GST pull-down验证新城疫病毒基质蛋白与鸡importinβ1蛋白的相互作用
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摘要
旨在利用GST pull-down技术验证新城疫病毒(Newcastle disease virus,NDV)基质(matrix,M)蛋白与鸡importinβ1蛋白在体外的相互作用。根据NDV ZJ1株M基因和鸡importinβ1基因序列设计引物,通过PCR扩增获得NDV M基因和鸡importinβ1基因,将其分别定向插入到原核表达载体pGEX-6p-1和pET-32a(+)构建重组原核表达载体pGEX-6p-M、pET-32a-importinβ1,然后转化至大肠杆菌BL21(DE3)后进行IPTG诱导表达,SDSPAGE检测重组蛋白的表达情况,并对包涵体重组蛋白进行变性和复性处理。然后以GST-M重组蛋白为诱饵蛋白,His-importinβ1重组蛋白为猎物蛋白,利用GST pull-down技术验证M蛋白与importinβ1蛋白的相互作用。结果表明,成功构建了重组原核表达载体pGEX-6p-M和pET-32a-importinβ1,将其转化至大肠杆菌BL21(DE3)获得了正确表达。SDS-PAGE电泳检测显示GST-M重组蛋白主要以包涵体形式存在,而His-importinβ1重组蛋白以可溶性和包涵体两种形式存在。利用蛋白重折叠试剂盒获得了有活性的GST-M重组蛋白,将其作为诱饵蛋白,可以捕获并检测到His-importinβ1重组蛋白,但是GST标签蛋白不能结合His-importinβ1重组蛋白。利用GST pull-down技术证实了NDV M蛋白与鸡importinβ1蛋白在体外具有直接的相互作用,这为深入研究importinβ1蛋白在NDV M蛋白细胞核定位的分子机制以及在NDV复制和致病中的作用奠定了基础。
The purpose of this study was to verify the in vitrointeraction between Newcastle disease virus(NDV)matrix(M)protein and chicken importin β1 protein.The products of NDV M gene and chicken importin β1 gene were obtained by PCR amplification,and then subcloned into the prokaryotic expression vectors pGEX-6p-1and pET-32a(+)to construct plasmids pGEX-6p-M and pET-32a-importin β1,respectively.The recombinant proteins were expressed by transforming the plasmids into BL21(DE3)under the condition of IPTG and then analyzed by SDS-PAGE.The obtained inclusion body weight histones were dealt with through denaturation and renaturation to recover activity.Then the interaction between GST-M(bait protein)and Hisimportin β1 (prey protein)was examined by GST pull-down assay.The results showed that the recombinant prokaryotic expression vectors pGEX-6p-M and pET-32a-importin β1 were successfully constructed,and the recombinant proteins were correctly expressed in BL21(DE3).However,GST-M existed in the form of inclusion bodies,but His-importin β1 existed in the form of both solubility and inclusion bodies.The activated GST-M recombinant protein was recovered by protein refolding kit and could capture the His-importin β1 protein when used as bait protein,while GST alone could not.The in vitrointeraction between NDV M protein and chicken importin β1 protein was verified by GST pull-down assay,which will provide foundation for further studying the role of importin β1 in the nuclear localization of NDV M protein and the replication and pathogenicity of NDV.
The purpose of this study was to verify the in vitrointeraction between Newcastle disease virus(NDV)matrix(M)protein and chicken importin β1 protein.The products of NDV M gene and chicken importin β1 gene were obtained by PCR amplification,and then subcloned into the prokaryotic expression vectors pGEX-6p-1and pET-32a(+)to construct plasmids pGEX-6p-M and pET-32a-importin β1,respectively.The recombinant proteins were expressed by transforming the plasmids into BL21(DE3)under the condition of IPTG and then analyzed by SDS-PAGE.The obtained inclusion body weight histones were dealt with through denaturation and renaturation to recover activity.Then the interaction between GST-M(bait protein)and Hisimportin β1 (prey protein)was examined by GST pull-down assay.The results showed that the recombinant prokaryotic expression vectors pGEX-6p-M and pET-32a-importin β1 were successfully constructed,and the recombinant proteins were correctly expressed in BL21(DE3).However,GST-M existed in the form of inclusion bodies,but His-importin β1 existed in the form of both solubility and inclusion bodies.The activated GST-M recombinant protein was recovered by protein refolding kit and could capture the His-importin β1 protein when used as bait protein,while GST alone could not.The in vitrointeraction between NDV M protein and chicken importin β1 protein was verified by GST pull-down assay,which will provide foundation for further studying the role of importin β1 in the nuclear localization of NDV M protein and the replication and pathogenicity of NDV.
