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以番鸭呼肠孤病毒σC表达蛋白为抗原的
ELISA检测方法的建立
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耿宏伟1，2，郭东春1，张云1，胡奇林3,刘明1,张序4，王立群2
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(1.中国农业科学院 哈尔滨兽医研究所 生物技术国家重点实验室,黑龙江 哈尔滨150001;
2.东北农业大学 生命科学院,黑龙江 哈尔滨150030； 3.福建省农业科学院 畜牧兽医研究所,
福建 福州350003； 4.哈尔滨市兽医卫生防疫站,黑龙江 哈尔滨150001)
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摘要：
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将鸭呼肠孤病毒小外壳σC蛋白编码基因克隆于原核表达载体pET32a上，经EcoRⅠ和SacⅠ双酶切鉴定及序列分析，获得了重组质粒pET32aσC，转化DH5α大肠埃希氏菌感受态细胞后，经SDS-PAGE和Western-blotting分析，融合蛋白能够与番鸭呼肠孤病毒感染康复鸭血清发生特异反应；以0.15mmol/L IPTG诱导，5h后融合蛋白σC表达量可达高峰，分子质量为50ku；融合蛋白纯化后被用作包被抗原，建立了检测鸭血清中呼肠孤病毒抗体的间接ELISA，经对检测条件优化，最佳包被浓度为5μg/mL，标准阳性血清的最适稀释倍数为1∶40。用该方法对50份鸭血清样品进行了检测，与琼脂扩散抗体检测法相比，ELISA具有良好的特异性和敏感性。 |
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关键词：
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番鸭呼肠孤病毒；σC编码基因；原核表达；酶联免疫吸附试验 |
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中图分类号：S 852.659.4:R 446.61 文献标识码：
A 文章编号：1673-4696(2006)03-0171-06 |
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Development of an indirect ELISA assay for the detection of
Muscovy duck reovirus using recombinant σC protein |
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GENG Hong-wei1,2, GUO
Dong-chun1, ZHANG Yun1, HU
Qi-lin3,
LIU Ming1, ZHANG Xu4，WANG
Li-qun2 |
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(1.National Key Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute, Chinese Academy of
Agricultural Sciences, Harbin 150001, China; 2.School of Life Science, Northeast Agricultural University, Harbin
150030，China; 3. Research Institute of Animal Science and Veterinary Medicine, Fujian Academy of Agricultural
Sciences, Fuzhou 350003, China; 4. Harbin Veterinary Station, Harbin 150001,China)
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Abstract: The gene encoding a minor core protein σC of Muscovy duck reovirus was cloned into a prokaryotic expression vector pET32a. The recombinant plasmid
pET32a-σC was amplified and extracted after being transformed into E.coli DH5α competent cells. Restriction analysis with EcoRⅠ+SacⅠ and sequence analysis indicated that the recombinant plasmid was inserted with a correct ORF. The fusion protein of approximately 50ku in size was produced in E.coli competent cells transformed with pET32a σC after induction with 0.15mmol/L of
IPTG. SDS-PAGE and Western blotting analyses showed that the fusion protein could react with the convalescent sera of duck infected with Muscovy duck reovirus. An indirect ELISA assay was developed by using the purified fusion σC protein as the coating antigen. The optimal concentration of σC was 5μg/mL and the dilution of serum sample was 1∶40. Examination of 50 serum samples from duck revealed that the ELISA assay was more sensitive and specific than the agar gel immunodiffusion(AGIP) test. |
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Key words: Muscovy duck reovirus; σC-encoding gene; prokaryotic expression; ELISA assay |
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