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| 5株鲆鲽鱼类病原菌多克隆抗体的制备与应用 |
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朱岩松1,2, 王秀华3, 韩雯1,2, 王锐1,2, 黄倢3
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1.中国海洋大学海洋生命学院，青岛266003;2.农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所，青岛266071;3.农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所，青岛266071
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| 摘要: |
| 本研究制备了鳗弧菌Vibrio anguillarum、杀鲑气单胞菌Aeromonas salmonicida、副溶血弧菌V. parahaemolyticus、哈维氏弧菌V. harveyi和腐败希瓦氏菌Shewanella putrefaciens 5株鲆鲽鱼类病原菌的兔抗血清，建立了5种菌的间接ELISA检测方法，并将该检测方法用于鱼类细菌分离物病原检测。人工感染结果显示，5株菌对大菱鲆的半数致死量(LD50)在102~107CFU/fish；制备的兔抗血清效价分别为1∶2 048 000、1∶16 000、1∶16 000、1∶1 024 000、1∶128 000；交叉反应结果显示，鳗弧菌、副溶血弧菌、哈维氏弧菌3株弧菌与抗血清相互之间存在交叉反应；抗血清特异检测灵敏度分别为104、108、107、105、106cell/ml；对13株海水鱼类细菌分离物进行检测，有1株腐败希瓦氏菌阳性，两株哈维氏弧菌阳性；1株副溶血弧菌和哈维氏弧菌均为阳性，该结果与16S rDNA序列的分子分析方法一致。 |
| 关键词: 鱼类病原菌 间接ELISA 多克隆抗体 交叉反应 病原检测 |
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| 基金项目:国家自然科学基金项目（31172440） |
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| Preparation and application of polyclonal antibodies against five pathogenic bacteria isolated from flounder |
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| Abstract: |
| Five marine fish pathogens, including Vibrio anguillarum, Aeromonas salmonicida, V.parahaemolyticus, V. harveyi, Shewanella putrefaciens were previously isolated and identified. In this study,virulence of these pathogens were determined by artificial infection experiments. The results showed that LD50 value of the five bacteria against turbot Scophthalmus maximus were 102~107CFU/fish. The polyclonal antibodies against them were produced from five New Zealand white rabbits respectively,and the titers of the polyclonal antibodies were 1∶2 048 000, 1∶16 000, 1∶16 000, 1∶1 024 000, 1∶128 000 respectively. The polyclonal antibodies were used in indirect ELISA as primary antibody, and the goat anti-rabbit IgG-HRP was used as a secondary antibody. The indirect ELISA method of rapid detection of five pathogens was developed. The sensitivity of the serum was tested, and the lowest concentrations of five pathogens that can be detected were 104, 108, 107, 105, and 106 cell/ml, respectively. Obvious cross reactions were present between three strains of vibrio and the antisera. By using the developed indirect ELISA method, 13 bacterial strains isolated from the infected fish were detected. One strain from the flounder and black rockfish Sebastodes fuscescens samples in the area of Jiaonan, Shandong Province, was positive for S. putrefaciens and two were positive for V. harveyi; one strain from Cynoglossus semilaevis in the area of Wudi, Shandong Province, was positive for both V. parahaemolyticus and V. harveyi. This result is consistent with the result of 16S rDNA method. |
| Key words: Fish pathogens Indirect ELISA Polyclonal antibody Cross reaction Pathogen detection |