利用体外定点诱变技术构建人突变型α-L-艾杜糖醛酸酶基因

Constructing Human Mutant IDUA Gene by in Vitro Site-Directed Mutagenesis Technique

【目的】构建突变型α L 艾杜糖醛酸酶基因 (IDUA)cDNA ,为体外表达、鉴定突变的性质提供物质基础。【方法】以野生型pcDNA3 IDUA为基础质粒 ,采用双引物法体外定点诱变技术 ,引入突变E40 4X。【结果】突变区域经PCR SSCP和测序证实诱导突变成功。【结论】本研究成功构建了突变型IDUAcDNA ,可用于下游的体外表达 ;此定点突变技术具有简便、诱变效率高等优点 ,是体外构建突变型基因的一种有效方法。

Objective The mutant IDUA gene (IDUA) cDNA was constructed in order to establish the foundation for in vitro expression and further identification. Methods Using double primer in vitro site-directed mutagenesis, the mutation was induced into the wild IDUA cDNA. Results The mutant IDUA cDNA gene was successfully constructed, which was proved by PCR-SSCP and sequencing. Conclusion The mutant IDUA cDNA is going to be used for further analysis of the mutation; this site-directed mutagenesis technique has the advantages of simplicity and high-efficiency.