OSF 1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

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。

Abstract:

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 plasmid was linearized by SacⅠand transformed into P. pastoris GS115 by electroporation. Recombinant P. pastoris GS115/ 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 ℃ 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-1.