绵羊肺炎支原体P113蛋白单克隆抗体的制备及其在免疫荧光检测中的应用研究
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摘要
P113蛋白是绵羊肺炎支原体重要的免疫原。为了制备针对P113蛋白的单克隆抗体,本研究以原核表达的重组P113蛋白免疫小鼠,制备了1株P113蛋白的特异性单克隆抗体C426,纯化后用异硫氰酸荧光素标记单克隆抗体,并建立了绵羊肺炎支原体的免疫荧光检测技术。结果显示,该株单克隆抗体的ELISA效价为1∶128 000,亚型为IgG2b,轻链类型为κ型,并具有良好的特异性。所建立的免疫荧光检测方法特异性强、灵敏度高,其对绵羊肺炎支原体培养物的检测下限为100 CCU/m L。该方法可以从感染动物的鼻拭子样品或肺组织中成功检测到绵羊肺炎支原体,与PCR方法的阳性符合率为86.25%,明显高于支原体分离培养的方法。本研究制备的P113蛋白单克隆抗体以及免疫荧光技术不仅为绵羊肺炎支原体感染的诊断提供了新的手段,同时为进一步研究P113的功能提供了良好的工具。
P113 protein is an important immunogen of Mycoplasma ovipneumoniae.In this study,a specific monoclonal antibody C426 against P113 protein was prepared by immunization of mice with a prokaryoticly-expressed recombinant protein.An immunofluorescence assay for the detection of Mycoplasma ovipneumoniae was developed by labeling the Mc Ab with fluorescein isothiocyanate.The results showed that the monoclonal antibody was specific to Mycoplasma ovipneumoniae with a high ELISA titer of1 ∶ 128 000,and the subtype of the monoclonal antibody was identified as IgG2 b with κ light chain.The established immunofluorescence assay hadhigh specificity and sensitivity.Its detection limit for Mycoplasma ovipneumoniae was100 CCU/m L.This method could successfully detect Mycoplasma ovipneumoniae in nasal swabs and lung tissues from infected animals.The coincidence rate between PCR and the immunofluorescence assay was 86.25%(69/80),which was significantly higher than that of mycoplasma isolation.In conclusion,the monoclonal antibody against P113 protein and the immunofluorescence assay developed in this study provide a new tool for the diagnosis of Mycoplasma ovpneumoniae infection and the further study of P113 functions.
P113 protein is an important immunogen of Mycoplasma ovipneumoniae.In this study,a specific monoclonal antibody C426 against P113 protein was prepared by immunization of mice with a prokaryoticly-expressed recombinant protein.An immunofluorescence assay for the detection of Mycoplasma ovipneumoniae was developed by labeling the Mc Ab with fluorescein isothiocyanate.The results showed that the monoclonal antibody was specific to Mycoplasma ovipneumoniae with a high ELISA titer of1 ∶ 128 000,and the subtype of the monoclonal antibody was identified as IgG2 b with κ light chain.The established immunofluorescence assay hadhigh specificity and sensitivity.Its detection limit for Mycoplasma ovipneumoniae was100 CCU/m L.This method could successfully detect Mycoplasma ovipneumoniae in nasal swabs and lung tissues from infected animals.The coincidence rate between PCR and the immunofluorescence assay was 86.25%(69/80),which was significantly higher than that of mycoplasma isolation.In conclusion,the monoclonal antibody against P113 protein and the immunofluorescence assay developed in this study provide a new tool for the diagnosis of Mycoplasma ovpneumoniae infection and the further study of P113 functions.
引文
[1] RIFATBEGOVI M,MAKSIMOVI Z,HULAJ B.Mycoplasma ovipneumoniae associated with severe respiratory disease in goats[J].The Veterinary Record,2011,168(21):565.
[2] BESSER T E,CASSIRER E F,POTTER K A,et al.Association of Mycoplasma ovipneumoniae infection with population-limiting respiratory disease in freeranging rocky mountain bighorn sheep(Ovis canadensis canadensis)[J].Journal of Clinical Microbiology,2008,46(2):423-430.
[3] KILIC A,KALENDER H,EROKSUZ H,et al.Identification by culture,PCR,and immunohistochemistry of mycoplasmas and their molecular typing in sheep and lamb lungs with pneumonia in Eastern Turkey[J].Tropical Animal Health and Production,2013,45(7):1525-1531.
[4] BESSER T E,FRANCES CASSIRER E,HIGHLAND M A,et al.Bighorn sheep pneumonia:Sorting out the cause of a polymicrobial disease[J].Preventive Veterinary Medicine,2013,108(2-3):85-93.
