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目的 :在巴斯德毕赤酵母中表达及纯化人ω干扰素 (interferonω ,IFN_ω)以对其功能进行深入研究。方法 :用PCR方法扩增人IFN_ω基因 ,与分泌型酵母表达载体pMEX9K重组 ,经酶切、PCR和序列测定证实重组表达载体pMEX9Kω构建正确。将重组载体pMEX9Kω用SalⅠ酶切后 ,转化毕赤酵母GS115。得到的转化子经诱导表达后在发酵上清中有人IFN_ω的表达 ,表达量为 4× 10 9U L。经过ButylSepharose 4FF疏水层析柱 ,SPSepharoseFF离子交换柱和Superdex 75凝胶过滤三步层析纯化 ,利用反相HPLC分析纯化的重组蛋白。结果 :得到了纯度 >99%的重组IFN_ω。N端氨基酸序列测定表明重组人IFN_ω具有正确的N端氨基酸残基顺序 ,相对分子质量为 2 2 0 0 0 ,并具有同天然蛋白相似的比活性。结论 :在毕赤酵母中成功地表达并纯化得到了同天然蛋白具有相似的比活性的人IFN_ω。
OBJECTIVE: To express and purify human interferon-ω (IFN-ω) in Pichia pastoris to further study its function. Methods: Human IFN-ω gene was amplified by PCR and recombined with secretory yeast expression vector pMEX9K. The recombinant plasmid pMEX9Kω was confirmed by restriction enzyme digestion, PCR and sequencing. The recombinant vector pMEX9Kω digested with SalI, transformed Pichia GS115. The resulting transformants were induced to express human IFN-omega in the fermentation supernatant after they were expressed, in an amount of 4 × 10 9 U L. Purification was performed on ButylSepharose 4FF hydrophobic column, SPSepharoseFF ion exchange column and Superdex 75 gel filtration by three-step chromatography. The purified recombinant protein was analyzed by reverse-phase HPLC. Results: A recombinant IFN_ω with> 99% purity was obtained. N-terminal amino acid sequence analysis showed that recombinant human IFN-ω has the correct N-terminal amino acid residue sequence with a relative molecular mass of 2200 and a specific activity similar to that of the native protein. CONCLUSION: Human IFN_ω with similar specific activity to native protein was successfully expressed and purified in Pichia pastoris.