3-酮基嘌呤还原酶(TSC10)基因表达载体的构建及其在毕赤酵母中的表达

摘要

TSC10基因所编码的3-酮基嘌呤还原酶是酵母中神经酰胺合成的重要因子.设计了一种从酵母中提取神经酰胺经济、便捷、高效的方法:①利用构建含有TSC10基因的毕赤酵母GS115表达载体pPIC3.5K-TSC10;②电转化法将该表达质粒转化到GS115感受态细胞中;③用G418筛选以及PCR鉴定;④使用qRT-PCR和SDS-PAGE进行检测.结果经过G418筛选以及PCR鉴定后确定获得了包含TSC10基因的毕赤酵母转化子,经过qRT-PCR和SDS-PAGE进行检测后发现TSC10基因在20个阳性菌株中均可以稳定、高效表达.成功构建了3-酮基嘌呤还原酶高表达的毕赤酵母菌株,并为后续获得高收率神经酰胺奠定了基础.%The 3-keto purine reductase encoded by TSC 10 gene is an important factor in the synthesis of ceram-ide in yeast.The economical,convenient and efficient method was designed for extracting ceramide from yeast.①Using the expression vector pPIC 3.5K-TSC10 containing the TSC10 gene of Pichia pastoris GS115.②The expres-sion plasmid was transformed into GS 115 competent cells by electroporation.③Screened with G418 and identified by PCR.④Detection was performed using qRT-PCR and SDS-PAGE.After purification by G418 and PCR identifi-cation,the Pichia pastoris transformants containing TSC 10 gene were identified and analyzed by qRT-PCR and SDS-PAGE.The TSC10 gene was stable and highly expressed in 20 positive strains.This method successfully con-structed the highly expressed Pichia pastoris of 3-ketopurine reductase and laid the foundation for the subsequent high yield of ceramide.