摘要
【目的】以单克隆抗体（monoclonal antibody,mAbs）为基础，建立检测膦丝菌素乙酰转移酶（the phosphinothricin acetyltransferase,PAT）的双抗体夹心ELISA方法,为转基因玉米PAT蛋白的检测提供参考。【方法】由P3301质粒扩增出膦丝菌素乙酰转移酶基因（blalaphos resistance gene）bar，并与表达载体pET28a构建重组质粒，诱导表达产生外源性PAT蛋白；以纯化后的PAT蛋白为免疫原，制备mAbs，建立双抗体夹心ELISA方法，并与PCR检测方法进行比较，确定该方法的准确性和可靠性。【结果】应用mAbs 4D5和3E8成功建立了检测PAT蛋白的双抗体夹心ELISA方法，其有效检测范围为3.125～50 ng/mL。采用该夹心ELISA方法对36份已知转基因背景的玉米样品进行了检测，根据阴阳性判定值OD450〉0.210，检测出含PAT蛋白的Bt176阳性样品4份，以bar基因为检测目标利用传统PCR方法也检测出相同的Bt176样品4份，2种检测方法的符合率为100%。【结论】利用建立的双抗体夹心ELISA免疫学检测方法，可以对转基因玉米中的PAT蛋白进行准确、特异、有效的检测。
【Objective】A monoclonal antibodybased sandwich enzyme-linked immunosorbent assay （ELISA） to detect phosphinothricin acetyltransferase （PAT） was developed and evaluated for determination of genetically modified （GM） maize.【Method】The coding sequence of the blalaphos resistance gene （bar） was amplified by PCR from the plasmid P3301,cloned into the plasmid pET28a and expressed in E.Coli BL21（DE3）.mAbs against PAT was prepared using purified recombinant PAT as immunogen.Based on mAbs 4D5 and 3E8,double-antibody sandwich ELISA method was develop and the accuracy and reliability of this assay were determined by comparing with the PCR method.【Result】The working range of sandwich ELISA was defined as a standard curve with a linear range of 3.125-50 ng/mL.Using the sandwich ELISA developed in this study,we could determine 4 GM maize Bt176 samples out of 36 corn samples analyzed with the threshold value of 0.210.PCR method targeting the bar gene showed the same results.【Conclusion】The sandwich ELISA developed in this study can be used as a specific and reliable immunoassay for screening PAT in GM maize.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2013年第11期45-50,共6页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家转基因重大专项(2009VX08012-003B)
作者简介
刘志浩（1986-），男，山东菏泽人，在读硕士，主要从事转基因作物检测研究。E—mail：liuzhihaow@163．com
[通信作者]柴同杰（1957-），男，山东泰安人，教授，博士生导师，主要从事环境微生物研究。Email：chaitj117@163．com
