OSF1在毕赤酵母菌中的分泌表达及其生物活性  

Expression and bioactivity of OSF-1 in Pichia pastoris

作　　者：康东红[1] 王燕[1] 张洪美[1] 冯潇雨[1] 曹维[1] 王萍[1] 

机构地区：[1]山东大学附属千佛山医院,山东济南250014

出　　处：《生物工程学报》2014年第2期274-283,共10页Chinese Journal of Biotechnology

基　　金：山东省优秀中青年科学家奖励基金(No.2004BS02020)资助~~

摘　　要：为了研究人成骨细胞刺激因子(Human osteoblast-stimulating factor,OSF-1)的生物学活性,构建OSF-1高效毕赤酵母表达菌株并表达纯化具有生物活性的OSF-1。首先通过全基因人工合成的方法合成人osf-1基因,并克隆至毕赤酵母分泌表达载体pPIC9K,构建重组表达质粒pPIC9K/osf-1,线性化后电转化酵母宿主菌GS115,构建P.pastoris GS115/pPIC9K/osf-1,经MD、G418-YPD平板和PCR法筛选,获得多拷贝转化子。阳性表达菌株在25℃,经1%的甲醇诱导表达96 h,重组蛋白的表达量达到最大。经SDS-PAGE电泳分析所表达的重组蛋白约为18 kDa,与人成骨细胞刺激因子相符。表达上清经SP-Sephadex C-50离子交换层析进行纯化,得到纯度达98%以上的OSF-1。Western blotting鉴定该蛋白对人成骨细胞刺激因子抗体具有良好的抗原性。生物活性测定表明提纯的OSF-1能促进鼠成骨细胞MC3T3-E1的增殖和分化。利用重组毕赤酵母高效分泌表达了有生物活性的OSF-1,为进一步开展其抗骨质疏松活性研究及产业化开发奠定了基础。In order to research the biologic activity of osteoblast-stimulating factor 1 （OSF-1）, the pPIC9K/osf-1 yeast expression vector was constructed to express and purify OSF-1. Firstly, the osf-1 gene sequence was obtained by artificial synthesis and cloned into Pichia pastoris expression vector pPIC9K to generate pPIC9K/osf-1. The recombinant plasmidwas linearized by Sac I and transformed into P. pastoris GSll5 by electroporation. Recombinant P. pastoris GSll5/ pPIC9K/osf-1 was screened by MD and G418-YPD plates and further identified by PCR. The positive P. pastoris was induced with 1% methanol at 25 ~C for 96 h. The target protein was analyzed by SDS-PAGE showing a special band about 18 kDa. The target protein was successfully purified from the supernatant of the broth using ion exchange chromatography of SP-Sephadex C-50. The purity of target protein was above 98%. Western blotting appeared a good antigenicity of the purified protein. Bioassay results show that the recombinant protein OSF-1 can promote the differentiation and proliferation of osteoblasts MC3T3-E1. We successfully expressed OSF-1 by recombinant P. pastoris for further development of anti-osteoporosis of research and industrial production of OSF-I.

关 键 词：OSF-1 毕赤酵母 蛋白表达 纯化 生物活性 

分 类 号：R580[医药卫生—内分泌] Q78[医药卫生—内科学]