人组织型纤溶酶原激活剂(t-PA)组合突变体FrGGl在CHO细胞中的高效表达  

High level Expression of a Combination Mutant (FrGGl) of Human Tissue type Plasminogen Activator (t PA) in Chinese Hamster Ovary Cells

作　　者：赵庆国[1,2] 黄培堂[1,2] 刘士辉[1,2] 

机构地区：[1]军事医学科学院生物工程研究所 [2]解放军白求恩国际和平医院

出　　处：《生物工程学报》1997年第1期98-101,共4页Chinese Journal of Biotechnology

基　　金：国家自然科学基金

摘　　要：为了得到ｔＰＡ组合突变体ＦｒＧＧＩ在ＣＨＯ细胞中的高效表达，将表达质粒筛选基因启动子上游的增强子（ｅｎｈａｎｃｅｒ）去除，构建了ＦｒＧＧＩ真核表达质粒ｐＺＬＦｒＧＧＩ。酶切线性化后，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－）。氨甲喋呤（ＭＴＸ）筛选转染细胞，混合加压，挑选克隆，在１×１０－７ｍｏｌ／ＬＭＴＸ压力下，获得表达水平达１５００～２５００ＩＵ／１０６细胞·２４ｈ的细胞株。此细胞株表达水平稳定，形态良好，倍增时间约为３６ｈ，且有进一步提高表达水平的潜能。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 dhfr selective gene was controlled only by SV40 promoter.Then the large amount of linearized pZLFrGGI was transfected into CHO dhfr - cells by electroporation.The expression clones appeared after two weeks of selection in selective medium plus MTX.Then mixed the clones 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 expression level reached to 1500～2500IU/10 6 cells·24h.The biological properties indicated this cell line may be used as a candidate for engineering purpose.

关 键 词：药物 组织型 纤溶酶原激活剂 突变体 基因表达 

分 类 号：R392-33[医药卫生—免疫学] R394.8[医药卫生—基础医学]