Prion protein gene expression in cultured cells.
Prion protein gene expression in cultured cells.
复制标题
培养细胞中朊病毒蛋白基因的表达。
DOI：
10.1093/protein/2.1.69
复制
发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Prusiner,SB
中科院分区：
文献类型：
--
作者：
Scott,MR;Butler,DA;Bredesen,DE;Wälchli,M;Hsiao,KK;Prusiner,SB
单拷贝基因编码朊病毒蛋白（PrP）的羊瘙痒病（PrPSc）和细胞（PrPC）亚型。培养的细胞系被发现表达内源性PrP mRNA的水平与成年啮齿动物的大脑中观察到的水平相当;然而，这些细胞总是被发现表达水平大大降低的PrP。在所有检查的细胞系中，PrP通过Western免疫印迹分析检测不到。这些细胞也是连接仓鼠PrP基因开放阅读框与几个强真核启动子的表达构建体的不良受体;通过转染这些表达载体获得的稳定克隆未能显示PrP的表达升高。当使用昆虫杆状病毒或哺乳动物SV 40为基础的载体产生极高水平的PrP mRNA时，产生了大量的PrP，尽管在这两种情况下，蛋白质的加工方式显然与大脑中观察到的PrPC不同。在猴COS-7细胞中，使用SV 40晚期启动子载体的表达系统中，PrP的显著部分被转运到细胞表面，PrPC在体内被发现。由杆状病毒载体合成的PrP未能诱导仓鼠瘙痒病，并且不具有与感染性相关的PrPScisoform特征。SV 40晚期启动子载体系统可用于阐明PrPC在羊瘙痒病感染过程中的作用以及PrPC在正常代谢中的功能。
A single copy gene encodes both the scrapie (PrPSc) and cellular (PrPC) isoforms of the prion protein (PrP). Cultured cell lines were found to express the endogenous PrP mRNA at levels comparable to those observed in the brains of adult rodents; however, these cells were invariably found to express greatly reduced levels of PrP. In all the cell lines examined, PrP was undetectable by Western immunoblot analysis. These cells were also poor recipients for expression constructs linking the hamster PrP gene open reading frame to several strong eukaryotic promoters; stable clones derived by transfection of these expression vectors failed to show elevated expression of PrP. When extremely high levels of PrP mRNA were produced using either an insect baculovirus or a mammalian SV40 based vector, significant quantities of PrP were produced, although in both cases the proteins were apparently processed differently from the PrPCobserved in brains. In an expression system using an SV40 late promoter vector in monkey COS-7 cells, a significant fraction of PrP was transported to the cell surface where PrPCis foundin vivo. PrP synthesized by the baculovirus vector failed to induce scrapie in hamsters and did not possess the characteristics of the PrPScisoform associated with infectivity. The SV40 late promoter vector system may permit experiments designed to elucidate the role of PrPScduring scrapie infection as well as the function of PrPCin normal metabolism.