人ω干扰素在巴斯德毕赤酵母中的表达及纯化  

Expression and purification of human interferon ω in yeast Pichia pastoris

作　　者：齐连权[1] 付玲[1] 于长明[1] 于婷[1] 徐春娥[1] 王海涛[1] 陈薇[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071

出　　处：《军事医学科学院院刊》2003年第4期268-270,共3页Bulletin of the Academy of Military Medical Sciences

摘　　要：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 yeast Pichia pastoris to evaluate its functions.Methods:Human IFN_ω gene, obtained by PCR_amplification, was cloned into the P.pastoris expression vector pMEX9K which has been cleaved by Xho Ⅰand Eco RⅠ.The recombinant vector pMEX9Kω was verified to be correctly constructed by enzyme digestion, PCR and sequencing.After linearization by Sal Ⅰ, the pMEX9Kω was transformed into GS115 by elctroporation. After being induced by methanol, the target protein IFN_ω was expressed in fermentation supernatant at high level. The purified protein was finally obtained with purity being higher than 99 per cent as determined by reverse phase HPLC by three_step chromatography: Butyl Sepharose 4 FF, SP Sepharose FF and Superdex 75.Results:The resulting protein was of at least 99% purity as determined by reverse phase HPLC. Analysis of the recombinant protein revealed that the protein had the same N_terminal sequence as the native one, its molecular weight was 22?000, and its specific activity was similar to natural human IFN_ω. Conclusion:The recombinant human IFN_ω was successfully expressed and purified in P.pastoris . [

关 键 词：ω干扰素 毕赤酵母 基因表达 纯化 

分 类 号：R346[医药卫生—基础医学]