血小板生成素基因的克隆及其初步表达

Gene Clone and Expression of Human Thrombopoietin

自 4月龄胎肝中提取总 RNA,经逆转录、多聚酶链反应 (PCR)获得了人血小板生成素 (TPO)全基因 ,长约 10 80 bp。将经限制性内切酶 Nde 和 Hind 双酶切后的 TPO基因与 p RSET A载体相连接 ,转化大肠杆菌后得到两个阳性重组质粒 p T4和 p T10。经核苷酸序列分析证实 ,所克隆的基因与文献报道一致。将 p T4转染 E. coli JM10 9(DE3) ,经 1mmol/ L IPTG诱导表达 ,SDS- PAGE分析 ,相对分子质量约 40 0 0 0处可见一条明显的表达带。

After the total RNA was extracted from fetal liver,the whole length gene(1 080 bp) of human thrombopoietin(TPO) was obtained by reverse transcription and PCR. The TPO gene was digested with Nde Ⅰ and Hind Ⅲ,then inserted into pRSET A vector and transformed to E.coli HMS174. The 2 positive clones obtained were identical in DNA sequences. On the basis of transfection of E.coli JM109(DE3) with positive plasmid, the expression of TPO was induced by 1 mmol/L IPTG. SDS PAGE showed that the molecular weight of the expressed TPO was about 40 000.