引用本文:	杜以帅,张丽娜,马晓娜,肖鹏,孙斌,蔡碧莹,王顺奎,于凯松,刘鹰.绿色荧光蛋白标记杀鲑气单胞菌的构建及其初步应用[J].海洋科学,2016,40(12):30-35.

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绿色荧光蛋白标记杀鲑气单胞菌的构建及其初步应用
杜以帅¹, 张丽娜¹, 马晓娜¹, 肖鹏¹, 孙斌², 蔡碧莹³, 王顺奎⁴, 于凯松⁴, 刘鹰¹
1.中国科学院海洋研究所;2.天津师范大学;3.青岛科技大学;4.山东东方海洋科技股份有限公司

摘要:

杀鲑气单胞菌(Aeromonas salmonicida)是引起疖疮病的典型致病细菌, 几乎可以侵染所有的鲑科(Aeromonas)鱼类, 为水产养殖业带来巨大的经济损失。本研究通过电击转化法将原核表达载体p^GFPuv成功导入杀鲑气单胞菌C4菌株(AS-C4)中, 获得了绿色荧光蛋白标记的杀鲑气单胞菌菌株AS-C4^GFP。AS-C4^GFP菌株在氨苄青霉素抗性平板上生长良好, 并在紫外光下激发出较强的绿色荧光。通过荧光显微镜观察菌体和PCR检测gfp和杀鲑气单胞菌vapA基因, 表明gfp基因已经成功导入AS-C4中。用AS-C4^GFP人工感染大西洋鲑(Salmo salar), 从死亡鱼的脾脏、肝脏、肾脏中均能分离出AS-C4^GFP, AS-C4^GFP在脾脏中最多。AS-C4^GFP具有特异性和可视性, 为研究杀鲑气单胞菌对大西洋鲑的侵染途径提供了一种良好的生物材料。

关键词: 杀鲑气单胞菌(Aeromonas salmonicida) 绿色荧光蛋白电转化大西洋鲑(Salmo salar)

DOI：10.11759/hykx20161206

分类号:

基金项目:第56批中国博士后科学基金(2014M560580); 山东省博士后创新基金(201402005); 国家自然科学基金(31472312, 41306152,31402283); 青岛市创新创业人才计划项目(13-CX-16)

Construction and application of green fluorescent protein for Aeromonas salmonicida strain

DU Yi-shuai,ZHANG Li-na,MA Xiao-na,XIAO Peng,SUN Bin,CAI Bi-ying,WANG Shun-kui,YU Kai-song,LIU Ying

Abstract:

Aeromonas salmonicida is a typical causative agent of furunculosis in salmonids and causes considerable economic loss in salmonid farms. In this study, we used electroporation to transform a p^GFPuv plasmid into the A. salmonicida C4 strain and successfully obtained a green fluorescent protein (GFP)-tagged strain AS-C4^GFP. The AS-C4^GFP strain grew well on plates with ampicillin and could be easily identified as bright green fluorescing colonies under UV light. The results from viewing AS-C4^GFP under a fluorescent microscope as well as our polymerase chain reaction (PCR) detection of gfp and vapA verified that we had successfully transformed p^GFPuv into A. salmonicida. In an artificial infection experiment, we used the streak plate method to isolate AS-C4^GFP from the spleen, liver, and kidney of dead Atlantic salmon (Salmo salar) and the results showed the spleen to have the most AS-C4^GFP. GFP-tagged AS-C4^GFP made A. salmonicida more easily and specifically detectable, thus providing good biological material for the study of the infection route of A. salmonicida in Atlantic salmon.

Key words: Aeromonas salmonicida green fluorescent protein electroporation Salmo salar