膦丝菌素乙酰转移酶抗原表位分析及快速ELISA定量检测方法的建立  

Antigen Epitope Analysis and Establishment of Rapid ELISA for Phosphinthricin Acetyltransferase

作　　者：梁雨欣 李忠鹏[1] 张春雨[1] 侯吉超 李小宇[1] 王永志[1,2] LIANG Yu-Xin;LI Zhong-Peng;ZHANG Chun-Yu;HOU Ji-Chao;LI Xiao-Yu;WANG Yong-Zhi(Institute of Plant Protection,Jilin Academy of Agricultural Sciences,Gongzhuling 136100,China;Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China;College of Plant Protection,Jilin Agricultural University,Changchun 130118,China)

机构地区：[1]吉林省农业科学院植物保护研究所,公主岭136100 [2]中国农业科学院植物保护研究所,北京100193 [3]吉林农业大学植物保护学院,长春130118

出　　处：《农业生物技术学报》2021年第3期591-598,共8页Journal of Agricultural Biotechnology

基　　金：吉林省农业科技创新工程项目(CXGC2018DC003);吉林省省级产业创新专项资金项目(2017C057-1);吉林省科技厅重点研发项目(20180201084SF)。

摘　　要：随着转基因大豆(Glycine max)市场的扩大,我国对转基因大豆的食品安全、环境风险等问题争议日渐加剧。越来越多的国家要求对转基因食品进行标识,部分国家对转基因成分含量亦有要求。为了建立转Bar基因大豆的快速、有效检测方法,对转Bar基因大豆进行定量检测,本研究以纯化的膦丝菌素乙酰转移酶(phosphinothricin acetyltransferase, PAT)蛋白为抗原免疫小鼠(Mus musculus),成功制备了18株特异性良好的PAT单克隆抗体(monoclonal antibody, MAb),利用在大肠杆菌(Escherichia coli)中表达的PAT分段蛋白筛选到一组能进行夹心ELISA的配对单克隆抗体9F5和3G12,并以这对单抗为基础建立了PAT快速定量双抗夹心ELISA (double antibody sandwich ELISA, DAS-ELISA)检测方法。该方法以9F5为捕获抗体包被酶标板,检测抗体3G12经辣根过氧化物酶(horseradish peroxidase, HRP)标记后以2μg/mL的工作浓度与抗原在37℃共同孵育30 min,室温避光显色15~20 min。该检测方法检测限为1.69 ng/mL,对PAT蛋白的检测范围为0.5~8.0μg/mL,板内板间变异系数均小于10%。该法灵敏度高、稳定性好,1 h即可完成检测,为转Bar基因大豆的快速定量检测提供了技术支撑。With the continuous expansion of genetically modified soybean market, Chinese citizens have become more and more disputable about the food safety and environmental risks of genetically modified soybean(Glycine max). More and more countries require labeling of genetically modified food, and some countries also have requirements on the content of genetically modified ingredients. Therefore, in order to establish a rapid and effective detection method for Bar-transgenic soybean quantitative detection, 18 strains of phosphinothricin acetyltransferase(PAT) monoclonal antibody(MAb) with good specificity were prepared in mice(Mus musculus) immunized with purified PAT protein, and a panel of monoclonal antibodies(9 F5 and3 G12) that can be used for sandwich enzyme linked immunosorbent assay(ELISA) were screened using the segmented expression peptides of PAT expressed in Escherichia coli, and a rapid quantitative double antibody sandwich ELSIA(DAS-ELISA) for PAT was established based on this panel of monoclonal antibodies. ELISA plate coated with the capture antibody 9 F5, and co-incubated with detector antibody 3 G12(2 μg/mL) labeled with HRP for 30 minutes at 37 ℃, color rendering at room temperature and away from light for 15~20 minutes. The detection limit was 1.69 ng/mL and detection range of PAT protein was 0.5~8.0 μg/mL. The coefficient of variation in the plate and between plates was less than 10%. With high sensitivity and stability,the detection could be completed in 50 minutes. This method provides technical support for detection of Bartransgenic soybean.

关 键 词：BAR基因 单克隆抗体 抗原表位 双抗夹心ELISA(DAS-ELISA) 

分 类 号：S412[农业科学—植物保护]