Molecular and Cell Biology, Process Sciences, Biotechnology, Bayer Corporation, Berkeley, CA, U.S.A.,
Cytotechnology. 2001 Sep;37(1):23-30. doi: 10.1023/A:1016124911071.
A mammalian gene expression vector based on cytomegalovirus (CMV)enhancer/promoter (CMVe/p) for the regulation of gene expression was further optimized by adding oriP elements derived from Epstein-Barr virus (EBV) and the Tat/TAR transactivation axisfrom human immunodeficiency virus type 1 (HIV-1). Using the Tat/TAR-oriP expression vector, a transient transfection system was optimized for an extended culture period to produce large amounts of secreted IL-2SA (an IL-2 mutein) in HKB11 cells. We observed a 4-fold increase in IL-2SA expression in cells transfected with vectors containing the HIV-1 transactivation axis (Tat/TAR) or oriP elements alone when compared to cells transfected with the control vector having a CMVe/p. Cells transfected with expression vectors equipped with both oriP and Tat/TAR showed an 18-fold increase in IL-2SA expression. This transient transfection system maintained high secretion of IL-2SA for a period of 10-day with no appreciable loss in expression. We demonstrate that during this 10-day culture period, it was possible to produce 1-100 mg of proteins using 500 mug of plasmid DNA.
基于巨细胞病毒（CMV）增强子/启动子（CMVe/p）的哺乳动物基因表达载体可用于调节基因表达，通过添加源自 EBV 的 oriP 元件和 HIV-1 的 Tat/TAR 反式激活轴，进一步对其进行了优化。使用 Tat/TAR-oriP 表达载体，优化了瞬时转染系统的延长培养期，以在 HKB11 细胞中大量产生分泌型 IL-2SA（IL-2 突变体）。与转染含有 CMVe/p 的对照载体的细胞相比，转染含有 HIV-1 反式激活轴（Tat/TAR）或 oriP 元件的载体的细胞中，IL-2SA 的表达增加了 4 倍。带有 oriP 和 Tat/TAR 的表达载体转染的细胞中，IL-2SA 的表达增加了 18 倍。该瞬时转染系统可在 10 天的时间内保持高水平的 IL-2SA 分泌，而表达水平没有明显下降。我们证明，在这 10 天的培养期内，使用 500µg 质粒 DNA 可以生产 1-100mg 蛋白质。
