鱼类病毒性出血性败血症病毒基质蛋白的原核表达及其亚细胞定位
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摘要
为了对鱼类病毒性出血性败血症病毒(viral hemorrhagic septicemia virus, VHSV)基质蛋白(matrix protein, M)进行功能研究,本实验通过PCR扩增了M基因全长序列,将其克隆至原核表达载体pET-32a(+),转化至大肠杆菌Rosetta(DE3)感受态细胞后进行IPTG诱导表达,将纯化后的重组蛋白免疫BALB/c小鼠制备多克隆抗体,采用间接ELISA检测抗体效价,并运用Westernblot和间接免疫荧光检测抗体特异性。结果显示,M基因全长为606bp,IPTG诱导得到的融合蛋白主要以包涵体的形式存在,大小约为36 kD,比预计略小。间接ELISA检测抗体效价大于1:102400,Westernblot检测显示该抗体可以特异性识别纯化的融合蛋白和VHSV感染的鲤上皮瘤(epithelioma papulosum cyprini, EPC)细胞中的M蛋白。间接免疫荧光结果显示M蛋白多抗能识别感染VHSV的EPC细胞中的M蛋白,且M蛋白主要定位于细胞质和细胞膜。本研究中M蛋白多克隆抗体的制备将有助于开展M蛋白的功能研究及疾病的免疫学诊断。
Viral hemorrhagic septicemia virus(VHSV) is one of the most serious pathogens of finfish that affects over 80 marine and freshwater species in North America, Europe, and Asia. The genome of VHSV is a negative-sense, single stranded RNA containing approximately 12000 base pairs that encode six proteins, which are the nucleoprotein(N), phosphoprotein(P), matrix protein(M), glycoprotein(G), non-virion protein(NV), and RNA polymerase protein(L) in order from 3? to 5?. The M protein functions in a variety of rhabdovirus infection processes such as assembly and budding, inhibiting host-cell directed transcription and gene expression, inducing apoptosis of the host cell, et al., which were all thoroughly investigated in other rhabdoviruses such as vesicular stomatitis virus and rabies virus, while less so in VHSV. In order to investigate the function of the M protein of VHSV, the M gene was amplified by PCR and cloned into the prokaryotic expression vector pET-32a(+), which was transformed into Escherichia coli Rosetta(DE3) competent cells. The recombinant protein was induced by IPTG, and the polyclonal antibody was prepared by immunizing BALB/c mice with purified recombinant protein. The titer of the antibody was detected by an indirect ELISA, and the antibody specificity was tested by western blot and indirect immunofluorescence. The results showed that the full-length M gene was 606 bp. The fusion protein induced by IPTG mainly existed in the form of an inclusion body, and the size was about 36 kDa, which was slightly smaller than expected. The indirect ELISA assay showed that the titer of the antibody was greater than 1 : 102400. The western blot showed that the antibody could specifically identify the purified fusion protein and M protein in VHSV infected Epithelioma papulosum cyprinid(EPC) cells. The indirect immunofluorescence showed that the M protein antibody can recognize the M protein in VHSV infected EPC cells, and the M protein was mainly localized in the cytoplasm and cell membrane. These results suggest that the prepared polyclonal antibody can be used as an effective tool to study the function of the M protein and for the diagnosis of VHSV.
Viral hemorrhagic septicemia virus(VHSV) is one of the most serious pathogens of finfish that affects over 80 marine and freshwater species in North America, Europe, and Asia. The genome of VHSV is a negative-sense, single stranded RNA containing approximately 12000 base pairs that encode six proteins, which are the nucleoprotein(N), phosphoprotein(P), matrix protein(M), glycoprotein(G), non-virion protein(NV), and RNA polymerase protein(L) in order from 3? to 5?. The M protein functions in a variety of rhabdovirus infection processes such as assembly and budding, inhibiting host-cell directed transcription and gene expression, inducing apoptosis of the host cell, et al., which were all thoroughly investigated in other rhabdoviruses such as vesicular stomatitis virus and rabies virus, while less so in VHSV. In order to investigate the function of the M protein of VHSV, the M gene was amplified by PCR and cloned into the prokaryotic expression vector pET-32a(+), which was transformed into Escherichia coli Rosetta(DE3) competent cells. The recombinant protein was induced by IPTG, and the polyclonal antibody was prepared by immunizing BALB/c mice with purified recombinant protein. The titer of the antibody was detected by an indirect ELISA, and the antibody specificity was tested by western blot and indirect immunofluorescence. The results showed that the full-length M gene was 606 bp. The fusion protein induced by IPTG mainly existed in the form of an inclusion body, and the size was about 36 kDa, which was slightly smaller than expected. The indirect ELISA assay showed that the titer of the antibody was greater than 1 : 102400. The western blot showed that the antibody could specifically identify the purified fusion protein and M protein in VHSV infected Epithelioma papulosum cyprinid(EPC) cells. The indirect immunofluorescence showed that the M protein antibody can recognize the M protein in VHSV infected EPC cells, and the M protein was mainly localized in the cytoplasm and cell membrane. These results suggest that the prepared polyclonal antibody can be used as an effective tool to study the function of the M protein and for the diagnosis of VHSV.
