稳定表达SLAM受体的Vero和BHK21细胞系的建立及其对犬瘟热病毒分离效果的比较
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摘要
旨在构建稳定表达SLAM受体的SLAM-Vero细胞系和SLAM-BHK21细胞系,并比较其对犬瘟热病毒(CDV)野毒株的敏感性,为CDV野毒株的快速分离与研究提供一种有效的工具。将真核表达载体Pcag-SLAM分别转染Vero细胞和BHK21细胞。经G418压力筛选结合有限稀释法筛选阳性克隆株,并通过RT-PCR、间接免疫荧光鉴定(IFA)和Western blot等方法对稳定表达SLAM受体的SLAM-Vero细胞系和SLAM-BHK21细胞系加以验证。应用这两种细胞系对3例临床犬瘟热病例的5种不同组织进行病毒分离,对分离得到的CDV进行RT-PCR及IFA鉴定。结果显示,经RT-PCR和Western blot验证,本研究成功构建出SLAM-Vero细胞系和SLAM-BHK21细胞系,SLAM-Vero细胞接种病毒12~24h即可出现典型的CDV导致的细胞融合体CPE,利用SLAM-Vero细胞从3只CDV阳性犬的肺和脾中分离得到2株CDV,而SLAM-BHK21细胞、亲本Vero细胞及亲本BHK21细胞未能分离出病毒。研究表明,与SLAM-BHK21细胞相比,SLAM-Vero细胞对CDV的分离率较高,并能产生明显的CPE。在分离CDV野毒株时,选择肺和脾更容易在SLAM-Vero细胞系上分离出病毒。
The objective of this work was to establish a SLAM-Vero cell line and a SLAM-BHK21 cell line that stanbly expressing SLAM receptor,and then to compare their sensitivities to the canine distemper virus(CDV)wild strains,aiming at providing another tools for the rapid isolation of CDV for further study.The eukaryotic expression vector Pcag-SLAM,which contains the SLAM gene,was transfected into the Vero cells and BHK-21 cells,respectively.Then the cells which harbor the SLAM gene were subsequently screened with G418 combined with end point dilution,RT-PCR,immunofluorescence assay,and Western blot.The two cell lines were subsequently applied to isolate virus from 5 different tissues in 3 clinical cases.The isolated CDV wasfurther identified by RT-PCR and Indirect Immunofluorescence assay.The results showed that the SLAM-Vero cells lines and SLAM-BHK21 cells lines were constructed successfully.SLAMVero inoculated virus for 12-24 hpresented a typical CDV-induced cell fusion of CPE.By using SLAM-Vero cells,two strains of CDV were obtained from three CDV positive canine lungs and spleens,while SLAM-BHK21 cell lines,parental Vero cells and parental BHK21 cells failed to isolate the virus.The results indicated that CDV was more readily to replicate and proliferate in SLAM-Vero cells and easier to produce visible CPE than that in SLAM-BHK21 cells.Canine lungs and spleens are more ideal candidates for isolation of CDV when using the newly established SLAM-Vero cell lines in this report.
The objective of this work was to establish a SLAM-Vero cell line and a SLAM-BHK21 cell line that stanbly expressing SLAM receptor,and then to compare their sensitivities to the canine distemper virus(CDV)wild strains,aiming at providing another tools for the rapid isolation of CDV for further study.The eukaryotic expression vector Pcag-SLAM,which contains the SLAM gene,was transfected into the Vero cells and BHK-21 cells,respectively.Then the cells which harbor the SLAM gene were subsequently screened with G418 combined with end point dilution,RT-PCR,immunofluorescence assay,and Western blot.The two cell lines were subsequently applied to isolate virus from 5 different tissues in 3 clinical cases.The isolated CDV wasfurther identified by RT-PCR and Indirect Immunofluorescence assay.The results showed that the SLAM-Vero cells lines and SLAM-BHK21 cells lines were constructed successfully.SLAMVero inoculated virus for 12-24 hpresented a typical CDV-induced cell fusion of CPE.By using SLAM-Vero cells,two strains of CDV were obtained from three CDV positive canine lungs and spleens,while SLAM-BHK21 cell lines,parental Vero cells and parental BHK21 cells failed to isolate the virus.The results indicated that CDV was more readily to replicate and proliferate in SLAM-Vero cells and easier to produce visible CPE than that in SLAM-BHK21 cells.Canine lungs and spleens are more ideal candidates for isolation of CDV when using the newly established SLAM-Vero cell lines in this report.
