摘要
从健康人外周血分离单核细胞,经LPS刺激后提取总RNA,用RT-PCR扩增hIFN-γ的cDNA。将扩增产物克隆后进行序列分析证明,克隆的hIFN-γcDNA除存在2个序列多态性位点外,与天然的干扰素氨基酸序列完全一致。利用温控表达系统在大肠杆菌中实现了hIFN-γ的高效表达,表达的目的蛋白可占菌体总蛋白的55%。用5L和50L发酵罐放量生产的产物,经高效疏水色谱HPHIC直接复性和纯化得到了重组hIFN-γ纯品,其比活性与天然蛋白相近。
Monocytes were obtained from healthy volunteers and total RNA was prepared from these cells after stimulation with LPS. Human interferonγ (γIFN) cDNA was amplified with RTPCR and cloned into plasmid vector. DNA sequencing showed that the amplified γIFN gene has two polimorphic sites. Highlevel expression of γIFN was achieved using temperaturecontroled E.coli expression system. The expressed protein is about 55% of total cellular protein after induction. Pure recombinant γIFN protein was prepared with simultaneous refolding and purification using high performance hydrophobic chromatography after fermentation of E.coli. The purified γIFN protein has comparable activity as natural product.
出处
《西北大学学报（自然科学版）》
CAS
CSCD
北大核心
2003年第5期540-544,共5页
Journal of Northwest University（Natural Science Edition）
基金
国家自然科学基金资助项目(No.29675017)
关键词
人干扰素-γ
反转录聚合酶链反应
重组蛋白表达
复性与纯化
高效疏水色谱
hIFN-γ
RT-PCR
high expression of recombinant protein
Hydrophobic interaction chromatography
renaturation and purification
