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鱼类病原性弧菌血清学诊断芯片技术构建

21129 2019-12-25 08:48:22 绳秀珍,李文涛,王欣欣,等 中国动物检疫
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针对鱼类致病性肠道弧菌(Vibrio ichthyoenteri)、鳗弧菌(Vibrio anguillarum)、副溶血弧菌(Vibrio parahaemolyticus)、溶藻弧菌(Vibrio alginolyticus)、河流弧菌(Vibrio fluvialis)和哈维氏弧菌(Vibrio harveyi),制备了牙鲆(Paralichthys olivaceus)抗血清,提取了各弧菌的外膜蛋白、胞外产物、鞭毛蛋白和全菌蛋白,分析了牙鲆抗血清与各抗原蛋白的免疫反应,发现各弧菌抗血清与各自的抗原反应最强,与其他弧菌抗原有不同程度的交叉反应。将6种弧菌的抗原组分固定于芯片载体形成微阵列,构建了其血清学诊断抗原芯片,使用辣根过氧化物酶标记的抗牙鲆IgM单抗2D8作为检测抗体,确定了检测结果判读方法;将抗原芯片应用于牙鲆和大菱鲆(Scophthalmus maximus)的弧菌病诊断,并利用ELISA技术,验证了该抗原芯片用于弧菌病诊断的准确性。本研究为鱼类弧菌病的血清学诊断提供了有力工具。

Construction of Serological Diagnostic Chip for Pathogenic Vibrio in Fish

The antiserum samples of Paralichthys olivaceus were prepared respectively against 6 kinds of pathogenic vibrios,including Vibrio ichthyoenteri,V. anguillarum,V. parahaemolyticus,V. alginolyticus,V. fluvialis and V. harveyi. The outer membrane protein,flagellum protein,extracellular products and whole-cell bacterial protein of the strains were extracted and purified,then the immune reactions between the antigen components and the antiserum samples were analyzed,it was found that,for all Vibrios,the immune reaction between its antigen and its own antiserum was the strongest,besides,there were also cross-reactions in different extent between the antiserum with the antigen of other Vibrios. Further,the antigen components of the 6 strains were fixed on the chip carrier to form an antigen microarray so as to construct the serological diagnostic antigen chip,the method for interpreting detection results was determined by using monoclonal antibody 2D8 of IgM marked with horseradish peroxidase as the detection antibody;the antigen chip was applied to diagnose vibriosis in Paralichthys olivaceus and Scophthalmus maximus,and its accuracy was verified by ELISA. As a conclusion,a useful tool was provided for serological diagnosis of fish vibriosis.

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