尿酸氧化酶与肌酐水解酶融合基因的克隆及其在大肠杆菌中的表达  

Cloning and its expression of fusion gene of urate oxidase and creatinine hydrolase in Escherichia coli

作　　者：程新[1] 刘芳[1] 张彦新[1] 蒋云生[2] 

机构地区：[1]广西中医学院附属瑞康医院肾内科,广西南宁530011 [2]中南大学湘雅二医院肾病研究所,湖南长沙410011

出　　处：《中国现代医学杂志》2013年第13期11-15,共5页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:30871168)

摘　　要：目的构建含尿酸氧化酶与肌酐水解酶融合基因的质粒并转化大肠杆菌,使其能同时分泌该两种酶分解尿酸及肌酐。方法根据GenBank数据库已知的尿酸氧化酶基因设计引物,扩增到去掉终止密码子的尿酸氧化酶基因中,并使其连接到质粒pGM36e上构建成pGM36e-U,然后根据已知肌酐水解酶基因设计引物扩增肌酐水解酶基因,把其连接到质粒pGM36e-U中尿酸氧化酶基因后构建成重组质粒pGM36e-U/C,转化大肠杆菌,筛选鉴定重组大肠杆菌,并进行SDS-PAGE分析。将工程菌于特定浓度肌酐和尿酸的培养基中培养24 h,取培养液测定肌酐和尿酸浓度。结果重组质粒得到大小约1.68 kb基因片段。为尿酸氧化酶及肌酐水解酶之合。转化大肠杆菌后尿酸氧化酶及肌酐水解酶活性得到表达。肌酐分解率提高43.50%,尿酸分解率提高42.32%。结论构建的含复合基因的重组质粒转化大肠杆菌后能表达尿酸氧化酶及肌酐水解酶活性。分解尿酸和肌酐。【Objective】 The plasmid of containing urate oxidase and creatinine hydrolase fusion gene were constructed and transformed into Escherichia coli for decomposing uric acid and creatinine.【Methods】 According to the GenBank data of uric urate oxidase gene,specific primers were designed,amplified to remove the stop codon of urate oxidase gene,and ligated to the plasmid pGM36e constructed pGM36e-U,followed by the GenBank database known creatinine hydrolase gene to design primers to amplify the creatinine hydrolase gene to construct plasmid pGM36e-U.Constructed recombinant plasmid pGM36e-U/C and transformed into E.coli,screened for recombinant E.coli,and SDS-PAGE analysis was conducted.Engineering bacteria was cultured at a particular concentration of creatinine and uric acid in the culture medium for 24 hours,the concentration of creatinine and uric acid in the culture fluid was determined.【Results】 The recombinant plasmid was about the size of 1.68 kb gene fragment containing uric acid oxidation enzymes and creatinine hydrolase.Activity of the creatinine hydrolase and uric acid oxidase transformed into E.colie were expressed.Creatinine decomposition rate increased by 43.5%,uric acid decomposition rate was 42.32%.【Conclusion】 The constructed recombinant plasmid containing complex gene of the creatinine hydrolase and uric acid oxidase was transformed into E.coli.The enzymes activity has been expressed.

关 键 词：基因克隆 基因工程菌 尿酸氧化酶 肌酐水解酶 融合基因 

分 类 号：Q78[生物学—分子生物学]