人内皮型一氧化氮合酶cDNA在COS-7细胞中的表达

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、Western blot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145ku；L-^14C-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。

Expression of human endothelial nitric oxide synthase cDNA in COS-7 product

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.