牛病毒性腹泻病毒单克隆抗体的制备及其应用
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摘要
为制备牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)单克隆抗体(MAb)及其直接荧光标记抗体,本试验用纯化的牛病毒性腹泻病毒Ht01株免疫雌性BALB/c小鼠。间接ELISA测其血清效价,取抗体效价达标小鼠的脾细胞与SP2/0细胞融合。用间接ELISA筛选阳性细胞株并进行2次亚克隆后注射入小鼠腹腔制备单抗腹水。利用辛酸-硫酸铵沉淀法纯化腹水,用间接免疫荧光试验测定其活性,用SDS-PAGE试验测定其纯度。经活性、纯度检测合格后,采用搅拌法与异硫氰酸荧光素(FITC)偶联;用葡聚糖G-25 Medium介质进行分离纯化。结果,本研究共获得2株能够稳定分泌IgG1类型的阳性杂交瘤细胞株和一种直接荧光抗体,分别被命名为BMAb11、BMAb20、DB11。稳定性试验表明,杂交瘤细胞分泌的抗体稳定性好。ELISA、间接免疫荧光试验和直接免疫荧光试验表明,单克隆抗体和直标荧光抗体均与BVDV发生特异性反应,说明它们的特异性较好。经ELISA鉴定,腹水抗体效价达到1∶12 800。直接荧光抗体荧光素与蛋白的摩尔比(F/P)为3.34,标准值在2.0～4.0之间,符合标准。上述研究结果表明,本研究制备的单克隆抗体和直标荧光抗体可用于BVDV的检测,为BVDV的临床诊断奠定了基础。
This study was intended to prepare the bovine viral diarrhea virus(BVDV)Monoclonal ntibodies(MAb) and its direct fluorescent labeled antibodies.Female BALB/c mice were immunized by purified bovine viral diarrhea virus Ht01 strain,in this study.The titers of serum were detected by indirect ELISA.Its spleen cells that were appropriate antibody titer,were fused with SP2/0 cells.The positive cell lines,screened by indirect ELISA,were sublimed twice,and put into the abdominal cavity of mice to prepare ascites.Ascites were purified by oceanic acid-ammonium sulfate precipitation.Its purity was determined by indirect immunofluorescence assay and SDS-PAGE.Then,they were coupled with fluorescein isothiocyanate(FITC) by agitation method,and separated and purified by the dextran G-25 medium.Two positive hybridoma cell lines and a direct fluorescent antibody,which could stably secrete IgG1 type of hybridoma cells,named BMAb11,BMAb20 and DB11,respectively,were obtained,in this study.The stability test showed that the antibodies secreted by hybridoma cells were compatible.ELISA,indirect and direct immunofluorescence tests showed that identify monoclonal antibodies and its direct fluorescent labeled antibodies reacted specifically with BVDV,showing their specificity was better.The titer of ascitic antibody was 1∶12 800 by ELISA.The direct fluorescent antibody fluorescein to molar ratio of protein(F/P) was 3.34.The standard ranged from 2.0 and 4.0,which was owned by the standard.Based on this,monoclonal antibodies(MAb) and direct fluorescent labeled antibodies,obtained from this study,lay a foundation for the clinical diagnosis of BVDV.
This study was intended to prepare the bovine viral diarrhea virus(BVDV)Monoclonal ntibodies(MAb) and its direct fluorescent labeled antibodies.Female BALB/c mice were immunized by purified bovine viral diarrhea virus Ht01 strain,in this study.The titers of serum were detected by indirect ELISA.Its spleen cells that were appropriate antibody titer,were fused with SP2/0 cells.The positive cell lines,screened by indirect ELISA,were sublimed twice,and put into the abdominal cavity of mice to prepare ascites.Ascites were purified by oceanic acid-ammonium sulfate precipitation.Its purity was determined by indirect immunofluorescence assay and SDS-PAGE.Then,they were coupled with fluorescein isothiocyanate(FITC) by agitation method,and separated and purified by the dextran G-25 medium.Two positive hybridoma cell lines and a direct fluorescent antibody,which could stably secrete IgG1 type of hybridoma cells,named BMAb11,BMAb20 and DB11,respectively,were obtained,in this study.The stability test showed that the antibodies secreted by hybridoma cells were compatible.ELISA,indirect and direct immunofluorescence tests showed that identify monoclonal antibodies and its direct fluorescent labeled antibodies reacted specifically with BVDV,showing their specificity was better.The titer of ascitic antibody was 1∶12 800 by ELISA.The direct fluorescent antibody fluorescein to molar ratio of protein(F/P) was 3.34.The standard ranged from 2.0 and 4.0,which was owned by the standard.Based on this,monoclonal antibodies(MAb) and direct fluorescent labeled antibodies,obtained from this study,lay a foundation for the clinical diagnosis of BVDV.
