5株鲆鲽鱼类病原菌多克隆抗体的制备与应用  

Preparation and application of polyclonal antibodies against five pathogenic bacteria isolated from flounder

作　　者：朱岩松[1,2] 王秀华[2] 韩雯[1,2] 王锐[1,2] 黄倢[2] 

机构地区：[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

出　　处：《渔业科学进展》2013年第3期68-74,共7页Progress in Fishery Sciences

基　　金：国家自然科学基金项目(31172440)资助

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 putrefaciens5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1:2048000、1:16000、1:16000、1:1024000、1:128000;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16SrDNA序列的分子分析方法一致。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 LD50value of the five bacteria against turbot Scophthal-musmaximuswere 102～107 CFU/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 000respectively.The poly-clonal antibodies were used in indirect ELISA as primary antibody,and the goat anti-rabbit IgGHRP 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 106cell/ml,respectively.Obvious cross reactions were present between three strains of vibrio and the antisera.By using the developed indirect ELISA method,13bacterial 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 16SrDNA method.

关 键 词：鱼类病原菌 间接ELISA 多克隆抗体 交叉反应 病原检测 

分 类 号：S917.1[农业科学—水产科学]