引文
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[2]APPEL M J,YATES R A,FOLEY G L,et al.Canine distemper epizootic in lions,tigers,and leopards in North America[J].J Vet Diagn Invest,1994,6(3):277-288.
[3]TAN B,WEN Y J,WANG F X,et al.Pathogenesis and phylogenetic analyses of canine distemper virus strain ZJ7isolate from domestic dogs in China[J].Virol J,2011,8:520.
[4]岳进华,刘鹤媛,周铭,等.大熊猫犬瘟热病毒LG株的分离鉴定[J].动物医学进展,2016,37(10):60-63.YUE J H,LIU H Y,ZHOU M,et al.Identification of canine distemper virus LG strain isolated from giant panda[J].Progress in Veterinary Medicine,2016,37(10):60-63.(in Chinese)
[5]TATSUO H,ONO N,YANAGI Y.Morbilliviruses use signaling lymphocyte activation molecules(CD150)as cellular receptors[J].J Virol,2001,75(13):5842-5850.
[6]LEDNICKY J A,MEEHAN T P,KINSEL M J,et al.Effective primary isolation of wild-type canine distemper virus in MDCK,MV1Lu and Vero cells without nucleotide sequence changes within the entire haemagglutinin protein gene and in subgenomic sections of the fusion and phospho protein genes[J].J Virol Methods,2004,118(2):147-157.
[7]蒙学莲,窦永喜,朱学亮,等.小反刍兽疫病毒血凝素蛋白与受体蛋白SLAM的相互作用[J].畜牧兽医学报,2014,45(3):426-433.MENG X L,DOU Y X,ZHU X L,et al.Interaction between hemagglutinin protein of Peste des petits ruminants virus and signalling lymphocyte activation molecule[J].Acta Veterinaria et Zootechnica Sinica,2014,45(3):426-433.(in Chinese)
[8]SEKI F,ONO N,YAMAGUCHI R,et al.Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM(CD150)and their adaptability to marmoset B95acells[J].J Virol,2003,77(18):9943-9950.
[9]AVERSA G,CHANG C C,CARBALLIDO J M,et al.Engagement of the signaling lymphocytic activation molecule(SLAM)on activated T cells results in IL-2-independent,cyclosporin A-sensitive T cell proliferation and IFN-gamma production[J].J Immunol,1997,158(9):4036-4044.
[10]KRUSE M,MEINL E,HENNING G,et al.Signaling lymphocytic activation molecule is expressed on mature CD83+dendritic cells and is up-regulated by IL-1β[J].J Immunol,2001,167(4):1989-1995.
[11]ROMERO X,MARTIN M,ZAPATER N.Expression of CD150on human leukocytes.Production and characterization of a new CD150monoclonal antibody[J].Original,2001,20(2):57-66.
[12]TANAKA K,MINAGAWA H,XIE M F,et al.The measles virus hemagglutinin downregulates the cellular receptor SLAM(CD150)[J].Arch Virol,2002,147(1):195-203.
[13]苏建青,褚秀玲,马秀亮,等.稳定表达犬信号淋巴细胞激活因子基因细胞株的建立与鉴定[J].安徽农业科学,2012,40(14):8077-8079.SU J Q,CHU X L,MA X L,et al.Construction and identification of a Vero cell line stably expressing canine SLAM gene[J].Journal of Anhui Agricultural Sciences,2012,40(14):8077-8079.(in Chinese)
[14]黄娟,王聪,张洪亮,等.水貂犬瘟热病毒受体——信号淋巴细胞激活因子的细胞定位及组织分布[J].畜牧兽医学报,2014,45(2):289-294.HUANG J,WANG C,ZHANG H L,et al.Cellular localization and immunohistochemical demonstration of a sort of canine distemper virus receptor named Signalling lymphocyte activation molecule in minks[J].Acta Veterinaria et Zootechnica Sinica,2014,45(2):289-294.(in Chinese)
[15]POSTE G.The growth and cytopathogenicity of virulent and attenuated strains of canine distemper virus in dog and ferret macrophages[J].J Comp Pathol,1971,81(1):49-54.
