人β干扰素与人血清白蛋白融合蛋白的表达及纯化.pdfVIP

生 物 工 程 学 报 Chin J Biotech 2009, November 25; 25(11): 1746-1752 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2009 Institute of Microbiology, CAS CSM, All rights reserved 生物技术与方法  张琦, 雷楗勇, 丁月娣, 陈蕴, 屈琳, 陈淑娴, 金坚 , 214122 : 为开展人干扰素-人血清白蛋白融合蛋白(IFN-HAS) 的临床前研究, 本研究采用免疫学方法对毕赤酵母转化 子进行筛选, 得到高产菌株 8 株。在 5 L 发酵罐上对高产菌株进行诱导表达, 产量达500 mg/L。发酵液经超滤浓缩、染 料亲和层析、镍离子螯合亲和层析和 DEAE 离子交换层析纯化后, 融合蛋白纯度达到96% 。Western blotting 杂交表明 融合蛋白具有人血清白蛋白和人干扰素的抗原性, 细胞病变抑制法测定IFN-HSA 比活性为1.96 ×107 IU/mg 。建立了 融合蛋白的生产工艺, 为IFN-HSA 的临床前研究奠定了基础。 : 干扰素, 融合蛋白, 毕赤酵母 Expression and purification of IFNβ-HSA fusion protein in Pichia pastoris Qi Zhang, Jianyong Lei, Yuedi Ding, Yun Chen, Lin Qu, Shuxian Chen, and Jian Jin Department of Cellular and Molecular Pharmacology, School of Medicine and Parmacology, Jiangnan University , Wuxi 214122, China Abstract: In order to obtain enough fusion protein for developing preclinical studies of IFN-HAS, we screened Pichia pastoris transformants expressing high-level protein by immunology method. The yield of IFNβ-HSA was about 500 mg/L by fed-batch fermentation. The purity of IFNβ-HSA reached 96% through the steps of ultrafiltration, Blue Sepharose FF, Ni2+-IMAC and DEAE Sepharose FF. Analysis of Western blotting showed that IFNβ-HSA had the antigenicity of IFNβ and HSA. The specific activity was about 1.96×107 IU/mg by standard survival activity test on WISH cells challenged with VSV virus. This study provided a method to produce IFNβ-HSA. Keywords: IFN, fusion protein, Pichia pastoris   ,  , 2~4 h, [1] 1993