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Development of Monoclonal Antibodies Recognizing Linear Epitope: Illustration by Three Bacillus thuringiensis Crystal Proteins of Genetically Modified Cotton, Maize, and Tobacco
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2017-11-09 00:00:00 , DOI: 10.1021/acs.jafc.7b03426 Zhen Cao 1, 2 , Wei Zhang 1 , Xiangxue Ning 1 , Baomin Wang 1 , Yunjun Liu 3 , Qing X. Li 2
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2017-11-09 00:00:00 , DOI: 10.1021/acs.jafc.7b03426 Zhen Cao 1, 2 , Wei Zhang 1 , Xiangxue Ning 1 , Baomin Wang 1 , Yunjun Liu 3 , Qing X. Li 2
Affiliation
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Bacillus thuringiensis Cry1Ac, Cry1Ia1, and Cry1Ie are δ-endotoxin insecticidal proteins widely implemented in genetically modified organisms (GMO), such as cotton, maize, and potato. Western blot assay integrates electrophoresis separation power and antibody high specificity for monitoring specific exogenous proteins expressed in GMO. Procedures for evoking monoclonal antibody (mAb) for Western blot were poorly documented. In the present study, Cry1Ac partially denatured at 100 °C for 5 min was used as an immunogen to develop mAbs selectively recognizing a linear epitope of Cry1Ac for Western blot. mAb 5E9C6 and 3E6E2 selected with sandwich ELISA strongly recognized the heat semidenatured Cry1Ac. Particularly, 3E6E2 recognized both E. coli and cotton seed expressed Cry1Ac in Western blot. Such strategy of using partially denatured proteins as immunogens and using sandwich ELISA for mAb screening was also successfully demonstrated with production of mAbs against Cry1Ie for Western blot assay in maize.
中文翻译：
识别线性表位的单克隆抗体的开发：转基因棉花，玉米和烟草的三种苏云金芽孢杆菌晶体蛋白插图
苏云金芽孢杆菌Cry1Ac，Cry1Ia1和Cry1Ie是δ-内毒素杀虫蛋白，广泛应用于转基因生物（GMO），例如棉花，玉米和马铃薯中。Western印迹分析整合了电泳分离能力和抗体高特异性，可监测转基因生物中表达的特定外源蛋白质。文献记载了唤起单克隆抗体（mAb）进行Western印迹的程序。在本研究中，将在100°C下变性5分钟的Cry1Ac用作免疫原，以开发可选择性识别Cry1Ac线性表位的mAb用于Western印迹。通过夹心ELISA选择的mAb 5E9C6和3E6E2强烈识别出热半变性Cry1Ac。特别是3E6E2识别两种大肠杆菌棉籽在Western blot中表达Cry1Ac。使用部分变性蛋白质作为免疫原并使用夹心ELISA进行mAb筛选的这种策略也已成功证明，针对玉米的针对Cry1Ie的mAb的生产可用于玉米的Western blot分析。
更新日期：2017-11-09
中文翻译：
识别线性表位的单克隆抗体的开发：转基因棉花，玉米和烟草的三种苏云金芽孢杆菌晶体蛋白插图
苏云金芽孢杆菌Cry1Ac，Cry1Ia1和Cry1Ie是δ-内毒素杀虫蛋白，广泛应用于转基因生物（GMO），例如棉花，玉米和马铃薯中。Western印迹分析整合了电泳分离能力和抗体高特异性，可监测转基因生物中表达的特定外源蛋白质。文献记载了唤起单克隆抗体（mAb）进行Western印迹的程序。在本研究中，将在100°C下变性5分钟的Cry1Ac用作免疫原，以开发可选择性识别Cry1Ac线性表位的mAb用于Western印迹。通过夹心ELISA选择的mAb 5E9C6和3E6E2强烈识别出热半变性Cry1Ac。特别是3E6E2识别两种大肠杆菌棉籽在Western blot中表达Cry1Ac。使用部分变性蛋白质作为免疫原并使用夹心ELISA进行mAb筛选的这种策略也已成功证明，针对玉米的针对Cry1Ie的mAb的生产可用于玉米的Western blot分析。
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