双抗夹心ELISA检测转Bar基因抗除草剂大豆  

Double-Antibody Sandwich ELISA for the Detection of Transgenic Bar Gene Herbicide-Tolerant Soybeans

作　　者：李小宇[1] 张春雨[1] 郭东全[1] 张淋淋[2] 尤晴 董英山[1] 王永志[1] 李启云[1] 

机构地区：[1]吉林省农业科学院植物保护研究所,东北作物有害生物综合治理重点实验室,吉林省农业微生物重点实验室,吉林公主岭136100 [2]东北农业大学植物保护学院,黑龙江哈尔滨150030 [3]吉林农业大学农学院,吉林长春130118

出　　处：《食品科学》2016年第4期222-225,共4页Food Science

基　　金：吉林省自然科学基金项目(20130101089JC);吉林省中青年科技领军人才及优秀创新团队项目(20121812);国家转基因生物新品种培育重大专项(2014ZX08004)

摘　　要：为快捷有效地检测转Bar基因抗除草剂大豆,利用已制备的抗除草剂Bar基因编码蛋白,膦丝菌素乙酰转移酶(phosphinothricin acetyltransferase,PAT)单克隆抗体和多克隆抗体,建立PAT蛋白双抗夹心酶联免疫吸附检测方法,对转Bar基因抗除草剂大豆不同组织材料进行定量检测。结果显示,最佳检测条件为捕获抗体质量浓度0.125μg/m L,包被酶标板,37℃孵育1 h后4℃静置过夜,检测样品37℃孵育1.5 h,检测抗体质量浓度6.25μg/m L,37℃孵育1.5 h;PAT蛋白的最低检测限为0.04 ng/m L,大豆蛋白体系中为8 ng/m L;重复性变异系数小于3%。利用上述检测条件,对实验建立的转Bar基因抗除草剂大豆进行PAT蛋白定量检测,成功地在根、茎、花、叶、种子不同部位检测到该蛋白的表达。To rapidly and efficiently detect transgenic Bar herbicide-tolerant soybeans,we produced a monoclonal antibody and a polyclonal antibody against phosphinothricin acetyltransferase（PAT） protein encoded by the Bar gene and then utilized them to established a double-antibody sandwich ELISA system for the detection of PAT protein,which could quantify PAT in different tissues and materials from transgenic Bar herbicide-tolerant soybeans. Reaction conditions were optimized as follows： 0.125 μg/m L capture antibody was coated onto the microtiter plates at 4 ℃ overnight after incubation at 37 ℃ for 1 h,the antigen was incubated at 37 ℃ for 1.5 h,and the detection antibody at 6.25 μg/m L was incubated at 37 ℃ for 1.5 h. The detection sensitivity was 0.04 ng/m L for purified PAT and 8 ng/m L for crude soybean protein. The coefficient of variation of reproducibility was less than 3%. This method has been successfully applied to detect the expression of PAT in the root,stem,leaf,flower and seed of transgenic soybean.

关 键 词：BAR 双抗夹心酶联免疫吸附 大豆 检测 

分 类 号：Q788[生物学—分子生物学]