草鱼Fibulin-4蛋白与草鱼呼肠孤病毒外衣壳蛋白相互作用的研究
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摘要
草鱼呼肠孤病毒(Grass carp reovirus,GCRV)是当前引起我国草鱼出血病的主要病原,其外衣壳蛋白通常在病毒的组织嗜性和细胞嗜性上发挥重要作用。本研究利用共定位、Dot-Blot技术和His-Pull Down技术,验证了草鱼Fibulin-4蛋白与草鱼呼肠孤病毒外衣壳蛋白的相互作用。将pDsRed1N1-VP7,pDsRed1N1-VP56分别与pEGFP-Fibulin-4共转染草鱼性腺细胞GCO。结果显示,病毒蛋白VP7,VP56和草鱼蛋白Fibulin-4均分布于细胞质中,两种蛋白都与Fibulin-4有部分重叠。应用Dot-Blot Overlay技术,将纯化获得的GST与GST-Fibulin-4蛋白倍比稀释杂交到PVDF膜上,经过His-VP7,His-VP56蛋白的孵育,结果表明随着杂交的蛋白量从低到高,蛋白信号由弱到强,His-VP7与His-VP56都能够吸附于GST-Fibulin-4蛋白上并发生相互作用,而不能吸附GST蛋白。利用Pull Down技术,裂解细胞表达的GFP蛋白与GFP-Fibulin-4蛋白,结合His-VP7,His-VP56的HisPur Cobalt Resin,在Elution中均能检测到GFP-Fibulin-4蛋白质,而检测不到对照蛋白质GFP,进一步证实了VP7,VP56与Fibulin-4蛋白之间的相互作用。
Grass carp reovirus is the main pathogen causing grass carp hemorrhage in China.The outer capsid protein usually plays an important role in the tissue tropism and cell tropism of the virus.In this study,co-localization,dot-blot overlay and His-pull down were used to further verify the interaction among grass Fibulin-4 protein and grass carp outer capsid reovirus protein.Recombinant plasmid pEGFP-Fibulin-4 and pDsRed1 N1-VP7 and pDsRed1 N1-VP56 were successfully constructed.pDsRed1 N1-VP7,pDsRed1 N1-VP56 and pEGFP-Fibulin-4 were transferred to GCO cells respectively.The result showed that virus proteinVP7,VP56 and host Fibulin-4 protein were distributed in cytoplasm and both of them overlapped with Fibulin-4.Using dot blot-overlay,the prokaryotic expressed and purified GST and GST-Fibulin-4 were immobilized on Poly vinylidene fluoride(PVDF),after His-VP7,His-VP56 protein incubation,positive signals were developed with anti-his monoclonal antibody.The results showed that GST-Fibulin-4 bound to His-VP7 and His-VP56,with a trend suggesting binding was dose-dependent.The pull down technology was carried out by using the PierceTMpull-down PolyHis Protein,Protein Interaction Kit,both of Elutions can be detected with GFP-Fibulin-4 proteins,not GFP,confirming the interactions between Fibulin-4 and VP7,as well as VP56.
Grass carp reovirus is the main pathogen causing grass carp hemorrhage in China.The outer capsid protein usually plays an important role in the tissue tropism and cell tropism of the virus.In this study,co-localization,dot-blot overlay and His-pull down were used to further verify the interaction among grass Fibulin-4 protein and grass carp outer capsid reovirus protein.Recombinant plasmid pEGFP-Fibulin-4 and pDsRed1 N1-VP7 and pDsRed1 N1-VP56 were successfully constructed.pDsRed1 N1-VP7,pDsRed1 N1-VP56 and pEGFP-Fibulin-4 were transferred to GCO cells respectively.The result showed that virus proteinVP7,VP56 and host Fibulin-4 protein were distributed in cytoplasm and both of them overlapped with Fibulin-4.Using dot blot-overlay,the prokaryotic expressed and purified GST and GST-Fibulin-4 were immobilized on Poly vinylidene fluoride(PVDF),after His-VP7,His-VP56 protein incubation,positive signals were developed with anti-his monoclonal antibody.The results showed that GST-Fibulin-4 bound to His-VP7 and His-VP56,with a trend suggesting binding was dose-dependent.The pull down technology was carried out by using the PierceTMpull-down PolyHis Protein,Protein Interaction Kit,both of Elutions can be detected with GFP-Fibulin-4 proteins,not GFP,confirming the interactions between Fibulin-4 and VP7,as well as VP56.
