一株新的大口黑鲈弹状病毒(YH01)的分离、鉴定及其糖蛋白的杆状病毒表达系统的构建（英文）
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摘要
目的:探究大口黑鲈弹状病毒株与其他已知弹状病毒的差异,构建该病毒糖蛋白的杆状病毒表达系统,用于后续口服疫苗的研发。创新点:基于获得的大口黑鲈弹状病毒的全基因组序列,并利用杆状病毒表达系统获得了高丰度的病毒糖蛋白,可结合家蚕用于后续口服疫苗的研发。方法:利用c DNA末端快速扩增法(RACE)及逆转录聚合酶链反应(RT-PCR)等技术获得了病毒的部分序列,拼接获得了病毒的全基因组序列。通过ClustalW比较该病毒与已知弹状病毒的3'leader及5'trailer序列差异,进一步利用MEGA软件比较该病毒全基因组序列与其他鱼类弹状病毒的差异,确定其系统进化树中的分支及地位。基于糖蛋白序列分析结果,利用杆状病毒表达载体构建该病毒的糖蛋白表达系统,感染昆虫卵巢细胞(SF9)后,借助荧光定量PCR(q RT-PCR)、免疫荧光及蛋白质印迹法(western blot)等技术确定病毒增殖及糖蛋白的表达情况。结论:该大口黑鲈弹状病毒株全长11 526 bp,且与鳜鱼弹状病毒为同一分支,属于水泡病毒属。构建的病毒糖蛋白杆状病毒表达系统成功在SF9细胞中表达了高丰度的糖蛋白。
As one of the most important aquatic fish, Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage. In this study, a new strain of M. salmoides rhabdovirus(MSRV) was isolated from Yuhang, Zhejiang Province, China, and named MSRV-YH01. The virus infected the grass carp ovary(GCO) cell line and displayed virion particles with atypical bullet shape, 300–500 nm in length and 100–200 nm in diameter under transmission electron microscopy. The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins. The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus. To explore the potential for a vaccine against MSRV, a glycoprotein(1–458 amino acid residues) of MSRV-YH01 was successfully amplified and cloned into the plasmid p FastBac1. The high-purity recombinant bacmid-glycoprotein was obtained from DH10 Bac through screening and identification. Based on polymerase chain reaction(PCR), western blot, and immunofluorescence assay, recombinant virus, including the MSRV-YH01 glycoprotein gene, was produced by transfection of SF9 cells using the pFastBac1-gE2, and then repeatedly amplified to express the glycoprotein protein. We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.
As one of the most important aquatic fish, Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage. In this study, a new strain of M. salmoides rhabdovirus(MSRV) was isolated from Yuhang, Zhejiang Province, China, and named MSRV-YH01. The virus infected the grass carp ovary(GCO) cell line and displayed virion particles with atypical bullet shape, 300–500 nm in length and 100–200 nm in diameter under transmission electron microscopy. The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins. The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus. To explore the potential for a vaccine against MSRV, a glycoprotein(1–458 amino acid residues) of MSRV-YH01 was successfully amplified and cloned into the plasmid p FastBac1. The high-purity recombinant bacmid-glycoprotein was obtained from DH10 Bac through screening and identification. Based on polymerase chain reaction(PCR), western blot, and immunofluorescence assay, recombinant virus, including the MSRV-YH01 glycoprotein gene, was produced by transfection of SF9 cells using the pFastBac1-gE2, and then repeatedly amplified to express the glycoprotein protein. We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.
引文
Alonso M,Leong JAC,2013.Licensed DNA vaccines against infectious hematopoietic necrosis virus(IHNV).Recent Pat DNA Gene Seq,7(1):62-65.https://doi.org/10.2174/1872215611307010009
Amarasinghe GK,Bào Y,Basler CF,et al.,2017.Taxonomy of the order Mononegavirales:update 2017.Arch Virol,162(8):2493-2504.https://doi.org/10.1007/s00705-017-3311-7
Anderson ED,Mourich DV,Fahrenkrug SC,et al.,1996.Genetic immunization of rainbow trout(Oncorhynchus mykiss)against infectious hematopoietic necrosis virus.Mol Mar Biol Biotechnol,5(2):114-122.