引文
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[2]宫晓炜,曹小安,郑福英,等.牛病毒性腹泻病毒感染的免疫应答研究进展[J].中国兽医科学,2012,42(11):1200-1205.GONG Xiao-wei,CAO Xiao-an,ZHENG Fu-ying,et al.Progress on the immune response caused by bovine viral diarrhea virus infection[J].Chinese Veterinary Science,2012,42(11):1200-1205.(in Chinese)
[3]杨俊兴,曹琛福,阮周曦,等.牛病毒性腹泻病毒NS3蛋白单克隆抗体的制备与鉴定[J].上海畜牧兽医通讯,2013(02):5-7.YANG Jun-xin,CAO Shen-fu,RUAN Zhou-xi,et al.Preparation and identification of monoclonal antibodies against bovine viral diarrhea virus NS3 protein[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2013(02):5-7.(in Chinese)
[4]黄美玲,吴鹏,李天森,等.牛病毒性腹泻病毒E2抗原表位表达及验证[J].生物技术,2016,26(02):152-157.HUANG Mei-ling,WU Peng,LI Tian-sen,et al.BVDV-E2epitope analysis,expression and antigen detection[J].Biotechnology,2016,26(02):152-157.(in Chinese)
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[6]段进刚,雷程红,葛婷,等.牛病毒性腹泻病诊断技术的研究进展[J].黑龙江畜牧兽医,2018(01):93-96.DUAN Jing-gang,LEI Cheng-hong,GE Ting,et al.Research progress in the diagnosis of bovine viral diarrhea[J].Heilongjiang Animal Husbandry and Veterinary Medicine,2018(01):93-96.(in Chinese)
[7]赵静虎,王华欣,朱战波.牛病毒性腹泻-黏膜病的流行状况及防控研究进展[J].黑龙江八一农垦大学学报,2016,28(06):122-125.ZHAO Jing-hu,WANG Hua-xin,ZHU Zhan-bo.Advances in epidemic situation and prevention of bovine viral diarrhea-mucosal disease[J].Journal of Heilongjiang August First Land Reclamation University,2016,28(06):122-125.(in Chinese)
[8]LANYON S R,HILL F I,REICHEL M P,et al.Bovine viral diarrhea:pathogenesis and diagnosis[J].Veterinary Journal,2014,199(2):201-209.
[9]Mahmoodi P,Shapouri M R,Ghorbanpour M,et al.Epitope mapping of bovine viral diarrhea virus nonstructural protein 3.[J].Veterinary Immunology&Immunopathology,2014,161(3-4):232-239.
[10]李新培,关平原,温永俊,等.牛病毒性腹泻病毒致病机制研究进展[J].中国畜牧兽医,2018,45(08):2303-2311.LI Xin-pei,GUAN Ping-yuan,WEN Yong-jun,et al.Research progress on pathogenesis of bovine viral diarrhea virus[J].China Animal Husbandry and Veterinary Medicine,2018,45(08):2303-2311.(in Chinese)
[11]薛麒,吴思捷,张莹辉,等.牛病毒性腹泻病毒检测研究进展[J].中国畜牧兽医文摘,2017,33(07):73-74.XUE Qi,WU Si-jie,ZHANG Ying-hui,et al.Research progress in detection of bovine viral diarrhea virus[J].Chinese Abstracts of Animal Husbandry and Veterinary Medicine,2017,33(07):73-74.(in Chinese)
[12]王隆柏,王晨燕,吴学敏,等.猪流行性腹泻病毒单克隆抗体的制备及特性分析[J].福建农业学报,2017,32(08):818-822.WANG Long-bo,WANG Chen-yan,WU Xue-min,et al.Preparation and characterization of monoclonal an tibodies against porcine epidemic diarrhea virus[J].Journal of Fujian Agricultural Sciences,2017,32(08):818-822.(in Chinese)
[13]徐树兰,李少英,赵俊,等.牛病毒性腹泻病毒荧光标记单克隆抗体的制备及鉴定[J].中国兽医科学,2011,41(10):1041-1046.XU Shu-lan,LI Shao-ying,ZHAO Jun,et al.Preparation and identification of fluorescein-labelled monoclonal antibodies against bovine viral diarrhea virus[J].Chinese Veterinary Sciences,2011,41(10):1041-1046.(in Chinese)
[14]范晴,谢芝勋,刘加波,等.牛病毒性腹泻病毒E2基因在昆虫细胞中的表达[J].动物医学进展,2013,34(04):9-12.FAN Qing,XIE Zhi-xun,LIU Jia-bo,et al.Expression of E2 gene of bovine viral diarrhea in insect cells[J].Progress in Animal Medicine,2013,34(04):9-12.(in Chinese)
[15]ALPAY G,YE,ILBA K.Serological relationships among subgroups in bovine viral diarrhea virus genotype 1(BVDV-1)[J].Veterinary Microbiology,2015,175(1):1-6.
