| 猪α干扰素在毕赤酵母中的高效表达和纯化 |
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| 引用本文： | 张存,叶伟成,王一成,袁秀芳,刘蔓雯,张燕.猪α干扰素在毕赤酵母中的高效表达和纯化[J].浙江农业学报,2010,22(1). |
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| 作者姓名： | 张存 叶伟成 王一成 袁秀芳 刘蔓雯 张燕 |
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| 作者单位： | 浙江省农业科学院,畜牧兽医研究所,浙江,杭州,310021 |
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| 摘 要： | 为了研制新型的猪用抗病毒制剂,开展了猪α干扰素(PoIFNα)在毕赤酵母中的表达和产物纯化研究。将PCR扩增的PoIFNα基因插入pPIC9k的SnaBⅠ和EcoRⅠ位点,构建了表达转移载体pPIC9k/PoIFNα;经酶切和测序鉴定,转移载体构建正确。pPIC9k/PoIFNα以限制性内切酶SalⅠ线性化后电转化毕赤酵母GS115菌株,经抗性筛选获得4拷贝PoIFNα基因的重组菌株GS115/PoIFNα;重组菌株经BMMY培养基诱导后上清中有目的蛋白,表达量达136mg/L。表达产物经过SP-Sepharose阳离子交换层析,可以得到纯度较高的rPoIFNα,纯化产物在MDBK细胞上抑制VSV的活性高达2.27×108U/mg。
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| 关 键 词： | 猪α干扰素 毕赤酵母 分泌表达 |
Expression and purification of porcine interferon-alpha in yeast Pichia pasteris |
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| ZHANG Cun,YE Wei-cheng,WANG Yi-cheng,YUAN Xiu-fang,LIU Man-wen,ZHANG Yan.Expression and purification of porcine interferon-alpha in yeast Pichia pasteris[J].Acta Agriculturae Zhejiangensis,2010,22(1). |
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| Authors: | ZHANG Cun YE Wei-cheng WANG Yi-cheng YUAN Xiu-fang LIU Man-wen ZHANG Yan |
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| Institution: | ZHANG Cun,YE Wei-cheng,WANG Yi-cheng,YUAN Xiu-fang,LIU Man-wen,ZHANG Yan(Institute of Animal Husb,ry Sciences , Veterinary Medicine,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China) |
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| Abstract: | Porcine interferon-alpha(PoIFNα)was expressed and purified to evaluate its functions.The PoIFNα gene,amplified by PCR.was cloned into P. pasteris expression vector pPIC9k which had been digested by SnaB I and EcoR I enzymes.The transfer plasmid pPIC9k/PoIFNα wag verified by enzyme digestion and sequencing.The pPIC9k/PoIFNα linearized by Sal I Wag electro-porated into P. paseris strain GS115.The recombinant yeast GS115/PoIFNα with four-copied PoIIFNα genes was isolated on the YPD agar plate with 1 mg·mL~(-1) G418.The recombinant protein PoIFNα was expressed in the fermentation supematant at a high level of 136 mg·L~(-1) after being induced by methanol.rPoIFNα was isolated at a purity of 81% by one step of SP-Sepharose chromatography.As a result,the purified product was verified to be of high cytokine activity by inhibiting the VSV viral cyto-pathogenic effeet on MDBK cell culture.which Wag about 2.27×10~8 U·mg~(-1). |
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| Keywords: | porcine interferon-alpha Pichia pasteris secreted expression |
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