用弱化dhfr双顺反子载体在CHO细胞中高效表达低分子量尿激酶突变体  

High-level expression of a low molecular weight urokinase variant with attenuated dhfr bicistronic vector in CHO cells

作　　者：潘淑媛[1] 高丽华[1] 胡显文[1] 杨波[1] 殷亮[1,2] 胥照平[1] 张正光[1] 郗永义[1] 陈惠鹏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]吉林大学生命科学院,长春130061

出　　处：《高技术通讯》2006年第8期834-840,共7页Chinese High Technology Letters

基　　金：863计划（Z18-03-12）资助项目.

摘　　要：将二氢叶酸还原酶基因（dhfr）克隆至pIRES载体中弱化的脑心肌炎病毒（ECMV）内部核糖体进入位点（IRES）的下游，构建了含有弱化dhfr筛选标记和可用氨甲蝶呤（MTX）加压提高表达水平的双顺反子真核表达载体pIRES—dhfr。利用该表达载体表达了一种无糖基化和具有凝血酶抗性的低分子量尿激酶型纤溶酶原激活剂（LMW—uPA）突变体（缺失尿激酶原的1—143位氨基酸，Arg156→Lys，Asn302→Ala），用脂质体转染方法将表达载体pIRES—dhfr／LMW—UK转染CHO—dhfr-细胞后经过一轮MTX筛选，几乎所有获得的单克隆细胞株都表达LMW—UK，其中约50％为表达水平较接近（500—5000IU／10^6cells／d）的高表达阳性克隆。用转瓶无血清培养表达水平约为17．5pg／cell／d的rCHO细胞系LB2-UK，收集的上清经过阳离子交换柱和凝胶过滤层析两步纯化后，获得的重组蛋白的纯度可以达到99％。用S-2444发色底物法检测，所获得的突变体双链UK比例大于98％。A attenuated selection marker bicistronic eukaryotic vector for stable and high-level expression of recombinant protein in mammalian cells by selective pressure of methotrexate （MTX）, designated pIRES-dhfr, was developed by inserting the dihydrofolate reductase （dhfr） gene into the downstream site of the attenuated encephalomyocarditis virus （ECMV） internal ribosome entry site （IRES） in pIRES vector. This vector was adopted to express a variant of low molecular weight urokinase-type plasminogen activator （LMW-uPA）, which is a unglycosylated and thrombin-resistant mutant of prourokinase that has no EGF-like domain and kringle domain （ 1-143 aa of pro-UK was deleted）, and has two amino acid substitutions in active site region （Arg156→Lys, Asn302→Ala）. The dhfr-deficient CHO cells were transfected by pIRES- dhfr/LMW-UK vector with lipofectine-mediated gene transfer technique, and the results showed that following one round of amplification under the selection of MTX and monoclonal culture, all of monoclonal cell lines were positive clone, and about 50% of recombinant cell lines expressed the similar and high levels of LMW-uPA （500-5000 1U/10^6cells/d）. The recombinant protein was purified with cation-exchange chromatography and gel-filtration from the culture supernatant, which was harvested from serum-free culture of a recombinant CHO cell line of expression level at about 17.5pg/cell/d, LB2-UK, in roler bottles. The purity of the recovered recombinant protein was about 99%. Analyzed by S-24A4 chro-/ mogenic assay, the active two-chain urokinase （tcu-PA） ratio of the LMW-uPA variant was about 98%.

关 键 词：低分子量尿激酶 突变体 内部核糖体进入位点 二氢叶酸还原酶基因 重组CHO细胞 

分 类 号：Q782[生物学—分子生物学]