引文
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[11]段志强,嵇辛勤,许厚强,等.KPNB1和Ran蛋白共同介导新城疫病毒基质蛋白的入核转运[J].微生物学报,2017,57(1):109-120.DUAN Z Q,JI X Q,XU H Q,et al.The nuclear import of Newcastle disease virus matrix protein depends on KPNB1and Ran protein[J].Acta Microbiologica Sinica,2017,57(1):109-120.(in Chinese)
[12]RICCA B L,VENUGOPALAN G,FLETCHER DA.To pull or be pulled:parsing the multiple modes of mechanotransduction[J].Curr Opin Cell Biol,2013,25(5):558-564.
[13]胡焱,段志强,嵇辛勤,等.鸡importinβ1基因的克隆及生物信息学分析[J].生物技术,2016,26(6):546-550,556.HU Y,DUAN Z Q,JI X Q,et al.Cloning and bioinformatics analysis of chicken importinβ1gene[J].Biotechnology,2016,26(6):546-550,556.(in Chinese)
[14]GHILDYAL R,HO A,WAGSTAFF K M,et al.Nuclear import of the respiratory syncytial virus matrix protein is mediated by importinβ1independent of importinα[J].Biochemistry,2005,44(38):12887-12895.
[15]GHILDYAL R,HO A,DIAS M,et al.The respiratory syncytial virus matrix protein possesses a Crm1-mediated nuclear export mechanism[J].J Virol,2009,83(11):5353-5362.
[16]PALMERI D,MALIM M H.Importinβcan mediate the nuclear import of an arginine-rich nuclear localization signal in the absence of importinα[J].Mol Cell Biol,1999,19(2):1218-1225.
[17]LI Q,ZHANG Z F,ZHENG Z H,et al.Identification and characterization of complex dual nuclear localization signals in human bocavirus NP1[J].J Gen Virol,2013,94(6):1335-1342.
[18]JOUBERT D A,BLASDELL K R,AUDSLEY MD,et al.Bovine ephemeral fever rhabdovirusα1protein has viroporin-like properties and binds importinβ1and importin 7[J].J Virol,2014,88(3):1591-1603.
[19]SEAL B S,KING D J,MEINERSMANN R J.Molecular evolution of the Newcastle disease virus matrix protein gene and phylogenetic relationships among the paramyxoviridae[J].Virus Res,2000,66(1):1-11.
[20]DUAN Z Q,LI Q H,HE L,et al.Application of green fluorescent protein-labeled assay for the study of subcellular localization of Newcastle disease virus matrix protein[J].J Virol Methods,2013,194(1-2):118-122.
[21]胡焱,熊建民,赵佳福,等.Importinβ1介导的病毒蛋白入核转运在病毒复制中作用的研究进展[J].中国细胞生物学学报,2017,39(8):1091-1098.HU Y,XIONG J M,ZHAO J F,et al.Advances in importinβ1-mediated nuclear transport of viral proteins in the replication of viruses[J].Chinese Journal of Cell Biology,2017,39(8):1091-1098.(in Chinese)
[22]LEE C,HODGINS D,CALVERT J G,et al.Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication[J].Virology,2006,346(1):238-250.
[23]GHILDYAL R,HO A,JANS D A.Central role of the respiratory syncytial virus matrix protein in infection[J].FEMS Microbiol Rev,2006,30(5):692-705.
[24]IWASAKI M,TAKEDA M,SHIROGANE Y,et al.The matrix protein of Measles virus regulates viral RNA synthesis and assembly by interacting with the nucleocapsid protein[J].J Virol,2009,83(20):10374-10383.
[25]MOK B W Y,LIU H L,CHEN P,et al.The role of nuclear NS1protein in highly pathogenic H5N1influenza viruses[J].Microbes Infect,2017,19(12):587-596.
[2]HARRISON M S,SAKAGUCHI T,SCHMITT AP.Paramyxovirus assembly and budding:building particles that transmit infections[J].Int J Biochem Cell Biol,2010,42(9):1416-1429.
[3]段志强,胡顺林,刘秀梵.新城疫病毒与其它副黏病毒基质蛋白功能的比较[J].微生物学报,2016,56(7):1070-1078.DUAN Z Q,HU S L,LIU X F.Function comparison of the matrix protein between Newcastle disease virus and other paramyxoviruses-A review[J].Acta Microbiologica Sinica,2016,56(7):1070-1078.(in Chinese)
[4]PEEPLES M E,WANG C,GUPTA K C,et al.Nuclear entry and nucleolar localization of the Newcastle disease virus(NDV)matrix protein occur early in infection and do not require other NDV proteins[J].JVirol,1992,66(5):3263-3269.
[5]DUAN Z Q,SONG Q Q,WANG Y Y,et al.Characterization of signal sequences determining the nuclear export of Newcastle disease virus matrix protein[J].Arch Virol,2013,158(12):2589-2595.
