3-酮基嘌呤还原酶(TSC10)表达载体的构建及其在毕赤酵母中的表达

目的：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,本文研究从酵母中提取神经酰胺经济、便捷、高效的方法。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。结论：本方法成功构建了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。

Construction of tsc10 gene expression vector and its expression in Pichia pastoris

Objective: The 3-keto purine reductase encoded by Tsc10 gene is an important factor in the synthesis of ceramide in yeast. This paper studies the economical, convenient and efficient method for extracting ceramide from yeast. Methods: 1.Using the expression vector pPIC3.5K-tsc10 containing the tsc10 gene of Pichia pastoris GS115.2.The expression plasmid was transformed into GS115 competent cells by electroporation.3.Screened with G418 and identified by PCR.4.Detection was performed using qRT-PCR and SDS-PAGE. Result: After purification by G418 and PCR identification, the Pichia pastoris transformants containing tsc10 gene were identified and analyzed by qRT-PCR and SDS-PAGE. The tsc10 gene was stable and highly expressed in 20 positive strains. Conclusion: This method successfully constructed the highly expressed Pichia pastoris of 3-ketopurine reductase and laid the foundation for the subsequent high yield of ceramide.