【摘要】 本研究从人脐静脉血管内皮细胞中提取总RNA ,采用逆转录PCR(RT PCR)方法 ,扩增出人内皮型一氧化氮合酶 (heNOS)cDNA ,总长度为 3731bp。将其克隆入pUCm T载体质粒 ,序列分析表明 ,克隆所得片段含有完整开放阅读框架 ,与GenBank中heNOScDNA序列同源性达 99 93% ,在此基础上发现有若干核酸多态性。将该基因片段亚克隆到真核表达载体pcDNA3 0上 ,用脂质体转染法将pcDNA3 0 heNOS转染到COS 7细胞株 ,RT PCR、Westernblot分别检测到外源性eNOS基因在mRNA水平和蛋白质水平的表达 ,分析表明所表达的heNOS蛋白质分子量为 14 5ku ;L 1 4C 精氨酸掺入同位素法检测证实所表达的eNOS蛋白具有生物学活性 ,能将精氨酸氧化为瓜氨酸更多还原

【Abstract】 Two pieces of cDNA fragments of heNOS were amplified by RT PCR from total RNA extracted from HUVEC, and were then cloned into pUCm T vector. The sequence of the cloned cDNA includes the whole ORF and is 99.93% identical to that of heNOS cDNA in GenBank. The heNOS cDNA was subcloned into eukaryotic expression vector pcDNA3.0. Recombinant expressing vector pcDNA3.0/heNOS was transfected into COS 7 cell lines. The protein of heNOS was identified by Western blot and the biological activity of heNOS was examined by L 14C Arginine corporation. Analysis demonstrated that the recombinant protein has oxidation activity in conversing L arginine to L citrulline in vitro with molecular weight was 145ku.These results have laid a foundation for further study of heNOS function.更多还原