间接竞争ELISA法对柞蚕微孢子虫的检测

Use of ICELISA to detect Nosema perny

姜义仁1，2王伯阳1孙影1王勇2石生林1杨瑞生1段玉玺2**秦利1**

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作者单位：1. 沈阳农业大学生物科学技术学院 辽宁省昆虫资源工程技术研究中心沈阳110866；2.沈阳农业大学植物保护学院沈阳110866

中文关键词：柞蚕微孢子虫，酶联免疫吸附测定，多克隆抗体，检测

英文关键词：Nosema pernyi, ELISA, polyclonal antibody, detection

中文摘要：

  柞蚕微孢子虫病是柞蚕唯一的检疫性病害，其致病病原物为柞蚕微孢子虫（Nosema pernyi Ding，Su & Wen），因此，柞蚕微孢子虫的检测对于该病的防治具有重要意义。本文通过制备柞蚕微孢子虫多克隆抗体，建立柞蚕微孢子虫间接竞争ELISA检测法。结果表明，柞蚕微孢子虫多克隆抗体效价为1∶104、浓度为3 mg·mL-1，主要由2条大小约50 ku和25 ku蛋白条带组成，可作为后续试验多克隆抗体材料。间接竞争ELISA法最佳抗原工作浓度为2.0 μg·mL-1微孢子虫孢壁蛋白溶液，最佳抗体工作浓度为兔抗血清按1∶102倍浓度稀释，酶标二抗最佳工作浓度为1∶5×104倍稀释，柞蚕微孢子虫间接竞争ELISA检测法的灵敏度为1.6×105 spores·mL-1。间接竞争ELISA法在柞蚕微孢子虫的检测方面具有一定的应用价值。

 

英文摘要：Microsporidiosis is the only quarantinable disease of Antheraea pernyi. Nosema pernyi is the lethal pathogen of microsporidiosis. Therefore, the detection of the spores of N. pernyi is important to prevent and treat this disease. In this paper, the effectiveness of an indirect competitive ELISA for detecting the spores of N. pernyi in A. pernyi was studied by preparing the polyclonal antibody for N. pernyi. The polyclonal antibody against N. pernyi was prepared by immunizing rabbits using a suspension containing spores of N. pernyi. The titre and antibody concentration were 1∶104 and 3 mg·mL-1 respectively, the antibody mainly contained two protein straps with molecular weights of 50 ku and 25 ku, respectively. The resultant polyclonal antibody can be kept for further study. The optimal antigen concentration for the ICELISA was 20 μg·mL-1 spore wall protein of N. pernyi, the optimal polyclonal antibody concentration was 1∶102, HRPIgG optimal concentration was 1∶5×104 and the sensitivity of the method was 1.6×105 spores·mL-1. The method had some value in the detection of N. pernyi.

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