Department of Virology, University of Istanbul, Veterinary Faculty, 34320, Istanbul, Turkey.
Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State, University, Manhattan, KS, USA.
Appl Biochem Biotechnol. 2019 Feb;187(2):506-517. doi: 10.1007/s12010-018-2815-2. Epub 2018 Jul 9.
The avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is recognized as an important avian pathogen, and new viral variants are a continuous threat to the poultry industry worldwide. Sensitive diagnostics and efficacious vaccines are necessary to combat IBV infections in chickens. The aim of this study was to produce recombinant N protein of IBV in the baculovirus system to use in ELISA diagnostic tests in order to enable the assessment of the sero-prevalence and risk of IBV infections in chickens in Turkey. For this, the gene encoding the N protein of the Beaudette strain of IBV was expressed using a recombinant baculovirus expression system. The recombinant N protein was purified using Ni-NTA affinity chromatography. An estimated 50-kDa recombinant protein corresponding to the expected molecular weight of IBV N including the 6xHis tag was detected using an anti-His monoclonal antibody. Specific immunoreactivity of the recombinant protein was confirmed by Western blot using antiserum obtained from vaccinated and naturally infected chicken from Turkey as well as using a monoclonal antibody raised against the N protein of the IBV Massachusetts strain. The results obtained with the in-house ELISA had high agreement with a commercial ELISA. Immunoreactivity analysis using antisera in Western blotting and the in-house ELISA suggests that the recombinant IBV N protein could be broadly cross-reactive with antisera produced against different IBV strains. We conclude that the recombinant baculovirus expressed IBV N protein could serve as a useful diagnostic antigen for detection of IBV infections in chickens by ELISA.
禽冠状病毒-传染性支气管炎病毒（AvCoV-IBV）被认为是一种重要的禽类病原体，新的病毒变体对全球家禽业构成持续威胁。为了在鸡中对抗 IBV 感染，需要敏感的诊断方法和有效的疫苗。本研究旨在使用杆状病毒系统生产 IBV 的重组 N 蛋白，用于 ELISA 诊断检测，以便评估 IBV 感染在土耳其鸡中的血清流行率和风险。为此，使用重组杆状病毒表达系统表达了 IBV Beaudette 株的 N 蛋白基因。使用 Ni-NTA 亲和层析法纯化重组 N 蛋白。使用抗 His 单克隆抗体检测到与预期的 IBV N 蛋白分子量（包括 6xHis 标签）相对应的约 50kDa 的重组蛋白。使用来自土耳其接种和自然感染鸡的抗血清以及针对 IBV 马萨诸塞株 N 蛋白的单克隆抗体通过 Western blot 确认了重组蛋白的特异性免疫反应性。内部 ELISA 获得的结果与商业 ELISA 高度一致。Western blot 中使用抗血清进行的免疫反应性分析和内部 ELISA 表明，重组 IBV N 蛋白可能与针对不同 IBV 株产生的抗血清广泛交叉反应。我们得出结论，重组杆状病毒表达的 IBV N 蛋白可用作 ELISA 检测鸡中 IBV 感染的有用诊断抗原。
