转基因作物pat蛋白的双抗体夹心酶联免疫检测方法研究  

Development of a Sandwich Enzyme-linked Immunosorbent Assay for Phosphinothricin Acethl Transferrse Protein of Genetically Modified Crops

作　　者：王敏[1,2] 张亮[2] 张威[2] 曹振[2] 谭桂玉[2] 南铁贵[2] 王保民[2] 

机构地区：[1]北京工商大学理学院生物技术系,北京市植物资源重点实验室,北京100048 [2]中国农业大学农学与生物技术学院,北京100093

出　　处：《分析化学》2013年第10期1555-1560,共6页Chinese Journal of Analytical Chemistry

基　　金：转基因生物新品种培育重大专项(2009ZX08012-017B)资助

摘　　要：将克隆到的pat基因构建原核表达载体pET30（＋）-pat并在大肠杆菌中进行表达，分离纯化得到高纯度pat蛋白。用所得到的pat蛋白制备了兔多克隆抗体和鼠单抗2F6。多抗效价〉8000，单抗效价为5×10^4；单抗为IgGl类，轻链为，（型。基于抗pat蛋白单克隆抗体和兔多克隆抗体建立了双抗夹心酶联免疫分析方法（SandwichELISA）。检测范围为1．6～100μg／L，线性回归方程y=0．6914x-2．572，决定系数为0．9951。用所建立的方法测定了5个转基因抗虫棉和2个非转基因棉花品种叶片中pat蛋白的含量，其中转基因抗虫棉中的3个品种检测出pat蛋白，其余均未检出pat蛋白。The phosphinothricin acethl transferase （pat） gene was cloned and constructed into a prokaryotic expression veetorpET30 （＋）-pat. The protein expressed by this gene was purified and used as immunogen to immunize NewZealand rabbits and mice for polyclonal antibody and monoclonal antibody separately. The polyclonal antibody titer was greater than 8000, the monoclonal antibody （mAb 2F6） titer was 5 x 104. The mAb was IgG1 isotype with s： light chains, pat protein sandwich enzyme-linked immunosorbent assay was developed with anti-pat protein mouse monoclonal antibody （mAb） and rabbit polyclonal antibody. The linear range of the method was 1. 56 - 100. 0 μg/L. The linear equation was y = 0. 6914x -2. 572, and the determinative coefficient was 0.9951. The assay was used to detect the content of pat protein in the leaf of five transgenic Bt cotton and two non-GM cotton varieties. The results showed that, three varieties of transgenic Bt cotton were positive and the others were negative.

关 键 词：转基因作物 pat基因 pat蛋白 双抗夹心酶联免疫检测法 

分 类 号：Q946.4[生物学—植物学] S184[农业科学—农业基础科学]