单核细胞增生李斯特菌σ~B启动子报告质粒的构建及其在应激研究中的应用  

Construction of a reporter plasmid carrying Listeria monocytogenes σ~B promoter and its application in stress studies

作　　者：夏叶[1] 辛永萍[1] 李肖梁[1] 方维焕[1] 江玲丽[2] 

机构地区：[1]浙江大学动物预防医学研究所/浙江省动物预防医学重点实验室,浙江杭州310058 [2]宁波卫生职业技术学院,浙江宁波315100

出　　处：《中国预防兽医学报》2016年第3期211-215,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：浙江省科技厅公益技术研究农业项目(2014C32047);"十二五"国家支撑计划项目(2013BAD19B09-2);2015年度留学人员科技活动项目择优资助经费项目

摘　　要：为探索人畜共患单核细胞增生李斯特菌的应激调控因子Sigma B（σ^B）在抗应激过程中的激活机制,本研究以pERL3为骨架,通过SOE-PCR技术构建携带3种不同σ^B依赖型启动子控制下的gfp基因的报告质粒,并利用荧光酶标仪分析携带这些报告质粒载体的单增李斯特菌在酸或盐应激条件下的σ^B活性以及不同σ^B调控子在应激条件下对σ^B激活能力的影响。结果显示：sigB自身启动子PsigB比P0880和P1602更为敏感,可以准确指示σ^B的活性;盐应激比酸应激对σ^B的激活程度高;5%的NaCl和30 min的应激时间足以对σ^B产生显著的激活效应;rsbS、rsbT和rsbV基因缺失显著降低σ^B在应激中的激活水平,而rsbX基因不介导应激诱导的σ^B激活。本研究所构建的GFP报告质粒可以准确指示单增李斯特菌的σ^B活性,可以用于应激条件下σ^B激活机制的研究。To construct a reliable reporter plasmid for analysis of σ^B activity of zoonotic Listeria monocytogenes exposed to stresses and effect of σ^B regulators on σ^Bactivation during stress, the sequence of gfp gene under the control of L.monocytogenes orB-promoter was amplified by SOE-PCR and cloned into pERL3 to construct the σ^B-promoter driven gfp gene reporter plasmid. The σ^Bactivity of L.monocytogenes strains were measured and expressed as relative fluorescence unites during stresses by the reporter plasmid. PsigB of sigB itself was proved to be the most sensitive promoter as an indicator of σ^B activity. Sodium stress was a more effective σ^B activator than acid stress in L.monocytogenes 10403S. Stress with 5% NaCl for duration of 30 rain would lead to effective σ^B activation. Deletion of rsbS, rsbT or rsbV significantly weakened σ^B activation during stress, while stress-induced σ^B activation occured in the absence of rsbX. The reporter plasmid constructed in this study is a reliable tool for measuring σ^B activity in L.monocytogenes as part of mechanistic studies of σ^B activation or regulation in response to stresses.

关 键 词：单核细胞增生李斯特菌 SIGMA B 绿色荧光蛋白 盐应激 

分 类 号：S852.61[农业科学—基础兽医学]