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制备大鼠血红素加氧酶 1(HO 1)变异体并测定该变异体的活力和抑制野生型大鼠HO 1作用 ,为临床防治新生儿高胆红素血症提供一条新途径。方法 分别构建含剪切的野生型大鼠HO 1cDNA和变异体大鼠HO 1cDNA的质粒pcDNA3HO1和pcDNA3HO1Δ2 5,转染、收集COS 1细胞并破膜。超速离心获取表达产物 (可溶性微粒体成份 ) ,最终检测表达产物的活力和变异体的竞争抑制性。结果 野生型大鼠HO 1在转染的COS 1细胞中高度表达 ,其活力为 136 88- 156 0 0单位 /毫克蛋白 /小时 ;而变异体HO 1的活力明显下降 ,仅为 194 8- 2 16 0单位 /毫克蛋白 /小时。野生型和变异体大鼠HO 1具有类似的泳动度 ,且其分子量均为 30kDa。当酶促反应中加入与野生型HO 1等量的变异体HO 1后 ,胆红素水平下降达 50 %。结论 变异体HO 1与野生型HO 1竞争性结合底物血红素 ,从而降低胆红素的形成。
Preparation of rat heme oxygenase 1 (HO 1) variant and determination of the mutant activity and inhibition of wild-type rat HO 1 role in the prevention and treatment of neonatal hyperbilirubinemia provide a new way. Methods Plasmids pcDNA3HO1 and pcDNA3HO1Δ2 5 containing the HO1 cDNA of the wild type and the HO1 cDNA of the mutant rat were constructed respectively and transfected. COS 1 cells were collected and ruptured. The expression product (soluble microsomal fraction) was obtained by ultracentrifugation to finally detect the viability of the expression product and the competitive inhibition of the variant. Results The wild-type rat HO 1 was highly expressed in the transfected COS 1 cells with a viability of 136 88- 156 0 0 units / mg protein / hr, whereas the activity of the mutant HO 1 was significantly decreased, only 194 8 2 16 0 units / mg protein / hour. HO 1 of wild-type and mutant rats had similar motility and both had a molecular weight of 30 kDa. Bilirubin levels were reduced by 50% when an equal amount of variant HO 1 was added to the wild-type HO 1 in the enzymatic reaction. Conclusion The variant HO 1 competes with the wild-type HO 1 for binding to the substrate heme, thereby reducing the formation of bilirubin.