[5] DASSANAYAKE R P,SHANTHALINGAM S,HERNDON C N,et al.Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia[J].Veterinary Microbiology,2010,145(3-4):354-359.
[6] GONCALVES R,MARIANO I,NNEZ A,et al.Atypical non-progressive pneumonia in goats[J].The Veterinary Journal,2010,183(2):219-221.
[7] WOOD M E,FOX K A,JENNINGS-GAINES J,et al.How respiratory pathogens contribute to lamb mortality in a poorly performing bighorn sheep(Ovis canadensis)herd[J].Journal of Wildlife Diseases,2015,53(1):126-130.
[8]谢秀兰,额尔和花,黎帅,等.宁夏不同绵羊品种中绵羊肺炎支原体的调查[J].中国兽医杂志,2017,53(6):11-13.XIE Xiu-lan,Eerhehua,LI Shuai,et al.The survey of Mycoplasma ovipneumoniae in different sheep breeds in Ningxia area[J].Chinese Journal of Veterinary Medicine,2017,53(6):11-13.(in Chinese)
[9] CHENG C,JUN Q,QINGLING M,et al.Serological and molecular survey of sheep infected with Mycoplasma ovipneumoniae,in Xinjiang,China[J].Tropical Animal Health and Production,2015,47(8):1641-1647.
[10]王华,杨发龙,岳华,等.四川省山羊支原体性肺炎流行病学调查[C].中国畜牧兽医学会兽医公共卫生学分会学术研讨会,2010.WANG Hua,YANG Fa-long,YUE Hua,et al.The epidemiological investigation of Mycoplasma pneumoniae of goats in Sichuan Province[C].Chinese Veterinary Public Health Association,CAAV,2010.(in Chinese)
[11] RONG G,ZHAO J M,HOU G Y,et al.Seroprevalence and molecular detection of Mycoplasma ovipneumoniae in goats in tropical China.[J].Tropical Animal Health and Production,2014,46(8):1491-1495.
[12] YANG F,DAO X,RODRIGUEZ-PALACIOS A,et al.A realtime PCR for detection and quantification of Mycoplasma ovipneumoniae[J].The Journal of Veterinary Medical Science,2014,76(12):1631-1634.
[13]韩瑞鑫,申捷,申之义.绵羊肺炎支原体TaqMan实时荧光定量PCR检测方法的建立与应用[J].中国兽医科学,2018,48(8):965-970.HAN Rui-xin,SHEN Jie,SHEN Zhi-yi.Development and application of the TaqMan real-time fluorescent quantitation PCR for the detection of Mycoplasma ovipneumoniae[J].Chinese Veterinary Science,2018,48(8):965-970.(in Chinese)
[14]黄坚,尹正军,岳华,等.绵羊肺炎支原体荧光定量PCR检测方法的建立与应用[J].中国兽医科学,2016,46(10):1270-1276.HUANG Jian,YIN Zheng-jun,YUE Hua,et al.Development and application of quantitative real-time PCR for the detection of Mycoplasma ovipneumoniae[J].Chinese Veterinary Science,2016,46(10):1270-1276.(in Chinese)
[15]冯旭飞,刘霜,张贤宇,等.绵羊肺炎支原体和多杀性巴氏杆菌双重PCR检测方法的建立及应用[J].中国兽医科学,2013,43(12):1257-1261.FENG Xu-fei,LIU Shuang,ZHANG Xian-yu,et al.Development of a duplex PCR assay for double detection of Mycoplasma ovipneumoniae and Pasteurella multocida[J].Chinese Veterinary Science,2013,43(12):1257-1261.(in Chinese)
[16]黄海碧,郑佳琪,王仁超,等.绵羊肺炎支原体P71蛋白分段表达及其间接ELISA方法的建立[J].中国预防兽医学报,2015,37(8):623-627.HUANG Hai-bi,ZHENG Jia-qi,WANG Ren-chao,et al.Development of an indirect ELISA method with the main antigentic region of Mycoplasma ovipneumoniae P71 protein expressed in E.coli[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(8):623-627.(in Chinese)
[17]王旭,张晓宇,张建华,等.绵羊肺炎支原体P130蛋白主要抗原域原核表达及其间接ELISA检测方法的建立[J].中国兽医科学,2018,48(3):281-287.WANG Xu,ZHANG Xiao-yu,ZHANG Jian-hua,et al.Development of an indirect ELISA with the main antigentic region of Mycoplasma ovipneumoniae P130 protein expressed in E.coli[J].Chinese Veterinary Science,2018,48(3):281-287.(in Chinese)
