中国人促红细胞生成素cDNA的克隆及其在COS—7细胞中的表达

利用PCR技术,以中国人促红细胞生成素(EPO)次全基因组为模板,进行了基因修补和重组,克隆出EPO cDNA全序列。同时发现中国人EPO cDNA与国外的克隆比较有一个核苷酸的差异,导致第62位氨基酸是丝氨酸,不是亮氨酸。将人EPO cDNA基因插入表达载体pSV2-dhfr中的不同克隆位点,构建了6种不同的转移载体质粒,即pSV2-dhfr/F1,pSV2/N2,pSV2-dhfr/F3,pSV2-dhfr/P4,pSV2-dhfr/G1和pSV2-dhfr/G3。将它们分别转染导入COS-7细胞,结果表明6种转移载体质粒转染的细胞上清液都有明显的EPO活性。人EPO cDNA基因转移载体质粒在COS-7细胞中的表达水平高于人次全EPO基因组转移载体质粒。

Cloning of a Human Erythropoietin cDNA and Its Expression in COS-7 Cells

The human erythropoietin(EPO)cDNA has been isolated from a human EPO subgenome established from a Chinese fetal liver by PCR method. Isolated EPO cDNA contains full coding sequence for mature erythropoietin gene. It contains five exons (582 base pairs). The human EPO cDNA is characterized by DNA sequencing of twice separately amplified fragments using dideoxy Sanger method. In comparison with foreign erythropoietin gene, the Leucine-62 is replaced with serine. The erythropoietin cDNA has been cloned in the pSV2-dhfr vector at different site to construct six transfection vector: pSV2-dhfr/F1, pSV2/F2, pSV2-dhfr/F3, pSV2-dhfr/F4, pSV2-dhfr/G1, pSV2-dhfr/G3. It is transfected into Cos-7 cells. Its product has erythropoietin activity which was checked by a EPO dependent cell line and ELISA assay. Results indicate that the activities of EPO cDNA coding for Erythropoietin was higher than that of EPO genome.