拟态弧菌的绿色荧光蛋白标记及其在感染草鱼体内的动态分布

EGFP marker of Vibrio mimicus and its dynamic distribution in infected Ctenopharyngodon idella

为了示踪研究拟态弧菌感染草鱼的动态过程,将增强型绿色荧光蛋白编码基因EGFP克隆至质粒p BAD24,并转化到拟态弧菌04-14菌株构建荧光标记重组菌。重组菌经阿拉伯糖诱导后,能高效表达EGFP蛋白;荧光显微镜观察和流式细胞仪检测均发现重组菌能够发出明显的绿色荧光信号,且传至30代后质粒稳定率仍为100%;生物学特性检测结果显示,与野生株相比,重组菌的形态、生长特性和细胞黏附性均未发生明显改变。用标记重组菌浸泡感染草鱼,定点采集鳃、肠道、肌肉、头肾、脾脏和肝脏,借助荧光信号检测4 d内细菌在不同组织脏器中的动态分布。结果发现感染4 h后即可在肠道和鳃中检测到绿色荧光信号,标记菌检出量分别为3.60×108和2.36×106CFU/g,直至10 h,其含量无明显变化,12 h后含菌量逐渐下降,但持续存在直至鱼死亡。标记菌在肌肉、头肾、脾脏和肝脏中呈现相似的动力学,感染24 h后才检测到荧光信号,24-85 h时间段含菌量呈现先增加后下降的变化,48 h达到峰值,检出量分别为9.58×104（肌肉）、8.75×104（头肾）、1.50×104（脾脏）和4.50×104CFU/g（肝脏）,但均低于肠道中的检出量,结果表明肠道是拟态弧菌黏附定植与繁殖的主要靶器官。

To detect Vibrio mimicus＇ dynamic distribution in infected C. idella,the gene encoding enhanced green fluorescent protein（EGFP）was cloned into the expression plasmid pBAD24 and transformed into the host cells V. mimicus strain 04-14 by electroporation to generate recombinant V. mimicus. The recombinant EGFP was highly expressed under induction with arabinose. The EGFP on the cell surface was also confirmed by fluorescence microscopy and flow cytometric analysis. Under nonselective condition, the stability of recombinant plasmid was 100% in EGFP-labeled V. mimicus after 30-generation time, and there was no significant difference between EGFP-labeled strain and wild strain in morphology, growth characteristics and adhesion ability to EPC cells. C. idella were immersed in water containing EGFP-labeled V. mimicus at the concentration of 5 x 10^7 CFU/mL. At least three fish per group were sacrificed at different time post infection, and in different tissues and organs, such as gill, intestine, muscle, head kidney, spleen and liver were sampled separately. EGFP-labeled V. mimicus in different tissues and organs was examined by the signal of fluorescence and the method of plate culture count within 4 days after infection. The green fluorescent signals appeared firstly in the intestine and gill at 4 h after infection, and the number of bacteria reached up to 3.60 x 108 and 2.36 x 10^6 CFU/g,respectively. The number of bacteria in the intestine and gill had no obvious changes from 4 to l0 h post infection, then declined gradually after 12 h, but continued to exist until fish death. Dynamics of bacteria in muscle, head kidney, spleen and liver were similar, the green fluorescent signals in these tissues and organs were found 24 h later after infection, and the number of bacteria increased at first but later decreased from 24 to 85 h. The number of bacteria in muscle, head kidney,spleen and liver reached their peaks 48 h post infection,being 9.58 x 10^4 ,8.75 x 10^4 ,1.50 x 10^4 and 4.50 x 10^4 CFU/g,respectively,but the number of bacteria which was detected in them was lower than that in the intestine. These results suggest that the intestine is the primary target organ for the adhesion, colonization and reproduction of V. mimicus.