【摘要】 为建立特异性和敏感性高的检测鸭坦布苏病毒感染的方法,采用原核表达系统对鸭坦布苏病毒(DTMUV)E基因进行了克隆与表达,SDS-PAGE电泳结果显示融合蛋白大小为54.3ku。Western blot结果表明,经亲和层析法纯化后的重组蛋白能与DTMUV阳性血清发生特异性反应。以纯化的重组E蛋白作为包被抗原,初步建立了检测E蛋白抗体的间接ELISA方法。对ELISA各种反应条件进行了优化,确定了最适工作条件。优化后确定的抗原最适包被浓度为7μg/mL,血清的最佳稀释度为1∶320,酶标抗体最适稀释度为1∶1 000。在优化条件下,阴性和阳性临界值判定标准为0.324 4。本研究建立的快速检测DT-MUV抗体的间接ELISA方法为该病的检测和流行病学调查提供了技术手段。更多还原

【Abstract】 The aim of this study is to establish a high specific and sensitive method for detecting duck tembusu virus.Envelop protein genes of duck tembusu virus were cloned and expressed.The molecular weight of E protein was 54.3 ku expressed in pET21a/BL21(DE3) system.Western-blot analysis showed that the purified fusion proteins could react with duck serum containing antibodies against DTMUV.An indirect ELISA was established for rapid detection of antibodies against DTMUV using the purified recombinant proteins as coating antigen.The reaction conditions were optimized,including 7 μg/mL coating antigen of the purified recombinant proteins,1∶320 dilution of testing serum and 1∶1 000 dilution of HRP conjugated anti-duck IgG with cut off-value of 0.324 4(OD450 nm).The results revealed the indirect ELISA could be used for laboratory diagnosis and serosurvey for DTMUV infection.更多还原