摘要
克隆了产朊假丝酵母 (Candidautilis)AS2 117尿酸氧化酶 (UrateOxidase,Uricase,EC1.7.3 .3)的基因。将此基因插入原核表达质粒pET2 1a后转化大肠杆菌BL2 1(DE3) ,获得高表达的重组转化子菌株。经IPTG诱导 ,重组尿酸酶基因表达量可达菌体可溶性蛋白的 40 %。重组尿酸氧化酶为有酶活性的可溶蛋白。Western印迹分析证实表达产物有免疫学活性。经DEAEDE5 2纤维素离子交换柱层析纯化 ,目的蛋白纯度可达 95 %。重组蛋白和天然蛋白的理化特征比较证明重组蛋白的热稳定性有较大提高。酶盒配制和临床应用实验表明重组蛋白可代替天然蛋白进行临床血清尿酸的分析。
Anurate oxidase (uricase,EC 1.7.3.3)gene from Candida utilis AS2.117 was cloned by PCR amplification with primers derived from conserved regions of published uicase DNA sequence.The DNA sequence of cloned uricase gene was determined and a high homology compared to the reported gene was found.The cloned gene was inserted into Bam HⅠ and Nde Ⅰ sites of pET21a to create the recombinant plasmid pURO.In Escherichia coli BL21(DE3)host,the expression lever of uricase reached to about 40% of total soluble proteins of the cell.The western blot analysis confirmed the result of expression.Properties of the enzyme protein produced by E.coli BL21(DE3)/pURO were determined and similar with those of original protein from Candida utilis AS2.117.Furthermore,the thermostability of the expressed protein was enhanced.The purified recombinant uricase was used in serum uric acid analysis.
出处
《生物工程学报》
CAS
CSCD
北大核心
2001年第1期68-72,共5页
Chinese Journal of Biotechnology
基金
国家自然科学基金资助项目!(39990 5 70 )&&
