鸭胚成纤维细胞上坦布苏病毒受体蛋白的鉴定
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摘要
【目的】拓展对坦布苏病毒受体的认知,为研究其在鸭体内的感染机制打下基础。【方法】取9日龄SPF鸭胚制备鸭胚成纤维细胞(DEF),待细胞长成单层后提取DEF膜蛋白,利用免疫共沉淀试验筛选出DEF膜蛋白中可与坦布苏病毒结合的蛋白,经病毒辅覆蛋白结合分析(VOPBA)验证后通过LC-MSMS质谱分析进行鉴定;同时人工合成鸭热休克蛋白70基因(HSP70)序列的开放阅读框(1905 bp),构建重组质粒pET32a-HSP70并转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达,以重组蛋白免疫小鼠制备抗鸭HSP70血清,与DEF细胞共孵育后接种坦布苏病毒,并采用实时荧光定量PCR检测抗血清对病毒感染的抑制作用。【结果】DEF膜蛋白中分子量约70 kD的蛋白可与坦布苏病毒结合,经LC-MSMS质谱分析和序列比对可确定该蛋白即为鸭热休克蛋白70(HSP70)。含重组质粒pET32aHSP70的BL21(DE3)感受态细胞经IPTG诱导4 h后,SDS-PAGE检测发现在分子量约85 kD处出现目的蛋白条带,获得的重组鸭HSP70蛋白主要以包涵体形式进行表达,且能与His标签抗体发生特异性反应。以重组鸭HSP70蛋白免疫小鼠获得的抗鸭HSP70血清可封闭DEF细胞上的HSP70,而竞争性抑制坦布苏病毒感染。【结论】HSP70是坦布苏病毒感染DEF细胞的受体,可作为新的靶点用于抗坦布苏病毒药物设计。
【Objective】The aim of the present study was to expand the understanding of the Tembusu virus receptor and lay the foundation for studying its pathogenesis in ducks.【Method】Duck embryo fibroblasts(DEF)were prepared from 9-day-old SPF duck embryo. Membrane protein was extracted from monolayer of DEF. Then proteins that could bind with Tembusu virus was screened by co-immunoprecipitation from DEF membrane proteins. Virus overlay protein binding assay(VOPBA)was used to validate the binding protein. The target protein band was analyzed by LC-MSMS mass spectrometry. The open reading frame(1905 bp)of duck heat shock protein 70 gene(HSP70)was synthesized artificially and the recombinant plasmid p ET32a-HSP70 was constructed. Then pET32a-HSP70 was transformed into Escherichia coli BL21(DE3)and the competent cell was induced by IPTG. In order to prepare anti duck HSP70 serum,mice were immunized by recombinant duck heat shock protein. Duck embryo fibroblasts were pre-incubated with anti-serum followed by Tembusu virus infection. Real-time fluorescence quantitative PCR was employed to determine the inhibition of anti-serum on infection of the virus.【Result】An approximately 70 kD protein in DEF membrane protein bound to Tembusu virus. LCMSMS mass spectrometry and sequence alignment demonstrated that the 70 kD protein was duck heat shock protein 70(HSP70). The recombinant plasmid pET32a-HSP70 containing BL21(DE3)competent cell was induced by IPTG for 4 h.The analysis of SDS-PAGE showed that the target protein band with molecular weight approximately 85 kD was observed.The recombinant duck HSP70 protein was expressed in inclusion bodies,and reacted specifically with antibody against His tag. Mouse anti duck HSP70 serum was prepared by immunization of recombinant duck HSP70. The infection of Tembusu virus was competitively inhibited by anti duck HSP70 serum which blocked the HSP70 in DEF cell.【Conclusion】HSP70 is identified as receptor of Tembusu virus infected DEF cell. It can be a new target for designing antiviral drug against Tembusu virus.
