尿酸酶基因工程菌的构建与分泌表达

Construction and secetory expression of gene engineering bacteria of uric acid enzyme

摘要:

目的：将产朊假丝酵母菌尿酸酶编码基因克隆至已改造的表达载体pET-28a中，使尿酸酶在大肠埃希菌（Escherichia coli，E.coli）中以分泌的形式持续表达，并具有生物学活性。方法：①利用重组PCR技术删除pET-28a中Lac O和Lac I序列构建表达载体；②金黄色葡萄球菌蛋白A（Staphylococal Protein A，SPA）信号肽编码基因与尿酸酶基因N端融合成目的基因；②将目的基因克隆至表达载体中，再转化至E.coli DH5ɑ中，使之表达；④利用SDS-PAGE鉴定并测定表达产物生物学活性。结果：尿酸酶在E.coli中以分泌的形式持续表达，并具有生物学活性。结论：利用肠道内正常菌群 E.coli构建可分泌、持续表达蛋白的载体pET-28a-sUOX，可将表达的尿酸酶作用于高尿酸血症模型上，为应用于动物实验通过肠道降低尿酸水平提供依据。

Abstract:

Objective：To clone uricase encoding gene in Candida utilis into the modified expression vector of pET-28a，and to make the uricase gene in Escherichia coli（E.coli） continuously express in secreted form with biological activity. Methods：To construct ex－pression vector pET-28a，RT-PCR method was used to delete the Lac O and Lac I sequence of pET-28a. The target gene was fused by the the N-terminal of uricase encoding gene and the signal peptide of protein A in Staphylococcus aureus. The fusion gene was cloned into the expression vector pET-28a，then transformed into E.coli DH5ɑ and expressed. The biological activity of expression product was identified and measured by SDS-PAGE gel electrophoresis. Results：The uricase gene in E.coli was continuously ex－pressed with biological activity in secreted form. Conclusion：As the normal flora in the gut，E.coli can be used to construct the ex－pression vector pET-28a-sUOX，which has secrete protein effect. Expression of uricase can be applied to the hyperuricemia model，which provide a basis for reducing uricase level through intestine.