Xu Wentao, Huang Kunlun, Zhao Heng, Luo Yunbo
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
J Agric Food Chem. 2005 Jun 1;53(11):4315-21. doi: 10.1021/jf050218a.
We have developed a new immunoassay method to detect genetically modified (GM) maize and rape containing phosphinothricin-N-acetyltransferase (PAT). PAT encoded by Bialaphos resistance gene (bar) was highly expressed in soluble form in Escherichia coli BL21(DE3) and purified to homogeneity by Ni2+ affinity chromatography. A simple and efficient extraction and purification procedure of PAT from GM maize and rape was developed by means of the immunoaffinity column (IAC) as a cleanup tool. Purified polyclonal antibodies against PAT was produced and coupled covalently to CNBr-activated Sepharose 4B. Both the binding conditions and elution protocols were optimized. The IAC was successfully employed to isolate and purify the PAT from the various tissues of GM maize (Bt11 and Bt176) and rapes (MS1/RF1 and MS8/RF3). Enzyme linked immunosorbent assay (ELISA) procedures were established further on to measure the PAT protein. GM maize cannot be differentiated from non-GM maize by ELISA. But IAC-ELISA allowed 0.5% GMOs to be detected in MS1/RF1 and MS8/RF3 and 10% GMOs to be detected in Bt11 and Bt176, which makes this method an acceptable method to access PAT protein in GM rapes and maize.
我们开发了一种新的免疫分析方法，用于检测含有膦丝菌素 - N - 乙酰转移酶（PAT）的转基因玉米和油菜。由双丙氨膦抗性基因（bar）编码的PAT在大肠杆菌BL21(DE3)中以可溶形式高效表达，并通过Ni2+亲和层析纯化至同质。借助免疫亲和柱（IAC）作为净化工具，开发了一种从转基因玉米和油菜中简单高效提取和纯化PAT的方法。制备了针对PAT的纯化多克隆抗体，并将其与溴化氰活化的琼脂糖4B共价偶联。对结合条件和洗脱方案进行了优化。成功使用IAC从转基因玉米（Bt11和Bt176）和油菜（MS1/RF1和MS8/RF3）的各种组织中分离和纯化PAT。进一步建立了酶联免疫吸附测定（ELISA）程序来测量PAT蛋白。通过ELISA无法区分转基因玉米和非转基因玉米。但IAC - ELISA能够检测出MS1/RF1和MS8/RF3中0.5%的转基因成分，以及Bt11和Bt176中10%的转基因成分，这使得该方法成为一种用于检测转基因油菜和玉米中PAT蛋白的可接受方法。
