(王娅琴)病原弧菌检测方法：免疫磁珠捕获-多重PCR技术的初步建立(终稿) 病原弧菌检测方法：免疫磁珠捕获-多重PCR技术 的初步建立 摘要 弧菌病是由弧菌属细菌引起的细菌性疾病，给海水养殖业带来了巨大的经济损失,同时对人类健康带来巨大的威胁。本研究旨在利用免疫磁珠捕获-多重PCR技术，建立一种快速、特异性强，同时检测多种病原弧菌的技术。通过亲和层析纯化的重组外膜蛋白K（OmpK）免疫新西兰大白兔制备抗血清，利用ELISA、 Western blotting、流式细胞仪检测OmpK抗血清与弧菌及外膜蛋白的结合，结果提示OmpK为副溶血弧菌、溶藻弧菌和河弧菌的交叉抗原。Sarkosyl（N-十二烷基肌氨酸钠）法提取弧菌外膜蛋白，以霍乱弧菌外膜蛋白免疫新西兰大白兔制备抗血清，Western blotting 结果显示，霍乱弧菌外膜蛋白抗血清与多种弧菌外膜蛋白在25kDa、38kDa、40kDa、58kDa、70kDa等处有较强结合，推测弧菌间存在新的交叉抗原。通过混合引物和混合模板的PCR体系，验证了副溶血弧菌、霍乱弧菌、溶藻弧菌、创伤弧菌和拟态弧菌5种病原弧菌的mPCR引物具有稳定性及特异性。实验结果为免疫磁珠捕获-多重PCR技术的建立提供研究基础。 关键词：病原弧菌、交叉抗原、免疫磁珠分离、mPCR I Primary study of detection of pathogenic vibrios by Magnetic immuuno capture- multiple polymerase chain reaction(MIC-mPCR) Abstract Vibrio is reported to be a universal marine pathogen that has caused vibriosis which bring huge economic damage to mariculture. Vibrio is also a well-known human pathogen, causing gastroenteritis through consumption of raw or undercooked seafood. Magnetic immuuno capture- multiple polymerase chain reaction (MIC-mPCR) was developed for detection of several kinds of vibrios at the same time. The recombinant outer membrane protein K were used to immunize New Zealand rabbits to obtain polyclonal antibodies. The coalescent among OmpK antibody, bacteria cell and outer membrane proteins were measured by ELISA, Western blotting and Flow Cytometry. The results indicate that OmpK is a sort of cross-reacting antigen among Vibrio parahaemolyticus, V. flurialis and V. alginolyticus. Western blot analysis of vibrio outer membrane protein with serum of V. cholerae showed that there were several cross-reacting bands in 25kDa, 38kDa, 40kDa, 58kDa and 70kDa. Its indicated that different cross-reacting antigens exist among vibrios. The multiple primer system and multiple template system were tested on 5 pathogenic vibrios, indicating the stability and specificity of mPCR primers. Key words: pathogenic vibrio, cross-reacting antigen, Magnetic immuuno capture, multiple polymerase chain reaction(MIC-mPCR). II 目录 摘要 . I Abstract . II 目录 . III 1. 前言 . 1 1.1 1.2 致病弧菌的危害 . 1 目前国内外弧菌检测方法研究进展 . 1 1.2.1 免疫学方法 . 1 1.2.2 分子生物学方法 . 2 1.3 1.4 免疫磁珠捕获-mPCR弧菌检测技术 . 3 免疫磁珠捕获系统 . 3 1.4.1 弧菌主要外膜蛋白及免疫原性 . 4 1.4.2 交叉抗原 . 4 1.5 2. 2.1 2.2 2.3 2.4 2.5 3. 3.1 多重PCR检测系统 . 4 实验菌株 . 5 实验动物 . 5 实验试剂 . 5 培养基 . 6 仪器 . 6 外膜蛋白的提取与纯化 . 6 3.1.1 弧菌总外膜蛋白的提取 . 6 3.1.2 重组OmpK的提取与纯化 . 7 3.1.3 蛋白浓度的测定（Bradford法） . 7 3.2 3.3 外膜蛋白的SDS-PAGE电泳 . 7 兔抗血清制备及效价测定 .