引文
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[2]Studer J,Janies D A.Global spread and evolution of viral haemorrhagic septicaemia virus[J].Journal of Fish Diseases,2011,34(10):741-747.
[3]Kim R,Faisal M.Emergence and resurgence of the viral hemorrhagic septicemia virus(Novirhabdovirus,Rhabdoviridae,Mononegavirales)[J].Journal of Advanced Research,2011,2(1):9-23.
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[8]Zhu R L,Zhang Q Y.Determination and analysis of the complete genome sequence of Paralichthys olivaceus rhabdovirus(PORV)[J].Archives of Virology,2014,159(4):817-820.
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[10]Rajani K R,Kneller E L P,Mckenzie M O,et al.Complexes of vesicular stomatitis virus matrix protein with host rae1and Nup98 involved in inhibition of host transcription[J].PLoS Pathogens,2012,8(9):e1002929.
[11]Mire C E,Whitt M A.The protease-sensitive loop of the vesicular stomatitis virus matrix protein is involved in virus assembly and protein translation[J].Virology,2011,416(1):16-25.
[12]Larrous F,Gholami A,Mouhamad S,et al.Two overlapping domains of a lyssavirus matrix protein that acts on different cell death pathways[J].Journal of Virology,2010,84(19):9897-9906.
[13]Cary Z D,Willingham M C,Lyles D S.Oncolytic vesicular stomatitis virus induces apoptosis in U87 glioblastoma cells by a type II death receptor mechanism and induces cell death and tumor clearance in vivo[J].Journal of Virology,2011,85(12):5708-5717.
[14]Pierce L R,Stepien C A.Evolution and biogeography of an emerging quasispecies:diversity patterns of the fish Viral Hemorrhagic Septicemia virus(VHSv)[J].Molecular Phylogenetics&Evolution,2012,63(2):327-341.
[15]He M,Yan X C,Liang Y,et al.Evolution of the viral hemorrhagic septicemia virus:divergence,selection and origin[J].Molecular Phylogenetics&Evolution,2014,77(1):34-40.
[16]Getchell R G,Cornwell E R,Bogdanowicz S,et al.Complete sequences of 4 viral hemorrhagic septicemia virus IVb isolates and their virulence in northern pike fry[J].Diseases of Aquatic Organisms,2017,126(3):211-227.
[17]Biacchesi S,Merour E,Chevret D,et al.NV proteins of fish novirhabdovirus recruit cellular PPM1Bb protein phosphatase and antagonize RIG-I-mediated IFN induction[J].Scientific Reports,2017,7:44025.
[18]Chinchilla B,Gomez-Casado E.Identification of the functional regions of the viral haemorrhagic septicaemia virus(VHSV)NV protein:Variants that improve function[J].Fish&Shellfish Immunology,2017,70:343-350.
[19]Baillon L,Mérour E,Cabon J,et al.A single amino acid change in the non-structural NV protein impacts the virulence phenotype of viral hemorrhagic septicemia virus in trout[J].Journal of General Virology,2017,98(6):1181-1184.
[20]Ke Q,Weaver W,Pore A,et al.Role of viral hemorrhagic septicemia virus matrix(M)protein in suppressing host transcription[J].Journal of Virology,2017,91(19):e00279-17.
[21]Chiou P P,Kim C H,Ormonde P,et al.Infectious hematopoietic necrosis virus matrix protein inhibits host-directed gene expression and induces morphological changes of apoptosis in cell cultures[J].Journal of Virology,2000,74(16):7619-7627.
[22]Walker P J,Blasdell K R,Calisher C H,et al.ICTV virus taxonomy profile:Rhabdoviridae[J].Journal of General Virology,2018,99:447-448.
[23]Mahy B W J,Van Regenmortel M H V.The Encyclopedia of Virology[M].3rd ed.Salt Lake:Academic Press,2008:221-227.
[24]Larrous F,Gholami A,Mouhamad S,et al.Two overlapping domains of a lyssavirus matrix protein that acts on different cell death pathways[J].Journal of Virology,2010,84(19):9897-9906.
