中文摘要：Cry1Ie是一种苏云金杆菌（Bt）毒蛋白，其与Cry1Ab和Cry1Ac相比具有不同的行为模式。转cry1Ie基因玉米在未来可能要实现商业化，因而急需开发出一种检测转基因植物及其产品中的Cry1Ie蛋白的方法。本文通过将Cry1Ie蛋白表达在大肠杆菌菌株Transetta DE3中，并用Ni-NTA离心柱纯化，再经过测序验证来建立ELISA检测方法。生物测定的结果显示，纯化的Cry1Ie蛋白对亚洲玉米螟有很高的毒性。作者分别利用免疫Cry1Ie蛋白的兔子和小鼠生成了多克隆抗体(pAb)和特异性单克隆抗体(mAb)1G(4)2D(6)。夹心ELISA方法对Cry1Ie蛋白的检测是通过用pAb涂层和mAb 1G(4)2D(6)检测实现的。不同玉米植株中的检测限（LOD）、定量限（LOQ）和分析定量范围分别为0.27-0.51、0.29-0.78和0.45-15.71 ng/mL。本文用转cry1Ie基因玉米对所研发的夹心ELISA方法进行了验证。研究结果表明，本文所研发的ELISA方法可用来检测转基因植物中的Cry1Ie蛋白。
外文摘要：Cry1Ie is a kind of Bacillus thuringiensis (Bt) toxin protein which has a different action model than the Cry1Ab and Cry1Ac protein. The transgenic maize expressing Cry1Ie might be commercially used in the near future and it is urgent to develop a method to detect Cry1Ie protein in transgenic plants and their products. To develop an ELISA method, Cry1Ie protein was expressed in Escherichia coli strain Transetta DE3, purified with the Ni-NTA spin columns, and then validated by sequencing. Bioassay results showed that the purified Cry1Ie protein was highly toxic to the Asian corn borer. The polyclonal antibody (pAb) and the specific monoclonal antibody (mAb) 1G(4)2D(6) were generated from rabbit and mice which were immunized with Cry1Ie protein, respectively. Western blotting of crude Cry1Ie protein extracts was established by employing mAb 1G(4)2D(6), whereas the mAb 1G(4)2D(6) negligibly recognized other Bt proteins. Sandwich ELISA against Cry1Ie protein was established by coating with pAb and detecting with mAb 1G(4)2D(6). The limit of detection (LOD), the limit of quantification (LOQ), and the quantification range of the assay in different matrices of maize plant were determined as 0.27-0.51, 0.29-0.78, and 0.45-15.71 ng/mL, respectively. Recoveries of Cry1Ie protein spiked in different maize tissues ranged from 75.1 to 99.5 %. The established sandwich ELISA was verified using transgenic maize overexpressing Cry1Ie. The results in this study suggested that the established ELISA method is effective for detecting Cry1Ie protein in transgenic plants.
外文关键词：Bacillus thuringiensis；Cry1Ie；Sandwich ELISA；Transgenic plant
作者：Zhang, Yuwen；Zhang, Wei；Liu, Yan；Wang, Jianhua；Wang, Guoying；Liu, Yunjun
作者单位：中国农业科学院
期刊名称：ANALYTICAL AND BIOANALYTICAL CHEMISTRY
期刊影响因子：3.125
出版年份：2016
出版刊次：9
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