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通过构建表达型质粒 pcDNA3HO1,并在 COS-1细胞中的瞬时表达,证实分离的 cDNA 编码血红素加氧酶-1(heme oxygenase-1,HO-1,以探讨 HO-1在新生期黄疸中的作用机制。实验中构建含编码 HO-1 cDNA 的表达型质粒 pcDNA3HO1,培养 COS-1细胞,表达型质粒 pcDNA3HO1转染 COS-1,收集细胞,超声破膜,超速离心得到微粒体颗粒,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE鉴定和酶活性检测。结果显示:HO-1 cDNA 在 COS-1细胞内高度表达,活性为对照组的5倍,分子量约为30 000。实验表明:分离的 cDNA 片段长1030碱基对,编码 HO-1,体外测活显示表达产物能显著增加胆红素水平。
The isolated cDNA was confirmed to encode heme oxygenase-1 (HO-1) by constructing the expression plasmid pcDNA3HO1 and transiently expressed in COS-1 cells to explore the role of HO-1 in neonatal jaundice The recombinant plasmid pcDNA3HO1 containing HO-1 cDNA was constructed and COS-1 cells were cultured.Expression plasmid pcDNA3HO1 was transfected into COS-1 cells and the cells were collected and sonicated by ultrasonication to obtain microsomal particles, SDS Polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme activity assay showed that HO-1 cDNA was highly expressed in COS-1 cells with 5-fold higher activity and a molecular weight of about 30 000. The results showed that: The isolated cDNA fragment was 1030 base pairs in length and encoded HO-1. The in vitro activity of the expressed product significantly increased the level of bilirubin.