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%.
出处
《高技术通讯》
CAS
CSCD
北大核心
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.
