鲤春病毒血症病毒核蛋白、磷蛋白与基质蛋白的表达、抗体制备及免疫原性比较
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摘要
鲤春病毒血症病毒(SVCV)能够引起鲤科鱼类大量死亡,被世界动物卫生组织(OIE)列为必须申报的重要疫病,也是我国唯一被列为一类疫病的鱼类传染病。为建立SVCV的快速免疫学诊断方法,研究其主要结构蛋白间的免疫原性差异,实验首先采用原核表达系统克隆并诱导表达,纯化SVCV的核蛋白(N)、磷蛋白(P)和基质蛋白(M),并进一步免疫新西兰白兔制备抗血清,抗血清经Protein A柱进一步纯化获得3种蛋白的多克隆抗体。利用间接酶联免疫吸附测定(ELISA)和免疫印迹实验(Western blot)对抗体效价和特异性进行分析验证。结果发现,SVCV的N、P和M重组蛋白均在原核表达系统获得大量表达,且表达的蛋白经纯化后免疫实验动物产生了相应的多克隆抗体。Western blot结果显示,3种蛋白抗体均与SVCV重组蛋白及天然蛋白发生特异性的免疫反应。ELISA结果显示,针对P蛋白制备的抗体效价最高,可达409 600;针对N和M蛋白制备的抗体效价也均大于204 800。同时,特异性检测实验结果显示,制备的3种蛋白抗体均仅与SVCV发生特异性免疫反应,而与SVCV宿主其他易感病毒均不发生交叉反应。实验结果将对SVCV的快速诊断及疫苗开发提供新的手段和思路。
Spring viremia of carp virus(SVCV) is an OIE listed highly pathogenic agent of several economically important Cyprinidae fish species. Currently, there is no effective vaccine or drug available for this virus, and prevention of SVC mostly relies on prompt diagnosis. Previously, detection methods based on the G gene or the G glycoprotein have been developed. However, the highly genetic diversity of the G gene seriously limits the reliability of those methods. To develop a rapid immunological detection method for SVCV, the N, P and M genes of SVCV were cloned into a p RSET-A prokaryotic expression vector. Recombinant proteins were then induced with 1 mmol/L IPTG at 37 °C. Next, recombinant proteins were purified and used to immunize New Zealand rabbits. Titers and specificities of antibodies were verified by indirect ELISA and Western blot. Our results indicated the obtained antibodies were highly specific to SVCV and the titers could reach at least as high as 204 800.Besides, indirect ELISA assays based on prepared antibodies showed there was no cross reaction with other aquatic viruses. Our results will be useful for the rapid immunological detection of SVCV and provide a new insight into the vaccine development of SVCV.
Spring viremia of carp virus(SVCV) is an OIE listed highly pathogenic agent of several economically important Cyprinidae fish species. Currently, there is no effective vaccine or drug available for this virus, and prevention of SVC mostly relies on prompt diagnosis. Previously, detection methods based on the G gene or the G glycoprotein have been developed. However, the highly genetic diversity of the G gene seriously limits the reliability of those methods. To develop a rapid immunological detection method for SVCV, the N, P and M genes of SVCV were cloned into a p RSET-A prokaryotic expression vector. Recombinant proteins were then induced with 1 mmol/L IPTG at 37 °C. Next, recombinant proteins were purified and used to immunize New Zealand rabbits. Titers and specificities of antibodies were verified by indirect ELISA and Western blot. Our results indicated the obtained antibodies were highly specific to SVCV and the titers could reach at least as high as 204 800.Besides, indirect ELISA assays based on prepared antibodies showed there was no cross reaction with other aquatic viruses. Our results will be useful for the rapid immunological detection of SVCV and provide a new insight into the vaccine development of SVCV.
引文
[1]Ahne W.Uptake and multiplication of spring viraemia of carp virus in carp,Cyprinus carpio L.[J].Journal of Fish Diseases,1978,1(3):265-268.
[2]Reschova S,Pokorova D,Nevorankova Z,et al.Detection of spring viraemia of carp virus(SVCV)with monoclonal antibodies[J].Veterinarni Medicina,2007,52(7):308-316.
[3]Zhang N Z,Zhang L F,Jiang Y N,et al.Molecular analysis of spring viraemia of carp virus in China:a fatal aquatic viral disease that might spread in East Asian[J].PLo S One,2009,4(7):e6337.
[4]Padhi A,Verghese B.Molecular evolutionary and epidemiological dynamics of a highly pathogenic fish rhabdovirus,the spring viremia of carp virus(SVCV)[J].Veterinary Microbiology,2012,156(1-2):54-63.
[5]Dikkeboom A L,Radi C,Toohey-Kurth K,et al.First report of spring viremia of carp virus(SVCV)in wild common carp in North America[J].Journal of Aquatic Animal Health,2004,16(4):169-178.