Assenberg R,Delmas O,Morin B,et al.,2010.Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription.Antiviral Res,87(2):149-161.https://doi.org/10.1016/j.antiviral.2010.02.322
Barr JN,Whelan SPJ,Wertz GW,2002.Transcriptional control of the RNA-dependent RNA polymerase of vesicular stomatitis virus.Biochim Biophys Acta,1577(2):337-353.https://doi.org/10.1016/S0167-4781(02)00462-1
Bauer EF,2013.Cascades of enemy release:impacts of an invasive species(Neogobius melanostomus)on the parasite communities of two native predators(Micropterus dolomieu and Micropterus salmoides).MS Thesis,University of New York College of Environmental Science and Forestry,Syracuse,New York,USA.https://doi.org/10.13140/RG.2.1.4146.6006
Castric J,Rasschaert D,Bernard J,1984.Evidence of lyssaviruses among rhabdovirus isolates from the European EEL Anguilla Anguilla.Ann Inst Pasteur/Virol,135(1):35-55.https://doi.org/10.1016/S0769-2617(84)80038-6
Chen ZY,Lei XY,Zhang QY,2012.The antiviral defense mechanisms in mandarin fish induced by DNA vaccination against a rhabdovirus.Vet Microbiol,157(3-4):264-275.https://doi.org/10.1016/j.vetmic.2011.12.025
Compans RW,Klenk HD,Caliguiri LA,et al.,1970.Influenza virus proteins:I.Analysis of polypeptides of the virion and identification of spike glycoproteins.Virology,42(4):880-889.https://doi.org/10.1016/0042-6822(70)90337-5
Dorson M,Torchy C,Chilmonczyk S,et al.,1984.A rhabdovirus pathogenic for perch,Perca fluviatilis L.:isolation and preliminary study.J Fish Dis,7(3):241-245.https://doi.org/10.1111/j.1365-2761.1984.tb00929.x
Emmenegger EJ,Kurath G,2008.DNA vaccine protects ornamental koi(Cyprinus carpio koi)against North American spring viremia of carp virus.Vaccine,26(50):6415-6421.https://doi.org/10.1016/j.vaccine.2008.08.071
Fu XZ,Lin Q,Liang HR,et al.,2017.The biological features and genetic diversity of novel fish rhabdovirus isolates in China.Arch Virol,162(9):2829-2834.https://doi.org/10.1007/s00705-017-3416-z
Galinier R,van Beurden S,Amilhat E,et al.,2012.Complete genomic sequence and taxonomic position of eel virus European X(EVEX),a rhabdovirus of European eel.Virus Res,166(1-2):1-12.https://doi.org/10.1016/j.virusres.2012.02.020
Gao EB,Chen GF,2018.Micropterus salmoides rhabdovirus(MSRV)infection induced apoptosis and activated interferon signaling pathway in largemouth bass skin cells.Fish Shellfish Immunol,76:161-166.https://doi.org/10.1016/j.fsi.2018.03.008
Grabowska AK,Lipińska AD,Rohde J,et al.,2009.New baculovirus recombinants expressing Pseudorabies virus(PRV)glycoproteins protect mice against lethal challenge infection.Vaccine,27(27):3584-3591.https://doi.org/10.1016/j.vaccine.2009.03.067
