多基因真核表达载体的构建及其产物的亚细胞定位

Construction of multi-gene eukaryote expression vector and sub-cellular location of its products

目的构建一种多基因表达载体,实现同一载体单一启动子的调控下表达多个基因以及这些基因在同一细胞不同细胞器中的定位。方法利用存在于某些病毒核酸序列中的不同2A肽,合成1段包括2个2A肽和不同限制性内切酶位点的多肽序列,形成单一的开放读码框,插入真核表达载体pcDNA3.1(-)Myc/His(B)相应的多克隆位点中,然后将带有膜定位的红色荧光蛋白(ERFP)、核定位的绿色荧光蛋白(EGFP)和胞浆定位的黄色荧光蛋白(EYFP)顺序插入2A肽之间的位点中,构建多基因真核表达载体pcDNA3.1 RGY。用脂质体转染小鼠成纤维NIH3T3细胞和人肾胚293细胞,利用流式细胞仪、荧光显微镜和共聚焦显微镜检测各种荧光蛋白的表达和亚细胞定位。结果转染24 h后用流式细胞仪检测,可同时检测到EGFP和ERFP的表达,且两种荧光蛋白的表达率十分接近;荧光显微镜下同时观察到绿色和红色荧光;共聚焦显微镜观察到在单一细胞上同时显示3种荧光,ERFP定位在细胞膜上,EGFP在细胞核中,而EYFP在细胞质中。结论通过串联方式将多个2A肽和基因置于同一个开放读码框中可实现在同一启动子的调控下表达多个基因。此外,在每个基因序列中加入不同的信号肽序列可将相应基因同时靶向不同的细胞器,实现同一细胞中不同的亚细胞定位。

Objective To construct a multi-gene eukaryote expression vector in which co-expression of multiple genes was achieved and their products were located in different organelles in a single cell under control of the same promoter.Methods Sequences of polypeptide including two different ＇2A＇ peptides and several restriction sites were synthesized to form a single open reading frame using the presence of different ＇2A＇ peptide sequences and inserted into the eukaryotic expression vector pcDNA3.1（-） Myc/His（B）.ERFP with membrane location signal,EGFP with nuclear location signal and cytoplasmic EYFP were respectively inserted into the sites between the ＇2A＇ peptides for the construction of eukaryotic expression vector pcDNA3.1 RGY.The vector was transfected to NIH3T3 cells or 293 cells by Liposome,and expression levels of the fluorescent proteins were measured and their sub-cellular locations were detected by flow cytometry,fluorescence and confocal microscopy,respectively.Results The expression levels of ERFP and EGFP were measured by flow cytometry in 24 h after the transfection,and the expression rates of the two proteins were quite similar.The green and red fluorescences were observed under fluorescence microscope and green,red and yellow fluorescences in a single cell were observed under confocal fluorescence microscope.ERFP,EGFP and EYFP were located on the cytomembrane,in the nuclei and the cytoplasm,respectively.Conclusion Expression of multiple genes can be achieved under control of the single promoter by inserting several 2A peptides and multiple genes into the same open reading frame.In addition,different sub-cellular locations can be observed in a single cell by adding signal peptide into the gene that makes it target the corresponding organelles.