FLP介导的酿酒酵母rDNA多拷贝定点整合  

FLP-mediated Multicopy Targeted Integration into the Ribosomal DNA of Saccharomyces cerevisiae

作　　者：潘辉[1] 邬向东[1] 袁汉英 李育阳 

机构地区：[1]复旦大学遗传学研究所遗传工程国家重点实验室,上海200433 [2]江西农业大学动物科学技术学院,南昌330045

出　　处：《高技术通讯》1999年第11期11-16,共6页Chinese High Technology Letters

基　　金：863计划资助

摘　　要：构建了一个带有ＦＲＴ位点以及启动子缺陷的ＵＲＡ３ｄ 选择标记的环状质粒，并通过位点特异性重组将其整合到酵母染色体ｒＤＮＡ中放置的一个ＦＲＴ位点上。Ｓｏｕｔｈｅｒｎ杂交分析表明若处于很强的选择压力下，该质粒的拷贝数会发生有效的扩增。为了证明该质粒能够作为表达载体， 将乙肝融合表面抗原ＳＡ－２８基因的表达单元插入该质粒后整合到ｒＤＮＡ位点。与ｒＤＮＡ位点整合了单个表达单元的菌株相比，ＳＡ－２８蛋白的表达量提高了３０～６０倍。A circular plasmid containing a FRT site as well as the promoter deficient selection marker URA3d was constructed and targeted into the yeast ribosomal DNA via site specific recombination between the plasmid contained and chromosomally placed FRT targets. By means of southern hybridization analysis, the efficient amplification of plasmid copy number was detected under a strong selection pressure. To explore the potential of the plasmid as expression vector, the expression cassette of a modified hepatitis B virus surface antigen SA 28 gene was inserted into the plasmid and targeted into the ribosomal DNA locus. The yield of the SA 28 protein was about 30～60 fold higher than that observed in the strain carrying only a single cassette integrated at the ribosomal DNA locus.

关 键 词：酿酒酵母 FLP重组酶 定点整合 染色体rDNA 

分 类 号：TS261.11[轻工技术与工程—发酵工程] Q783[轻工技术与工程—食品科学与工程]