Ho PY,Chen YC,Maekawa S,et al.,2018.Efficacy of recombinant protein vaccines for protection against Nocardia seriolae infection in the largemouth bass Micropterus salmoides.Fish Shellfish Immunol,78:35-41.https://doi.org/10.1016/j.fsi.2018.04.024
Hoffmann B,Beer M,Schütze H,et al.,2005.Fish rhabdoviruses:molecular epidemiology and evolution.In:Fu ZF(Ed.),The World of Rhabdoviruses.Springer,Berlin,Heidelberg,p.81-117.https://doi.org/10.1007/3-540-27485-5_5
Kim CH,Johnson MC,Drennan JD,et al.,2000.DNA vaccines encoding viral glycoproteins induce nonspecific immunity and Mx protein synthesis in fish.J Virol,74(15):7048-7054.https://doi.org/10.1128/JVI.74.15.7048-7054.2000
Ksenofontov AL,Badun GA,Fedorova NV,et al.,2008.Quantitation of the glycoprotein spike area on the surface of enveloped viruses.Mol Biol,42(6):973-975.https://doi.org/10.1134/s0026893308060204
Lei Y,Qi RR,Cui LB,et al.,2015.Diagnosis of rhabdovirus disease in juvenile largemouth bass Micropterus salmonides.J Dalian Fish Univ,30(3):305-308(in Chinese).https://doi.org/10.16535/j.cnki.dlhyxb.2015.03.013
Lorenzen N,Lorenzen E,Einer-Jensen K,et al.,1998.Protective immunity to VHS in rainbow trout(Oncorhynchus mykiss,Walbaum)following DNA vaccination.Fish Shellfish Immunol,8(4):261-270.https://doi.org/10.1006/fsim.1997.0134
Lorenzoni M,D?rr AJM,Erra R,et al.,2002.Growth and reproduction of largemouth bass(Micropterus salmoides Lacépède,1802)in Lake Trasimeno(Umbria,Italy).Fish Res,56(1):89-95.https://doi.org/10.1016/S0165-7836(01)00309-5
Luo X,Deng GC,Zhao CC,et al.,2013.Isolation and preliminary identification of rhabdovirus from Channa maculata cultured in pond.Acta Hydrobiol Sin,37(4):620-625(in Chinese).https://doi.org/10.7541/2013.72
Lv SJ,Lu BJ,Xu JH,et al.,2015.Immune response of peroxinectin of Chinese mitten crab Eriocheir sinensis to exterior stimulation.Dev Comp Immunol,51(1):56-64.https://doi.org/10.1016/j.dci.2015.02.016
Ma DM,Deng GC,Bai JJ,et al.,2013.A strain of Siniperca chuatsi rhabdovirus causes high mortality among cultured largemouth bass in south China.J Aquat Anim Health,25(3):197-204.https://doi.org/10.1080/08997659.2013.799613
Sakai K,Hagiwara K,Omatsu T,et al.,2015.Isolation and characterization of a novel rhabdovirus from a wild boar(Sus scrofa)in Japan.Vet Microbiol,179(3-4):197-203.https://doi.org/10.1016/j.vetmic.2015.05.013
Sano T,Nishimura T,Okamoto N,et al.,1976.Isolation of rhabdovirus from European eels(Anguilla anguilla)at Japanese port of entry.Fish Health News,5:5-6.
