鸡源志贺菌IpaC基因变构体重组酵母表达载体的构建及初步表达

Construction and preliminary expression of recombinant Pichia pastoris vector of IpaC gene allosteric from chicken Shigella

为使IpaC基因表达产物保持天然的生物学活性,实现在酵母表达系统中高效表达,本研究首先采用反向PCR将IpaC基因358～360位和361～363位酵母菌低频密码子突变为偏嗜性密码子,并进一步构建了克隆载体pMD18-T-IpaC＊和酵母表达载体pPICZαA-IpaC＊,然后将线性化重组质粒pPICZαA-IpaC＊电转化至毕赤酵母X-33中,对阳性转化子进行诱导表达,并用SDS-PAGE检测其表达产物。结果表明SDS-PAGE在大约63 000处检测出目的条带,实现了鸡源志贺菌IpaC基因在毕赤酵母中的初步表达。这为进一步研究IpaC生物学功能奠定了基础。

In order to maintain the biological activity of natural IpaC gene expression product,and achieve efficient expression in yeast expression systems,using inverse PCR to mutate low frequency yeast codons at IpaC gene 358-360 bit and 361-363 bit into bias tropism codons,and further build the cloning vector pMD18-T-IpaC＊and yeast expression vector pPICZαA-IpaC＊,then linearized plasmid pPICZαA-IpaC＊,electric transformed into Pichia pastoris X-33.The positive clones were induced by methanol,and detect the expression results by SDS-PAGE.SDS-PAGE results shows the purpose of strip approximately 63 000,indicating that chicken ShigellaIpaC gene was preliminary expressed in Pichia pastoris,which laid the foundation for further study on IpaC biological functions.