大鼠肝类固醇激素和异质素受体基因Nrli2过表达载体的构建和表达  

Construction and expression of the overexpression vector of steroid xenobiotic receptor gene Nrli2 in rat liver

作　　者：徐浩[1] 李明意[1] 胡敏[1] 张谷裕[1] 

机构地区：[1]广东医学院附属医院肝胆外科,广东湛江524001

出　　处：《中华实验外科杂志》2013年第8期1646-1648,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81041099）;广东省自然科学基金资助项目（S2011010003750）;广东医学院附属医院博士科研基金资助项目;广东医学院青年基金资助项目;广东医学院附属医院青年基金资助项目（2009K04）

摘　　要：目的构建类固醇激素和异质素受体（SXR）基因Nrli2过表达质粒，并通过HEK-293细胞系验证其表达。方法大鼠肝组织中的总RNA逆转录后将含相应酶切位点的引物扩增到Nrli2编码框，并将Nrli2片段亚克隆至pcDNA3．1（＋），同时对其进行酶切和测序鉴定，挑选pcDNA3．1（＋）．Nrli2转染至293细胞系中，48h收集细胞进行荧光定量聚合酶链反应（FQ—PCR），72h后Westernblot法检测。结果成功扩增Nrli2编码区，并将其克隆至载体pcDNA3．1中。HEK-293细胞系、pcDNA-3．1（＋）、pcDNA3．1（＋）-Nrli2中SXR的FQ—PCR结果分别为1．00±0．09、4．88±0．94、473274．04±28784．24，后者分别与前两者比较，差异有统计学意义（P〈0．01）。结论成功构建Nrli2过表达载体，并在HEK-293细胞中成功表达。Objective To construct the overexpression plasmid of steroid xenobiotic receptor （SXR） gene Nrli2, and verify its expression by HEK-293 cells. Methods The inverse transcription of total RNA in rat liver was amplified to Nrli2 coding frame with enzyme loci primer, and the Nrli2 was sub- cloned into the pcDNA3.1, which was subjected to enzyme digestion and verified by sequencing. pcDNA3.1 （ ＋ ） -Nrl i2 was transfected into 293 cells, and 48 h later, the cells were harvested for fluores- cent quantitative polymerase chain reaction （ FQ-PCR）, and 72 h later for Western blotting. Results The Nrl i2 coding region was successfully amplified, and cloned into the vector pcDNA3.1. The expression of SXR gene Nrli2 in HEK-293 cells and pcDNA-3. 1 （ ＋ ） transfected group was significantly lower than in pcDNA3.1 （ ＋ ） -Nrl i2 transfected group （ both P 〈 0. 01 ）. Conclusion We have successfully constructed Nrli2 eukaryotic overexpression vector, which was successfully expressed in 293 cells.

关 键 词：类固醇激素 异质素受体 载体构建 基因克隆 

分 类 号：R363[医药卫生—病理学]