抗牛传染性鼻气管炎病毒gD蛋白单克隆抗体的制备及鉴定
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摘要
目的制备牛传染性鼻气管炎病毒(infectious bovine rhinotracheities virus,IBRV)gD蛋白单克隆抗体,并鉴定其生物学特性。方法采用超速离心纯化的IBRV全病毒免疫BALB/c小鼠,取免疫后血清抗体呈阳性的小鼠脾细胞,通过融合剂PEG与骨髓瘤细胞SP2/0融合,采用间接免疫荧光法(IFA)筛选杂交瘤细胞,并制备腹水,分析单抗的生物学特性。结果筛选出1株稳定分泌抗IBRV gD蛋白单抗的杂交瘤细胞,命名为3C1。单抗的重链为IgG1亚类,轻链为kappa链;上清和腹水效价分别为1∶40和1∶12 800;单抗可与IBRV及gD重组蛋白分别在相对分子质量71 000和96 000处发生特异性反应,可与感染IBRV的MDBK细胞发生特异性结合,产生荧光。结论成功制备了抗IBRV gD蛋白的单克隆抗体,为深入研究gD蛋白功能及IBR的临床诊断方法筛选等提供了数据材料。
Objective To prepare the monoclonal antibody(MAb)against gD protein of infectious bovine rhinotracheities virus(IBRV)and identify its biological characteristics. Methods BALB/c mice were immunized with the complete IBRV purified by ultracentrifugation. The spleen cells of immunized mice positive for serum antibody were fused with myeloma SP2/0 cells by using PEG. Hybridoma cells were screened by indirect immunofluorescence(IFA),with which the ascites was prepared,and the biological characteristics of MAb was analyzed. Results A hybridoma cell line stably secreting MAb against IBRV gD protein was screened and named as 3 C1. The heavy chain of the MAb was of IgG1 subclass while the light chain was kappa chain. The titers of MAb in supernatant and ascite were 1 ∶ 40 and 1 ∶ 12 800 respectively. The MAb showed specific reaction bands,with relative molecular masses of 71 000 and 96 000,with IBRV and gD protein,respectively,as well as the specific binding to the MDBK cells infected with IBRV. Conclusion The MAb against IBRV gD protein was prepared successfully,which provided data for further study on the function of gD protein and clinical diagnosis of IBR.
Objective To prepare the monoclonal antibody(MAb)against gD protein of infectious bovine rhinotracheities virus(IBRV)and identify its biological characteristics. Methods BALB/c mice were immunized with the complete IBRV purified by ultracentrifugation. The spleen cells of immunized mice positive for serum antibody were fused with myeloma SP2/0 cells by using PEG. Hybridoma cells were screened by indirect immunofluorescence(IFA),with which the ascites was prepared,and the biological characteristics of MAb was analyzed. Results A hybridoma cell line stably secreting MAb against IBRV gD protein was screened and named as 3 C1. The heavy chain of the MAb was of IgG1 subclass while the light chain was kappa chain. The titers of MAb in supernatant and ascite were 1 ∶ 40 and 1 ∶ 12 800 respectively. The MAb showed specific reaction bands,with relative molecular masses of 71 000 and 96 000,with IBRV and gD protein,respectively,as well as the specific binding to the MDBK cells infected with IBRV. Conclusion The MAb against IBRV gD protein was prepared successfully,which provided data for further study on the function of gD protein and clinical diagnosis of IBR.
引文
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[9] HUANG H,WU X,CAO L,et al. Preparation and identify cation of monoclonal antibody against C1q/TNF-related protein4[J]. Monoclon Antib Immunodiagn Immunother,2016,35(6):280-284.
[10] CHEN T. Prokaryotic expression of gD gene of bovine infectious rhinotracheitis virus and establishment of indirect ELISA assay[D]. Yangling:Northwest University of Agriculture and Forestry University,2012.(in Chinese)陈婷.牛传染性鼻气管炎病毒gD基因原核表达及间接ELISA检测方法的建立[D].陕西杨凌:西北农林科技大学,2012.
[11] ZHOU Y H. Identification of an antigen epitope on the glycoprotein D with monoclonal antibody and establishment of a double antibody sandwich ELISA for detection for infectious bovine rhinotracheitis virus[D]. Beijing:Chinese Academy of Agricultural Sciences,2015.(in Chinese)周跃辉.牛传染性鼻气管炎病毒糖蛋白gD单抗制备及其抗原表位鉴定与双抗夹心ELISA的建立[D].北京:中国农业科学院,2015.
[12] WANG H Y. Establishment of IBR indirect ELISA and development of recombinant gE glycoprotein and monoclonal antibody[D]. Changchun:Jilin University,2006.(in Chinese)王海燕. IBR间接ELISA的建立及重组gE糖蛋白与单抗的研制[D].吉林长春:吉林大学,2006.