[18]麻昌姣.绵羊肺炎支原体p60、p65蛋白分段表达及基于串联蛋白p60(N)-p65(C)间接ELISA检测方法的建立[D].呼和浩特:内蒙古农业大学, 2017.MA Chang-jiao.Expression of fragments of MO p60and p65 proteins and establishment of a recombinant protein p60(N)-p65(C)based indirect ELISA[D].Huhhot:Inner Mongolia Agricultural University, 2017.(in Chinese)
[19]孙喜安,韩敏,刘晓松,等.绵羊肺炎支原体分离株交叉反应性的测定[J].畜牧与饲料科学,2012,33(1):112-113,118.SUN Xi-an,HAN Min,LIU Xiao-song,et al.Measurement of cross reactivity of Mycoplasma ovipneumoniae isolates[J].Animal Husbandry and Feed Science,2012,33(1):112-113,118.(in Chinese)
[20]赵萍,逯忠新,储岳峰,等.绵羊肺炎支原体间接血凝诊断方法的建立[J].甘肃农业大学学报,2008,43(1):29-32.ZHAO Ping,LU Zhong-xin,CHU Yue-feng,et al.Development of the indirect haemagglutination test of M.ovipneumoniae[J].Journal of Gansu Agricultural University,2008,43(1):29-32.(in Chinese)
[21]杨发龙,张贤宇,汤承,等.绵羊肺炎支原体P113蛋白C端重复区的表达及其免疫原性[J].中国兽医科学,2013,43(7):733-737.YANG Fa-long,ZHANG Xian-yu,TANG Cheng,et al.Expression and immunogenicity of a C terminal repeat region of Mycoplasma ovipneumoniae P113protein[J].Chinese Veterinary Science,2013,43(7):733-737.(in Chinese)
[22] FONTES L V Q,CAMPOS G S,BECK P A,et al.Precipitation of bovine rotavirus by polyethylen glycol(PEG)and its application to produce polyclonal and monoclonal antibodies[J].Journal of Virological Methods,2005, 123(2):147-153.
[23]姜畔.鸭肝炎病毒单克隆抗体的制备及ELISA病毒检测方法的建立[D].北京:中国兽医药品监察所,2011.JIANG Pan.Reparation of monoclonal antibody against DHV and development of ELISA for detecting the virus[D].Beijing:China Institute of Veterinary Drug Control,2011.(in Chinese)
[24] WILKINS M R,PASQUALI C,APPEL R D,et al.From proteins to proteomes:large scale protein identification by two-dimensional electrophoresis and amino acid analysis[J].Bio/technology(Nature Publishing Company),1996,14(1):61.
[25]苏建青,褚秀玲,杨松涛,等.抗犬瘟热病毒荧光标记单抗的制备及初步鉴定[J].安徽农业科学,2009,10(1):115-118.SU Jian-qing,CHU Xiu-ling,YANG Song-tao,et al.Preparation and preliminary identification of fluoresce in labeled monoclonal antibody against canine distemper virus[J].Journal of Anhui Agricultural Sciences,2009,10(1):115-118.(in Chinese)
[26]杨发龙,王华,岳华,等.山羊中绵羊肺炎支原体的分离及鉴定[J].中国畜牧兽医,2010,37(8):177-180.YANG Fa-long,WANG Hua,YUE Hua,et al.Isolation and identification of Mycoplasma ovipneumoniae in goats[J].China Animal Husbandry&Veterinary Medicine,2010,37(8):177-180.(in Chinese)
[27]张建华,黄海碧,王晓晖,等.绵羊肺炎支原体免疫组织化学及间接免疫荧光检测方法的建立[J].中国兽医科学, 2017,47(2):172-178.ZHANG Jian-hua,HUANG Hai-bi,WANG Xiao-hui,et al.Establishment of indirect immunofluorescence assay and immunohistochemistry assay for detection of Mycoplasma ovipneumonia[J].Chinese Veterinary Science,2017,47(2):172-178.(in Chinese)
[2] BESSER T E,CASSIRER E F,POTTER K A,et al.Association of Mycoplasma ovipneumoniae infection with population-limiting respiratory disease in freeranging rocky mountain bighorn sheep(Ovis canadensis canadensis)[J].Journal of Clinical Microbiology,2008,46(2):423-430.
