一种酿酒酵母附加型分泌表达载体pYES2/CT/α-factor的构建及鉴定  

Construction and Determination of an Episomal Secreting Expression Vector:pYES2/CT/α-factor for Saccharomyces cerevisiae

作　　者：胡亚冬[1,2] 姚冬生[2] 

机构地区：[1]广州科仁生物工程有限公司,广州510600 [2]暨南大学微生物技术研究所,广州510632

出　　处：《中国生物工程杂志》2009年第12期49-53,共5页China Biotechnology

基　　金：国家十一五"863"计划重点项目(2007AA100605)资助项目

摘　　要：以pPIC9为模板,通过PCR扩增获得酿酒酵母的α-交配因子(α-factor),并克隆至酿酒酵母胞内表达载体pYES2/CT中,构建了一种新型酿酒酵母附加型分泌表达载体pYES2/CT/α-factor(pYCα)。再将甘露聚糖酶基因(mannase,man)通过酶切、连接克隆至pYCα的α-factor的下游,构建了pYCα-man重组载体检验pYCα的分泌性能和稳定性。结果显示α-factor可引导甘露聚糖酶基因在胞外分泌表达,在曲利本兰培养基上形成明显的水解圈,进一步分析重组菌胞外和胞内酶活,结果显示对照INVSc1/pYCα的两种酶活都未检测到,而INVSc1/pYCα-man具有明显的胞外酶活,未检测到胞内酶活,说明构建的pYCα具有良好的分泌性能;稳定性实验表明重组质粒连续培养150 h仍具有良好的稳定性。pPIC9 was used as a template and α-factor gene of Saccharomyces cerevisiae was amplified by PCR. The gene was cloned in the intracellular expression vector pYES2/CT for Saccharomyces cerevisiae and the secreting expression vector named pYES2/CT/α-factor（pYCα） was constructed. The gene of mannase （man）from recombinant vector of pKLACl-man （pKLman） was cut by restriction enzymes and linked with pYCα. This recombinant vector pYCα-man was used to determine the secretory ability and stability of pYCα. The excellent secretory ability of pYCα was proved by two experiments. One showed that INVScl/pYCα-man clones formed the clear rings around the clones on the medium contained trypan blue,while INVScl/pYCα clones had no rings. Further analysis of mannase activity of extracellular supernatant and intracellular extracts showed that both extracellular and intracellular mannase activities of INVScl/pYCα were not detected,while INVScl/pYCα-man had evident extracellular mannase activities and no intracellular mannase activities. The stability of pYCct was also very good proved by continuous cultivation for about 150h.

关 键 词：酿酒酵母 信号肽 分泌表达 

分 类 号：Q782[生物学—分子生物学]