【作者】 黄志清；

【导师】 林爱星；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 猪干扰素-γ(PoIFN-γ)是由激活的T细胞和NK细胞产生的一类细胞因子,它具有高效抗病毒和免疫调节作用,有广阔的应用前景。为了更好地利用基因工程技术来开发重组猪干扰素-γ制剂,本研究将去除信号肽的猪干扰素-γ cDNA基因置于酿酒酵母α因子分泌信号的DNA序列后,构建成能精确分泌表达PoIFN-γ蛋白的重组表达载体pPIC9K-α-PoIFN-γ,通过电转化导入毕赤酵母GS115中,采用G418进行筛选,获得了两株能够高效分泌表达PoIFN-Y的多拷贝插入重组子。提取重组子的基因组DNA,采用PCR方法对重组子进行鉴定,结果表明PoIFN-γ成熟多肽基因成功整合到宿主菌的基因组中。重组子经甲醇诱导表达96h后,离心培养物,取上清用75%硫酸铵4℃沉淀过夜,再用PBS(pH7.4)透析24h,然后进行SDS-PAGE和Western blot分析,结果表明所获得的两株重组子都能分泌表达出17kD和23kD左右的PoIFN-γ特异蛋白,表达量分别为105mg/L和108mg/L,约占培养液总蛋白的60%。采用PK-15/HCV系统对其中一株重组子的分泌表达产物进行抗病毒活性研究,结果表明所获得的重组PoIFN-γ能够很好地保护猪肾细胞(PK-15株)免受猪瘟病毒HCV(石门系)的感染。 本研究首次在毕赤酵母表达系统中以分泌形式表达出重组PoIFN-γ,为利用基因工程手段开发重组猪干扰素-γ制剂打下了良好的基础。更多还原

【Abstract】 Porcine interferon-gamma (PoIFN-γ), produced by activated cytotoxic T cells and natural killer cells, plays important roles on anti-infectious and immunomodulatory effects. In order to develop the application of recombinant PoIFN-y, a recombinant expression vector was constructed in this study. The porcine interferon-gamma gene, in which the sequence encoding signal peptide was replaced by that of the a -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. PCR analysis of recombinants showed that the porcine interferon-gamma gene had successfully integrated into the Pichia pastoris GS115 genome. SDS-PAGE and Western blot assays of culture broth from this two methanol-induced expression strains demonstrated that recombinant PoIFN-γ 17kD and 23kD proteins, were secreted into the culture medium. Target proteins, about 60% of total proteins, were obtained in the culture medium at the concentration of 105mg/L and 108mg/L respectively under shake cultivation. The recombinant PoIFN-y can well protect PK-15 cells against virulent hog cholera virus (HCV) infection in vitro.This is the first secreted expression of porcine interferon-gamma gene in Pichia pastoris, which establish a good foundation for large-scale production of the recombinant PoIFN-γ.更多还原