摘要
结核病是目前全世界最具威胁性的细菌感染病,迫切需要新型有效的预防和治疗手段。在研究中,为了更直接方便地探究结核发病机制以及研制新型疫苗,故需获得能表达绿色荧光蛋白的海分枝杆菌。通过PCR方法,以pEGFP-N1为模版扩增出增强型绿色荧光蛋白基因序列,将扩增产物克隆到载体pMV261上,成功构建出重组质粒pMV261-EGFP。利用电穿孔技术将pMV261-EGFP转化入海分枝杆菌,并用卡那霉素抗性筛选重组海分枝杆菌克隆,在激光共聚焦显微镜下能正确观察到EGFP的表达。能自发释放荧光的海分枝杆菌的成功构建,将为研究结核发病机制以及研制新型疫苗奠定实验基础。
Tuberculosis is the most threatening bacterial infection in the world, and recently new and effective methods of prevention and treatment are urgently needed. For more immediate and convenient detection and research on the pathogenesis of tuberculosis and the development of new vaccines, it is necessary to obtain the Mycobacterium Marinum expressing green fluorescent protein. In this study, enhanced green fluorescent protein gene sequence was implified by polymerase chain reaction（PCR） from the template of pEGFP-N1 and cloned into Eukaryotic expression shuttle vector pMV261, successfully constructed the recombinant plasmid pMV261-EGFP. pMV261-egfpwas transformed into M.Marinum by electropuncture technique, and the recombinant M.Marinum clones were selected by kanamycin resistance. The expression of EGFP was observed correctly under the laser confocal microscope. The established recombinant M.Marinum expressing EGFP may lay the foundation for the study of the pathogenesis of tuberculosis and the development of a new vaccine.
作者
刘梓健
彭宝洲
粟海波
Liu Zijian;Peng Baozhou;Su Haibo(GMU-GIBH Joint School of Life Sciences,Guangzhou Medical University,Guangdong Guangzhou 511436)
出处
《生物化工》
2018年第4期44-46,50,共4页
Biological Chemical Engineering
关键词
结核病
结核分枝杆菌
海分枝杆菌
绿色荧光蛋白
Tuberculosis
Mycobacterium tuberculosis
Mycobacterium Marinum
Green fluorescent protein
