强化表达分泌型酵母载体YEp5101和YEp5102的构建  

Construction of enhancing gene expression and secretion vectors YEp5101 and YEp5102 in yeast

作　　者：余焱林[1] 刘飞鹏[1] 茅善芝 

机构地区：[1]暨南大学生物学系

出　　处：《第一军医大学学报》1995年第2期143-146,共4页Journal of First Military Medical University

摘　　要：采用ＰＣＲ二段扩增法，从酵母基因组中获取６３５ｂｐ完整的ＭＦα（α─ｍａｔｉｎｇｆａｃｔｏｒ）基因及１８０ｂｐ的ＵＡＳＰＧＫ（３─磷酸甘油酸激酶基因的上游激活序列）ＤＮＡ片段。首先，将４３０ｂｐ的ＭＦα基因启动子和信号序列与１８０ｂｐＵＡＳｐＧＫ片段分别克隆到ＰＵＣ１８载体上；然后，从含有ＭＦα基因启动子和信号序列的阳性重组子ｐＭＦα１与含有ＵＡＳＰＧＫ阳性重组子ｐＵＡＳ８质粒中，分别获得ＭＦα基因启动子和信号序列及ＵＡＳＰＧＫ片段，构建成含ＭＦα基因启动子和信号序列的表达分泌型载体ＹＥｐ５１０１及含ＭＦα启动子和信号序列与ＵＡＳＰＧＫ的强化表达分泌型载体ＹＥｐ５１０２。这两种载体可用于外源基因表达的比较研究，探讨ＵＡＳ对外源基因表达的调控作用。We successfully isolated the 180 bp UASPGK and 635bp MFαgenes from the genomic DNA of haploidSaccharomyces cerivisiae by “PCR two grade amplification”,and cloned the 180bp UASPGK and the 430bppromoter and signal sequence from the 635bp MFαgene into pUC18 respectively. Recombinant pUAS8 car-rying UASPGK and recombinant pMFα1 containing MFα promoter and signal sequence were selected by insitu colony hybridization. Then,the 430 bp Sal 1 to HindⅢ fragment of pMF α1 was inserted into YEp51 di-gested by Sal 1 and Hind Ⅲ,and vector YEp5101 was generated. In the meantime,we connected 180bpsal I to BamH I fragment of pUAS8, 430bp Sal I to Hind Ⅲfragment of pMFα1 and large fragment ofYEp51 digested by BamH1 and HindⅢ,and constructed the vector YEp5102.Vector YEp5101 carries thepromoter and signal sequence of MFα gene,and vector YEp5102 contains UASPGK,promoter and signal se-quence of MFα gene. The vectors were all shuttle vectors,and can replicate in both.E. coli and yeast.Weinfer that these vectors may be expression and secretion vectors of a very high level.

关 键 词：酵母 载体 MFα基因 UASPGK 分泌型载体 

分 类 号：Q949.326[生物学—植物学]