食品科学

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双抗夹心ELISA检测转Bar基因抗除草剂大豆

李小宇1,张春雨1,郭东全1,张淋淋2,尤 晴3,董英山1,王永志1,*,李启云1,*   

  1. 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
  • 出版日期:2016-02-25 发布日期:2016-02-23
  • 通讯作者: 王永志,李启云
  • 基金资助:

    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)

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

LI Xiaoyu1, ZHANG Chunyu1, GUO Dongquan1, ZHANG Linlin2, YOU Qing3, DONG Yingshan1, WANG Yongzhi1,*, LI Qiyun1,*   

  1. 1. Jilin Key Laboratory of Agricultural Microbiology, Key Laboratory of Integrated Pest Management on Crops in Northeast,
    Institute of Plant Protection, Jilin Academy of Agricultural Sciences, Gongzhuling 136100, China;
    2. College of Plant Protection, Northeast Agricultural University, Harbin 150030, China;
    3. College of Agronomy, Jilin Agricultural University, Changchun 130118, China
  • Online:2016-02-25 Published:2016-02-23
  • Contact: WANG Yongzhi, LI Qiyun

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0.125 μg/mL,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6.25 μg/mL,37 ℃孵育1.5 h;PAT蛋白的最低检测限为0.04 ng/mL,大豆蛋白体系中为8 ng/mL;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。

关键词: Bar, 双抗夹心酶联免疫吸附, 大豆, 检测

Abstract:

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/mL 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/mL was incubated
at 37 ℃ for 1.5 h. The detection sensitivity was 0.04 ng/mL for purified PAT and 8 ng/mL 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

Key words: Bar, double-antibody sandwich ELISA, soybeans, detection

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