[16]孔君,刘箐,韩跃武,等.单克隆抗体制备技术的最新进展及应用前景[J].免疫学杂志,2011,27(02):170-173.KONG Jun,LIU Jing,HAN Yue-wu,et al.The advances and application prospects of monoclonal antibody preparation technology[J].Journal of Immunology,2011,27(02):170-173.(in Chinese)
[17]杨立新.牛病毒性腹泻病毒p80蛋白单抗制备与表位鉴定及LJ36/14毒株的分离鉴定[D].北京:中国农业科学院研究生院,2016.YANG Li-xin.Identification of an antigen epitope on the p80 protein with monoclonal antibody and isolation and characterization of the virus strain LJ36/14 for bovine viral diarrhea virus[D].Beijing:Graduate School of Chinese Academy of Agricultural Sciences,2016.(in Chinese)
[18]王雪枝.牛病毒性腹泻病毒E0蛋白单抗与多抗制备及实验感染小鼠的研究[D].北京:中国农业科学院研究生院,2014.WANG Xue-zhi.Preparations of monoclonal antibody and polyclonal antibody against EO protein of bovine viral diarrhea virus and experimental infection of mice with the virus[D].Beijing:Graduate School of Chinese Academy of Agricultural Sciences,2014.(in Chinese)
[19]徐树兰,李少英,冷雪,等.鉴别牛病毒性腹泻病毒基因型单克隆抗体的制备及其特性[J].农业生物技术学报,2010,18(05):1001-1007.XU Shu-lan,LI Shao-ying,LENG Xue,et al.Preparation and preliminary analysis of monoclonal antibodies against different genotypes of bovine viral diarrhea virus[J].Journal of Agricultural Biotechnology,2010,18(05):1001-1007.(in Chinese)
[20]ZHANG X,DIRAVIYAM T,LI X,et al.Preparation of chicken IgY against recombinant E2 protein of bovine viral diarrhea virus(BVDV)and development of ELISA and ICA for BVDV detection[J].Journal of the Agricultural Chemical Society of Japan,2016,80(12):2467-2472.
[21]杨立新,朱远茂,周跃辉,等.牛病毒性腹泻病毒p80蛋白的表达及其单克隆抗体的制备[J].黑龙江畜牧兽医,2016(7):1-4.YANG Li-xin,ZHU Yuan-mao,ZHOU Yue-hui,et al.Expression of the p80 protein of bovine viral diarrhea virus and preparation of its monoclonal antibody[J].Heilongjiang Animal Husbandry and Veterinary Medicine,2016(7):1-4.(in Chinese)
[22]何民辉,李真光,温永俊,等.PRRSV-N蛋白单抗制备与直接免疫荧光方法建立[J].基因组学与应用生物学,2017,36(12):5077-5083.HE Min-hui,LI Zhen-guang,WEN Yong-jun,et al.Preparation of PRRSV-N protein monoclonal antibody and establishment of DIF techniques[J].Genomics and Biology Applied,2017,36(12):5077-5083.(in Chinese)
[23]王姝,朱远茂,薛飞,等.牛病毒性腹泻病毒E2蛋白单克隆抗体的制备及初步鉴定[J].中国生物工程杂志,2013,33(04):40-45.WANG Shu,ZHU Yuan-mao,XUE Fei,et al.Preparation and preliminary characterization of monoclonal antibodies against E2 protein of bovine viral diarrhea virus[J].Chinese Journal of Bioengineering,2013,33(04):40-45.(in Chinese)
[2]宫晓炜,曹小安,郑福英,等.牛病毒性腹泻病毒感染的免疫应答研究进展[J].中国兽医科学,2012,42(11):1200-1205.GONG Xiao-wei,CAO Xiao-an,ZHENG Fu-ying,et al.Progress on the immune response caused by bovine viral diarrhea virus infection[J].Chinese Veterinary Science,2012,42(11):1200-1205.(in Chinese)
[3]杨俊兴,曹琛福,阮周曦,等.牛病毒性腹泻病毒NS3蛋白单克隆抗体的制备与鉴定[J].上海畜牧兽医通讯,2013(02):5-7.YANG Jun-xin,CAO Shen-fu,RUAN Zhou-xi,et al.Preparation and identification of monoclonal antibodies against bovine viral diarrhea virus NS3 protein[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2013(02):5-7.(in Chinese)
