Matsumoto S, Ishii A, Ikura K, Ueda M, Sasaki R
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Biosci Biotechnol Biochem. 1993 Aug;57(8):1249-52. doi: 10.1271/bbb.57.1249.
Human erythropoietin (Epo) cDNA was engineered for expression in cultured tobacco cells (Nicotiana tabacum L. cv. BY2). Two plasmid DNAs were constructed: pCEP, which contained Epo cDNA under control of the cauliflower mosaic virus-derived 35S RNA promoter and terminator, and pNSEP, which contained signal sequence-deleted Epo cDNA under control of the 35S RNA promoter and terminator. By using the electroporation method, each of these plasmid DNAs was transferred into the protoplasts of BY2 cells together with a plasmid, pNR35, which conferred G418-resistance on the cells. Four G418-resistant clones were obtained from protoplasts transfected with pNSEP and pNR35, and only one of them, named 11N, survived in suspension culture. Integration of pNSEP DNA into the genome of 11N cells was confirmed by Southern blot and PCR analyses. Production of Epo mRNA was shown by Northern blot analysis. Epo protein was shown to be expressed in 11N cells by colorimetric enzyme immunoassay. The productivity of Epo in the 11N cells (1 pg/g of wet cells) was very low.
对人促红细胞生成素（Epo）cDNA进行改造，使其在培养的烟草细胞（烟草品种BY2）中表达。构建了两种质粒DNA：pCEP，其在花椰菜花叶病毒衍生的35S RNA启动子和终止子的控制下包含Epo cDNA；pNSEP，其在35S RNA启动子和终止子的控制下包含缺失信号序列的Epo cDNA。通过电穿孔法，将这些质粒DNA中的每一种与赋予细胞G418抗性的质粒pNR35一起转入BY2细胞的原生质体中。从用pNSEP和pNR35转染的原生质体中获得了四个G418抗性克隆，其中只有一个命名为11N的克隆在悬浮培养中存活下来。通过Southern印迹和PCR分析证实了pNSEP DNA整合到11N细胞的基因组中。通过Northern印迹分析显示了Epo mRNA的产生。通过比色酶免疫测定法显示Epo蛋白在11N细胞中表达。11N细胞中Epo的生产率（1 pg/g湿细胞）非常低。
