猪α干扰素在毕赤酵母中的高效表达和纯化  

Expression and purification of porcine interferon-alpha in yeast Pichia pasteris

作　　者：张存[1] 叶伟成[1] 王一成[1] 袁秀芳[1] 刘蔓雯[1] 张燕[1] 

机构地区：[1]浙江省农业科学院畜牧兽医研究所,浙江杭州310021

出　　处：《浙江农业学报》2010年第1期6-9,共4页Acta Agriculturae Zhejiangensis

基　　金：浙江省科技项目(2005C22034)

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。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 SnaBⅠ and EcoRⅠ enzymes. The transfer plasmid pPIC9k/PoIFNα was verified by enzyme digestion and sequencing. The pPIC9k/PoIFNα linearized by SalⅠ was 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 supernatant 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 effect on MDBK cell culture,which was about 2.27×10^8 U·mg^-1.

关 键 词：猪Α干扰素 毕赤酵母 分泌表达 

分 类 号：Q78[生物学—分子生物学]