[6]付峰,刘荭,黄健,等.鲤春病毒血症病毒(SVCV)的研究进展[J].中国水产科学,2006,13(2):328-334.Fu F,Liu H,Huang J,et al.Study progress of spring viremia of carp virus[J].Journal of Fishery Sciences of China,2006,13(2):328-334(in Chinese).
[7]王姝,张利峰,徐立蒲,等.鲤春病毒核酸检测方法研究进展[J].检验检疫学刊,2012,22(3):49-51.Wang S,Zhang L F,Xu L P,et al.Progress in the nucleic acid testing of carp virus[J].Journal of Inspection and Quarantine,2012,22(3):49-51(in Chinese).
[8]Shao L,Xiao Y,He Z K,et al.An N-targeting real-time PCR strategy for the accurate detection of spring viremia of carp virus[J].Journal of Virological Methods,2016,229:27-34.
[9]Lyles D S,Kuzmin I V,Rupprecht C E.Rhabdoviridae[C]//Knipe D M,Howley P M.Fields Virology.6th ed.Philadelphia,PA:Lippincott Williams&Wilkins,2013:885-922.
[10]Wertz G W,Perepelitsa V P,Ball L A.Gene rearrangement attenuates expression and lethality of a nonsegmented negative strand RNA virus[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(7):3501-3506.
[11]Hoffmann B,Schütze H,Mettenleiter T C.Determination of the complete genomic sequence and analysis of the gene products of the virus of Spring Viremia of Carp,a fish rhabdovirus[J].Virus Research,2002,84(1-2):89-100.
[12]Ahne W,Bjorklund H V,Essbauer S,et al.Spring viremia of carp(SVC)[J].Diseases of Aquatic Organisms,2002,52(3):261-272.
[13]高隆英,史秀杰,刘荭,等.用RT-PCR法快速检测鲤春病毒血症病毒基因[J].水生生物学报,2002,26(5):452-456.Gao L Y,Shi X J,Liu H,et al.Detection of spring viremia of carp virus(SVCV)gene using reversepolymerase chain-reaction(RT-PCR)[J].Acta Hydrobiologica Sinica,2002,26(5):452-456(in Chinese).
[14]KoutnáM,Vesely T,Psikal I,et al.Identification of spring viraemia of carp virus(SVCV)by combined RT-PCR and nested PCR[J].Diseases of Aquatic Organisms,2003,55(3):229-235.
[15]耿波,孙效文,牟振波,等.鲤春病毒糖蛋白(G)基因的分离及同源性比较[J].生物技术通报,2006(S1):450-454.Geng B,Sun X W,Mu Z B,et al.The isolation of glycoprotein g gene of spring viraemia of carp virus and the comparation of sequence homologous[J].Biotechnology Bulletin,2006(S1):450-454(in Chinese).
[16]Xiao Y,Shao L,Zhang C W,et al.Genomic evidence of homologous recombination in spring viremia of carp virus:a negatively single stranded RNA virus[J].Virus Research,2014,189:271-279.
[17]Reed L J,Müench H.A simple method of estimating fifty percent endpoints[J].The American Journal of Hygiene,1938,27(3):493-497.
[18]王宏华,侯竹美,张全启,等.病毒性出血性败血症病毒单链抗体的原核表达及其功能鉴定[J].水产学报,2016,40(1):128-134.Wang H H,Hou Z M,Zhang Q Q,et al.Prokaryotic expression and functional verification of Sc Fv antibody against viral hemorrhagic septicemia virus[J].Journal of Fisheries of China,2016,40(1):128-134(in Chinese).
[19]Dixon P F,Longshaw C B.Assessment of commercial test kits for identification of spring viraemia of carp virus[J].Diseases of Aquatic Organisms,2005,67(1-2):25-29.
[20]Miller O,Fuller F J,Gebreyes W A,et al.Phylogenetic analysis of spring virema of carp virus reveals distinct subgroups with common origins for recent isolates in North America and the UK[J].Diseases of Aquatic Organisms,2007,76(3):193-204.
[21]宗乾坤,张也,吕利群.Ⅱ型草鱼呼肠孤病毒VP4、VP35蛋白多克隆抗体制备及其免疫原性分析[J].水产学报,2016,40(3):355-362.Zong Q K,Zhang Y,LüL Q.Preparation and immunogenicity of polyclonal antibodies against VP4,VP35 protein of typeⅡgrass carp reovirus[J].Journal of Fisheries of China,2016,40(3):355-362(in Chinese).
[2]Reschova S,Pokorova D,Nevorankova Z,et al.Detection of spring viraemia of carp virus(SVCV)with monoclonal antibodies[J].Veterinarni Medicina,2007,52(7):308-316.
[3]Zhang N Z,Zhang L F,Jiang Y N,et al.Molecular analysis of spring viraemia of carp virus in China:a fatal aquatic viral disease that might spread in East Asian[J].PLo S One,2009,4(7):e6337.