[3] KILIC A,KALENDER H,EROKSUZ H,et al.Identification by culture,PCR,and immunohistochemistry of mycoplasmas and their molecular typing in sheep and lamb lungs with pneumonia in Eastern Turkey[J].Tropical Animal Health and Production,2013,45(7):1525-1531.
[4] BESSER T E,FRANCES CASSIRER E,HIGHLAND M A,et al.Bighorn sheep pneumonia:Sorting out the cause of a polymicrobial disease[J].Preventive Veterinary Medicine,2013,108(2-3):85-93.
[5] DASSANAYAKE R P,SHANTHALINGAM S,HERNDON C N,et al.Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia[J].Veterinary Microbiology,2010,145(3-4):354-359.
[6] GONCALVES R,MARIANO I,NNEZ A,et al.Atypical non-progressive pneumonia in goats[J].The Veterinary Journal,2010,183(2):219-221.
[7] WOOD M E,FOX K A,JENNINGS-GAINES J,et al.How respiratory pathogens contribute to lamb mortality in a poorly performing bighorn sheep(Ovis canadensis)herd[J].Journal of Wildlife Diseases,2015,53(1):126-130.
[8]谢秀兰,额尔和花,黎帅,等.宁夏不同绵羊品种中绵羊肺炎支原体的调查[J].中国兽医杂志,2017,53(6):11-13.XIE Xiu-lan,Eerhehua,LI Shuai,et al.The survey of Mycoplasma ovipneumoniae in different sheep breeds in Ningxia area[J].Chinese Journal of Veterinary Medicine,2017,53(6):11-13.(in Chinese)
[9] CHENG C,JUN Q,QINGLING M,et al.Serological and molecular survey of sheep infected with Mycoplasma ovipneumoniae,in Xinjiang,China[J].Tropical Animal Health and Production,2015,47(8):1641-1647.
[10]王华,杨发龙,岳华,等.四川省山羊支原体性肺炎流行病学调查[C].中国畜牧兽医学会兽医公共卫生学分会学术研讨会,2010.WANG Hua,YANG Fa-long,YUE Hua,et al.The epidemiological investigation of Mycoplasma pneumoniae of goats in Sichuan Province[C].Chinese Veterinary Public Health Association,CAAV,2010.(in Chinese)
[11] RONG G,ZHAO J M,HOU G Y,et al.Seroprevalence and molecular detection of Mycoplasma ovipneumoniae in goats in tropical China.[J].Tropical Animal Health and Production,2014,46(8):1491-1495.
[12] YANG F,DAO X,RODRIGUEZ-PALACIOS A,et al.A realtime PCR for detection and quantification of Mycoplasma ovipneumoniae[J].The Journal of Veterinary Medical Science,2014,76(12):1631-1634.
[13]韩瑞鑫,申捷,申之义.绵羊肺炎支原体TaqMan实时荧光定量PCR检测方法的建立与应用[J].中国兽医科学,2018,48(8):965-970.HAN Rui-xin,SHEN Jie,SHEN Zhi-yi.Development and application of the TaqMan real-time fluorescent quantitation PCR for the detection of Mycoplasma ovipneumoniae[J].Chinese Veterinary Science,2018,48(8):965-970.(in Chinese)
[14]黄坚,尹正军,岳华,等.绵羊肺炎支原体荧光定量PCR检测方法的建立与应用[J].中国兽医科学,2016,46(10):1270-1276.HUANG Jian,YIN Zheng-jun,YUE Hua,et al.Development and application of quantitative real-time PCR for the detection of Mycoplasma ovipneumoniae[J].Chinese Veterinary Science,2016,46(10):1270-1276.(in Chinese)
[15]冯旭飞,刘霜,张贤宇,等.绵羊肺炎支原体和多杀性巴氏杆菌双重PCR检测方法的建立及应用[J].中国兽医科学,2013,43(12):1257-1261.FENG Xu-fei,LIU Shuang,ZHANG Xian-yu,et al.Development of a duplex PCR assay for double detection of Mycoplasma ovipneumoniae and Pasteurella multocida[J].Chinese Veterinary Science,2013,43(12):1257-1261.(in Chinese)
[16]黄海碧,郑佳琪,王仁超,等.绵羊肺炎支原体P71蛋白分段表达及其间接ELISA方法的建立[J].中国预防兽医学报,2015,37(8):623-627.HUANG Hai-bi,ZHENG Jia-qi,WANG Ren-chao,et al.Development of an indirect ELISA method with the main antigentic region of Mycoplasma ovipneumoniae P71 protein expressed in E.coli[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(8):623-627.(in Chinese)
