Construction and secetory expression of gene engineering bacteria of uric acid enzyme
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Construction and secetory expression of gene engineering bacteria of uric acid enzyme
Journal of Chongqing Medical UniversityVol. 43, Issue 1, Pages: 127(2018)
作者机构:
重庆医科大学生物化学与分子生物学教研室,重庆,400016
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Published:2018,
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Tang Lixia, Liu Jie, Liu Xianjun, et al. Construction and secetory expression of gene engineering bacteria of uric acid enzyme. [J]. Journal of Chongqing Medical University 43(1):127(2018)
DOI:
Tang Lixia, Liu Jie, Liu Xianjun, et al. Construction and secetory expression of gene engineering bacteria of uric acid enzyme. [J]. Journal of Chongqing Medical University 43(1):127(2018)DOI:
Construction and secetory expression of gene engineering bacteria of uric acid enzyme
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.
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