[16]ROCKBORN G.An attenuated strain of canine distemper virus in tissue culture[J].Nature,1959,184(S11):822.
[17]METZLER A E,HIGGINS R J,KRAKOWKA S,et al.Virulence of tissue culture-propagated canine distemper virus[J].Infect Immun,1980,29(3):940-944.
[18]HASHIGUCHI T,OSE T,KUBOTA M,et al.Structure of the measles virus hemagglutinin bound to its cellular receptor SLAM[J].Nat Struct Mol Biol,2011,18(2):135-141.
[19]SARKAR J,BALAMURUGAN V,SEN A,et al.Sequence analysis of morbillivirus CD150receptor-signaling lymphocyte activation molecule(SLAM)of different animal species[J].Virus Genes,2009,39(3):335-341.
[20]SATO H,YONEDA M,HONDA T,et al.Morbillivirus receptors and tropism:Multiple pathways for infection[J].Front Microbiol,2012,3:75.
[21]SAWATSKY B,BENTE D A,CZUB M,et al.Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression,fusion support and particle assembly[J].J Gen Virol,2016,97(5):1066-1076.
[22]AVILA M,KHOSRAVI M,ALVES L,et al.Canine distemper virus envelope protein interactions modulated by hydrophobic residues in the fusion protein globular head[J].J Virol,2015,89(2):1445-1451.
[23]QIAO J,MENG Q,CHEN C,et al.Characterization of a new dog isolate of canine distemper virus from China[J].Acta Virol,2011,55(4):303-310.
[24]赵建军,闫如勋,张海玲,等.稳定表达貉犬瘟热病毒细胞受体SLAM的Vero细胞系的建立及应用[J].微生物学报,2012,52(12):1515-1523.ZHAO J J,YAN R X,ZHANG H L,et al.Establishment and application of a Vero cell line stably expressing raccoon dog SLAM,the cellular receptor of canine distemper virus[J].Acta Microbiologica Sinica,2012,52(12):1515-1523.(in Chinese)
[2]APPEL M J,YATES R A,FOLEY G L,et al.Canine distemper epizootic in lions,tigers,and leopards in North America[J].J Vet Diagn Invest,1994,6(3):277-288.
[3]TAN B,WEN Y J,WANG F X,et al.Pathogenesis and phylogenetic analyses of canine distemper virus strain ZJ7isolate from domestic dogs in China[J].Virol J,2011,8:520.
[4]岳进华,刘鹤媛,周铭,等.大熊猫犬瘟热病毒LG株的分离鉴定[J].动物医学进展,2016,37(10):60-63.YUE J H,LIU H Y,ZHOU M,et al.Identification of canine distemper virus LG strain isolated from giant panda[J].Progress in Veterinary Medicine,2016,37(10):60-63.(in Chinese)
[5]TATSUO H,ONO N,YANAGI Y.Morbilliviruses use signaling lymphocyte activation molecules(CD150)as cellular receptors[J].J Virol,2001,75(13):5842-5850.
[6]LEDNICKY J A,MEEHAN T P,KINSEL M J,et al.Effective primary isolation of wild-type canine distemper virus in MDCK,MV1Lu and Vero cells without nucleotide sequence changes within the entire haemagglutinin protein gene and in subgenomic sections of the fusion and phospho protein genes[J].J Virol Methods,2004,118(2):147-157.
[7]蒙学莲,窦永喜,朱学亮,等.小反刍兽疫病毒血凝素蛋白与受体蛋白SLAM的相互作用[J].畜牧兽医学报,2014,45(3):426-433.MENG X L,DOU Y X,ZHU X L,et al.Interaction between hemagglutinin protein of Peste des petits ruminants virus and signalling lymphocyte activation molecule[J].Acta Veterinaria et Zootechnica Sinica,2014,45(3):426-433.(in Chinese)
[8]SEKI F,ONO N,YAMAGUCHI R,et al.Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM(CD150)and their adaptability to marmoset B95acells[J].J Virol,2003,77(18):9943-9950.