[25]Ameyama S,Toriumi H,Takahashi T,et al.Monoclonal antibody#3-9-16 recognizes one of the two isoforms of rabies virus matrix protein that exposes its N-terminus on the virion surface[J].Microbiology and Immunology,2003,47(9):639-651.
[26]Solon J,Gareil O,Bassereau P,et al.Membrane deformations induced by the matrix protein of vesicular stomatitis virus in a minimal system.[J].Journal of General Virology,2005,86(12):3357-3363.
[27]Dancho B,Mckenzie M O,Connor J H,et al.Vesicular stomatitis virus matrix protein mutations that affect association with host membranes and viral nucleocapsids[J].Journal of Biological Chemistry,2009,284(7):4500-4509.
[28]Zhao X C,Zhang X W,Zhang S F,et al.Prokaryotic expression and purification of matrix protein of bd-06 strain rabies virus and preparation of its polyclonal antibody[J].China Animal Husbandry&Veterinary Medicine,2013,40(4):36-39.[赵雪超,张学炜,张守峰,等.狂犬病病毒BD-06株基质蛋白的原核表达、纯化及其多克隆抗体的制备[J].中国畜牧兽医,2013,40(4):36-39.]
[29]Zhang S K,Chen B J,Xiao X,et al.Preparation of recombinant VSV Matrix protein and establishment of ELISA for antibody detection[J].Journal of Shanghai Jiaotong University(Agricultural Science),2014,32(1):89-94.[张世宽,陈备娟,肖贤,等.重组水泡性口炎病毒基质蛋白制备及ELISA检测方法的建立[J].上海交通大学学报(农业科学版),2014,32(1):89-94.]
[30]Gong M M,Zeng N,Cheng C F,et al.Development of indirect ELISA for detection of antibodies against rabies virus based on prokaryotic expression of matrix protein[J].Chinese Journal of Zoonoses,2013,29(1):17-22.[宫苗苗,曾妮,程朝飞,等.狂犬病病毒基质蛋白的原核表达及其间接ELISA方法的建立[J].中国人兽共患病学报,2013,29(1):17-22.]
[2]Studer J,Janies D A.Global spread and evolution of viral haemorrhagic septicaemia virus[J].Journal of Fish Diseases,2011,34(10):741-747.
[3]Kim R,Faisal M.Emergence and resurgence of the viral hemorrhagic septicemia virus(Novirhabdovirus,Rhabdoviridae,Mononegavirales)[J].Journal of Advanced Research,2011,2(1):9-23.
[4]Faisal M,Shavalier M,Kim R K,et al.Spread of the emerging viral hemorrhagic septicemia virus strain,genotype IVb,in Michigan,USA[J].Viruses,2012,4(5):734-760.
[5]Duesund H,Nylund S,Watanabe K,et al.Characterization of a VHS virus genotype III isolated from rainbow trout(Oncorhychus mykiss)at a marine site on the west coast of Norway[J].Virology Journal,2010,7(1):19.
[6]Ju J B,Takano T,Hirono I,et al.Genome analysis of viral hemorrhagic septicemia virus isolated from Japanese flounder Paralichthys olivaceus,in Japan[J].Fisheries Science,2006,72(4):906-908.
[7]Wisik K,Seokryel K,Duwoon K,et al.An outbreak of VHSV(viral hemorrhagic septicemia virus)infection in farmed olive flounder Paralichthys olivaceus in Korea[J].Aquaculture,2009,296(1-2):165-168.
[8]Zhu R L,Zhang Q Y.Determination and analysis of the complete genome sequence of Paralichthys olivaceus rhabdovirus(PORV)[J].Archives of Virology,2014,159(4):817-820.
[9]Ahmadivand S,Soltani M,Mardani K,et al.Isolation and identification of viral hemorrhagic septicemia virus(VHSV)from farmed rainbow trout(Oncorhynchus mykiss)in Iran[J].Acta Tropica,2016,156:30-36.
[10]Rajani K R,Kneller E L P,Mckenzie M O,et al.Complexes of vesicular stomatitis virus matrix protein with host rae1and Nup98 involved in inhibition of host transcription[J].PLoS Pathogens,2012,8(9):e1002929.
[11]Mire C E,Whitt M A.The protease-sensitive loop of the vesicular stomatitis virus matrix protein is involved in virus assembly and protein translation[J].Virology,2011,416(1):16-25.
[12]Larrous F,Gholami A,Mouhamad S,et al.Two overlapping domains of a lyssavirus matrix protein that acts on different cell death pathways[J].Journal of Virology,2010,84(19):9897-9906.
[13]Cary Z D,Willingham M C,Lyles D S.Oncolytic vesicular stomatitis virus induces apoptosis in U87 glioblastoma cells by a type II death receptor mechanism and induces cell death and tumor clearance in vivo[J].Journal of Virology,2011,85(12):5708-5717.