[17]王旭,张晓宇,张建华,等.绵羊肺炎支原体P130蛋白主要抗原域原核表达及其间接ELISA检测方法的建立[J].中国兽医科学,2018,48(3):281-287.WANG Xu,ZHANG Xiao-yu,ZHANG Jian-hua,et al.Development of an indirect ELISA with the main antigentic region of Mycoplasma ovipneumoniae P130 protein expressed in E.coli[J].Chinese Veterinary Science,2018,48(3):281-287.(in Chinese)
[18]麻昌姣.绵羊肺炎支原体p60、p65蛋白分段表达及基于串联蛋白p60(N)-p65(C)间接ELISA检测方法的建立[D].呼和浩特:内蒙古农业大学, 2017.MA Chang-jiao.Expression of fragments of MO p60and p65 proteins and establishment of a recombinant protein p60(N)-p65(C)based indirect ELISA[D].Huhhot:Inner Mongolia Agricultural University, 2017.(in Chinese)
[19]孙喜安,韩敏,刘晓松,等.绵羊肺炎支原体分离株交叉反应性的测定[J].畜牧与饲料科学,2012,33(1):112-113,118.SUN Xi-an,HAN Min,LIU Xiao-song,et al.Measurement of cross reactivity of Mycoplasma ovipneumoniae isolates[J].Animal Husbandry and Feed Science,2012,33(1):112-113,118.(in Chinese)
[20]赵萍,逯忠新,储岳峰,等.绵羊肺炎支原体间接血凝诊断方法的建立[J].甘肃农业大学学报,2008,43(1):29-32.ZHAO Ping,LU Zhong-xin,CHU Yue-feng,et al.Development of the indirect haemagglutination test of M.ovipneumoniae[J].Journal of Gansu Agricultural University,2008,43(1):29-32.(in Chinese)
[21]杨发龙,张贤宇,汤承,等.绵羊肺炎支原体P113蛋白C端重复区的表达及其免疫原性[J].中国兽医科学,2013,43(7):733-737.YANG Fa-long,ZHANG Xian-yu,TANG Cheng,et al.Expression and immunogenicity of a C terminal repeat region of Mycoplasma ovipneumoniae P113protein[J].Chinese Veterinary Science,2013,43(7):733-737.(in Chinese)
[22] FONTES L V Q,CAMPOS G S,BECK P A,et al.Precipitation of bovine rotavirus by polyethylen glycol(PEG)and its application to produce polyclonal and monoclonal antibodies[J].Journal of Virological Methods,2005, 123(2):147-153.
[23]姜畔.鸭肝炎病毒单克隆抗体的制备及ELISA病毒检测方法的建立[D].北京:中国兽医药品监察所,2011.JIANG Pan.Reparation of monoclonal antibody against DHV and development of ELISA for detecting the virus[D].Beijing:China Institute of Veterinary Drug Control,2011.(in Chinese)
[24] WILKINS M R,PASQUALI C,APPEL R D,et al.From proteins to proteomes:large scale protein identification by two-dimensional electrophoresis and amino acid analysis[J].Bio/technology(Nature Publishing Company),1996,14(1):61.
[25]苏建青,褚秀玲,杨松涛,等.抗犬瘟热病毒荧光标记单抗的制备及初步鉴定[J].安徽农业科学,2009,10(1):115-118.SU Jian-qing,CHU Xiu-ling,YANG Song-tao,et al.Preparation and preliminary identification of fluoresce in labeled monoclonal antibody against canine distemper virus[J].Journal of Anhui Agricultural Sciences,2009,10(1):115-118.(in Chinese)
[26]杨发龙,王华,岳华,等.山羊中绵羊肺炎支原体的分离及鉴定[J].中国畜牧兽医,2010,37(8):177-180.YANG Fa-long,WANG Hua,YUE Hua,et al.Isolation and identification of Mycoplasma ovipneumoniae in goats[J].China Animal Husbandry&Veterinary Medicine,2010,37(8):177-180.(in Chinese)
[27]张建华,黄海碧,王晓晖,等.绵羊肺炎支原体免疫组织化学及间接免疫荧光检测方法的建立[J].中国兽医科学, 2017,47(2):172-178.ZHANG Jian-hua,HUANG Hai-bi,WANG Xiao-hui,et al.Establishment of indirect immunofluorescence assay and immunohistochemistry assay for detection of Mycoplasma ovipneumonia[J].Chinese Veterinary Science,2017,47(2):172-178.(in Chinese)
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