大鼠寡霉素敏感相关蛋白的克隆及真核表达载体的构建.pdfVIP

第13卷4期 天 津 医 科 大 学 学 报 Vo1．13．No．4 2007年 12月 JOURNAL OF TIANJIN MEDICAL UNIVERSITY Dec．2007 479 大鼠寡霉素敏感相关蛋白的克隆 及真核表达载体的构建 张建红 ，刘艳霞 ，王宝利2范海鸣 ，吴艳娜 (天津医科大学1．药理学教研室；2内分泌研究所，天津 300070) [摘要]目的：克隆大鼠寡霉素敏感相关蛋白(OSCP)的基因并构建pEGFP—N 真核表达载体。方法：采用逆转录聚合 酶链式反应 (RT—PCR)从大鼠脑组织中扩增出OSCP基因，将其插入到pTA2克隆载体中，测序鉴定后亚克隆至 pEGFP—N．真核表达载体并进行酶切、测序鉴定。结果：PCR扩增出约为700 bp的基因片段；重组克隆载体pTA2一 OSCP经测序鉴定证明pTA2载体中插入了目的基因片段；重组表达载体pEGFP—N．一OSCP经酶切、测序鉴定证明其 构建成功。结论：OSCP基因克隆和真核表达载体构建成功，这为下一步开展该基因的重组蛋白表达及功能研究奠定 了基础。 [关键词]寡霉素敏感相关蛋白；克隆；pEGFP—N1真核表达载体；大鼠 [中图分类号]Q78 [文献标识码]A [文章编号]1006—8147(2007)04—0479—04 Cloning of rat oligomycin sensitivity-conferring protein and construction of its eukaryotic expression vector ZHANG Jian—hong’，LIU Yan—xia ，WANG Bao-li ，FAN Hai—ming ，WU Yan-na’ (1．Department of Pharmacology；2．Institute of Endocrinology，Tianjin Medical University，Tianjin 300070，China) ABSTRACT Objective：To clone rat oligomycin sensitivity-conferring protein and construct its eukaryotic expression vector pEGFP—N1．Methods：OSCP gene was amplified from the tissues of rat brain using reverse transcription PCR (RT—PCR)and inserted into pTA2 cloning vector．After DNA sequencing， the target DNA was subcloned into eukaryotic expression vector pEGFP—N1 which was confirmed by restriction endonucleases and DNA sequencing．Results：The amplified gene was about 700 bp in length； the recombinant clone vector pTA2-OSCP identified by DNA sequencing proved that the target DNA fragment was inserted into pTA2 vector；the recombinant eukaryotic expression vector pEGFP-NI identified by restriction endonucleases and DNA sequencing proved that pEGFP-N1——OSCP vector was successfully constructed．Conc