【摘要】 将去除信号肽的猪干扰素γ(PoIFNγ)基因置于酿酒酵母α因子分泌信号的DNA序列后,构建成pPIC9KαPoIFNγ分泌型重组表达载体,电转化导入毕赤酵母GS115中,经G418筛选后获得2株多拷贝插入的重组子。SDSPAGE和Westernblot分析结果表明,所获得的重组子能够分泌表达出17kD和23kD左右的PoIFNγ特异蛋白,其表达量为108mgL,占培养液总蛋白的60%。实验首次在毕赤酵母表达系统中实现了PoIFNγ基因的分泌表达。更多还原

【Abstract】 The porcine interferon-gamma(PoIFN-γ) gene, in which the sequence encoding signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was cloned into Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-α-PoIFN-γ was then transformed into Pichia pastoris GS115 cells by electroporation and stable multicopy recombinant Pichia pastoris strains were selected by G418 resistance. Two recombinants of multiple inserts were obtained. SDS-PAGE and Western blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant PoIFN-γ, 17kD and 23kD proteins, were secreted into the culture medium. Target proteins, 60% of total proteins, were obtained in the culture medium at the concentration of 108mg/L. This is the first secreted expression of porcine interferon-gamma gene in Pichia pastoris.更多还原