| 猪乙型脑炎病毒囊膜E蛋白间接ELISA诊断方法的建立与初步应用 |
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| 引用本文： | 张宁,金洪涛,丁壮,张瑞岩,刘雯,李勇,薛会亮,李丹,夏志平.猪乙型脑炎病毒囊膜E蛋白间接ELISA诊断方法的建立与初步应用[J].中国畜牧兽医,2010,37(7):170-175. |
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| 作者姓名： | 张宁 金洪涛 丁壮 张瑞岩 刘雯 李勇 薛会亮 李丹 夏志平 |
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| 作者单位： | （1.吉林大学畜牧兽医学院，长春 130062；2.中国人民解放军军事医学科学院军事兽医研究所，长春 130062） |
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| 基金项目： | 猪流行性乙型脑炎防控技术研究与示范 |
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| 摘 要： | 本研究利用纯化的原核表达乙型脑炎囊膜E蛋白作为包被抗原,建立了乙型脑炎间接ELISA诊断方法。对检测的各种条件进行了优化,优化反应条件后确定的抗原最适包被浓度为2μg/mL,抗原最佳包被条件为37℃包被2 h,血清的最适稀释度为1∶160,酶标抗体最适稀释度为1∶5000,最佳封闭条件为1%BSA,阴阳性临界值判定标准为D492 nm=0.254。该方法不与猪瘟、猪繁殖与呼吸综合征、猪圆环病毒2型、猪伪狂犬病毒阳性血清反应,其D492 nm0.254,说明该方法具有良好的特异性。采用该方法对150份疑似乙型脑炎血清样品进行检测,结果显示,与某猪乙型脑炎试剂盒相比符合率为90.77%,表明建立的间接ELISA方法具有较高的敏感性和特异性,因此,本研究成功建立了能特异性检测抗乙型脑炎血清抗体的ELISA检测方法。
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| 关 键 词： | 乙型脑炎病毒 E蛋白 间接ELISA |
Development of an Indirect ELISA for the Detection of Japanese Encephalitis Virus Using Envelope E Protein |
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| ZHANG Ning,JIN Hong-tao,DING Zhuang,ZHANG Rui-yan,LIU Wen,LI Yong,XUE Hui-liang,LI Dan,XIA Zhi-ping.Development of an Indirect ELISA for the Detection of Japanese Encephalitis Virus Using Envelope E Protein[J].China Animal Husbandry & Veterinary Medicine,2010,37(7):170-175. |
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| Authors: | ZHANG Ning JIN Hong-tao DING Zhuang ZHANG Rui-yan LIU Wen LI Yong XUE Hui-liang LI Dan XIA Zhi-ping |
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| Institution: | (1.College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China;2.Military Veterinary Institute, Academy of Military Medical Sciences of PLA, Changchun 130062, China) |
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| Abstract: | An indirect ELISA was established for the rapid detection of specific Japanese encephalitis virus (JEV) antibodies in swine using a recombinant E protein as an antigen. The recombinant E protein was expressed from recombinant plasmid pET28a-E in Escherichia coli. The various factors affected the experiments were optimized. This assay was optimized using 2 μg/mL E protein as coat antigen, the optimal coating condition of JEV for ELISA was incubated at 37 ℃ for 2 hours, 1∶160 dilution of testing serum and 1∶5000 dilution of HRP-labeled goat anti-swine IgG, the best blocking buffer was BAS with 1% consent ration. The critical value of positive and negative of ELISA was D492 nm=0.254, this method is not with the serums of CSFV, PRRSV, PCV2, PRV reaction, D492 nm<0.254, explain its specificity. Test against 150 serum samples from Jilin province by this assay detected 43.33% positive, which showed 90.77% agreement with ELISA kit test. Therefore, this study indicated that an indirect ELISA method for specifically detection JEV in serum was successfully established. |
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| Keywords: | Japanese encephalitis virus recombinant E protein indirect ELISA |
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