用逆转录-聚合酶链反应(RT-PCR)以人肝细胞总RNA为模板, 扩增了人锰超氧化物歧化酶(hMnSOD)的cDNA片段, 将此cDNA克隆到载体pGEM-T中.对重组质粒进行限制酶切分析和序列测定, 确定为含hMnSODcDNA的重组质粒将该hMnSODcDNA重组到表达载体pBV220内, 重组质粒在大肠杆菌DH5-α中表达hMnSOD, 表达产物占菌体总蛋白的14%, 具有持异性SOD酶活性.
The cDNA encoding human manganese superoxide dismutase was amplified from the human liver total RNA by RT-PCR, ligated into pBV220. The cloned gene was analyzed by restriction enzymes EcoR l, Sal Ⅰ, and BamHⅠ. The sequence of the cloned gene was determined. hMnSOD was induced and expressed upon temperature shift from 32℃ to 42℃.Mn2+ supplementation in the bacterial growth media resulted in about 3-fold increasing of SOD activity. The special protein expressed accounts for 14% of the total protein of the bacteria.
骆训懿,周丽君,陈晓穗,王欲晓,谢邦铁,王晶翼,陈宇萍,何菱.人锰超氧化物歧化酶cDNA的克隆、测序及表达[J].生物化学与生物物理进展,1996,23(4):349-352
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