Seo JY,Kim KH,Kim SG,et al.,2006.Protection of flounder against hirame rhabdovirus(HIRRV)with a DNA vaccine containing the glycoprotein gene.Vaccine,24(7):1009-1015.https://doi.org/10.1016/j.vaccine.2005.07.109
Serena MS,Geisler C,Metz GE,et al.,2013.Expression and purification of Suid Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose Aujeszky’s disease in infected pigs.Protein Expr Purif,90(1):1-8.https://doi.org/10.1016/j.pep.2013.04.008
Toth AM,Geisler C,Aumiller JJ,et al.,2011.Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system.Protein Expr Purif,80(2):274-282.https://doi.org/10.1016/j.pep.2011.08.002
Walker PJ,Wang YH,Cowley JA,et al.,1994.Structural and antigenic analysis of the nucleoprotein of bovine ephemeral fever rhabdovirus.J Gen Virol,75(8):1889-1899.https://doi.org/10.1099/0022-1317-75-8-1889
Xu XG,Wang ZS,Zhang Q,et al.,2011.Baculovirus surface display of E envelope glycoprotein of Japanese encephalitis virus and its immunogenicity of the displayed proteins in mouse and swine models.Vaccine,29(4):636-643.https://doi.org/10.1016/j.vaccine.2010.11.045
Xu XG,Wang ZS,Zhang Q,et al.,2012.Baculovirus as a PRRSV and PCV2 bivalent vaccine vector:baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2.J Virol Methods,179(2):359-366.https://doi.org/10.1016/j.jviromet.2011.11.023
Zeng WW,Wang Q,Wang YY,2013.Isolation and characterization of a rhabdovirus from snakehead fish(Ophicephalus striatus).J Fish China,37(9):1416-1424(in Chinese).https://doi.org/10.3724/SP.J.1231.2013.38588
Zhang LJ,Li NQ,Lin Q,et al.,2018.An avirulent Micropterus salmoides rhabdovirus vaccine candidate protects Chinese perch against rhabdovirus infection.Fish Shellfish Immunol,77:474-480.https://doi.org/10.1016/j.fsi.2018.03.047
Zhang S,Huang YH,Zhu JL,et al.,2018.Expression of hNeuritin protein in a baculovirus expression system and the analysis of its activity.Gene,647:129-135.https://doi.org/10.1016/j.gene.2018.01.026
Zhang XL,Xiao F,Li SM,et al.,2018.China Fishery Statistical Yearbook 2018.China Agriculture Press,Beijing,China,p.33(in Chinese).
Zhang ZY,You Y,Meng FL,et al.,2011.A primary research of using silkworm larvae as substitutes of feed proteins.JChangsha Univ,25(2):68-69(in Chinese).https://doi.org/10.3969/j.issn.1008-4681.2011.02.025
Amarasinghe GK,Bào Y,Basler CF,et al.,2017.Taxonomy of the order Mononegavirales:update 2017.Arch Virol,162(8):2493-2504.https://doi.org/10.1007/s00705-017-3311-7
Anderson ED,Mourich DV,Fahrenkrug SC,et al.,1996.Genetic immunization of rainbow trout(Oncorhynchus mykiss)against infectious hematopoietic necrosis virus.Mol Mar Biol Biotechnol,5(2):114-122.
Assenberg R,Delmas O,Morin B,et al.,2010.Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription.Antiviral Res,87(2):149-161.https://doi.org/10.1016/j.antiviral.2010.02.322
Barr JN,Whelan SPJ,Wertz GW,2002.Transcriptional control of the RNA-dependent RNA polymerase of vesicular stomatitis virus.Biochim Biophys Acta,1577(2):337-353.https://doi.org/10.1016/S0167-4781(02)00462-1
Bauer EF,2013.Cascades of enemy release:impacts of an invasive species(Neogobius melanostomus)on the parasite communities of two native predators(Micropterus dolomieu and Micropterus salmoides).MS Thesis,University of New York College of Environmental Science and Forestry,Syracuse,New York,USA.https://doi.org/10.13140/RG.2.1.4146.6006
Castric J,Rasschaert D,Bernard J,1984.Evidence of lyssaviruses among rhabdovirus isolates from the European EEL Anguilla Anguilla.Ann Inst Pasteur/Virol,135(1):35-55.https://doi.org/10.1016/S0769-2617(84)80038-6
Chen ZY,Lei XY,Zhang QY,2012.The antiviral defense mechanisms in mandarin fish induced by DNA vaccination against a rhabdovirus.Vet Microbiol,157(3-4):264-275.https://doi.org/10.1016/j.vetmic.2011.12.025
Compans RW,Klenk HD,Caliguiri LA,et al.,1970.Influenza virus proteins:I.Analysis of polypeptides of the virion and identification of spike glycoproteins.Virology,42(4):880-889.https://doi.org/10.1016/0042-6822(70)90337-5