【Objective】The aim of the present study was to expand the understanding of the Tembusu virus receptor and lay the foundation for studying its pathogenesis in ducks.【Method】Duck embryo fibroblasts(DEF)were prepared from 9-day-old SPF duck embryo. Membrane protein was extracted from monolayer of DEF. Then proteins that could bind with Tembusu virus was screened by co-immunoprecipitation from DEF membrane proteins. Virus overlay protein binding assay(VOPBA)was used to validate the binding protein. The target protein band was analyzed by LC-MSMS mass spectrometry. The open reading frame(1905 bp)of duck heat shock protein 70 gene(HSP70)was synthesized artificially and the recombinant plasmid p ET32a-HSP70 was constructed. Then pET32a-HSP70 was transformed into Escherichia coli BL21(DE3)and the competent cell was induced by IPTG. In order to prepare anti duck HSP70 serum,mice were immunized by recombinant duck heat shock protein. Duck embryo fibroblasts were pre-incubated with anti-serum followed by Tembusu virus infection. Real-time fluorescence quantitative PCR was employed to determine the inhibition of anti-serum on infection of the virus.【Result】An approximately 70 kD protein in DEF membrane protein bound to Tembusu virus. LCMSMS mass spectrometry and sequence alignment demonstrated that the 70 kD protein was duck heat shock protein 70(HSP70). The recombinant plasmid pET32a-HSP70 containing BL21(DE3)competent cell was induced by IPTG for 4 h.The analysis of SDS-PAGE showed that the target protein band with molecular weight approximately 85 kD was observed.The recombinant duck HSP70 protein was expressed in inclusion bodies,and reacted specifically with antibody against His tag. Mouse anti duck HSP70 serum was prepared by immunization of recombinant duck HSP70. The infection of Tembusu virus was competitively inhibited by anti duck HSP70 serum which blocked the HSP70 in DEF cell.【Conclusion】HSP70 is identified as receptor of Tembusu virus infected DEF cell. It can be a new target for designing antiviral drug against Tembusu virus.
引文
黄欣梅,赵冬敏,刘宇卓,杨婧,韩凯凯,李银.2015.鹅坦布苏病毒E蛋白结构域III的原核表达及抗原性分析[J].南方农业学报,46(1):150-154.[Huang X M,Zhao D M,Liu Y Z,Yang J,Han K K,Li Y.2015.Prokaryotic expression and antigenic analysis of E protein domain III in goose tembusu virus[J].Journal of Southern Agriculture,46(1):150-154.]
任宝波,王玉艳,王纯净,李士泽,杨焕民.2005.HSP70家族的分类及基因结构与功能[J].动物医学进展,26(1):98-101.[Ren B B,Wang Y Y,Wang C J,Li S Z,Yang HM.2005.Classification,gene structure and function of HSP70 family[J].Progress in Veterinary Medicine,26(1):98-101.]
张杰,单宝来,田永生,彭日荷,姚泉洪,汪良驹.2017.葡萄HSP17基因的合成与功能分析[J].江苏农业学报,33(3):503-509.[Zhang J,Shan B L,Tian Y S,Peng R H,Yao Q H,Wang L J.2017.Synthesis and functional analysis of HSP17 gene in grape[J].Jiangsu Journal of Agricultural Sciences,33(3):503-509.]
张响英,唐现文,陈静波,李腾腾.2017.热应激诱导奶牛乳腺上皮细胞凋亡及其对HSP70基因表达的影响[J].河南农业科学,46(8):142-146.[Zhang X Y,Tang X W,Chen J B,Li T T.2017.Effects of heat stress on apoptosis and expression of HSP70 mRNA in bovine mammary epithelial cells[J].Journal of Henan Agricultural Sciences,46(8):142-146.]
赵冬敏,黄欣梅,刘宇卓,韩凯凯,杨婧,谢星星,刘晓燕,李银.2014.鹅坦布苏病毒E蛋白结构域I的原核表达及免疫原性鉴定[J].南方农业学报,45(12):2259-2263.[Zhao D M,Huang X M,Liu Y Z,Han K K,Yang J,Xie X X,Liu X Y,Li Y.2014.Prokaryotic expression and immunogenicity identification of goose tembusu virus Eprotein domain I[J].Journal of Southern Agriculture,45(12):2259-2263.]
