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Stable expression of ICOS/Fc in CHO cells
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 11
- DOI:
- 10.3321/j.issn:1000-2790.2005.11.004
- Key Word:
- 可诱导共刺激分子Fc;融合蛋白;亲和层析;纯化;Western blotting
Abstract： 目的:探讨Picos/Fc融合蛋白基因在中国仓鼠卵巢细胞(CHO)中的稳定表达及其表达产物的纯化和初步鉴定,为其功能研究和临床应用奠定基础.方法:将Picos/Fc表达质粒及含G418抗性基因neor的质粒Pegfp共转染CHO细胞,经G418抗性培养基筛选、克隆化培养、夹心ELISA检测,挑选100%强阳性且表达均一的细胞克隆,扩大培养取上清经Protein A亲和层析纯化,SDS-PAGE和Western印迹等进行分子质量、纯度、抗原特异性的初步鉴定.结果:获得稳定表达ICOS/Fc蛋白的CHO细胞单克隆,制备并经Protein A亲和层析柱获得纯化的ICOS/Fc融合蛋白,SDS-PAGE胶可见一条Mr约70×103的与预期分子质量大小相符的蛋白条带,Western印迹法可在同一位置检测到该蛋白与HRP酶标抗人Fc mAb特异结合.结论:在CHO细胞中成功地建立了稳定表达ICOS/Fc融合蛋白的细胞系,表达并纯化了有抗原特异性的重组ICOS/Fc融合蛋白.
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