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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。
Total RNA was extracted from 4-month-old fetal liver and the whole gene of human thrombopoietin (TPO) was obtained by reverse transcription and polymerase chain reaction (PCR). The TPO gene double-digested with Nde and Hind restriction enzymes was ligated with p RSET A vector and transformed into E. coli to obtain two positive recombinant plasmids p T4 and p T10. Nucleotide sequence analysis confirmed that the cloned gene was reported in the literature. P T4 was transfected into E. coli JM109 (DE3) and induced by 1 mmol / L IPTG. SDS-PAGE analysis showed that a significant expression band was found at a molecular weight of about 40 000.