赵冬敏,刘宇卓,黄欣梅,韩凯凯,杨婧,刘青涛,毕可然,李银.2017.坦布苏病毒E蛋白结构域I/II蛋白免疫保护效果[J].江苏农业学报,33(2):379-383.[Zhao D M,Liu Y Z,Huang X M,Han K K,Yang J,Liu Q T,Bi K R,Li Y.2017.Immunoprotective effect of tembusu virus E protein domains I and II[J].Jiangsu Journal of Agricultural Sciences,33(2):379-383.]
朱文姣,常琛,朱玉虎,周国丽,尹志安,杨国庆,刘慧敏.2018.猪流行性腹泻病毒coe基因的克隆表达及卵黄抗体的制备[J].河南农业大学学报,52(4):560-565.[Zhu W J,Chang C,Zhu Y H,Zhou G L,Yin Z A,Yang G Q,Liu H M.2018.Clone and expression of coe gene and preparation of egg yolk antibody against COE of porcine epidemic diarrhea virus[J].Journal of Henan Agricultural University,52(4):560-565.]
Chen S T,Lin Y L,Huang M T,Wu M F,Cheng S C,Lei HY,Lee C K,Chiou T W,Wong C H,Hsieh S L.2008.CLEC5A is critical for dengue-virus-induced lethal disease[J].Nature,453(7195):672-676.
Chen Y C,Wang S Y,King C C.1999.Bacterial lipopolysaccharide inhibits dengue virus infection of primary human monocytes/macrophages by blockade of virus entry via a CD14-dependent mechanism[J].Journal of Viorology,73(4):2650-2657.
Chu J J,Ng M L.2004.Interaction of West Nile virus withαVβ3integrin mediates virus entry into cells[J].The Journal of Biological Chemistry,279(52):54533-54541.
Das S,Laxminarayana S V,Chandra N,Ravi V,Desai A.2009.Heat shock protein 70 on Neuro2a cells is a putative receptor for Japanese encephalitis virus[J].Virology,385(1):47-57.
Fang S,Wu Y,Wu N,Zhang J,An J.2013.Recent advances in DENV receptors[J].The Scientific World Journal,684690.doi:10.1155/2013/684690.
Jindadamrongwech S,Thepparit C,Smith D R.2004.Identification of GRP 78(BiP)as a liver cell expressed receptor element for dengue virus serotype 2[J].Archives of Virology,149(5):915-927.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod[J].Methods,25(4):402-408.
Liu Q,Huang X,Zhao D,Han K,Liu Y,Yang J,Bi K,Li Y.2017.Identification of heat shock protein A9 as a tembusu virus binding protein on DF-1 cells[J].Virus Research,227:110-114.
Pattnaik P,Babu J P,Verma S K,Tak V,Rao P V.2007.Bacterially expressed and refolded envelope protein(domain III)of dengue virus type-4 binds heparan sulfate[J].Journal of Chromatography,846(1-2):184-194.
Pokidysheva E,Zhang Y,Battisti A J,Bator-Kelly C M,Chipman P R,Xiao C,Gregorio G G,Hendrickson W A,Kuhn R J,Rossmann M G.2006.Cryo-EM reconstruction of dengue virus in complex with the carbohydrate recognition domain of DC-SIGN[J].Cell,124(3):485-493.
Reyes-Del Valle J,Chávez-Salinas S,Medina F,Del Angel RM.2005.Heat shock protein 90 and heat shock protein70 are components of dengue virus receptor complex in human cells[J].Journal of Virology,79(8):4557-4567.
Smit J M,Moesker B,Rodenhuis-Zybert I,Wilschut J.2011.Flavivirus cell entry and membrane fusion[J].Viruses,3(2):160-171.
Su J,Li S,Hu X,Yu X,Wang Y,Liu P,Lu X,Zhang G,Hu X,Liu D,Li X,Su W,Lu H,Mok N S,Wang P,Wang M,Tian K,Gao G F.2011.Duck egg-drop syndrome caused by BYD virus,a new tembusu-related flavivirus[J].PLoS One,6(3):e18106.