[6]WULAN W N,HEYDET D,WALKER E J,et al.Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA viruses[J].Front Microbiol,2015,6:553.
[7]AUDSLEY M D,JANS D A,MOSELEY G W,et al.Roles of nuclear trafficking in infection by cytoplasmic negative-strand RNA viruses:paramyxoviruses and beyond[J].J Gen Virol,2016,97(10):2463-2481.
[8]PRYOR M J,RAWLINSON S M,BUTCHER R E,et al.Nuclear localization of dengue virus nonstructural protein 5through its importinα/β-recognized nuclear localization sequences is integral to viral infection[J].Traffic,2007,8(7):795-807.
[9]PUMROY R A,KE S,HART D J,et al.Molecular determinants for nuclear import of influenza A PB2by importinαisoforms 3and 7[J].Structure,2015,23(2):374-384.
[10]CAI M S,WANG S,XING J J,et al.Characterization of the nuclear import and export signals,and subcellular transport mechanism of varicella-zoster virus ORF9[J].J Gen Virol,2011,92(3):621-626.
[11]段志强,嵇辛勤,许厚强,等.KPNB1和Ran蛋白共同介导新城疫病毒基质蛋白的入核转运[J].微生物学报,2017,57(1):109-120.DUAN Z Q,JI X Q,XU H Q,et al.The nuclear import of Newcastle disease virus matrix protein depends on KPNB1and Ran protein[J].Acta Microbiologica Sinica,2017,57(1):109-120.(in Chinese)
[12]RICCA B L,VENUGOPALAN G,FLETCHER DA.To pull or be pulled:parsing the multiple modes of mechanotransduction[J].Curr Opin Cell Biol,2013,25(5):558-564.
[13]胡焱,段志强,嵇辛勤,等.鸡importinβ1基因的克隆及生物信息学分析[J].生物技术,2016,26(6):546-550,556.HU Y,DUAN Z Q,JI X Q,et al.Cloning and bioinformatics analysis of chicken importinβ1gene[J].Biotechnology,2016,26(6):546-550,556.(in Chinese)
[14]GHILDYAL R,HO A,WAGSTAFF K M,et al.Nuclear import of the respiratory syncytial virus matrix protein is mediated by importinβ1independent of importinα[J].Biochemistry,2005,44(38):12887-12895.
[15]GHILDYAL R,HO A,DIAS M,et al.The respiratory syncytial virus matrix protein possesses a Crm1-mediated nuclear export mechanism[J].J Virol,2009,83(11):5353-5362.
[16]PALMERI D,MALIM M H.Importinβcan mediate the nuclear import of an arginine-rich nuclear localization signal in the absence of importinα[J].Mol Cell Biol,1999,19(2):1218-1225.
[17]LI Q,ZHANG Z F,ZHENG Z H,et al.Identification and characterization of complex dual nuclear localization signals in human bocavirus NP1[J].J Gen Virol,2013,94(6):1335-1342.
[18]JOUBERT D A,BLASDELL K R,AUDSLEY MD,et al.Bovine ephemeral fever rhabdovirusα1protein has viroporin-like properties and binds importinβ1and importin 7[J].J Virol,2014,88(3):1591-1603.
[19]SEAL B S,KING D J,MEINERSMANN R J.Molecular evolution of the Newcastle disease virus matrix protein gene and phylogenetic relationships among the paramyxoviridae[J].Virus Res,2000,66(1):1-11.
[20]DUAN Z Q,LI Q H,HE L,et al.Application of green fluorescent protein-labeled assay for the study of subcellular localization of Newcastle disease virus matrix protein[J].J Virol Methods,2013,194(1-2):118-122.
[21]胡焱,熊建民,赵佳福,等.Importinβ1介导的病毒蛋白入核转运在病毒复制中作用的研究进展[J].中国细胞生物学学报,2017,39(8):1091-1098.HU Y,XIONG J M,ZHAO J F,et al.Advances in importinβ1-mediated nuclear transport of viral proteins in the replication of viruses[J].Chinese Journal of Cell Biology,2017,39(8):1091-1098.(in Chinese)
[22]LEE C,HODGINS D,CALVERT J G,et al.Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication[J].Virology,2006,346(1):238-250.
[23]GHILDYAL R,HO A,JANS D A.Central role of the respiratory syncytial virus matrix protein in infection[J].FEMS Microbiol Rev,2006,30(5):692-705.
[24]IWASAKI M,TAKEDA M,SHIROGANE Y,et al.The matrix protein of Measles virus regulates viral RNA synthesis and assembly by interacting with the nucleocapsid protein[J].J Virol,2009,83(20):10374-10383.
[25]MOK B W Y,LIU H L,CHEN P,et al.The role of nuclear NS1protein in highly pathogenic H5N1influenza viruses[J].Microbes Infect,2017,19(12):587-596.