双荧光标记伪结核棒状杆菌的构建与评价  

Construction and evaluation of double fluorescence labeled Corynebacterium pseudotuberculosis

作　　者：刘娉婷 牟诗颖 牛禄婷 雷佳莹 李鑫灿 王芝英 周作勇[1] LIU Pingting;MOU Shiying;NIU Luting;LEI Jiaying;LI Xincan;WANG Zhiying;ZHOU Zuoyong(College of Veterinary Medicine,Southwest University,Rongchang,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆荣昌402460

出　　处：《中国兽医学报》2024年第12期2572-2578,共7页Chinese Journal of Veterinary Science

基　　金：重庆市级大学生创新训练计划资助项目(S202310635102)。

摘　　要：为建立荧光标记的伪结核棒状杆菌(Corynebacterium pseudotuberculosis,Cp)菌株,本研究将超折叠绿色荧光蛋白编码基因(sfGFP)与红色荧光蛋白编码基因(mCherry)融合,克隆进pXMJ19并在Cp中表达。评价不同pH值条件下培养的Cp重组菌的荧光信号,以及Cp重组菌感染巨噬细胞J774A.1和小鼠的可视化检测效果。结果显示,成功将sfGFP和mCherry融合并克隆进pXMJ19质粒(pXMJ19-sfGFPmCherry),将其电转入Cp宣汉株(XH02)获得双荧光标记菌株XH02-pXMJ19-sfGFPmCherry(XH02-sfGFPmCherry)。在鲜血琼脂平板上培养,XH02-sfGFPmCherry呈粉红色单菌落;在荧光显微镜下观察,分别可见绿色、红色2种荧光;与在pH7.0下培养相比,在pH5.0下培养时,XH02-sfGFPmCherry的绿色荧光强度显著降低,但红色荧光强度显著增强;体外感染J774A.1、体内感染小鼠的肝脏和肾脏中均能观察到发绿色和红色荧光的Cp,且2种荧光的重合性好。本研究表明构建的双荧光标记Cp可高效表达红、绿2种荧光蛋白,在不同pH值条件下表达优势荧光信号,可用于Cp体外感染巨噬细胞和体内感染小鼠的监测,为研究Cp感染提供了一种定位和示踪潜在工具。To establish a fluorescence-labeled Corynebacterium pseudotuberculosis(Cp),the superfolded green fluorescent protein gene(sfGFP)fused to the red fluorescent protein gene(mCherry)was cloned into pXMJ19and expressed in Cp.The fluorescent signals of recombinant Cp cultured under different pH values,and the visualized detection of bacteria in the macrophages J774A.1 or mice infected with recombinant Cp were evaluated.The results showed that a double fluorescence labeled XH02-pXMJ19-sfGFPmCherry(XH02-sfGFPmCherry)was successfully constructed by electrotransformation of Cp with pXMJ19-sfGFPmCherry,which containing sfGFPfused to mCherry.The colony morphology of XH02-sfGFPmCherry on fresh blood agar plates was pink.The XH02-sfGFPmCherryshowed green and red fluorescence when observed under the fluorescence microscope.Compared with cultivation at pH7.0,XH02-sfGFPmCherryshowed a significant decrease in green fluorescence intensity(fluorescence intensity/D600)at pH5.0,but significantly increased in red fluorescence intensity at pH5.0.Green and red fluorescences of Cp were observed in the XH02-sfGFPmCherry-infected J774A.1 in vitro or in the liver and kidney of XH02-sfGFPmCherry-infected mice in vivo,and with good overlap of two kinds of fluorescences.This study demonstrates that the constructed dual fluorescent labeled Cp can efficiently express both red and green fluorescent proteins,and express the dominant fluorescent signals under different pH value conditions,which can be used for Cp monitoring in vitroinfection of macrophages and in vivoinfection of mice by this pathogen,and providing potential tool for the localization and tracing research of Cp infection.

关 键 词：sfGFPmCherry双荧光标记 伪结核棒状杆菌 构建 评价 

分 类 号：S852.61[农业科学—基础兽医学]