Ⅱ型鲤疱疹病毒ORF121蛋白的多克隆抗体制备及鉴定
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摘要
针对CyHV-2病毒ORF121基因(GenBank:AFJ20543.1)进行原核表达系统的构建,将纯化重组蛋白作为抗原来免疫BALB/c小鼠获得多克隆抗体,应用该抗体开展CyHV-2病毒诊断及其感染机制研究。以CyHV-2病毒感染细胞上清液为扩增模板,扩增ORF121基因构建至pGEX-4T原核表达载体,经异丙基硫代半乳糖苷(IPTG)诱导表达rORF121重组蛋白,利用尿素纯化后免疫6周龄BALB/c小鼠制备多克隆抗体。结果显示,CyHV-2病毒ORF121基因可在原核表达系统中高效表达目的重组蛋白rORF121,经SDS-PAGE分析大小约为60 ku,主要以不可溶的包涵体存在。利用尿素溶解rORF121蛋白免疫BALB/c小鼠获得抗ORF121蛋白的多克隆抗体,Western Blot实验显示,该抗体可特异性识别CyHV-2病毒感染RyuF-2细胞样品。研究表明,利用CyHV-2感染RyuF-2细胞后,本研究制备的抗ORF121蛋白的多克隆抗体能够通过间接免疫荧光实验特异性识别CyHV-2病毒感染的细胞样品。本研究制备的抗ORF121蛋白的多克隆抗体,能够为CyHV-2病毒诊断技术的构建以及深入开展CyHV-2病毒感染机制提供良好的技术基础。
Cyprinid herpesvirus 2(CyHV-2) is an emerging pathogen of the crucian carp, Carassius carassius,which has caused huge economic losses in China and appears to be spreading worldwide. The objective of this study was to produce and characterize new polyclonal antibodies against CyHV-2 ORF121 that were suitable for diagnostic use and the investigation of ORF121 function. The open reading frame(ORF) of 121 was amplified from the culture supernatant of the CyHV-2. ORF121 was subcloned into the pGEX-4 T vector and recombinant protein was expressed in Escherichia coli. The purified recombinant protein ORF121 was used to immunize mice and polyclonal antibodies were obtained. The recombinant protein ORF121 was purified under urea conditions then used to 6-week-old BALA/c mice to prepare its polyclonal antibody. Western Blot and indirect fluorescence assay(IFA) assay were used to validate the ORF121 polyclonal antibody. We found the recombinant protein ORF121(rORF121) could be expressed in E. coli. aggregated in the form of inclusion body. The specificity of the anti-ORF121 polyclonal antibody was confirmed by Western Blot. IFA with anti-ORF121 polyclonal antibody for the detection of CyHV-2 infected RyuF-2 cells was developed in this study. In conclusion, the anti-ORF121 polyclonal antibody will be a valuable tool in further studies to elucidate the mechanisms of viral infection and CyHV-2 diagnosis.
Cyprinid herpesvirus 2(CyHV-2) is an emerging pathogen of the crucian carp, Carassius carassius,which has caused huge economic losses in China and appears to be spreading worldwide. The objective of this study was to produce and characterize new polyclonal antibodies against CyHV-2 ORF121 that were suitable for diagnostic use and the investigation of ORF121 function. The open reading frame(ORF) of 121 was amplified from the culture supernatant of the CyHV-2. ORF121 was subcloned into the pGEX-4 T vector and recombinant protein was expressed in Escherichia coli. The purified recombinant protein ORF121 was used to immunize mice and polyclonal antibodies were obtained. The recombinant protein ORF121 was purified under urea conditions then used to 6-week-old BALA/c mice to prepare its polyclonal antibody. Western Blot and indirect fluorescence assay(IFA) assay were used to validate the ORF121 polyclonal antibody. We found the recombinant protein ORF121(rORF121) could be expressed in E. coli. aggregated in the form of inclusion body. The specificity of the anti-ORF121 polyclonal antibody was confirmed by Western Blot. IFA with anti-ORF121 polyclonal antibody for the detection of CyHV-2 infected RyuF-2 cells was developed in this study. In conclusion, the anti-ORF121 polyclonal antibody will be a valuable tool in further studies to elucidate the mechanisms of viral infection and CyHV-2 diagnosis.
引文
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[15]Shibata T,Nanjo A,Saito M,et al.In vitro characteristics of Cyprinid herpesvirus 2:effect of kidney extract supplementation on growth[J].Diseases of Aquatic Organisms,2015,115(3):223-232.
