摘要
【目的】建立一种能够对Wilson病(WD)基因突变进行分析的方法,为众多的WD基因突变进行致病性和致病程度的分析。【方法】本试验应用的酿酒酵母是YPH252,及其CCC2基因突变型ccc2酵母细胞。应用酶切方法构建了寡拷贝酵母表达质粒pMA92。将ATP7B亚克隆到酵母表达质粒pMA92上,进行ccc2酵母的转化和筛选,Westernblot分析转化后酵母ATP7B的表达情况,在铜、铁、色氨酸缺陷型的酵母SD培养基上培养转化的细胞,进行酵母生长曲线的分析,并进行Fet3p氧化酶活性的测定。【结果】在ccc2酵母细胞内表达人的ATP7B可以弥补ccc2的运铜功能,使ccc2酵母细胞在铜、铁及亮氨酸缺陷型培养基上生长良好。【结论】ccc2酵母细胞是研究ATP7B功能的一种良好的细胞模型,单拷贝ATP7B的表达就可以代偿ATP7B的功能缺陷,从而建立了可以对ATP7B进行分析的酵母分析系统。
Objective] To establish a method that allows detailed structure function analysis for mutations of ATP7B. The yeast strains were YPH252, and CCC2 knock out type ccc2. The pMA92 yeast single copy expression plasmid was constructed using the enzyme digestion method based on pMA91. Normal ATP7B was cloned to pMA92, and transformed to ccc2 cells and proceeded screening. Western blot was applied to test the expression of ATP7B. The transformed ccc2 cells were cultured in the SD medium lacking copper, iron and leucine. The growth rate of yeast was analyzed and the activity of fet3p oxidase was assayed. Ccc2 cells could grow well in the SD medium lacking copper, iron and leucine if normal ATP7B was expressed in the cells. [ Conclusion] Our experiments confirm that ATP7B can completely compensate the function of ccc2p to deliver copper. Single copper of ATP7B can make the yeast grow well in SD medium lacking copper and iron. The system that can be used to analyze the function of ATP7B is successfully established.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2003年第5期455-458,462,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
广东省自然科学基金资助项目(010705和21894)
211工程重点建设基金资助项目(中山医科大学98138)
