA mimotope displayed generated. Results OTA mimotope phage was successful expressed and OTA mimotope phage which digested by Enterokinase had significantly higher binding rate with antibody than phage which not digested by Enterokinase. Non-toxic OTA competitive ELISA was established by using this digested OTA mimotope phage, the detecting limitation was 100pg/ml, the linear range of theinhibition curves was between 250pg/ml and 1000pg/ml. Spiked recoveries of the farina tritici blank samples, the recovery rate of OTA were 99.8% - 112. 3% and coefficients of variation were 8. 19% - 11.64% , then 16 commercially available samples were tested and the positive rate was 31.25%. Conclusion OTA mimotope phage were successfully expressed and non-toxic OTA competitive ELISA was established.

关 键 词：噬菌体展示 噬菌粒 赭曲霉毒素A 酶联免疫吸附测定 

分 类 号：TS207.4[轻工技术与工程—食品科学] R155.31[轻工技术与工程—食品科学与工程]