Tang Y,Diao Y,Yu C,Gao X,Ju X,Xue C,Liu X,Ge P,Qu J,Zhang D.2013.Characterization of a tembusu virus isolated from naturally infected house sparrows(Passer domesticus)in northern China[J].Transboundary and Emerging Disease,60(2):152-158.
Thepparit C,Smith D R.2004.Serotype-specific entry of dengue virus into liver cells:identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor[J].Journal of Virology,78(22):12647-12656.
Zhang W,Chen S,Mahalingam S,Wang M,Cheng A.2017.An updated review of avian-origin tembusu virus:A newly emerging avian Flavivirus[J].The Journal of General Virology,98(10):2413-2420.
Zhao D,Liu Q,Han K,Wang H,Yang J,Bi K,Liu Y,Liu N,Tian Y,Li Y.2018.Identification of Glucose-regulated protein 78(GRP78)as a receptor in BHK-21 cells for duck tembusu virus infection[J].Frontiers in Microbiology,9:694.doi:10.3389/fmicb.2018.00694.
任宝波,王玉艳,王纯净,李士泽,杨焕民.2005.HSP70家族的分类及基因结构与功能[J].动物医学进展,26(1):98-101.[Ren B B,Wang Y Y,Wang C J,Li S Z,Yang HM.2005.Classification,gene structure and function of HSP70 family[J].Progress in Veterinary Medicine,26(1):98-101.]
张杰,单宝来,田永生,彭日荷,姚泉洪,汪良驹.2017.葡萄HSP17基因的合成与功能分析[J].江苏农业学报,33(3):503-509.[Zhang J,Shan B L,Tian Y S,Peng R H,Yao Q H,Wang L J.2017.Synthesis and functional analysis of HSP17 gene in grape[J].Jiangsu Journal of Agricultural Sciences,33(3):503-509.]
张响英,唐现文,陈静波,李腾腾.2017.热应激诱导奶牛乳腺上皮细胞凋亡及其对HSP70基因表达的影响[J].河南农业科学,46(8):142-146.[Zhang X Y,Tang X W,Chen J B,Li T T.2017.Effects of heat stress on apoptosis and expression of HSP70 mRNA in bovine mammary epithelial cells[J].Journal of Henan Agricultural Sciences,46(8):142-146.]
赵冬敏,黄欣梅,刘宇卓,韩凯凯,杨婧,谢星星,刘晓燕,李银.2014.鹅坦布苏病毒E蛋白结构域I的原核表达及免疫原性鉴定[J].南方农业学报,45(12):2259-2263.[Zhao D M,Huang X M,Liu Y Z,Han K K,Yang J,Xie X X,Liu X Y,Li Y.2014.Prokaryotic expression and immunogenicity identification of goose tembusu virus Eprotein domain I[J].Journal of Southern Agriculture,45(12):2259-2263.]
赵冬敏,刘宇卓,黄欣梅,韩凯凯,杨婧,刘青涛,毕可然,李银.2017.坦布苏病毒E蛋白结构域I/II蛋白免疫保护效果[J].江苏农业学报,33(2):379-383.[Zhao D M,Liu Y Z,Huang X M,Han K K,Yang J,Liu Q T,Bi K R,Li Y.2017.Immunoprotective effect of tembusu virus E protein domains I and II[J].Jiangsu Journal of Agricultural Sciences,33(2):379-383.]
朱文姣,常琛,朱玉虎,周国丽,尹志安,杨国庆,刘慧敏.2018.猪流行性腹泻病毒coe基因的克隆表达及卵黄抗体的制备[J].河南农业大学学报,52(4):560-565.[Zhu W J,Chang C,Zhu Y H,Zhou G L,Yin Z A,Yang G Q,Liu H M.2018.Clone and expression of coe gene and preparation of egg yolk antibody against COE of porcine epidemic diarrhea virus[J].Journal of Henan Agricultural University,52(4):560-565.]
