Samanta H K, Kim Y J
Eugene Tech International, Inc., Allendale, New Jersey.
J Biotechnol. 1991 Aug;20(1):1-16. doi: 10.1016/0168-1656(91)90031-p.
We have expressed human tissue plasminogen activator (t-PA) gene at high levels in a mouse cell line. The t-PA cDNA with deletion of the long 3' untranslated region was inserted into a bovine papilloma virus (BPV) derived vector under the control of a mouse metallothionein promoter. The mouse metallothionein (mMT) gene also provided signals for splicing and polyadenylation. Mouse C127 cells transfected with this construct secreted t-PA at high levels into the cell culture medium. When an SV40 polyadenylation signal was inserted between the t-PA cDNA and the mMT splicing signals, the expression level increased by several fold. The expression levels did not increase further upon either introduction of Rous sarcoma virus LTR into the plasmid or mutation of the translation initiation context sequence to conform with the consensus one. Most of the plasmid appears to be integrated into the host chromosome. Cells producing high levels of t-PA tend to detach from the dish in a few days after passage. When grown on porous microcarriers, however, such cells can be maintained in culture for months and t-PA can be harvested continuously.
我们已在小鼠细胞系中高水平表达了人组织纤溶酶原激活物（t-PA）基因。将缺失长3'非翻译区的t-PA cDNA插入到受小鼠金属硫蛋白启动子控制的源自牛乳头瘤病毒（BPV）的载体中。小鼠金属硫蛋白（mMT）基因还提供了剪接和聚腺苷酸化信号。用该构建体转染的小鼠C127细胞将t-PA高水平分泌到细胞培养基中。当在t-PA cDNA和mMT剪接信号之间插入SV40聚腺苷酸化信号时，表达水平提高了几倍。在质粒中引入劳氏肉瘤病毒LTR或使翻译起始上下文序列突变以符合共有序列后，表达水平均未进一步提高。大多数质粒似乎整合到了宿主染色体中。产生高水平t-PA的细胞在传代后几天往往会从培养皿中脱离。然而，当在多孔微载体上生长时，此类细胞可在培养中维持数月，并且可以持续收获t-PA。
