| 鸭肠炎病毒NP蛋白的原核表达及间接ELISA方法的建立 |
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| 引用本文： | 李敏,陈彦希,代淑燕,刘忠伟,周碧君,文明.鸭肠炎病毒NP蛋白的原核表达及间接ELISA方法的建立[J].畜牧与兽医,2008,40(12). |
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| 作者姓名： | 李敏 陈彦希 代淑燕 刘忠伟 周碧君 文明 |
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| 作者单位： | 1. 贵州大学动物科学学院,贵州,贵阳,550025
2. 贵州大学生命科学学院,贵州,贵阳,550025 |
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| 摘 要： | 设计一对特异性引物扩增出鸭肠炎病毒(DEV)核衣壳蛋白(NP)基因,并将其定向插入到原核表达载体pET32a上,构建了NP基因的原核表达载体pET-NP;将重组载体pET-NP转化表达宿主菌BL21后,经SDS-PAGE分离后行Western blot显示,获得的表达产物具有良好的免疫原性;应用His.Bind亲和层析柱纯化重组NP蛋白,并以此作为包被抗原,初步建立了检测鸭肠炎病毒抗体的iNP-ELISA;经方阵滴定确定,重组蛋白抗原的最佳包被浓度为5.0μg/L,血清最佳稀释度为1∶80,阳性判定标准为:待检血清OD405值≥1.2,且待检血清OD405和阴性血清OD405的比值≥2.0;应用iNP-ELISA对450份鸭血清样本进行检测,结果iNP-ELISA与全病毒包被的iDEV-ELISA符合率达90.9%。
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| 关 键 词： | 鸭肠炎病毒 核衣壳蛋白 原核表达 间接ELISA |
Prokaryotic expression of nucleocapsid protein of duck enteritis virus and development of indirect ELISA |
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| LI Min,CHEN Yan-xi,DAI Shu-yan,LIU Zhong-wei,ZHOU Bi-jun,WEN Ming.Prokaryotic expression of nucleocapsid protein of duck enteritis virus and development of indirect ELISA[J].Animal Husbandry & Veterinary Medicine,2008,40(12). |
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| Authors: | LI Min CHEN Yan-xi DAI Shu-yan LIU Zhong-wei ZHOU Bi-jun WEN Ming |
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| Abstract: | A pair of primers was designed to amplify the nuleocapsid protein(NP)gene from duck enteritis virus(DEV)genome.The cloned fragment was digested with BamH I and Hind Ⅲ,and then inserted into pET32a vector to obtain the recombinant pET-NP plasmid.The recombinant plasmid was transformed into E.coli BL21,and expressed in a high level after induced with IPTG.The SDS-PAGE and Western blotting analysis of expressed product indicated that the fusion protein was about 48 ku in molecular weight and showed specific immunoreactivity with anti-DEV sera.The recombinant protein was purified with His.Bind resin protein purification procedure,and then iNP-ELISA was established using the purified protein as antigen.The optimal concentration of the coated antigen was 5.0 μg/mL and the optimal dilution of serum was 1∶80.The positive criterion of this ELISA assay was OD405≥1.2 in the tested serum and OD405(tested serum)/OD405(negative serum)≥2.0.Four hundreds and fifty serum samples from Guizhou province were detected by iNP-ELISA and iDEV-ELISA with DEV as the coating antigen,respectively,and the agreement ratio between the two methods was up to 90.9%. |
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| Keywords: | duck enteritis virus nuleocapsid protein prokaryotic expression indirect ELISA |
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