甲醇酵母表达系统高拷贝数整合型表达载体的构建

Construction of Expression Vector for High copy number Integration of the Methylotrophic Yeast Hansenula polymorpha

以甲醇酵母（Ｈａｎｓｅｎｕｌａｐｏｌｙｍｏｒｐｈａ）表达系统的ＹＩｐ型表达载体ｐＪＦ５１为基础，通过引入宿主来源的一个ｒＤＮＡ随机顺序和由ＳＶ４０早期启动子控制的Ｇ４１８抗性基因（ｎｅｏ）及对野生型标记基因Ｈｐｌｅｕ２启动子大部分上游序列的缺失，构建了一个高拷贝整合型表达载体ｐＭＩＲＨ。有关转化、筛选和拷贝分析等试验结果表明，由ｐＭＩＲＨ可产生含高拷贝数载体的转化子，并可通过由ｌｅｕ２＋筛选和Ｇ４１８抗性筛选所组成的二级筛选方法富集这些含高拷贝数载体的转化子。有关完整ＤＮＡ和消化ＤＮＡＳｏｕｔｈｅｒｎ杂交分析结果进一步表明，上述高拷贝数的载体以串联重复排列的方式整合于宿主基因组。

Based on the YIp type expression vector pJF5 1 of the methylotrophic yeast Hansenula polymorpha , a novel vector, pMIRH , for high copy number integration was constructed, which contains a random sequence from the ribosomal DNA of the host chromosome, a defficient maker gene Hp leu 2d derivated from the wild gene with a partial upstream sequence deleted and a G418 resistance gene ( neo ) controled by the SV40 early promotor. Results from experiments of transformation, screening and Dot and Southern hybridization showed that pMIRH can result in transformants with high copy number vectors, which can be screened by a secondary G418 resistance selection, and these vectors were integrated into the chromosome in a tandem arrangement.