| 检测鸭呼肠孤病毒抗体间接ELISA方法的建立 |
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| 引用本文： | 孙陈莉,杨瑞,王义敏,赵娜,张小荣,彭大新,吴艳涛.检测鸭呼肠孤病毒抗体间接ELISA方法的建立[J].兽医大学学报,2014(1):26-29,38. |
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| 作者姓名： | 孙陈莉 杨瑞 王义敏 赵娜 张小荣 彭大新 吴艳涛 |
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| 作者单位： | 扬州大学农业部畜禽传染病学重点开放实验室,江苏扬州225009 |
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| 基金项目： | 公益性行业（农业）专项（201003012） |
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| 摘 要： | RT—PCR扩增鸭呼肠孤病毒（Duckreovirus，DRV）的dB基因，克隆到pET-32a（＋）表达载体，再转化大肠杆菌Transetta（DE3）；含有重组质粒pET—σB的大肠杆菌经IPTG诱导，获得大小为55000的以包涵体形式表达的σB重组蛋白。Westernblotting显示，σB重组蛋白能够与兔抗DRV多抗血清特异性结合。以高亲和NI—NTA树脂在变性条件下纯化、梯度尿素复性的σB重组蛋白为包被抗原，建立了检测DRV抗体的间接ELSIA方法。分别以间接ELSIA方法和血清中和试验对DRV感染鸭血清和SPF鸭血清进行检测，两者的符合率为100％。该间接ELSIA方法对鸭瘟病毒、鸭病毒性肝炎病毒和禽流感病毒阳性血清均无交叉反应。
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| 关 键 词： | 鸡呼肠孤病毒 σB重组蛋白 原核表达 间接ELISA |
Establishment of an indirect ELISA for detecting antibodies against duck reovirus |
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| Institution: | SUN Chen-li, YANG Rui, WANG Yi-min, ZHAO Na, ZHANG Xiao-rong, PENG Da-xin, WU Yan- tao (Key Laboratory of Animal Infectious Diseases of Ministry of Agriculture ,Yangzhou Universit y , Yangzhou , J iangsu 225009, China) |
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| Abstract: | The aB gene of duck reovirus （DRV） was amplified by RT-PCR and cloned into pro- karyotic expression vector pET-32a （＋）, and then the aB protein was expressed in Transetta （DE3） with IPTG induction. Western-blotting analysis showed that the 55 000 recombinant σB in the form of inclusion body could be recognized by polyclonal antibodies of rabbit against DRV. The recombinant σB was purified with high-affinity Ni-NTA resin under denaturing and renatured by gradient urea. By using the purified σB protein, the indirect ELISA assay for the detection of DRV antibodies in duck serum was applied after optimized the working conditions. By detecting serums from infected ducks or specific pathogen-free duck, the agreement ratio reached at 100% between the indirect ELISA and neutralization test. The indirect ELISA showed no cross reaction with the positive sera of duck plague virus,duck virus hepatitis and avian influenza virus. |
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| Keywords: | duck reovirus recombination protein σB prokaryotic expression indirect ELISA |
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