人Neuritin真核表达载体的构建及其在细胞中的定位

Construction of the eukaryotic expression vector carrying encoding gene of human neuritin and its location in AD293 cells

目的获得人突触生长素Neuritin基因并构建pEGFP-C1-neuritin真核表达载体,观察Neuritin在人AD293细胞中的定位。方法利用PCR获得Neuritin基因,然后克隆到pEGFP-C1载体中,利用脂质体将构建的表达载体转染AD293细胞,荧光观察融合蛋白的表达。结果从人类cDNA文库中得到Neuritin的完整开放阅读框序列,将其重组到pEGFP-C1载体中并转染AD293细胞,荧光显示Neuritin蛋白定位在细胞浆中。结论成功构建Neuritin真核表达载体并在人AD293细胞表达,证明Neuritin蛋白定位于细胞浆,为进一步研究Neuritin与其他蛋白的相互作,探讨Neuritin功能及作用机制奠定理论基础。

【Objective】To construct the recombinant eukaryotic expression vector pEGFP-C1-neuritin carrying encoding gene of human neuritin and test its location in AD293 cells.【Methods】The full length of neuritin cDNA sequence was amplified from human cDNA library by PCR.Then neuritin completed ORF sequence was ligated into plasmid pEGFP-C1.AD293 cells were transfected with pEGFP-C1-neuritin vector.The expression of neuritin was observed by fluorescence microscope.【Results】Neuritin sequence obtained by PCR was recombined into pEGFP-C1 vector.AD293 cells were transfected with pEGFP-C1-neuritin vector and neuritin protein was found locating in cytoplasm.【Conclusions】The eukaryotic expression plasmid pEGFP-C1-neuritin has been successfully constructed and expressed in the AD293 cells.Neuritin protein has been proved locating in cytoplasm.