[13] LI Y,VAN DRUNEN LITTLE-VAN DEN HURK B,LIANG L A. Characterization of cell-binding properties of bovine herpes virus 1 glycoproteins B,C,and D:identification of a dual cell-binding function of gB[J]. J Virol,1995,69:4758-4768.
[14] HUTCHINGS D L,VAN DRUNEN LITTLE-VAN DEN HURK S,BABIUK L A. Lymphocyte proliferative responses to separated bovine herpesvirus 1 proteins in immune cattle[J]. J Virol,1990,64(10):5114-5122.
[2] MAYFIELD J E,GOOD P J,VANOORT H J,et al. Cloning and cleavage site mapping of DNA from bovine herpes virus 1(Cooper strain)[J]. J Virol,1983,47:259-264.
[3] LEMAIRE M,SEHYNTS F,MEYER Q,et al.. Latency and reactivation of a glycoprotein E negative bovine herpes virus type-1 vaccine:influence of virus load and effect of specific maternal antibodies[J]. Vaccine,2001,19:4795-4804.
[4] JONES C. Herpes simplex vitrus type 1 and bovine herpes virus1 latency[J]. Clin Microbiol Rev,2003,16:79-95.
[5] WU S Y,LU Z,DING Y J,et al. Prevention and treatment of bovine infectious nasal bronchitis in Henan Province[J]. Henan Husbandry and Animal Husbandry,2004,25:35-36.(in Chinese)吴胜耀,鲁哲,丁亚军,等.河南省首例牛传染性鼻气管炎防治体会[J].河南畜牧兽医,2004,25:35-36.
[6] BI Y,WEN X B,NI H B. Preparation and identification of monoclonal antibody against gD protein of bovine infectious rhinotracheitis virus[J]. Chin J Biologicals,2017,30(10):1050-1054.(in Chinese)毕莹,闻晓波,倪宏波.牛传染性鼻气管炎病毒gD蛋白单克隆抗体的制备及鉴定[J].中国生物制品学杂志,2017,30(10):1050-1054.
[7] ZHAO W,BI Y,WEN X B,et al. Preparation and identification of monoclonal antibody against bovine infectious rhino-tracheitis virus gE protein[J]. Chin J Biologicals,2018,31(2):155-159.(in Chinese)赵微,毕莹,闻晓波,等.牛传染性鼻气管炎病毒gE蛋白单克隆抗体的制备及鉴定[J].中国生物制品学杂志,2018,31(2):155-159.
[8] HOU Q,PENG W P,SUN Y,et al. Prokaryotic expression of the coding region of the major antigenic region of classical swine fever virus E2 protein and preparation of its monoclonal antibody[J]. Chin Veter Sci,2008,38(1):1-5.(in Chinese)侯强,彭伍平,孙元,等.猪瘟病毒E2蛋白主要抗原区编码基因的原核表达及其单克隆抗体的制备[J].中国兽医科学,2008,38(1):1-5.
[9] HUANG H,WU X,CAO L,et al. Preparation and identify cation of monoclonal antibody against C1q/TNF-related protein4[J]. Monoclon Antib Immunodiagn Immunother,2016,35(6):280-284.
[10] CHEN T. Prokaryotic expression of gD gene of bovine infectious rhinotracheitis virus and establishment of indirect ELISA assay[D]. Yangling:Northwest University of Agriculture and Forestry University,2012.(in Chinese)陈婷.牛传染性鼻气管炎病毒gD基因原核表达及间接ELISA检测方法的建立[D].陕西杨凌:西北农林科技大学,2012.
[11] ZHOU Y H. Identification of an antigen epitope on the glycoprotein D with monoclonal antibody and establishment of a double antibody sandwich ELISA for detection for infectious bovine rhinotracheitis virus[D]. Beijing:Chinese Academy of Agricultural Sciences,2015.(in Chinese)周跃辉.牛传染性鼻气管炎病毒糖蛋白gD单抗制备及其抗原表位鉴定与双抗夹心ELISA的建立[D].北京:中国农业科学院,2015.
[12] WANG H Y. Establishment of IBR indirect ELISA and development of recombinant gE glycoprotein and monoclonal antibody[D]. Changchun:Jilin University,2006.(in Chinese)王海燕. IBR间接ELISA的建立及重组gE糖蛋白与单抗的研制[D].吉林长春:吉林大学,2006.
[13] LI Y,VAN DRUNEN LITTLE-VAN DEN HURK B,LIANG L A. Characterization of cell-binding properties of bovine herpes virus 1 glycoproteins B,C,and D:identification of a dual cell-binding function of gB[J]. J Virol,1995,69:4758-4768.
[14] HUTCHINGS D L,VAN DRUNEN LITTLE-VAN DEN HURK S,BABIUK L A. Lymphocyte proliferative responses to separated bovine herpesvirus 1 proteins in immune cattle[J]. J Virol,1990,64(10):5114-5122.