原核生物pPLacZ报道分子系统的构建及鉴定  

Construction and Identification of Prokaryotic Molecule Reporter System pPlacZ

作　　者：黄勇[1] 张晓楠[1] 王丽[1] 宋庆贺[1] 王涛[1] 陈南春[1] 陈苏民[1] 

机构地区：[1]第四军医大学生物化学与分子生物学教研室,西安710032

出　　处：《科学技术与工程》2006年第20期3268-3271,3275,共5页Science Technology and Engineering

摘　　要：构建可以鉴定原核基因启动子和检测基因转录活性的报道分子系统pPLacZ。用PCR方法从野生型大肠杆菌基因组DNA中扩增lacZ基因的全基因序列(3258bp)和结构基因序列(3089bp)克隆入经改建的含有pUC19的多克隆位点的pBR322载体中,经酶切和测序鉴定正确的两个质粒分别命名为pPLacZ-Control和pPLacZ-Basic。用PCR从野生型大肠杆菌基因组DNA中扩增热休克基因htpX的启动子序列并克隆入pPLacZ-Basic中,经酶切测序鉴定正确的报道载体pPLacZ-htpX转入细菌中,检测高温应激时β-半乳糖苷酶活性水平。成功构建了原核生物报道分子系统pPLacZ以及检测基因htpX的报道质粒pPLacZ-htpX。应用此系统通过检测LacZ的比活性成功测定了大肠杆菌htpX基因高温应激时其转录活性的动态变化。原核生物质粒报道分子系统pPLacZ操作简便,可替代国际通用的原核噬菌体报道分子系统对原核基因的启动子进行鉴定和转录活性检测。To construct a reporter system pPlacZ that can be used to identify prokaryotic gene promoter and analyze the transcriptional activity of genes. The complete gene sequence （3 258 bp） and encode DNA sequence （3 089 bp） of the E. coli lacZ was amplified from the genomic DNA of E. coli W3110 by PCR. The amplified lacZ fragments was cloned into the reconstructed plasmid of pBR322 containing the MCS sequence of pUC19 to pPLacZ-Control and pPLacZ-Basic. The promoter sequence of the heat shock gene htpX was amplified from the E. coli genomic DNA by PCR and was cloned into the plasmid of pPLacZ-Basic to form the reporter vector pPLacZ-htpX. Then induced pPLacZ-htpX into E. coli W3110 cells and to detect the activity of β-Galactosidase after heat shock induction. The Prokaryotic Molecule Reporter System pPlacZ was constructed successfully, and was identified by analysis of the transcriptional activity of htpX after heat shock induction. This reporter system can be used to identify the promoter of prokaryotic genes and analyze the transcriptional activity of prokaryotic genes.

关 键 词：原核基因 报道分子系统 LACZ 

分 类 号：R392.33[医药卫生—免疫学]