| 蓝舌病病毒重组VP7蛋白单克隆抗体制备及竞争ELISA检测方法的建立 |
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| 引用本文： | 耿宏伟,秦永丽,李俊平,杨涛,孙恩成,刘霓红,白安斌,徐青元,王凌凤,赵晶,覃绍敏,林俊,郭怡璠,吴东来.蓝舌病病毒重组VP7蛋白单克隆抗体制备及竞争ELISA检测方法的建立[J].中国预防兽医学报,2012(5):388-392. |
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| 作者姓名： | 耿宏伟 秦永丽 李俊平 杨涛 孙恩成 刘霓红 白安斌 徐青元 王凌凤 赵晶 覃绍敏 林俊 郭怡璠 吴东来 |
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| 作者单位： | 东北农业大学动物医学学院;中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部兽医公共卫生重点开放实验室;黑龙江省农业科学院;广西兽医研究所;广西大学 |
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| 基金项目： | 国家863项目(2011AA10A212);黑龙江省自然科学基金项目(ZJN-0602-01);中央级公益性科研院所基本科研业务费专项(ZGKJ201105) |
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| 摘 要： | 为了建立蓝舌病(BT)的血清学诊断方法,本研究利用原核表达的蓝舌病病毒(BTV)血清型12型VP7纯化蛋白免疫BALB/c小鼠,制备2株单克隆抗体(MAb),分别命名为BTV-2D10和BTV-4H7。IFA试验表明,2株MAb均能与BTV 24个血清型发生特异性反应,而与茨城病病毒(IBAV)、中山病病毒(CV)、赤羽病病毒(AKAV)、牛病毒性腹泻病毒(BVDV)、牛传染性鼻气管炎病毒(IBRV)、牛轮状病毒(BRV)、牛肠道病毒(BEV)、牛呼肠孤病毒(RV)及口蹄疫病毒(FMDV)无交叉反应,表明2株MAb均为BTV群特异性抗体。采用重组表达的VP7蛋白作为包被抗原建立的竞争ELISA方法证明,BTV-4H7 MAb对不同血清型BTV阳性血清具有良好的阻断效果,而对AKAV、IBAV、BRV和FMDV阳性血清无阻断作用。本研究建立的竞争ELISA方法与IDEXX公司的试剂盒检测包括65份已知背景血清和322份采自广西省的山羊血清样品,检测结果符合率分别达100%和98%。该竞争ELISA方法的建立为BTV抗体的监测提供了安全、快速、准确的技术手段。
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| 关 键 词： | 蓝舌病病毒 重组VP7蛋白 群特异性单克隆抗体 竞争ELISA |
Development of monoclone antibody competitive ELISA for detection of Bluetongue virus antibodies |
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| GENG Hong-wei,QIN Yong-li,LI Jun-ping,YANG Tao,SUN En-cheng,LIU Ni-hong,BAI An-bin,XU Qing-yuan,WANG Ling-feng,ZHAO Jing,QIN Shao-min,LIN Jun,GUO Yi-fan,WU Dong-lai.Development of monoclone antibody competitive ELISA for detection of Bluetongue virus antibodies[J].Chinese Journal of Preventive Veterinary Medicine,2012(5):388-392. |
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| Authors: | GENG Hong-wei QIN Yong-li LI Jun-ping YANG Tao SUN En-cheng LIU Ni-hong BAI An-bin XU Qing-yuan WANG Ling-feng ZHAO Jing QIN Shao-min LIN Jun GUO Yi-fan WU Dong-lai |
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| Institution: | 1,2(1.College of Animal Medicine,Northeast Agricultural University,Harbin 150030,China;2.Key Laboratory of Veterinary Public Health,Ministry of Agricultural,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,China;3.Heilongjiang Academy of Agricultural Sciences,Harbin 150086,China; 4.Guangxi Veterinary Research Institute,Nanning 530001,China;5.Guangxi University,Nanning 530004,China) |
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| Abstract: | To development ELISA method for detecting antibodies against bluetongue virus(BTV),two monoclonal antibodies(MAbs) against BTV12 VP7 protein were produced by fusing SP2/0 myeloma cells with spleen cells of BALB/c mice immunized with purified recombinant BTV12 VP7 protein and screened by ELISA coated by BTV12.The indirect immunofluorescence assay showed that both of the MAbs were reacted positive with 24 serotypes of BTV,but not reacted with other related viruses.These results indicated that the two MAbs were group-specific antibodies to BTV.Furthermore,the competitive ELISA with the MAbs was established and the MAb 4H7 had the group specific blocking effect.Clinical sample detectionss showed the method had 100% agreement with IDEXX kit to detect of 65 known background serum samples and 98% agreement with IDEXX kit to detect 322 serum samples from Guangxi.Therefore,the competitive ELISA assay established in this study provided a low-cost,effective,quick and accurate method for the detection of antibodies against BTV. |
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| Keywords: | bluetongue virus recombinant VP7 protein group specificity monoclonal antibody competitive ELISA |
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