[4]黄美玲,吴鹏,李天森,等.牛病毒性腹泻病毒E2抗原表位表达及验证[J].生物技术,2016,26(02):152-157.HUANG Mei-ling,WU Peng,LI Tian-sen,et al.BVDV-E2epitope analysis,expression and antigen detection[J].Biotechnology,2016,26(02):152-157.(in Chinese)
[5]孟庆森,王炜,黄慧文,等.牛病毒性腹泻病毒的研究进展[J].当代畜牧,2017(33):16-18.MENG Qing-sen,WANG Wei,HUANG Hui-wen,et al.Research progress of bovine viral diarrhea virus[J].Contemporary Animal Husbandry,2017(33):16-18.(in Chinese)
[6]段进刚,雷程红,葛婷,等.牛病毒性腹泻病诊断技术的研究进展[J].黑龙江畜牧兽医,2018(01):93-96.DUAN Jing-gang,LEI Cheng-hong,GE Ting,et al.Research progress in the diagnosis of bovine viral diarrhea[J].Heilongjiang Animal Husbandry and Veterinary Medicine,2018(01):93-96.(in Chinese)
[7]赵静虎,王华欣,朱战波.牛病毒性腹泻-黏膜病的流行状况及防控研究进展[J].黑龙江八一农垦大学学报,2016,28(06):122-125.ZHAO Jing-hu,WANG Hua-xin,ZHU Zhan-bo.Advances in epidemic situation and prevention of bovine viral diarrhea-mucosal disease[J].Journal of Heilongjiang August First Land Reclamation University,2016,28(06):122-125.(in Chinese)
[8]LANYON S R,HILL F I,REICHEL M P,et al.Bovine viral diarrhea:pathogenesis and diagnosis[J].Veterinary Journal,2014,199(2):201-209.
[9]Mahmoodi P,Shapouri M R,Ghorbanpour M,et al.Epitope mapping of bovine viral diarrhea virus nonstructural protein 3.[J].Veterinary Immunology&Immunopathology,2014,161(3-4):232-239.
[10]李新培,关平原,温永俊,等.牛病毒性腹泻病毒致病机制研究进展[J].中国畜牧兽医,2018,45(08):2303-2311.LI Xin-pei,GUAN Ping-yuan,WEN Yong-jun,et al.Research progress on pathogenesis of bovine viral diarrhea virus[J].China Animal Husbandry and Veterinary Medicine,2018,45(08):2303-2311.(in Chinese)
[11]薛麒,吴思捷,张莹辉,等.牛病毒性腹泻病毒检测研究进展[J].中国畜牧兽医文摘,2017,33(07):73-74.XUE Qi,WU Si-jie,ZHANG Ying-hui,et al.Research progress in detection of bovine viral diarrhea virus[J].Chinese Abstracts of Animal Husbandry and Veterinary Medicine,2017,33(07):73-74.(in Chinese)
[12]王隆柏,王晨燕,吴学敏,等.猪流行性腹泻病毒单克隆抗体的制备及特性分析[J].福建农业学报,2017,32(08):818-822.WANG Long-bo,WANG Chen-yan,WU Xue-min,et al.Preparation and characterization of monoclonal an tibodies against porcine epidemic diarrhea virus[J].Journal of Fujian Agricultural Sciences,2017,32(08):818-822.(in Chinese)
[13]徐树兰,李少英,赵俊,等.牛病毒性腹泻病毒荧光标记单克隆抗体的制备及鉴定[J].中国兽医科学,2011,41(10):1041-1046.XU Shu-lan,LI Shao-ying,ZHAO Jun,et al.Preparation and identification of fluorescein-labelled monoclonal antibodies against bovine viral diarrhea virus[J].Chinese Veterinary Sciences,2011,41(10):1041-1046.(in Chinese)
[14]范晴,谢芝勋,刘加波,等.牛病毒性腹泻病毒E2基因在昆虫细胞中的表达[J].动物医学进展,2013,34(04):9-12.FAN Qing,XIE Zhi-xun,LIU Jia-bo,et al.Expression of E2 gene of bovine viral diarrhea in insect cells[J].Progress in Animal Medicine,2013,34(04):9-12.(in Chinese)
[15]ALPAY G,YE,ILBA K.Serological relationships among subgroups in bovine viral diarrhea virus genotype 1(BVDV-1)[J].Veterinary Microbiology,2015,175(1):1-6.
