Ward T, Pipkin P A, Clarkson N A, Stone D M, Minor P D, Almond J W
School of Animal and Microbial Sciences, University of Reading, Whiteknights, UK.
EMBO J. 1994 Nov 1;13(21):5070-4. doi: 10.1002/j.1460-2075.1994.tb06836.x.
Using an anti-receptor mAb that blocks the attachment of echovirus 7 and related viruses (echoviruses 13, 21, 29 and 33), we have isolated a complementary DNA clone that encodes the human decay-accelerating factor (CD55). Mouse cells transfected with the CD55 clone bind echovirus 7, and this binding is blocked by the anti-receptor mAb. The method used (CELICS) allows rapid and direct cloning of genes encoding cell surface receptors. It is based on episomal replication and high efficiency expression of complementary DNA clones in the vector pCDM8 in COS or WOP cells, in conjunction with a sensitive immuno-focal screen that uses antibody probes linked to beta-galactosidase. Receptor positive cells were identified by a colour change and isolated individually using a micromanipulator. DNA extracted from a small number of cells was then cloned directly in Escherichia coli.
利用一种能阻断埃可病毒7及相关病毒(埃可病毒13、21、29和33)附着的抗受体单克隆抗体,我们分离出了一个编码人衰变加速因子(CD55)的互补DNA克隆。用CD55克隆转染的小鼠细胞能结合埃可病毒7,且这种结合可被抗受体单克隆抗体阻断。所使用的方法(CELICS)能快速直接地克隆编码细胞表面受体的基因。它基于附加型复制以及互补DNA克隆在载体pCDM8中于COS或WOP细胞内的高效表达,同时结合一种敏感的免疫聚焦筛选,该筛选使用与β - 半乳糖苷酶相连的抗体探针。通过颜色变化鉴定出受体阳性细胞,并使用显微操作器逐个分离。然后将从少量细胞中提取的DNA直接克隆到大肠杆菌中。