雪莲类PEBP基因在毕赤酵母中的表达及鉴定

Expression and Identification of PEBP-like Gene from Saussurea involucrate Kar. er kir in Pachia pastoris

为了构建雪莲类PEBP基因真核表达载体,在毕赤酵母中表达并鉴定。将雪莲类PEBP基因开放阅读框序列克隆到真核表达载体pPIC9k上,转化到毕赤酵母(Pichia pastoris)GS115细胞内,利用筛选到的Muts(methanol utilization slow)重组酵母株进行表达,经SDS-PAGE分析,表达产物置于低温条件下进行抗冻试验。结果在培养基上清液中含有一明显的特异性蛋白条带,大小为20 kD,与预期相符,上清液抗冻检测表明PEBP具有一定的抗冻性,表明该基因在毕赤酵母GS115中得到了表达。该研究成功构建了真核表达载体pPIC9k-PEBP,获得了表达产物,并为进一步研究雪莲类PEBP基因的抗冻功能打下基础。

This assay was designed to construct the eukaryotic expression vector,investigate the expression of PEBP-like in yeast Pichia pastoris GS115 cells and identify its product.The PEBP gene was inserted into the vector pPIC9k.The recombinant vector was transferred into Pichia pastoris GS115.A Muts（Methanol utilization slow）recombinant strain GS115 was selected and induced the expression of protein by methanol and low temperature overnight.The supematants were analyzed by SDS-PAGE and identified by low temperature.After methanol induction,a new anticipating protein of 20kD appeared as an expected size.Activity tests revealed that this protein has been definite anti-freeze character by the biological activity detection of the supematant.The eukaryotic expression system of PEBP-like is successfully constructed.It lays the foundation for the further application study on the antifreeze characters of the PEBP.