[4]Padhi A,Verghese B.Molecular evolutionary and epidemiological dynamics of a highly pathogenic fish rhabdovirus,the spring viremia of carp virus(SVCV)[J].Veterinary Microbiology,2012,156(1-2):54-63.
[5]Dikkeboom A L,Radi C,Toohey-Kurth K,et al.First report of spring viremia of carp virus(SVCV)in wild common carp in North America[J].Journal of Aquatic Animal Health,2004,16(4):169-178.
[6]付峰,刘荭,黄健,等.鲤春病毒血症病毒(SVCV)的研究进展[J].中国水产科学,2006,13(2):328-334.Fu F,Liu H,Huang J,et al.Study progress of spring viremia of carp virus[J].Journal of Fishery Sciences of China,2006,13(2):328-334(in Chinese).
[7]王姝,张利峰,徐立蒲,等.鲤春病毒核酸检测方法研究进展[J].检验检疫学刊,2012,22(3):49-51.Wang S,Zhang L F,Xu L P,et al.Progress in the nucleic acid testing of carp virus[J].Journal of Inspection and Quarantine,2012,22(3):49-51(in Chinese).
[8]Shao L,Xiao Y,He Z K,et al.An N-targeting real-time PCR strategy for the accurate detection of spring viremia of carp virus[J].Journal of Virological Methods,2016,229:27-34.
[9]Lyles D S,Kuzmin I V,Rupprecht C E.Rhabdoviridae[C]//Knipe D M,Howley P M.Fields Virology.6th ed.Philadelphia,PA:Lippincott Williams&Wilkins,2013:885-922.
[10]Wertz G W,Perepelitsa V P,Ball L A.Gene rearrangement attenuates expression and lethality of a nonsegmented negative strand RNA virus[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(7):3501-3506.
[11]Hoffmann B,Schütze H,Mettenleiter T C.Determination of the complete genomic sequence and analysis of the gene products of the virus of Spring Viremia of Carp,a fish rhabdovirus[J].Virus Research,2002,84(1-2):89-100.
[12]Ahne W,Bjorklund H V,Essbauer S,et al.Spring viremia of carp(SVC)[J].Diseases of Aquatic Organisms,2002,52(3):261-272.
[13]高隆英,史秀杰,刘荭,等.用RT-PCR法快速检测鲤春病毒血症病毒基因[J].水生生物学报,2002,26(5):452-456.Gao L Y,Shi X J,Liu H,et al.Detection of spring viremia of carp virus(SVCV)gene using reversepolymerase chain-reaction(RT-PCR)[J].Acta Hydrobiologica Sinica,2002,26(5):452-456(in Chinese).
[14]KoutnáM,Vesely T,Psikal I,et al.Identification of spring viraemia of carp virus(SVCV)by combined RT-PCR and nested PCR[J].Diseases of Aquatic Organisms,2003,55(3):229-235.
[15]耿波,孙效文,牟振波,等.鲤春病毒糖蛋白(G)基因的分离及同源性比较[J].生物技术通报,2006(S1):450-454.Geng B,Sun X W,Mu Z B,et al.The isolation of glycoprotein g gene of spring viraemia of carp virus and the comparation of sequence homologous[J].Biotechnology Bulletin,2006(S1):450-454(in Chinese).
[16]Xiao Y,Shao L,Zhang C W,et al.Genomic evidence of homologous recombination in spring viremia of carp virus:a negatively single stranded RNA virus[J].Virus Research,2014,189:271-279.
[17]Reed L J,Müench H.A simple method of estimating fifty percent endpoints[J].The American Journal of Hygiene,1938,27(3):493-497.
[18]王宏华,侯竹美,张全启,等.病毒性出血性败血症病毒单链抗体的原核表达及其功能鉴定[J].水产学报,2016,40(1):128-134.Wang H H,Hou Z M,Zhang Q Q,et al.Prokaryotic expression and functional verification of Sc Fv antibody against viral hemorrhagic septicemia virus[J].Journal of Fisheries of China,2016,40(1):128-134(in Chinese).
[19]Dixon P F,Longshaw C B.Assessment of commercial test kits for identification of spring viraemia of carp virus[J].Diseases of Aquatic Organisms,2005,67(1-2):25-29.
[20]Miller O,Fuller F J,Gebreyes W A,et al.Phylogenetic analysis of spring virema of carp virus reveals distinct subgroups with common origins for recent isolates in North America and the UK[J].Diseases of Aquatic Organisms,2007,76(3):193-204.
[21]宗乾坤,张也,吕利群.Ⅱ型草鱼呼肠孤病毒VP4、VP35蛋白多克隆抗体制备及其免疫原性分析[J].水产学报,2016,40(3):355-362.Zong Q K,Zhang Y,LüL Q.Preparation and immunogenicity of polyclonal antibodies against VP4,VP35 protein of typeⅡgrass carp reovirus[J].Journal of Fisheries of China,2016,40(3):355-362(in Chinese).