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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 To construct an inducible expression vector for Salmonella in vivo with good genetic stability. Methods Based on the cloning vector pGB2, PnirB and EGFP genes of Salmonella anaerobic promoters were connected in series, and the rrnbT1T2 transcription termination sequence was introduced downstream of MCS in its multiple cloning site to construct the inducible expression vector pGnirB-EGFP-rrnb, Electroporation into Salmonella typhimurium phoP / phoQ strain, the plasmid stability and protein expression were detected. Results Salmonellae containing low-copy recombinant plasmids had higher than 95% plasmid stability after 100 passages of selective antibiotic-deficient antibiotics, and obvious green fluorescence was observed under confocal laser scanning microscope after 72 hours anaerobic resting. Conclusion The highly stable Salmonella inducible expression vector was constructed successfully, which laid the foundation for the development of a new oral vaccine against Salmonella typhimurium.