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埃博拉病毒糖蛋白GP1亚基融合蛋白的真核表达与纯化 被引量:1

Eukaryotic Expression and Purification of Ebola Virus Glycoprotein GP1 Subunit Fusion Protein
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摘要 埃博拉病毒糖蛋白与病毒黏附、入侵宿主细胞等过程密切相关,为了更好地研究该糖蛋白与宿主细胞的相互作用,拟通过哺乳动物细胞表达系统,构建GP1亚基Fc标签融合蛋白。经鉴定,成功构建了埃博拉病毒糖蛋白GP1亚基的真核表达载体pFUSE-GP1-hIgG1-Fc2,并利用该载体及293T细胞构建了表达GP1亚基融合蛋白的哺乳动物细胞系。该融合蛋白N端带有IL2分泌信号肽,C端带有人IgG1-Fc2标签,可在293T细胞以分泌表达的方式存在于细胞培养上清中,而后利用Protein A亲和层析进行融合蛋白纯化。经Western Blots鉴定,成功制备GP1-Fc融合蛋白。这不仅为埃博拉病毒糖蛋白的研究提供了技术支撑,也为其他种类蛋白的哺乳动物细胞真核表达纯化提供了新思路。 Ebola virus glycoprotein(EBOV_GP) plays important roles in viral Adhesion invasion in host cells. In order to explore the interaction effects of EBOV-GP in host cells, GP1 subunit fusion protein was constructed by mammalian cell expression system in this study. It was identified that eukaryotic expression vector pFUSE-GP1-hIgG1-Fc2 was constructed and 293 T cell lines expressing GP1-Fc were established for producing fusion protein. The fusion protein GP1-Fc, which has IL-2 secreting signal peptides in its N-terminal and a IgG1-Fc2 tag in its C-terminal, can be expressed in 293 T cells by secreting into the cell culture supernatant. GP1-Fc fusion protein was successfully prepared by Protein A affinity chromatography for purification and identified by Western Blots. This study could not only provide a technical support for other studies on EBOV_GP, but also show a new perspective for other eukaryotic expression and purification of fusion proteins.
作者 王中一 李佳明 付莹莹 郭振东 陆兵 钱军 刘林娜 WANG Zhongyi;LI Jiaming;FU Yingying;GUO Zhendong;LU Bing;QIAN Jun;LIU Linna(Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China)
出处 《吉林农业大学学报》 CAS CSCD 北大核心 2019年第1期92-96,共5页 Journal of Jilin Agricultural University
基金 国家重点研发计划专项(2016YFC1202604) 国家自然科学基金项目(31502077)
关键词 埃博拉病毒病 糖蛋白 真核表达 Protein A亲和层析 Ebola virus glycoprotein eukaryotic expression Protein A affinity chromatography
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