phoA为报告基因的甲型副伤寒沙门氏菌启动子文库的建立及初步鉴定  

Construction of a Promoter Library of Salmonella Paratyphi A with phoA as a Reporter Gene and Its Preliminary Identification

作　　者：许泽仰 洪愉[2] 冯梦蝶[1] 毛普加 宋武战[2] 杨洪文[2] 赵继华[2] 黄芬[1] 井申荣[1] 曾韦锟[1] 

机构地区：[1]昆明理工大学医学院,昆明市650500 [2]成都军区昆明总医院核医学科,昆明市650032

出　　处：《医学分子生物学杂志》2014年第4期187-191,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.31160193）,云南省教育厅科学研究基金（No.2010Y398）,云南省应用基础研究面上项目（No.2010ZC055,2012FB135）

摘　　要：目的：构建以phoA为报告基因的甲型副伤寒沙门氏菌启动子文库。方法构建了以phoA为报告基因的“启动子陷阱载体” pPhoA，将甲型副伤寒沙门氏菌基因组DNA的随机酶切片段（500～1500 bp）连接到载体上，启动phoA表达产物与BCIP产生蓝色反应筛选启动子，经过PCR、测序、比对等分析文库质量。结果库容量达到30000个，覆盖基因组全长的2.6倍，同时验证了4个能够在加入BCIP的平板中表现出蓝色的菌落，发现了4个启动子插入片段。结论成功构建高质量的甲型副伤寒沙门氏菌启动子文库，为进一步研究甲型副伤寒沙门氏菌的基因表达与调控奠定了基础。Objective A promoter library of Salmonella paratyphi A was built with phoA as the report gene.Methods The “promoter trap vector” pPhoA was constructed with phoA as the report gene.DNA fragments of Salmonella paratyphi A were connected to the vector , which enabled phoA to express products that could react with BCIP , producing blue color and screening the promoter.The quality of the library was analyzed by PCR , sequencing and comparison.Results The library had 30000 clones, 2.6 times of the whole length of the genome of Salmonella paratyphi A.Four clones were verified , appearing blue in the plate with BCIP and 4 promoter insert fragments were identified.Conclusions A high-quality promoter library of Salmonella paratyphi A was successfully constructed , which lays a foundation for further studying gene expression and regulation of Salmonella paratyphi A.

关 键 词：甲型副伤寒沙门氏菌 PHOA BCIP 启动子库 

分 类 号：R378.2[医药卫生—病原生物学]