[14]Pierce L R,Stepien C A.Evolution and biogeography of an emerging quasispecies:diversity patterns of the fish Viral Hemorrhagic Septicemia virus(VHSv)[J].Molecular Phylogenetics&Evolution,2012,63(2):327-341.
[15]He M,Yan X C,Liang Y,et al.Evolution of the viral hemorrhagic septicemia virus:divergence,selection and origin[J].Molecular Phylogenetics&Evolution,2014,77(1):34-40.
[16]Getchell R G,Cornwell E R,Bogdanowicz S,et al.Complete sequences of 4 viral hemorrhagic septicemia virus IVb isolates and their virulence in northern pike fry[J].Diseases of Aquatic Organisms,2017,126(3):211-227.
[17]Biacchesi S,Merour E,Chevret D,et al.NV proteins of fish novirhabdovirus recruit cellular PPM1Bb protein phosphatase and antagonize RIG-I-mediated IFN induction[J].Scientific Reports,2017,7:44025.
[18]Chinchilla B,Gomez-Casado E.Identification of the functional regions of the viral haemorrhagic septicaemia virus(VHSV)NV protein:Variants that improve function[J].Fish&Shellfish Immunology,2017,70:343-350.
[19]Baillon L,Mérour E,Cabon J,et al.A single amino acid change in the non-structural NV protein impacts the virulence phenotype of viral hemorrhagic septicemia virus in trout[J].Journal of General Virology,2017,98(6):1181-1184.
[20]Ke Q,Weaver W,Pore A,et al.Role of viral hemorrhagic septicemia virus matrix(M)protein in suppressing host transcription[J].Journal of Virology,2017,91(19):e00279-17.
[21]Chiou P P,Kim C H,Ormonde P,et al.Infectious hematopoietic necrosis virus matrix protein inhibits host-directed gene expression and induces morphological changes of apoptosis in cell cultures[J].Journal of Virology,2000,74(16):7619-7627.
[22]Walker P J,Blasdell K R,Calisher C H,et al.ICTV virus taxonomy profile:Rhabdoviridae[J].Journal of General Virology,2018,99:447-448.
[23]Mahy B W J,Van Regenmortel M H V.The Encyclopedia of Virology[M].3rd ed.Salt Lake:Academic Press,2008:221-227.
[24]Larrous F,Gholami A,Mouhamad S,et al.Two overlapping domains of a lyssavirus matrix protein that acts on different cell death pathways[J].Journal of Virology,2010,84(19):9897-9906.
[25]Ameyama S,Toriumi H,Takahashi T,et al.Monoclonal antibody#3-9-16 recognizes one of the two isoforms of rabies virus matrix protein that exposes its N-terminus on the virion surface[J].Microbiology and Immunology,2003,47(9):639-651.
[26]Solon J,Gareil O,Bassereau P,et al.Membrane deformations induced by the matrix protein of vesicular stomatitis virus in a minimal system.[J].Journal of General Virology,2005,86(12):3357-3363.
[27]Dancho B,Mckenzie M O,Connor J H,et al.Vesicular stomatitis virus matrix protein mutations that affect association with host membranes and viral nucleocapsids[J].Journal of Biological Chemistry,2009,284(7):4500-4509.
[28]Zhao X C,Zhang X W,Zhang S F,et al.Prokaryotic expression and purification of matrix protein of bd-06 strain rabies virus and preparation of its polyclonal antibody[J].China Animal Husbandry&Veterinary Medicine,2013,40(4):36-39.[赵雪超,张学炜,张守峰,等.狂犬病病毒BD-06株基质蛋白的原核表达、纯化及其多克隆抗体的制备[J].中国畜牧兽医,2013,40(4):36-39.]
[29]Zhang S K,Chen B J,Xiao X,et al.Preparation of recombinant VSV Matrix protein and establishment of ELISA for antibody detection[J].Journal of Shanghai Jiaotong University(Agricultural Science),2014,32(1):89-94.[张世宽,陈备娟,肖贤,等.重组水泡性口炎病毒基质蛋白制备及ELISA检测方法的建立[J].上海交通大学学报(农业科学版),2014,32(1):89-94.]
[30]Gong M M,Zeng N,Cheng C F,et al.Development of indirect ELISA for detection of antibodies against rabies virus based on prokaryotic expression of matrix protein[J].Chinese Journal of Zoonoses,2013,29(1):17-22.[宫苗苗,曾妮,程朝飞,等.狂犬病病毒基质蛋白的原核表达及其间接ELISA方法的建立[J].中国人兽共患病学报,2013,29(1):17-22.]
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