产尿酸氧化酶基因工程菌的构建

Construction of gene engineering bacterium to produce urate oxidase

目的将尿酸氧化酶基因克隆到保加利亚乳酸杆菌中,使之能产生尿酸氧化酶,为开发能够分解尿酸、对高尿酸血症有治疗作用的可食用基因工程菌奠定基础。方法根据genbank上已知的产朊假丝酵母菌尿酸氧化酶基因序列(Uricase,E12709),PCR扩增尿酸氧化酶基因片段,将其插入质粒pMG36e,构建成重组质粒pMG36e-U,电转化保加利亚乳酸杆菌,SDS-PAGE鉴定基因工程菌体裂解液中的尿酸氧化酶,测定尿酸氧化酶活性。结果从产朊假丝酵母基因组中PCR扩增到尿酸氧化酶基因,并构建成含有尿酸氧化酶基因的重组质粒pMG36e-U,重组质粒pMG36e-U电转化保加利亚乳酸杆菌成功构建了含尿酸氧化酶的基因工程乳酸杆菌。SDS-PAGE测定基因工程菌合成的尿酸氧化酶亚基分子量约为34 kD,在体外测定菌酶液活性可达0.33 U.mL-1。结论尿酸氧化酶基因克隆入保加利亚乳酸杆菌中,并具有酶分解能力。

Objective To clone urate oxidase gene into lactobacillus Bulgaria so as to produce urate oxidase, and to lay basis for the development of edible genetic engineering bacteria to treat hyperuricemia. Methods According to genbank＇s known candida utilis urate oxidase gene sequences （Uricase, E12709） in the genbank, PCR amplification of urate oxidase gene fragments was done and inserted into plasmid pMG36e to construct the recombinant plasmid pMG36e- U. Electrotransformation of lactobacillus Bulgaria, SDS-PAGE analysis of the urate oxidase in the genetic engineering cell lysate urate oxidase activity were done. Results Genome which suspended animation production yeast prion was amplified to uric acid oxidase gene by polymerase chain reaction （PCR）, and its recombinant plasmid pMG36e-U con- structed. The pMG36e-U constructed genetically engineered lactobacillus which contained uric acid oxidase gene by electrotransformation of lactobacillus Bulgaria. The molecular weight of uric acid oxidase subunit was 34 kD by SDS- PAGE. The activity of bacteria enzyme was up to 0. 33 U·mL^-1 in vitro. Conclusion The uric acid oxidase gene which is cloned in lactobacillus Bulgaria has an ability of enzyme decomposition.