Kim D W, Uetsuki T, Kaziro Y, Yamaguchi N, Sugano S
Department of Virology, Institute of Medical Science, University of Tokyo, Japan.
Gene. 1990 Jul 16;91(2):217-23. doi: 10.1016/0378-1119(90)90091-5.
We have characterized the promoter region of the human elongation factor 1 alpha-encoding gene (EF-1 alpha) and developed a versatile expression system which has a wide host range and a high efficiency of gene expression. To identify the promoter region of the EF-1 alpha gene necessary for efficient gene expression, we constructed four pEF-CAT plasmids that have the bacterial cat gene fused to four different sites of the human EF-1 alpha gene: (i) ligated to the end of the TATA box (pEF220-CAT); (ii) ligated in exon 1 (pEF204-CAT and pEF233-CAT), and (iii) ligated in exon 2 (pEF321-CAT). All the pEF-CAT plasmids were highly expressed in all the cell types tested, including fibroblasts and lymphoid cells. Plasmid pEF321-CAT, which contains the first exon and the first intron, gave the highest level of cat expression. Plasmids pEF204- and pEF233-CAT, which contain part of the first exon but do not contain the first intron, were less efficient in cat expression than was pEF321-CAT. Plasmid pEF220-CAT, which lacks both the first exon and the first intron, was the least efficient. Plasmid pEF321-CAT was several- to 100-fold more efficient in cat expression than plasmid pSV2-CAT depending on the recipient cell types. The promoter of pEF321 plasmid also directed the stable expression of the bacterial neo gene more efficiently than the promoter of the simian virus 40 (SV40) early gene or the long terminal repeat of Rous sarcoma virus. Using this system, the SV40 early gene and the cDNA encoding human CD4 were also expressed efficiently.
我们已经对人类延伸因子1α编码基因（EF-1α）的启动子区域进行了特征分析，并开发了一种通用的表达系统，该系统具有广泛的宿主范围和高效的基因表达能力。为了鉴定高效基因表达所需的EF-1α基因启动子区域，我们构建了四个pEF-CAT质粒，这些质粒将细菌cat基因与人类EF-1α基因的四个不同位点融合：（i）连接到TATA框末端（pEF220-CAT）；（ii）连接到外显子1（pEF204-CAT和pEF233-CAT），以及（iii）连接到外显子2（pEF321-CAT）。所有pEF-CAT质粒在所有测试的细胞类型中都有高表达，包括成纤维细胞和淋巴细胞。包含第一个外显子和第一个内含子的质粒pEF321-CAT产生了最高水平的cat表达。包含第一个外显子部分但不包含第一个内含子的质粒pEF204-和pEF233-CAT在cat表达方面比pEF321-CAT效率低。既缺乏第一个外显子又缺乏第一个内含子的质粒pEF220-CAT效率最低。根据受体细胞类型，质粒pEF321-CAT在cat表达方面比质粒pSV2-CAT效率高几倍至100倍。pEF321质粒的启动子也比猿猴病毒40（SV40）早期基因的启动子或劳氏肉瘤病毒的长末端重复序列更有效地指导细菌neo基因的稳定表达。使用该系统，SV40早期基因和编码人类CD4的cDNA也能高效表达。
