人促红细胞生成素基因组基因和cDNA的克隆及其在COS－7细胞中的表达

利用ＰＣＲ技术，从正常人胎肝染色体ＤＮＡ中克隆到长度为１５７２ｂｐ的人促红细胞生成素（ＥＰＯ）基因组基因片段，它包含除第一个外显子和第一个内含子外所有外显子及内含子。再人工合成１３ｂｐ外显子１的编码区，并与１５７２ｂｐ片段拼接，从而得到除第一个内含子的人促红细胞生成素基因组基因。将克隆得到的ＥＰＯ基因插入载体ｐＳＶ２－ｄｈｆｒ得到ｐＳＶ２－ＥＰＯ表达载体，转染ＣＯＳ－７细胞后获得高效表达。利用自行研制的小鼠抗人ＥＰＯ单抗及兔抗人ＥＰＯ多抗，对表达产物进行ＥＬＩＳＡ定量测定，细胞分泌ＥＰＯ量高达２５１±７Ｕ／ｍｌ．Ｋｒｙｓｔａｌ法测得体外生物活性２４１．５±６．５Ｕ／ｍｌ．用ＥＰＯ单抗免疫沉淀结合ＳＤＳ－ＰＡＧＥ对转染细胞的表达产物做了进一步鉴定，清晰地看到了ＥＰＯ条带。从高效表达ＥＰＯ的转染细胞中分离纯化ｍＲＮＡ，用ＲＴ－ＰＣＲ方法扩增并克隆到ＥＰＯ的ｃＤＮＡ，这为ＥＰＯ在其它系统中的表达及ＥＰＯ的功能与结构的研究打下了基础。

Cloning of Human Erythropoietin Genomic Gene and cDNA and Expression in COS-7 Cells

A 13bp coding region of the human erythropoietin gene exon Ⅰwas synthesized and ligated to a 1572 bp fragment corresponding to the sequence from exon Ⅱ to exon Ⅴ of the gene which was cloned from human fetal liver DNA by using PCR techique. The resulting fragment, containing all exons and introns of the EPO gene except intron Ⅰ,was ligated to the Hind Ⅲ and BglⅡsites of pSV2-dhfr,yielding the plasmid PSVZ-EPO. COS-7 cells transfected with the constucted pSV2-EPO secreted hEPO up to 251±7 U/ml of the supernatant. RNA was prepared from the transfected COS-7 cells. mRNA was isolated through polyAT tract mRNA isolation systems. EPO cDNA was cloned by using RT-PCR technique.