转译优化对EPO基因在COS7细胞中表达的影响  

Effect of Optimized Translational Initiation on EPO cDNA Expression in COS7 Cells

作　　者：童涌[1] 周向军[1] 曹韫旭[1] 温肇荣[1] 陆德如[1] 

机构地区：[1]第二军医大学医学生物技术和分子遗传研究所,上海200433

出　　处：《生物工程学报》1994年第4期342-345,共4页Chinese Journal of Biotechnology

摘　　要：利用COS7细胞暂时表达系统,研究转译起始序列对EPO-cDNA表达的影响。通过DNA重组技术,构建了原EPO-cDNA表达载体pCSV-EPO(1),其转译起始序列为5＇AATTCATGG3＇。同时通过定点突变技术,将起始序列改变成5＇CCACCATGG3＇,而构建了另一表达载体pCSV-EPO(2)。后者经序列分析证明无误后和前者均通过DEAE-dextran法转染COS7细胞,用ELISA法定量测量EPO表达水平,转染后48小时及72小时收集含pCSV-EPO(1)的COS7细胞上清,测定结果为1580pg/ml及954pg/ml,而含pCSV-EPO(2)的上清则为25300pg/ml和17450pg/ml。这表明经优化起始序列的基因表达远高于未经优化的。Using DNA recombination technique, we constructed a EPO-cDNA expression vector pCSV-EPO (1), its sequence around the ATG initiator codon was 5' AATTCATGG 3'. By applying site-directed mutagenesis to the EPO-cDNA, another EPO-cDNA expression vector was constructed, mutations were created near the ATG initiator codon, its sequence was changed to 5' CCACCATGG 3'. It was demonstrated by DNA sequencing. After that, the two expression vectors were introduced into COS7 cells by DEAE-dextran-mediated transfection. The EPO concentration in culture was determined by EPO-ELISA kit. The results showed that the pCSV-EPO (2) had a higher EPO expression level than the pCSV-EPO (1) had.

关 键 词：促红细胞生成素 基因表达 COS7细胞 

分 类 号：Q75[生物学—分子生物学]