Dorson M,Torchy C,Chilmonczyk S,et al.,1984.A rhabdovirus pathogenic for perch,Perca fluviatilis L.:isolation and preliminary study.J Fish Dis,7(3):241-245.https://doi.org/10.1111/j.1365-2761.1984.tb00929.x
Emmenegger EJ,Kurath G,2008.DNA vaccine protects ornamental koi(Cyprinus carpio koi)against North American spring viremia of carp virus.Vaccine,26(50):6415-6421.https://doi.org/10.1016/j.vaccine.2008.08.071
Fu XZ,Lin Q,Liang HR,et al.,2017.The biological features and genetic diversity of novel fish rhabdovirus isolates in China.Arch Virol,162(9):2829-2834.https://doi.org/10.1007/s00705-017-3416-z
Galinier R,van Beurden S,Amilhat E,et al.,2012.Complete genomic sequence and taxonomic position of eel virus European X(EVEX),a rhabdovirus of European eel.Virus Res,166(1-2):1-12.https://doi.org/10.1016/j.virusres.2012.02.020
Gao EB,Chen GF,2018.Micropterus salmoides rhabdovirus(MSRV)infection induced apoptosis and activated interferon signaling pathway in largemouth bass skin cells.Fish Shellfish Immunol,76:161-166.https://doi.org/10.1016/j.fsi.2018.03.008
Grabowska AK,Lipińska AD,Rohde J,et al.,2009.New baculovirus recombinants expressing Pseudorabies virus(PRV)glycoproteins protect mice against lethal challenge infection.Vaccine,27(27):3584-3591.https://doi.org/10.1016/j.vaccine.2009.03.067
Ho PY,Chen YC,Maekawa S,et al.,2018.Efficacy of recombinant protein vaccines for protection against Nocardia seriolae infection in the largemouth bass Micropterus salmoides.Fish Shellfish Immunol,78:35-41.https://doi.org/10.1016/j.fsi.2018.04.024
Hoffmann B,Beer M,Schütze H,et al.,2005.Fish rhabdoviruses:molecular epidemiology and evolution.In:Fu ZF(Ed.),The World of Rhabdoviruses.Springer,Berlin,Heidelberg,p.81-117.https://doi.org/10.1007/3-540-27485-5_5
Kim CH,Johnson MC,Drennan JD,et al.,2000.DNA vaccines encoding viral glycoproteins induce nonspecific immunity and Mx protein synthesis in fish.J Virol,74(15):7048-7054.https://doi.org/10.1128/JVI.74.15.7048-7054.2000
Ksenofontov AL,Badun GA,Fedorova NV,et al.,2008.Quantitation of the glycoprotein spike area on the surface of enveloped viruses.Mol Biol,42(6):973-975.https://doi.org/10.1134/s0026893308060204
Lei Y,Qi RR,Cui LB,et al.,2015.Diagnosis of rhabdovirus disease in juvenile largemouth bass Micropterus salmonides.J Dalian Fish Univ,30(3):305-308(in Chinese).https://doi.org/10.16535/j.cnki.dlhyxb.2015.03.013
Lorenzen N,Lorenzen E,Einer-Jensen K,et al.,1998.Protective immunity to VHS in rainbow trout(Oncorhynchus mykiss,Walbaum)following DNA vaccination.Fish Shellfish Immunol,8(4):261-270.https://doi.org/10.1006/fsim.1997.0134
Lorenzoni M,D?rr AJM,Erra R,et al.,2002.Growth and reproduction of largemouth bass(Micropterus salmoides Lacépède,1802)in Lake Trasimeno(Umbria,Italy).Fish Res,56(1):89-95.https://doi.org/10.1016/S0165-7836(01)00309-5
Luo X,Deng GC,Zhao CC,et al.,2013.Isolation and preliminary identification of rhabdovirus from Channa maculata cultured in pond.Acta Hydrobiol Sin,37(4):620-625(in Chinese).https://doi.org/10.7541/2013.72
Lv SJ,Lu BJ,Xu JH,et al.,2015.Immune response of peroxinectin of Chinese mitten crab Eriocheir sinensis to exterior stimulation.Dev Comp Immunol,51(1):56-64.https://doi.org/10.1016/j.dci.2015.02.016
Ma DM,Deng GC,Bai JJ,et al.,2013.A strain of Siniperca chuatsi rhabdovirus causes high mortality among cultured largemouth bass in south China.J Aquat Anim Health,25(3):197-204.https://doi.org/10.1080/08997659.2013.799613
Sakai K,Hagiwara K,Omatsu T,et al.,2015.Isolation and characterization of a novel rhabdovirus from a wild boar(Sus scrofa)in Japan.Vet Microbiol,179(3-4):197-203.https://doi.org/10.1016/j.vetmic.2015.05.013
Sano T,Nishimura T,Okamoto N,et al.,1976.Isolation of rhabdovirus from European eels(Anguilla anguilla)at Japanese port of entry.Fish Health News,5:5-6.
