一种诱导表达绿色荧光蛋白穿梭质粒的构建及其在荧光示踪中的应用  

Construction of A Shuttled Plasmid Inducibly Expressing Green Fluorescent Protein and Its Application in Fluorescence Tracing

作　　者：左东 李子晨 尹伊 胡海 王少辉[1] 祁晶晶[1] 田明星 于圣青[1] ZUO Dong;LI Zichen;YIN Yi;HU Hai;WANG Shaohui;QI Jingjing;TIAN Mingxing;YU Shengqing(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2024年第4期25-31,共7页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(32072828,31972723);中国农业科学院科技创新工程(SHVRI-ASTIP-2014-8)。

摘　　要：细菌的荧光标记是一种重要的常用实验标记技术,用于研究细菌生理生化特性、感染宿主体内示踪、感染细胞示踪等研究。细菌的荧光标记通常通过质粒表达荧光蛋白来实现,而不同类型的细菌,由于菌种特性不同,构建的质粒往往不能通用。本研究构建了一种可诱导表达增强型绿色荧光蛋白的穿梭质粒pBT-iEGFP,该质粒可通过添加无水四环素诱导表达绿色荧光蛋白。诱导表达和传代稳定性试验表明,pBT-iEGFP质粒可在禽致病性大肠杆菌、鼠伤寒沙门菌和马耳他布鲁菌中成功诱导表达绿色荧光蛋白,在五代盲传中该质粒能在细菌中稳定遗传。胞内布鲁菌诱导表达试验证实,pBT-iEGFP质粒可成功示踪细胞感染过程中活的布鲁菌。总之,本研究构建的pBT-iEGFP质粒可作为细菌的荧光示踪研究工具,应用于多种实验研究。Fluorescence labeling of bacteria is an important and commonly used experimental technique,which is used to study the physiological and biochemical characteristics of bacteria and trace bacteria in infected hosts and cells.Bacterial fluorescent labeling is usually achieved by the expression of fluorescent protein in plasmid.However,the constructed plasmid cannot be used in common due to different species and characteristics of bacteria.In this study,we constructed a shuttled plasmid pBT-iEGFP that induced expression of enhanced green fluorescent protein by anhydrous tetracycline.The results showed that the pBT-iEGFP plasmid was successfully induced for expression of green fluorescent protein in avian pathogenic Escherichia coli,Salmonella typhimurium and Brucella melitensis.The plasmid was stably inherited in bacteria for 5 blind passages.Induced expression of EGFP in intracellular Brucella confirmed that the pBT-iEGFP could be used to trace live Brucella in the process of cell infection.In summary,the plasmid pBT-iEGFP constructed in the study could be used in a variety of scientific experiments,which provided a tool for the research of bacterial fluorescence tracing.

关 键 词：诱导表达质粒 绿色荧光蛋白 大肠杆菌 沙门菌 布鲁菌 

分 类 号：Q782[生物学—分子生物学]