[16]孔君,刘箐,韩跃武,等.单克隆抗体制备技术的最新进展及应用前景[J].免疫学杂志,2011,27(02):170-173.KONG Jun,LIU Jing,HAN Yue-wu,et al.The advances and application prospects of monoclonal antibody preparation technology[J].Journal of Immunology,2011,27(02):170-173.(in Chinese)
[17]杨立新.牛病毒性腹泻病毒p80蛋白单抗制备与表位鉴定及LJ36/14毒株的分离鉴定[D].北京:中国农业科学院研究生院,2016.YANG Li-xin.Identification of an antigen epitope on the p80 protein with monoclonal antibody and isolation and characterization of the virus strain LJ36/14 for bovine viral diarrhea virus[D].Beijing:Graduate School of Chinese Academy of Agricultural Sciences,2016.(in Chinese)
[18]王雪枝.牛病毒性腹泻病毒E0蛋白单抗与多抗制备及实验感染小鼠的研究[D].北京:中国农业科学院研究生院,2014.WANG Xue-zhi.Preparations of monoclonal antibody and polyclonal antibody against EO protein of bovine viral diarrhea virus and experimental infection of mice with the virus[D].Beijing:Graduate School of Chinese Academy of Agricultural Sciences,2014.(in Chinese)
[19]徐树兰,李少英,冷雪,等.鉴别牛病毒性腹泻病毒基因型单克隆抗体的制备及其特性[J].农业生物技术学报,2010,18(05):1001-1007.XU Shu-lan,LI Shao-ying,LENG Xue,et al.Preparation and preliminary analysis of monoclonal antibodies against different genotypes of bovine viral diarrhea virus[J].Journal of Agricultural Biotechnology,2010,18(05):1001-1007.(in Chinese)
[20]ZHANG X,DIRAVIYAM T,LI X,et al.Preparation of chicken IgY against recombinant E2 protein of bovine viral diarrhea virus(BVDV)and development of ELISA and ICA for BVDV detection[J].Journal of the Agricultural Chemical Society of Japan,2016,80(12):2467-2472.
[21]杨立新,朱远茂,周跃辉,等.牛病毒性腹泻病毒p80蛋白的表达及其单克隆抗体的制备[J].黑龙江畜牧兽医,2016(7):1-4.YANG Li-xin,ZHU Yuan-mao,ZHOU Yue-hui,et al.Expression of the p80 protein of bovine viral diarrhea virus and preparation of its monoclonal antibody[J].Heilongjiang Animal Husbandry and Veterinary Medicine,2016(7):1-4.(in Chinese)
[22]何民辉,李真光,温永俊,等.PRRSV-N蛋白单抗制备与直接免疫荧光方法建立[J].基因组学与应用生物学,2017,36(12):5077-5083.HE Min-hui,LI Zhen-guang,WEN Yong-jun,et al.Preparation of PRRSV-N protein monoclonal antibody and establishment of DIF techniques[J].Genomics and Biology Applied,2017,36(12):5077-5083.(in Chinese)
[23]王姝,朱远茂,薛飞,等.牛病毒性腹泻病毒E2蛋白单克隆抗体的制备及初步鉴定[J].中国生物工程杂志,2013,33(04):40-45.WANG Shu,ZHU Yuan-mao,XUE Fei,et al.Preparation and preliminary characterization of monoclonal antibodies against E2 protein of bovine viral diarrhea virus[J].Chinese Journal of Bioengineering,2013,33(04):40-45.(in Chinese)