柞蚕微孢子虫孢壁蛋白多克隆抗体的制备及鉴定

Preparation and Identification of a Polyclonal Antibody Against Spore Wall Proteins from Nosema pernyi

柞蚕微孢子虫(Nosema pernyi)孢壁蛋白在侵染宿主的过程中扮演重要角色,制备柞蚕微孢子虫孢壁蛋白多克隆抗体可为建立病害的快速诊断技术提供免疫试剂,并有利于研究孢壁蛋白的功能。采用沸水浴法提取柞蚕微孢子虫孢壁蛋白作为抗原免疫新西兰兔,收集纯化血清后得到柞蚕微孢子虫孢壁蛋白抗体。间接ELISA法检测制备的多克隆抗体效价达到1∶2.56×104。采用Western blotting方法在该多克隆抗体与柞蚕微孢子虫孢壁蛋白的免疫反应中检测到分子质量为32 kD的孢壁蛋白。该多克隆抗体对纯化后的柞蚕微孢子虫有较好的检测灵敏度,用于检测感染柞蚕微孢子虫的柞蚕蛹和蛾均呈现阳性结果。

The spore wall proteins of Nosema pernyi play an important role in infection to host. Preparation of polyclonal antibody against N. pernyi spore wall proteins could provide immunological agent for rapid diagnose of the disease and facilitate investigations on biological functions of spore wall proteins. In our study, the spore wall proteins of N. pernyi were extracted by boiling water method and were used as antigen to immunize New Zealand rabbit for collection of serum to prepare polyclonal antibody against the spore wall proteins. Indirect-ELISA assay showed that titer of the prepared polyclonal antibody reached 1：2.56 ×10^4, Western blotting result displayed a spore wall protein with molecular weight of 32 kD in the immunological reaction between spore wall proteins and the polyclonal antibody, The polyclonal antibody ex- hibited a high diagnostic sensitivity against purified N. pernyi spores, and also showed positive reaction to the pupae and moths infected N. pernyi.