人组织型纤溶酶原激活剂(t-PA)突变体在CHO细胞中的高效表达

High-level Expression of A Combination Mutant (FrGGI) of Human Tissue-type Plasminogen Activator (t-PA) In Chinese Hamster Ovary Cells

构建了二氢叶酸还原酶(dhfr)选择基因启动子上游无增强子(cnhancer)的组织型纤溶酶原激活剂(t-PA)组合突变体FrGGI真核表达质粒pZLFrGGI。将pZLFrGGI酶切线性化,采用大剂量DNA电击介导法,转染dhfr基因缺陷型中国仓鼠卵巢细胞系(CHO-dhfr^-)。用氨甲喋呤(MTX)筛选转染细胞。混合加压后,挑选克隆,在1×10^(-7)mol/L MTX压力下,得到了表达水平达1500～2500IU/10~6细胞·24小时的细胞株。此细胞株表达水平稳定,形态良好,倍增时间为36小时,且有进一步提高表达水平的潜能,有望发展为工程细胞株。

In this paper, pZLFrGGI, the eukaryotic expression plasmid of t-PA combination mutant FrGGI, was constructed, in which the transcription of foreign gene was controlled by adenovirus major later promoter (AdMLP) and SV40 enhancer and the transcription of selective gene dhfr was controlled only by SV40 promoter. Then the large amount of linearized pZLFrGGI was transfected into CHO-dhfr cells by electroporation. The expression c ones appeared after two weeks of selection in selective medium plus MTX. Then the clones mixed and continued to co-amplify the foreign gene in the selective medium with stepwise increasing concentrations of MTX to 1 × 10-7 mol/L. After two round of cloning of the expression cells, a high-level, stable expression cell line was obtained with exppression level reached to 1500-2500IU/106 cells · 24h. The biological properties indicated this cell line might be used as a candidate for engineering purpose.