Construction Of A Multi-gene Eukaryote Expression Vector And Sub-cellular Localization Of Its Products

[Objective] To construct a multi-gene eukaryote expression vector in which thecoexpression of multiple genes were accomplished and their products were located indifferent organelles in a single cell under control of the same promoter. It can provide theexperimental basis and methods for the multiple genetherapy of tumors and autoimmunediseases.[Methods] A artificial nucleotide sequence including two different2A‘peptideoriginated from FMDV and ERAV and several restriction sites （5-ATG-Nhe I-KpnI-F2A-Xho I-E2A-EcoR I-TAA-BamH I-3） were synthesized to form a single openreading frame.The sequence that contain initiation codon ATG, Nhe I, Kpn I, F2A, Xho I,E2A, EcoR I, termination codon TAA and BamH I is168bp and inserted into theeukaryotic expression vector pcDNA3.1(-) Myc/His(B) by digestion with Nhe Iå'ŒBamH I.ERFP with membrane localization signal, EGFP with nuclear localization signal andcytoplasmic EYFP were in order inserted into the sites between the2A‘peptide forconstructing the eukaryotic expression vector pcDNA3.1RGY. The vector was transfectedto NIH3T3cells by Liposome, and expression levels of the fluorescent proteins and theirsubcellular localizations were detected after24hour and48hour by flow cytometry,fluorescence microscope and confocal microscope, respectively.[Results] The expression of ERFP and EGFP were detected by flow cytometry24hafter the transfection. The everage expression rates of EGFP is68.85%and the everageexpression rates of ERFP is62.59%. the expression rates of the two proteins were quitesimilar and didn‘t have statistics significance. EGFP and ERFP fluorescences wereobserved by fluorescence microscopy. Three fluorescences were observed in one cell byconfocal fluorescence microscope that ERFP was targeted to the membrane, EGFP wastargeted to the nucleus and EYFP was targeted to the cytoplasm.[Conclusion] It was successfully accomplished that express multiple genes with singlepromoter by inserting several2A and multiple genes to the same open reading frame. In addition, different subcellular localization was observed in a single cell by adding signalpeptides into the every gene that makes every gene targeting to the correspondingorganelles and the transmembrane localization need more time than cytoplasm and nucleuslocalization. The study layed the foundation for the multiple genes coexpression andsubcellular localization.