[16]Krogh A,Larsson B,von Heijne G,et al.Predicting transmembrane protein topology with a hidden Markov model:application to complete genomes[J].Journal of Molecular Biology,2001,305(3):567-580.
[17]桂建芳.异育银鲫养殖新品种--“中科3号”简介[J].科学养鱼,2009(5):21.Gui J F.Introduction of a new breed of Carassius auratus-"Zhongke No.3"[J].Scientific Fish Farming,2009(5):21.
[18]Hedrick R P,Waltzek T B,McDowell T S.Susceptibility of koi carp,common carp,goldfish,and goldfish×common carp hybrids to Cyprinid herpesvirus2and herpesvirus3[J].Journal of Aquatic Animal Health,2006,18(1):26-34.
[19]Doszpoly A,Benko M,Csaba G,et al.Introduction of the family Alloherpesviridae:the first molecular detection of herpesviruses of cyprinid fish in Hungary[J].Magyar Allatorvosok Lapja,2011,133(3):174-181.
[20]罗丹,梁利国,谢骏,等.鲤疱疹病毒Ⅰ、Ⅱ、Ⅲ型研究进展[J].水生态学杂志,2014,35(3):94-400.Luo D,Liang L G,Xie J,et al.Research progress of Cyprinid herpesvirus I,Ⅱ,Ⅲ[J].Journal of Hydroecology,2014,35(3):94-400(in Chinese).
[21]Eide K E,Miller-Morgan T,Heidel J R,et al.Investigation of koi herpesvirus latency in koi[J].Journal of Virology,2011,85(10):4954-4962.
[22]Prescott M A,Reed A N,Jin L,et al.Rapid detection of Cyprinid herpesvirus 3 in latently infected koi by recombinase polymerase amplification[J].Journal of Aquatic Animal Health,2016,28(3):173-180.
[23]郑树城,王庆,李莹莹,等.鲤疱疹病毒3型研究进展[J].病毒学报,2016,32(1):108-120.Zheng S C,Wang Q,Li Y Y,et al.Research advances in Cyprinid herpesvirus 3[J].Chinese Journal of Virology,2016,32(1):108-120(in Chinese).
[24]向骏,程安春,汪铭书.疱疹病毒衣壳蛋白及其组装研究进展[J].中国农业科学,2010,43(22):4739-4745.Xiang J,Cheng A C,Wang M S.Advance in capsid proteins and assembly of herpesvirus[J].Scientia Agricultura Sinica,2010,43(22):4739-4745(in Chinese).
[25]Lu J F,Xu D,Lu L Q.A novel cell line established from caudal fin tissue of Carassius auratus gibelio is susceptible to Cyprinid herpesvirus 2 infection with the induction of apoptosis[J].Virus Research,2018,258:19-27.
[2]Groff J M,LaPatra S E,Munn R J,et al.A viral epizootic in cultured populations of juvenile goldfish due to a putative herpesvirus etiology[J].Journal of Veterinary Diagnostic Investigation,1998,10(4):375-378.
[3]Jung S J,Miyazaki T.Herpesviral haematopoietic necrosis of goldfish,Carassius auratus[J].Journal of Fish Diseases,1995,18(3):211-220.
[4]Waltzek T B,Kurobe T,Goodwin A E,et al.Development of a polymerase chain reaction assay to detect Cyprinid herpesvirus 2 in goldfish[J].Journal of Aquatic Animal Health,2009,21(1):60-67.
[5]Wu T,Ding Z F,Ren M,et al.The histo-and ultrapathological studies on a fatal disease of Prussian carp(Carassius gibelio)in mainland China associated with Cyprinid herpesvirus 2(CyHV-2)[J].Aquaculture,2013,412-413:8-13.
[6]陈昌福,陈辉,胡明,等.异育银鲫疱疹病毒病及其防控对策(下)[J].当代水产,2015(5):77-79.Chen C F,Chen H,Hu M,et al.Carassius auratus herpes virus disease and its prevention and control measures(below)[J].Current Fisheries,2015(5):77-79.
[7]He J Q,Shi X J,Yu L,et al.Development and evaluation of a loop-mediated isothermal amplification assay for diagnosis of Cyprinid herpesvirus 2[J].Journal of Virological Methods,2013,194(1-2):206-210.
[8]Kong S Y,Jiang Y S,Wang Q,et al.Detection methods of Cyprinid herpesvirus 2 infection in silver crucian carp(Carassius auratus gibelio)via a pORF72 monoclonalantibody[J].Journal of Fish Diseases,2017,40(12):1791-1798.
