摘要
将IL-2cDNA和二氢叶酸还原酶(DHFR)基因插入质粒pSVL,组建了pSVLD/IL-2,转化CHOdhfr^-细胞,在选择培养基中筛选出表达阳性的细胞克隆,为了提高表达量,在DMEM培养基中逐渐增加氨甲喋呤(MTX)的浓度,使已整合的IL-2基因同DHFR基因一同扩增,最后得到了能耐受50μmol/L MTX的4个细胞株,表达活性大于5.1×10~5U/L,转化的细胞株传30代仍能稳定地分泌IL-2。同时,对CHO细胞分泌的糖基化IL-2进行了初步的纯化研究,将含有IL-2的细胞培养液用硫酸铵沉淀浓缩后,再经分子筛柱(Sepharose 4B)、离子交换柱(DEAE-SepharoseCL 6B)以及反向柱ProRPC3步纯化后,总回收率大于40％,比活性达9.6×10~6U/mg。
A plasmid pSVLD/IL-2 containing SV40 promoters huma n IL-2 cDNA and DHFR gene was constructed and was transfected into CHO celis by lipofectin method. In order to amplify IL-2 cDNA, the transformants were grown in selective medium with increasing concentrations of MTX. Four cell clones resistant to 50μmol/L MTX were obtained. Biologically active IL-2 was found in supernatant with a titer of 5.1×106 U/L.These cloned cell lines were shown to constitutively produce IL-2 after passa-ging for more than 30 times.The rIL-2 was purified by consecutive ion-exchange chromatography gel filtration and ProRPC reversed phase column, resulting in a specific activity of 9.6×106 U/mg and 46% recovery.
出处
《病毒学报》
CAS
CSCD
北大核心
1993年第3期203-208,共6页
Chinese Journal of Virology
基金
国家863计划高科技生物领域资助
关键词
白细胞介素2
CHO细胞
提纯
Interleukin-2, Recombinant DNA, Chinese Hamster Ovary Cells
