绿色荧光蛋白标记杀鲑气单胞菌的构建及其初步应用

Construction and application of green fluorescent protein for Aeromonas salmonicida strain

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vap A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。

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 pGFPuv plasmid into the A.salmonicida C4 strain and successfully obtained a green fluorescent protein (GFP)-tagged strain AS-C4GFP. The AS-C4GFP 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-C4GFP under a fluorescent microscope as well as our polymerase chain reaction (PCR) detection of gfp and vapA verified that we had successfully transformed pGFPuv into A. salmonicda. In an artificial infection experiment, we used the streak plate method to isolate AS-C4GFP from the spleen, liver, and kidney of dead Atlantic salmon (Salmo salar) and the results showed the spleen to have themost AS-C4GFP. GFP-tagged AS-C4GFP 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