[9]AVERSA G,CHANG C C,CARBALLIDO J M,et al.Engagement of the signaling lymphocytic activation molecule(SLAM)on activated T cells results in IL-2-independent,cyclosporin A-sensitive T cell proliferation and IFN-gamma production[J].J Immunol,1997,158(9):4036-4044.
[10]KRUSE M,MEINL E,HENNING G,et al.Signaling lymphocytic activation molecule is expressed on mature CD83+dendritic cells and is up-regulated by IL-1β[J].J Immunol,2001,167(4):1989-1995.
[11]ROMERO X,MARTIN M,ZAPATER N.Expression of CD150on human leukocytes.Production and characterization of a new CD150monoclonal antibody[J].Original,2001,20(2):57-66.
[12]TANAKA K,MINAGAWA H,XIE M F,et al.The measles virus hemagglutinin downregulates the cellular receptor SLAM(CD150)[J].Arch Virol,2002,147(1):195-203.
[13]苏建青,褚秀玲,马秀亮,等.稳定表达犬信号淋巴细胞激活因子基因细胞株的建立与鉴定[J].安徽农业科学,2012,40(14):8077-8079.SU J Q,CHU X L,MA X L,et al.Construction and identification of a Vero cell line stably expressing canine SLAM gene[J].Journal of Anhui Agricultural Sciences,2012,40(14):8077-8079.(in Chinese)
[14]黄娟,王聪,张洪亮,等.水貂犬瘟热病毒受体——信号淋巴细胞激活因子的细胞定位及组织分布[J].畜牧兽医学报,2014,45(2):289-294.HUANG J,WANG C,ZHANG H L,et al.Cellular localization and immunohistochemical demonstration of a sort of canine distemper virus receptor named Signalling lymphocyte activation molecule in minks[J].Acta Veterinaria et Zootechnica Sinica,2014,45(2):289-294.(in Chinese)
[15]POSTE G.The growth and cytopathogenicity of virulent and attenuated strains of canine distemper virus in dog and ferret macrophages[J].J Comp Pathol,1971,81(1):49-54.
[16]ROCKBORN G.An attenuated strain of canine distemper virus in tissue culture[J].Nature,1959,184(S11):822.
[17]METZLER A E,HIGGINS R J,KRAKOWKA S,et al.Virulence of tissue culture-propagated canine distemper virus[J].Infect Immun,1980,29(3):940-944.
[18]HASHIGUCHI T,OSE T,KUBOTA M,et al.Structure of the measles virus hemagglutinin bound to its cellular receptor SLAM[J].Nat Struct Mol Biol,2011,18(2):135-141.
[19]SARKAR J,BALAMURUGAN V,SEN A,et al.Sequence analysis of morbillivirus CD150receptor-signaling lymphocyte activation molecule(SLAM)of different animal species[J].Virus Genes,2009,39(3):335-341.
[20]SATO H,YONEDA M,HONDA T,et al.Morbillivirus receptors and tropism:Multiple pathways for infection[J].Front Microbiol,2012,3:75.
[21]SAWATSKY B,BENTE D A,CZUB M,et al.Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression,fusion support and particle assembly[J].J Gen Virol,2016,97(5):1066-1076.
[22]AVILA M,KHOSRAVI M,ALVES L,et al.Canine distemper virus envelope protein interactions modulated by hydrophobic residues in the fusion protein globular head[J].J Virol,2015,89(2):1445-1451.
[23]QIAO J,MENG Q,CHEN C,et al.Characterization of a new dog isolate of canine distemper virus from China[J].Acta Virol,2011,55(4):303-310.
[24]赵建军,闫如勋,张海玲,等.稳定表达貉犬瘟热病毒细胞受体SLAM的Vero细胞系的建立及应用[J].微生物学报,2012,52(12):1515-1523.ZHAO J J,YAN R X,ZHANG H L,et al.Establishment and application of a Vero cell line stably expressing raccoon dog SLAM,the cellular receptor of canine distemper virus[J].Acta Microbiologica Sinica,2012,52(12):1515-1523.(in Chinese)