Chen S T,Lin Y L,Huang M T,Wu M F,Cheng S C,Lei HY,Lee C K,Chiou T W,Wong C H,Hsieh S L.2008.CLEC5A is critical for dengue-virus-induced lethal disease[J].Nature,453(7195):672-676.
Chen Y C,Wang S Y,King C C.1999.Bacterial lipopolysaccharide inhibits dengue virus infection of primary human monocytes/macrophages by blockade of virus entry via a CD14-dependent mechanism[J].Journal of Viorology,73(4):2650-2657.
Chu J J,Ng M L.2004.Interaction of West Nile virus withαVβ3integrin mediates virus entry into cells[J].The Journal of Biological Chemistry,279(52):54533-54541.
Das S,Laxminarayana S V,Chandra N,Ravi V,Desai A.2009.Heat shock protein 70 on Neuro2a cells is a putative receptor for Japanese encephalitis virus[J].Virology,385(1):47-57.
Fang S,Wu Y,Wu N,Zhang J,An J.2013.Recent advances in DENV receptors[J].The Scientific World Journal,684690.doi:10.1155/2013/684690.
Jindadamrongwech S,Thepparit C,Smith D R.2004.Identification of GRP 78(BiP)as a liver cell expressed receptor element for dengue virus serotype 2[J].Archives of Virology,149(5):915-927.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod[J].Methods,25(4):402-408.
Liu Q,Huang X,Zhao D,Han K,Liu Y,Yang J,Bi K,Li Y.2017.Identification of heat shock protein A9 as a tembusu virus binding protein on DF-1 cells[J].Virus Research,227:110-114.
Pattnaik P,Babu J P,Verma S K,Tak V,Rao P V.2007.Bacterially expressed and refolded envelope protein(domain III)of dengue virus type-4 binds heparan sulfate[J].Journal of Chromatography,846(1-2):184-194.
Pokidysheva E,Zhang Y,Battisti A J,Bator-Kelly C M,Chipman P R,Xiao C,Gregorio G G,Hendrickson W A,Kuhn R J,Rossmann M G.2006.Cryo-EM reconstruction of dengue virus in complex with the carbohydrate recognition domain of DC-SIGN[J].Cell,124(3):485-493.
Reyes-Del Valle J,Chávez-Salinas S,Medina F,Del Angel RM.2005.Heat shock protein 90 and heat shock protein70 are components of dengue virus receptor complex in human cells[J].Journal of Virology,79(8):4557-4567.
Smit J M,Moesker B,Rodenhuis-Zybert I,Wilschut J.2011.Flavivirus cell entry and membrane fusion[J].Viruses,3(2):160-171.
Su J,Li S,Hu X,Yu X,Wang Y,Liu P,Lu X,Zhang G,Hu X,Liu D,Li X,Su W,Lu H,Mok N S,Wang P,Wang M,Tian K,Gao G F.2011.Duck egg-drop syndrome caused by BYD virus,a new tembusu-related flavivirus[J].PLoS One,6(3):e18106.
Tang Y,Diao Y,Yu C,Gao X,Ju X,Xue C,Liu X,Ge P,Qu J,Zhang D.2013.Characterization of a tembusu virus isolated from naturally infected house sparrows(Passer domesticus)in northern China[J].Transboundary and Emerging Disease,60(2):152-158.
Thepparit C,Smith D R.2004.Serotype-specific entry of dengue virus into liver cells:identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor[J].Journal of Virology,78(22):12647-12656.
Zhang W,Chen S,Mahalingam S,Wang M,Cheng A.2017.An updated review of avian-origin tembusu virus:A newly emerging avian Flavivirus[J].The Journal of General Virology,98(10):2413-2420.
Zhao D,Liu Q,Han K,Wang H,Yang J,Bi K,Liu Y,Liu N,Tian Y,Li Y.2018.Identification of Glucose-regulated protein 78(GRP78)as a receptor in BHK-21 cells for duck tembusu virus infection[J].Frontiers in Microbiology,9:694.doi:10.3389/fmicb.2018.00694.
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