pIRES2-NGF-NT-3真核表达载体的构建与鉴定  

Construction and Identification of pIRES2-NGF-NT-3 Bicistronic Eukaryotic Expression Vector

作　　者：栗炳南[1] 李卫东[1] 林俊堂[1] 丰慧根[1] 原志庆[1] 

机构地区：[1]新乡医学院生命科学技术学院,中国河南新乡453003

出　　处：《生命科学研究》2014年第4期315-322,共8页Life Science Research

基　　金：河南省教育厅科学技术研究重点项目(13A180850);新乡医学院重点领域招标课题(ZD2011-16);新乡医学院科研培育基金项目(2013QN117);新乡医学院研究生科研创新支持计划项目(YJSCX201231Y);新乡医学院博士启动金项目(2011);2010年度河南省省院科技合作项目(102106000017);2012年度河南省重大科技攻关项目(122101310100)

摘　　要：构建双基因共表达载体pIRES2-NGF-NT-3并检测其在HEK293细胞中的表达。人神经生长因子(nerve growth factor,NGF)和神经营养素3(neurotrophin-3,NT-3)是采用直接PCR的方法从人外周血单个核细胞的基因组DNA中获取,将人神经生长因子的cDNA片段插入到pIRES2-EGFP多克隆位点构建成为pIRES2-NGF-EGFP。神经营养素3 cDNA片段通过替换绿色荧光蛋白基因(EGFP)的方式插入到pIRES2-NGFEGFP中构建成为pIRES2-NGF-NT-3双基因共表达载体,将pIRES2-NGF-NT-3用脂质体转染HEK293细胞并采用RT-PCR与Western-blot的方法检测其表达。人神经生长因子和神经营养素3被克隆,通过测序和酶切鉴定的得知与基因库报道序列一致。pIRES2-NGF-NT-3转染HEK293细胞后双基因在mRNA和蛋白水平均得到了表达。人神经生长因子和神经营养素3双基因真核表达载体成功构建,它提供了一个新的表达系统,为进一步研究双基因的功能奠定了基础。Construction of double gene co-expression vector pIRES2-NGF-NT-3 and detection of its expression in HEK293 cells were performed. Human nerve growth factor（NGF） and neurotrophin-3（NT-3）genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by direct PCR.The NGF cDNA fragment was inserted into the multiple cloning sites of pIRES2-EGFP to generate the bicistronic eukaryotic expression plasmid pIRES2-NGF-EGFP. Then NT-3 cDNA fragment was cloned into the pIRES2-NGF-EGFP to replace EGFP creating plasmid pIRES2-NGF-NT-3. Then the recombinant plasmid was transfected into HEK293 cells with the cationic liposome. RT-PCR and Western blotting were used to detect NGF and NT-3 mRNA and protein expression in HEK293 cells. Construction of pIRES2-NGF-NT-3 was identified by means of double digestion and sequencing. The expression of mRNA and protein of NGF and NT-3 was significantly up-regulated after transfection. The NGF and NT-3 co-expression plasmid is successfully constructed. It provides a novel expression system, which enable to further study on the functions of NGF and NT-3 genes.

关 键 词：人神经生长因子 神经营养素3 真核双表达载体 内部核糖体进入位点 

分 类 号：Q812[生物学—生物工程]