猪干扰素-γ基因在毕赤酵母中的分泌表达

将去除信号肽的猪干扰素-γ（PoIFN-γ）基因置于酿酒酵母α因子分泌信号的DNA序列后, 构建成pPIC9K-α-PoIFN-γ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。SDS-PAGE和Western blot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PoIFN-γ特异蛋白，其表达量为108mg/L，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PoIFN-γ基因的分泌表达。

Secreted Expression of Porcine Interferon-gamma Gene in Pichia pastoris

The porcine interferon-gamma (PoIFN-gamma) gene, in which the sequence encoding signal peptide was replaced by that of the alpha-factor of Saccharomyces cerevisiae, was cloned into Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-alpha-PoIFN-gamma 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-gamma, 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 108 mg/L. This is the first secreted expression of porcine interferon-gamma gene in Pichia pastoris.