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建立并优化了转基因油菜(Brassicacampestris)中的PAT蛋白酶联免疫检测技术。首先通过Western杂交和酶活性分析鉴定了PAT蛋白的纯度和活性。然后以其为抗原免疫新西兰大白兔制备了PAT蛋白的多克隆抗体,并采用硫铵沉淀法和proteinA-Sepharose4B对其进行了纯化。所得抗体对PAT蛋白的检测限为2×10-5mg/mL,且与植物源的几种结构功能相关蛋白均无交叉反应。然后对植物蛋白进行初步提取,应用酶联免疫检测技术对转基因油菜(MS1/RF1andMS8/RF3)中的PAT蛋白进行了检测,结果表明ELISA能高效地鉴别转基因和非转基因油菜。
PAT protein enzyme immunoassay was established and optimized in Brassicacampestris. The purity and activity of the PAT protein was first identified by Western blotting and enzyme activity analysis. Then polyclonal antibody of PAT protein was prepared by immunizing New Zealand white rabbits with antigen and purified by ammonium sulfate precipitation and protein A-Sepharose 4B. The detection limit of the obtained antibody to PAT protein was 2 × 10-5mg / mL, and there was no cross-reaction with several structural protein related to plant origin. Then the preliminary extraction of plant protein was carried out. The PAT protein in transgenic rapeseed (MS1 / RF1andMS8 / RF3) was detected by enzyme-linked immunosorbent assay. The results showed that ELISA could effectively identify both transgenic and non-transgenic rapeseed.