中国人红细胞生成素cDNA克隆与高效表达

CDNA CLONING AND HIGH EXPRESSION OF CHINESE ERYTHROPOIETIN

以中国人正常胎肝为原料提取总RNA，逆转录合成cDNA与pCR3载体相连接，构建成pE／C表达质粒。序列鉴定结果表明，cDNA序列与国内外已报导的相比，除第2个密码子的第3个碱基系引物设计所致差异外，其余部分完全相同。利用脂质转染法将质粒转入中国白鼠卵巢细胞，获得能高效稳定表达重组人红细胞生成素的工程细胞。

Total RNA was extracted from Chinese fetal liver. Erythropietin cDNA was synthesized from the total RNA by reverse transcription. After PCR amplification, pE/C expressing plasmid was constructed by conjugating the PCR product with PCR3 plasmid. The results of EPO cDNA Sequence analysis showed no difference with those already reported except for the third base of the Second codon which was due to the design of the primer. The PE/C and pDHFR were transfected together into ovary cells of Chinese hamster by lipofectin. Engineering cells could express recombinant human erythropoietin highly and stable. The expression was about 8000 IU/ml media.