引文
[1]张超,王庆,石存斌,曾伟伟,刘永奎,江海明,吴淑勤.草鱼呼肠孤病毒HZ08株的分离与鉴定[J].中国水产科学,2010,17(6):1257-1263.
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[8]Lu J,Wang H,Zhang Y,Li Y,Lu L.Grass carp reovirus NS26interacts with cellular lipopolysaccharide-induced tumor necrosis factor-alpha factor,LITAF[J].Virus Genes,2016,1-8.
[9]喻飞,王浩,宗乾坤,张娅楠,刘海云,陈百朋,吕利群.草鱼呼肠孤病毒GCRV VP56相互作用蛋白的鉴定[J].水产学报,2016,40(3):371-378.
[10]Tian Y,Jiao Z,Dong J,Sun C,Jiang X,Ye X.Grass carp reovirus-gd108fiber protein is involved in cell attachment[J].Virus Genes,2017,53(4):1-10.
[11]Yu F,Wang H,Wang L,Lu L.Orthoreovirus outerfiber proteins are substrates for sumo-conjugating enzyme Ubc9[J].Oncotarget,2016,7(48):79814-79827.
[12]Yu F,Wang H,Liu W,Lu L.Grass carp ctenopharyngodon idella Fibulin-4as a potential interacting partner for grass carp reovirus outer capsid proteins[J].Fish Shellfish Immunol,2015,(48)169-174.
[13]王土,许丹,吕利群.应用dsRNA测序技术检测草鱼呼肠孤病毒的混合感染[J].上海海洋大学学报,2012,21(5):756-762.
[14]Liu W,Hao W,Fei Y,Lu L.Grass carp reovirus outer capsid proteins VP5and VP7interact in vitro[J].Arch Virol,2017,162(8):2375-2380.
[15]Wojterakwiczor J,Gro F,Leffers H M,Kang M,Schneider M,Scheibe R.Transfer of a redox-signal through the cytosol by redox-dependent microcompartmentation of glycolytic enzymes at mitochondria and actin cytoskeleton[J].Front Plant Sci,2012,3(1):284.
[16]De Vega S,Iwamoto T,Yamada Y.Fibulins:multiple roles in matrix structures and tissue functions[J].Cell Mol Life Sci,2009,66(11-12):1890-1902.
[17]Giltay R,Timpl R,Kostka G.Sequence,recombinant expression and tissue localization of two novel extracellular matrix proteins,fibulin-3and fibulin-4[J].Matrix Biol,1999,18(5):469-480.
[18]McLaughlin P J,Chen Q,Horiguchi M,Starcher B C,Stanton J B,Broekelmann T J,Marmorstein A D,McKay B,Mecham R,Nakamura T,Marmorstein L Y.Targeted disruption of fibulin-4abolishes elastogenesis and causes pperinatal lethality in mice[J].Mol Cell Biol,2006,26(5):1700.
[19]Li R,Wang L.Fibulin-4is a novel wnt/β-Catenin pathway activator in human osteosarcoma[J].Biochem Biophys Res Commun,2016,474(4):730-735.
[20]Heine H,Delude R L,Monks B G,Espevik T,Golenbock D T.Bacterial lipopolysaccharide induces expression of the stress response genes hop and H411[J].J Biol Chem,1999,274(30):21049-21055.
[21]Djokic J,Fagotto-Kaufmann C,Bartels R,Nelea V,Reinhardt D P.Fibulin-3,-4,and-5are highly susceptible to proteolysis,interact with cells and heparin,and form multimers[J].J Biol Chem,2013,288(31):22821-22835.
[2]王方华,李安兴.草鱼病毒性出血病研究进展[J].南方水产科学,2006,2(3):66-71.
[3]马贵华,陈道印,刘六英,刘正有,李志强.草鱼出血病的免疫学研究进展[J].渔业现代化,2008,35(1):45-49.
