NirB启动子调控下鼠沙门氏菌体内诱导型表达载体的构建  

Construction of the recombinant attenuated Salmonella typhimurium under the control of the in vivo inducible nirB promoter

作　　者：郭恒[1] 王学林[1] 李慧萍[1] 刘学[1] 张凌怡[2] 唐艺芝[1] 高鹤[1] 杨秀丽[1] 徐德启[1,3] 刘明远[1] 王光明[1] 

机构地区：[1]吉林大学人兽共患病研究所,吉林长春130062 [2]吉林大学第二临床医学院妇产科 [3]美国食品与药品管理局(FDA),美国马里兰20892

出　　处：《中国实验诊断学》2011年第7期1041-1044,共4页Chinese Journal of Laboratory Diagnosis

摘　　要：目的构建遗传稳定性良好的沙门氏菌体内诱导型表达载体。方法以克隆载体pGB2为基础,将沙门氏菌厌氧启动子PnirB和EGFP基因串联,并在其多克隆位点MCS下游引入rrnbT1T2转录终止序列,构建沙门氏菌低拷贝体内诱导型表达载体pGnirB-EGFP-rrnb,电转化入鼠伤寒沙门氏菌phoP/phoQ株,对质粒的稳定性及蛋白表达情况进行检测。结果含有低拷贝重组质粒的沙门氏菌在缺失抗生素选择压力下盲传100代后质粒稳定性高于95%,在厌氧静置72 h培养后激光共聚焦显微镜下可观察到明显绿色荧光。结论高度稳定的沙门氏菌体内诱导型表达载体构建成功,为研制以鼠伤寒沙门氏菌为活载体的新型口服疫苗奠定了基础。Objective Construction of a stable expression vector under the control of the in vivo inducible nirB promoter in the salmonella typhimurium（S.typhimurium）.Methods Anaerobic promoter PnirB of salmonella and EGFP were connected into cloning vector pGB2,rrnbT1T2 transcription termination sequence was inserted into the downstream of multiply clone sites（MCS）.This plasmid was introduced into S.typhimurium strain phoP/phoQ.The stability of pGB2-based vaccine constructs was determined.The expression of report gene EGFP was detected.Results Salmonella contained pGB2-based plasmid was cultivated for 100 generations without antibiotic selection,the stability of plasmid was up to 95%.Strong green fluorescent can be observed after stationary cultivation for 72 hours under laser scanning confocal microscope.Conclusion The low-copy-number internal-induced eukaryotic expression vector f pGnirB-EGFP-rrnb was constructed successfully and stably expressed in salmonella under the control of the in vivo inducible PnirB promoter.It laid a foundation of further study on a new type pre-exposure oral vaccine by using attenuated salmonella as vector.

关 键 词：沙门氏菌 体内诱导型启动子 疫苗 暴露前 

分 类 号：R346[医药卫生—基础医学]