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目的制备人血红素加氧酶_1(humanhemeoxygenase_1,hHO_1)突变体,探讨酶的作用机制并应用突变体降低胆红素水平,为防治新生儿期高未结合胆红素血症的发生提供新途径。方法采用PCR方法制备hHO_1His25Ala突变体(hHO_1)cDNA,构建突变体表达载体pBHO_1(M)。野生型和突变体表达载体pBHO_1和pBHO_1(M)分别转化大肠杆菌DH5α,IPTG诱导表达目的蛋白。经30%～60%(NH4)2SO4盐析、Q_SepharoseFastFlow阴离子交换树脂分离纯化,并进行酶活性测定。结果构建的载体能分别表达产物,经30%～60%(NH4)2SO4盐析后纯度提高3.6倍,再经2次Q_SepharoseFastFlow阴离子交换树脂分离纯度提高30倍,酶活性测定显示,hHO_1较野生型hHO_1(whHO_1)活性下降了91.21%。结论hHO_1的第25位组氨酸在酶与底物血红素氧化反应中起着重要作用,制备的hHO_1能明显降低胆红素水平。
Objective To prepare human hemeoxygenase 1 (hHO_1) mutants, to explore the mechanism of action of enzymes and reduce the level of bilirubin with mutants, to provide new information for the prevention and treatment of neonatal hyperbilirubinemia way. Methods The hHO_1His25Ala mutant (hHO_1) cDNA was prepared by PCR and the mutant expression vector pBHO_1 (M) was constructed. The wild-type and mutant expression vectors pBHO_1 and pBHO_1 (M) were transformed into E. coli DH5α respectively, and the target protein was induced by IPTG. After 30% ~ 60% (NH4) 2SO4 salting out, Q_SepharoseFastFlow anion exchange resin was isolated and purified, and enzyme activity determination. Results The constructed vector was able to express the product respectively. After being purified by 30% -60% (NH4) 2SO4 salting-out, the purity of the product increased by 3.6-fold, and the purity of Q_SepharoseFastFlow anion exchange resin was increased by 30 times. The enzyme activity assay showed that hHO_1 Activity of hHO_1 (whHO_1) decreased by 91.21%. Conclusion Histidine at position 25 of hHO_1 plays an important role in the heme oxidation of substrate and substrate. The prepared hHO_1 can significantly reduce the level of bilirubin.