脱氧雪腐镰刀菌烯醇模拟表位的2种融合蛋白的表达及其在无毒酶联免疫吸附方法中的应用  

Application in Non-Toxic ELISA of Expression of Deoxynivalenol Mimotope Fusion Protein

作　　者：徐富勇[1,2] 孟玮[1] 刘仁荣[1] 徐玲[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学院,江西南昌330013 [2]南昌大学生命科学与食品工程学院,江西南昌330029

出　　处：《食品科学》2014年第8期198-203,共6页Food Science

基　　金：江西省自然科学基金项目(20122BAB214006);江西省教育厅科技项目(GJJ13573)

摘　　要：脱氧雪腐镰刀菌烯醇（deoxynivalenol,DON）的模拟表位（CDON）,是从噬菌体展示随机肽库中淘选出来的七肽,可模拟DON与抗DON抗体结合.为了在酶联免疫吸附方法（enzyme linked immunosorbent assay,ELISA）中用重组蛋白替代DON偶联物,以期开发出能代替偶联DON人工抗原的无毒检测DON的ELISA方法,通过构建重组表达载体pGEX-CDON和pC89S4-CDON,并在大肠杆菌表达系统中分别表达和纯化GST-CDON和pⅧ-CDON融合蛋白,比较测定2种融合蛋白与抗DON抗体的结合效果.ELISA方阵滴定结果显示：纯化的融合蛋白具有良好的反应原性.在间接竞争性ELISA中,当以融合蛋白GST-CDON为包被抗原时,检测限为31 ng/mL,线性范围为31～500 ng/mL,IC50为194 ng/mL,加标回收率为54.1％～65.4％,变异系数为6.28％～13.37％;当以融合蛋白pⅧ-CDON为包被抗原时,检测限为15 ng/mL,线性范围为15～500 ng/mL,IC50为94 ng/mL,加标回收率为81.7％～89.0％,变异系数为3.15％～7.55％.Deoxynivalenol （DON） mimotope,designated as CDON,is a mimicking epitope （CMRPWLQ） screened from a phagedisplayed random peptide library.In order to replace DON conjugated toxin with non-toxic recombinant protein in ELISA,two novel expression vectors,which were designated as plasmid pGEX-CDON and phagemid pC89S4-CDON,were used to produce GST-CDON and pⅧ-CDON fusion proteins in E.coli.After purification,both GST-CDON and pⅧ-CDON fusion proteins showed good reactogenicity with an anti-DON antibody in a competitive inhibition ELISA test.When GST-CDON was used as coating antigen,the linear range of the competitive inhibition ELISA was 31-500 ng/mL with an IC50 value of 194 ng/mL,and spiked recoveries were 54.1％-65.4％,with coefficient of variation of 6.28％-13.37％.The detection limit was 31 ng/inL.Upon using pⅧ-CDON as coating protein,the linear range of the competitive inhibition ELISA was 15-500 ng/mL with an IC50 value of 94 ng/mL,and spiked recoveries were 81.7％-89.0％,with coefficient of variation of 3.15％-7.55％.The detection limit was 15 ng/mL.ELISA analysis and comparison showed that the reactogenicity and specificity of pⅧ-CDON binding to anti-DON antibody were better than that of GST-CDON fusion protein.Therefore,pⅧ-CDON is promising for establishing an ELISA without the application of the toxic mycotoxin conjugate.

关 键 词：脱氧雪腐镰刀菌烯醇 模拟表位 质粒 噬菌粒 

分 类 号：Q819[生物学—生物工程]