【摘要】 目的观察K562细胞分泌的外泌体exosomes。构建CD63与绿色荧光蛋白融合蛋白载体,并在K562细胞中表达。方法采用梯度离心的方法分离K562细胞培养上清中的exosomes,并用扫描电镜观察。构建pEGFP-N1-CD63重组质粒,以不同方式转染至K562细胞,观察表达效率的差异。结果质粒pEGFP-N1-CD63经测序鉴定,通过激光共聚焦显微镜观察,能够在K562细胞中表达。Amaxa核转染的效率高于脂质体转染的方式。结论成功构建pEGFP-N1-CD63质粒并在K562细胞中表达,可对exosomes进行示踪,为后续实验奠定基础。更多还原

【Abstract】 Objective To obtain the exosomes from the culture media of K562 cells. To construct and express CD63-GFP fusion gene in K562 cells. Methods Exosomes from K562 culture medium were obtained by ultracentrifugation, and the morphology of exosomes were observed under a transmission electron microscope. CD63 was amplified by RT-PCR and the amplified fragments were inserted into pEGFP-N1 vector. Recombinant plasmid pEGFP-N1-CD63 was transfected into K562 cells. Results CD63-GFP protein was found to be expressed by K526 cells. The transfection efficiency of the Amaxa Nucleofector system was higher than the transfection with Fugene 6 and Lipofectamin2000. Conclusion The CD63-GFP fusion gene was successfully cloned and expressed in K562 cells. The expressed recombinant proteins could be used as a tracer of exosomes.更多还原