[4]Attoui H,Fang Q,Mohd J F,Cantaloube J F,Biagini P,de Micco P,de Lamballerie X.Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus,Grass carp reovirus,Striped bass reovirus and golden ide reovirus(genus Aquareovirus,family Reoviridae)[J].J Gen Virol,2002,83(8):1941-1951.
[5]Wang Q,Zeng W,Liu C,Zhang C,Wang Y,Shi C,Wu S.Complete genome sequence of a reovirus isolated from grass carp,indicating different genotypes of GCRV in china[J].J Virol,2012,86(22):12466.
[6]Fan Y,Rao S,Zeng L,Ma J,Zhou Y,Xu J,Zhang H.Identification and genomic characterization of a novel fish reovirus,Hubei grass carp disease reovirus,isolated in 2009in China[J].J Gen Virol,2013,94(10):2266-2277.
[7]Wang H,Yu F,Li J,Lu L.Laminin receptor is an interacting partner for viral outer capsid protein VP5in grass carp reovirus infection[J].Virology,2016,490:59-68.
[8]Lu J,Wang H,Zhang Y,Li Y,Lu L.Grass carp reovirus NS26interacts with cellular lipopolysaccharide-induced tumor necrosis factor-alpha factor,LITAF[J].Virus Genes,2016,1-8.
[9]喻飞,王浩,宗乾坤,张娅楠,刘海云,陈百朋,吕利群.草鱼呼肠孤病毒GCRV VP56相互作用蛋白的鉴定[J].水产学报,2016,40(3):371-378.
[10]Tian Y,Jiao Z,Dong J,Sun C,Jiang X,Ye X.Grass carp reovirus-gd108fiber protein is involved in cell attachment[J].Virus Genes,2017,53(4):1-10.
[11]Yu F,Wang H,Wang L,Lu L.Orthoreovirus outerfiber proteins are substrates for sumo-conjugating enzyme Ubc9[J].Oncotarget,2016,7(48):79814-79827.
[12]Yu F,Wang H,Liu W,Lu L.Grass carp ctenopharyngodon idella Fibulin-4as a potential interacting partner for grass carp reovirus outer capsid proteins[J].Fish Shellfish Immunol,2015,(48)169-174.
[13]王土,许丹,吕利群.应用dsRNA测序技术检测草鱼呼肠孤病毒的混合感染[J].上海海洋大学学报,2012,21(5):756-762.
[14]Liu W,Hao W,Fei Y,Lu L.Grass carp reovirus outer capsid proteins VP5and VP7interact in vitro[J].Arch Virol,2017,162(8):2375-2380.
[15]Wojterakwiczor J,Gro F,Leffers H M,Kang M,Schneider M,Scheibe R.Transfer of a redox-signal through the cytosol by redox-dependent microcompartmentation of glycolytic enzymes at mitochondria and actin cytoskeleton[J].Front Plant Sci,2012,3(1):284.
[16]De Vega S,Iwamoto T,Yamada Y.Fibulins:multiple roles in matrix structures and tissue functions[J].Cell Mol Life Sci,2009,66(11-12):1890-1902.
[17]Giltay R,Timpl R,Kostka G.Sequence,recombinant expression and tissue localization of two novel extracellular matrix proteins,fibulin-3and fibulin-4[J].Matrix Biol,1999,18(5):469-480.
[18]McLaughlin P J,Chen Q,Horiguchi M,Starcher B C,Stanton J B,Broekelmann T J,Marmorstein A D,McKay B,Mecham R,Nakamura T,Marmorstein L Y.Targeted disruption of fibulin-4abolishes elastogenesis and causes pperinatal lethality in mice[J].Mol Cell Biol,2006,26(5):1700.
[19]Li R,Wang L.Fibulin-4is a novel wnt/β-Catenin pathway activator in human osteosarcoma[J].Biochem Biophys Res Commun,2016,474(4):730-735.
[20]Heine H,Delude R L,Monks B G,Espevik T,Golenbock D T.Bacterial lipopolysaccharide induces expression of the stress response genes hop and H411[J].J Biol Chem,1999,274(30):21049-21055.
[21]Djokic J,Fagotto-Kaufmann C,Bartels R,Nelea V,Reinhardt D P.Fibulin-3,-4,and-5are highly susceptible to proteolysis,interact with cells and heparin,and form multimers[J].J Biol Chem,2013,288(31):22821-22835.