干酪乳杆菌同源整合载体的构建与鉴定  

Construction and Identification of Lactobacillus Case Homologous Recombination Vector

作　　者：郭建军[1] 龚小华[1] 袁林[1] 曾静[1] 付锦楠[1] 何顺华[1] 

机构地区：[1]江西省科学院微生物研究所,南昌330096

出　　处：《江西科学》2015年第4期491-494,518,共5页Jiangxi Science

基　　金：江西省科技支撑计划项目(20142BBF60036);江西省自然(青年)科学基金项目(2013BAB214014)

摘　　要：构建乳酸菌同源重组载体,以实现外源基因在乳酸菌中的整合型表达。PCR扩增lacZ基因表达盒lacZ-cassette,和upp基因表达盒upp-cassette,SOE-PCR扩增lacZ-P-upp,产物回收后连接到pORI28载体中,构建载体pORZP;根据干酪乳杆菌Lactobacillus casei 393全基因组中lai基因序列设计引物,PCR扩增lai基因两端同源重组臂lai-up和lai-down基因序列,产物经回收后,连接至pORZP载体中,得同源重组载体pORZPlai。双酶切及DNA测序结果证实,成功构建了同源重组载体pORZP-lai,为实现外源基因在乳酸菌中的非抗性、整合型表达奠定了基础。The purpose of this study was to construct homologous integration vector of Lactobacillus Case and to implement the ectogenic genome integration into Lactobacillus Case genome successfully.The lac Z gene with its promoter and catabolite activator protein（ CAP） binding site was mplified from the pUC19 vector,and the Lactobacillus Casei 393 upp gene with its putative promoter region was PCR amplified,Both purified PCR amplicons were joined by SOE-PCR,The resulting lac Z-Pupp fusion products were ligated into the blunt-ended pORI28 backbone. Using chromosome DNA of Lactobacillus Casei 393 as template,the left and right arm of lai were amplified by PCR and purified by purification kit. The PCR product was cloned into pORZP vector,then the homologous recombination pORZP-lai vector was obtained. The results showed that the target genes were amplified successfully,double enzyme digestion and DNA sequencing confirmed that homologous recombination vector pORZP-lai was successfully constructed.

关 键 词：干酪乳杆菌 同源重组 表达载体 

分 类 号：Q936[生物学—微生物学]