埃博拉病毒糖蛋白GP1亚基融合蛋白的真核表达与纯化
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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.
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.
引文
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[2] Yan J, Gao G F. MicroRNAs: the novel targets for Ebola drugs [J]. Science China Life Sciences, 2014, 57(10): 985-986.
[3] Wang Z Y, Guo Z D, Li J M, et al. Genome-wide search for competing endogenous RNAs responsible for the effects induced by Ebola virus replication and transcription using a trVLP system [J]. Frontiers in Cellular and Infection Microbiology, 2017, 7:479.
[4] Becker S, Spiess M, Klenk H D. The asialoglycoprotein receptor is a potential liver-specific receptor for Marburg virus [J]. The Journal of General Virology, 1995, 76 (Pt2):393-399.
[5] Chan S Y, Empig C J, Welte F J, et al. Folate receptor-alpha is a cofactor for cellular entry by Marburg and Ebola viruses [J]. Cell, 2001, 106(1): 117-126.
[6] Alvarez C P, Lasala F, Carrillo J, et al. C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans [J]. Journal of Virology, 2002, 76(13): 6841-6814.
[7] Takada A, Fujioka K, Tsuiji M, et al. Human macrophage C-type lectin specific for galactose and N-acetylgalactosamine promotes filovirus entry [J]. Journal of Virology, 2004, 78(6): 2943-2947.
[8] Chandran K, Sullivan N J, Felbor U, et al. Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection [J]. Science, 2005, 308(5728): 1643-1645.
[9] Shimojima M, Takada A, Ebihara H, et al. Tyro3 family-mediated cell entry of Ebola and Marburg viruses [J]. Journal of Virology, 2006, 80(20): 10109-10116.
[10] Kondratowicz A S, Lennemann N J, Sinn P L, et al. T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus [J]. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(20): 8426-8431.
[11] Carette J E, Raaben M, Wong A C, et al. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1 [J]. Nature, 2011, 477(7364): 340-343.
[12] Cote M, Misasi J, Ren T, et al. Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection [J]. Nature, 2011, 477(7364): 344-348.
[13] Miller E H, Obernosterer G, Raaben M, et al. Ebola virus entry requires the host-programmed recognition of an intracellular receptor [J]. The EMBO Journal, 2012, 31(8): 1947-1960.
[14] Salvador B, Sexton N R, Carrion R, et al. Filoviruses utilize glycosaminoglycans for their attachment to target cells [J]. Journal of Virology, 2013, 87(6): 3295-3304.
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