[9]彭俊杰,张琪,贾路路,等.鲤疱疹病毒Ⅱ型(CyHV-2)ORF25蛋白的原核表达及单克隆抗体的制备[J].华中农业大学学报,2017,36(2):96-101.Peng J J,Zhang Q,Jia L L,et al.Prokaryotic expression and preparation of monoclonal antibody against ORF25protein of Cyprinid herpesvirusⅡ[J].Journal of Huazhong Agricultural University,2017,36(2):96-101(in Chinese).
[10]Davison A J,Kurobe T,Gatherer D,et al.Comparative genomics of carp herpesviruses[J].Journal of Virology,2013,87(5):2908-2922.
[11]Li L J,Luo Y Z,Gao Z X,et al.Molecular characterisation and prevalence of a new genotype of Cyprinid herpesvirus 2 in mainland China[J].Canadian Journal of Microbiology,2015,61(6):381-387.
[12]Zeng X T,Chen Z Y,Deng Y S,et al.Complete genome sequence and architecture of crucian carp Carassius auratus herpesvirus(CaHV)[J].Archives of Virology,2016,161(12):3577-3581.
[13]Liu B,Zhou Y,Li K,et al.The complete genome of Cyprinid herpesvirus 2,a new strain isolated from Allogynogenetic crucian carp[J].Virus Research,2018,256:6-10.
[14]Wang H,Xu L J,Lu L Q.Detection of Cyprinid herpesvirus 2 in peripheral blood cells of silver crucian carp,Carassius auratus gibelio(Bloch),suggests its potential in viral diagnosis[J].Journal of Fish Diseases,2016,39(2):155-162.
[15]Shibata T,Nanjo A,Saito M,et al.In vitro characteristics of Cyprinid herpesvirus 2:effect of kidney extract supplementation on growth[J].Diseases of Aquatic Organisms,2015,115(3):223-232.
[16]Krogh A,Larsson B,von Heijne G,et al.Predicting transmembrane protein topology with a hidden Markov model:application to complete genomes[J].Journal of Molecular Biology,2001,305(3):567-580.
[17]桂建芳.异育银鲫养殖新品种--“中科3号”简介[J].科学养鱼,2009(5):21.Gui J F.Introduction of a new breed of Carassius auratus-"Zhongke No.3"[J].Scientific Fish Farming,2009(5):21.
[18]Hedrick R P,Waltzek T B,McDowell T S.Susceptibility of koi carp,common carp,goldfish,and goldfish×common carp hybrids to Cyprinid herpesvirus2and herpesvirus3[J].Journal of Aquatic Animal Health,2006,18(1):26-34.
[19]Doszpoly A,Benko M,Csaba G,et al.Introduction of the family Alloherpesviridae:the first molecular detection of herpesviruses of cyprinid fish in Hungary[J].Magyar Allatorvosok Lapja,2011,133(3):174-181.
[20]罗丹,梁利国,谢骏,等.鲤疱疹病毒Ⅰ、Ⅱ、Ⅲ型研究进展[J].水生态学杂志,2014,35(3):94-400.Luo D,Liang L G,Xie J,et al.Research progress of Cyprinid herpesvirus I,Ⅱ,Ⅲ[J].Journal of Hydroecology,2014,35(3):94-400(in Chinese).
[21]Eide K E,Miller-Morgan T,Heidel J R,et al.Investigation of koi herpesvirus latency in koi[J].Journal of Virology,2011,85(10):4954-4962.
[22]Prescott M A,Reed A N,Jin L,et al.Rapid detection of Cyprinid herpesvirus 3 in latently infected koi by recombinase polymerase amplification[J].Journal of Aquatic Animal Health,2016,28(3):173-180.
[23]郑树城,王庆,李莹莹,等.鲤疱疹病毒3型研究进展[J].病毒学报,2016,32(1):108-120.Zheng S C,Wang Q,Li Y Y,et al.Research advances in Cyprinid herpesvirus 3[J].Chinese Journal of Virology,2016,32(1):108-120(in Chinese).
[24]向骏,程安春,汪铭书.疱疹病毒衣壳蛋白及其组装研究进展[J].中国农业科学,2010,43(22):4739-4745.Xiang J,Cheng A C,Wang M S.Advance in capsid proteins and assembly of herpesvirus[J].Scientia Agricultura Sinica,2010,43(22):4739-4745(in Chinese).
[25]Lu J F,Xu D,Lu L Q.A novel cell line established from caudal fin tissue of Carassius auratus gibelio is susceptible to Cyprinid herpesvirus 2 infection with the induction of apoptosis[J].Virus Research,2018,258:19-27.