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首页> 外文期刊>International Journal of Molecular Sciences >Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis
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Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis

机译：通过外显子置换/修复和定点诱变开发治疗性嵌合尿酸酶。

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The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more “human-like” uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine–human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1–2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7–8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu , the resulting chimera H 1-2 P 3 H 4 P 5-6 H 7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H 1-2 P 3 H 4 P 5-6 H 7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H 1-2 P 3 H 4 P 5-6 H 7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine–baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5–11.0 and temperature range of 20–40 °C.
机译：在类人猿进化过程中尿酸氧化酶的活性丧失，导致人类对高尿酸血症和痛风的高度敏感性。为了开发一种更具“人样”性的尿酸酶用于治疗，进行了外显子置换/修复和定点诱变，从而获得了与推导的人尿酸酶（dHU）具有更高同源性并增加了溶尿活性的猪人尿酸酶。在一项外显子替代研究中，野生猪尿酸酶（wPU）基因中的第6外显子被dhu中的相应外显子替代，完全消除了其活性。替换外显子5、3和1-2分别导致活性降低85％，60％和45％。但是，外显子4和7-8的替换并没有显着改变酶的活性。当dhu中的外显子5、6和3替换为wpu中的对应物时，所得嵌合体H 1-2 P 3 H 4 P 5-6 H 7-8是有活性的，但仅占wPU的28％。多序列比对和同源性建模预测，H 1-2 P 3 H 4 P 5-6 H 7-8中的E24D和E83G突变有利于其活性的进一步提高。定点诱变后，H 1-2 P 3 H 4 P 5-6 H 7-8（E24D＆E83G）与dHU的同源性增加（91.45％），并且活性和催化效率均高于FDA批准的猪狒狒获得嵌合体（PBC）。它在pH 8.5和35°C时显示最佳活性，在pH范围6.5–11.0和温度范围20–40°C下稳定。

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