新基因PRR11的克隆、原核表达及鉴定  

Cloning,Prokaryotic Expression and Identification of Novel Human Gene PRR11

作　　者：崔涛[1,2] 兰欢[1,2] 杜刚[1,2] 刘革力[2] 易发平[1,2] 卜友泉[1,2] 宋方洲[1,2] 

机构地区：[1]重庆医科大学生物化学与分子生物学教研室,重庆400016 [2]重庆医科大学分子医学与肿瘤研究中心,重庆400016

出　　处：《生物技术通报》2009年第3期98-101,105,共5页Biotechnology Bulletin

基　　金：国家自然科学基金(30801356;30871237;30872758);重庆市科委自然科学基金(2007BB5302);重庆医科大学重点课题(XBZD200707)

摘　　要：克隆新基因PRR11的开放阅读框区,构建其原核表达载体,并进行表达检测及鉴定。以Hela细胞cDNA为模板,RT-PCR扩增PRR11基因,克隆入原核表达载体PET-28a中,酶切、测序鉴定确认获得PRR11基因的重组原核表达载体PET-28a-PRR11。然后把PET-28a-PRR11重组载体转化到BL21中,经IPTG诱导蛋白表达,提取细胞蛋白并采用SDS-PAGE和蛋白质免疫印迹法检测目的蛋白的表达情况。结果表明成功扩增了PRR11基因,双酶切、测序鉴定证实目的基因成功克隆到原核表达载体PET-28a中,目的蛋白成功表达。成功构建的PRR11基因的原核表达载体,及PRR11的重组蛋白表达产物,为进一步研究PRR11的基因功能奠定了基础。It was to clone PRR11 gene,construct recombinant prokaryotic expression vector,and identify its exogenous expression in E.eoli. PRR11 gene was amplified by RT-PCR from Hela cell cDNA,and cloned into prokaryotic expression vector PET-28a to construct PET28a-PRR11. The recombinant plasmid was subsequently transducted into BL21 and the exogenous protein expression was induced By IPTG. The exogenous PRR11 expression was examined by SDS-PAGE and Western immunoblotting. Results showed that PRR11 gene was successfully amplified by PCR and cloned into PET-28a vector by restriction endonuclease and sequencing analysis. SDS-PAGE and Western blotting demonstrated that the PRR11 was exogenously expressed in E.eoli BL21 with a relatively high level. Thus,the successfull expression of PRRII gene in E.coli could provides the basis for further research on PRR11 function.

关 键 词：PRR11基因 PET-28a 原核表达 

分 类 号：Q78[生物学—分子生物学] Q255