Seo JY,Kim KH,Kim SG,et al.,2006.Protection of flounder against hirame rhabdovirus(HIRRV)with a DNA vaccine containing the glycoprotein gene.Vaccine,24(7):1009-1015.https://doi.org/10.1016/j.vaccine.2005.07.109
Serena MS,Geisler C,Metz GE,et al.,2013.Expression and purification of Suid Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose Aujeszky’s disease in infected pigs.Protein Expr Purif,90(1):1-8.https://doi.org/10.1016/j.pep.2013.04.008
Toth AM,Geisler C,Aumiller JJ,et al.,2011.Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system.Protein Expr Purif,80(2):274-282.https://doi.org/10.1016/j.pep.2011.08.002
Walker PJ,Wang YH,Cowley JA,et al.,1994.Structural and antigenic analysis of the nucleoprotein of bovine ephemeral fever rhabdovirus.J Gen Virol,75(8):1889-1899.https://doi.org/10.1099/0022-1317-75-8-1889
Xu XG,Wang ZS,Zhang Q,et al.,2011.Baculovirus surface display of E envelope glycoprotein of Japanese encephalitis virus and its immunogenicity of the displayed proteins in mouse and swine models.Vaccine,29(4):636-643.https://doi.org/10.1016/j.vaccine.2010.11.045
Xu XG,Wang ZS,Zhang Q,et al.,2012.Baculovirus as a PRRSV and PCV2 bivalent vaccine vector:baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2.J Virol Methods,179(2):359-366.https://doi.org/10.1016/j.jviromet.2011.11.023
Zeng WW,Wang Q,Wang YY,2013.Isolation and characterization of a rhabdovirus from snakehead fish(Ophicephalus striatus).J Fish China,37(9):1416-1424(in Chinese).https://doi.org/10.3724/SP.J.1231.2013.38588
Zhang LJ,Li NQ,Lin Q,et al.,2018.An avirulent Micropterus salmoides rhabdovirus vaccine candidate protects Chinese perch against rhabdovirus infection.Fish Shellfish Immunol,77:474-480.https://doi.org/10.1016/j.fsi.2018.03.047
Zhang S,Huang YH,Zhu JL,et al.,2018.Expression of hNeuritin protein in a baculovirus expression system and the analysis of its activity.Gene,647:129-135.https://doi.org/10.1016/j.gene.2018.01.026
Zhang XL,Xiao F,Li SM,et al.,2018.China Fishery Statistical Yearbook 2018.China Agriculture Press,Beijing,China,p.33(in Chinese).
Zhang ZY,You Y,Meng FL,et al.,2011.A primary research of using silkworm larvae as substitutes of feed proteins.JChangsha Univ,25(2):68-69(in Chinese).https://doi.org/10.3969/j.issn.1008-4681.2011.02.025