血清4型禽腺病毒Fiber蛋白在杆状病毒中的表达及其在抗体检测中的应用

为建立检测血清4型禽腺病毒（FAdV-4）抗体快捷特异方法，本研究首先分别构建含FAdV-4纤突蛋白Fiber-1和Fiber-2的两个重组杆状病毒rBv-Fiber-1和rBv-Fiber-2。在利用IFA以及Western blot鉴定重组杆状病毒分别高效表达Fiber-1和Fiber-2蛋白的基础上，以Ni柱纯化蛋白，并作为特异性识别FAdV-4抗体的包被抗原构建间接ELISA方法。特异性试验表明，间接ELISA仅特异地检出FAdV-4阳性血清，而不与Ⅰ群其他血清型禽腺病毒及其他禽源病毒阳性血清反应。间接ELISA检测灵敏度高于常规IFA方法，批间和批内重复性变异系数均小于10%。临床血清检测结果表明，间接ELISA可有效检出感染FAdV-4或免疫FAdV-4灭活疫苗的鸡群中抗Fiber抗体，且检测结果与BioChek商品化ELISA试剂盒一致。结果表明，本研究利用杆状病毒系统表达的Fiber蛋白及基于表达产物所构建的间接ELISA方法在FAdV-4感染预警和免疫评估有良好的应用前景

Expression of Fiber Protein of Serotype 4 Fowl Adenovirus in Baculovirus and Its Application in Antibody Detection

In order to develop rapid and specific approach for antibody detection of serotype 4 fowl adenovirus (FAdV-4), two recombinant baculovirus containing Fiber-1 or Fiber-2 gene of FAdV-4, named as rBv-Fiber-1 and rBv-Fiber-2, was first constructed respectively. IFA and Western blot identification revealed that rBv-Fiber-1 and rBv-Fiber-2 could express the corresponding protein efficiently. The recombinant protein was then purified by Ni and used as FAdV-4-Specific coating antigen to develop Fiber-1 or Fiber-2 based indirect ELISA, respectively. Specificity analysis showed that the ELISA only reacted with the serum against FAdV-4, not with the serum against other avian pathogens or other serotypes of FAdV tested. Sensitivity analysis demonstrated that the ELISA was more sensitive than IFA. The inter-batch and intra-batch assay coefficient of variation (CV) of the ELISA were both lower than 10%. The ELISA had good compatibility with Biochek commercial ELISA in detecting the antibodies in chickens vaccinated with an inactivated FAdV-4 vaccine or infected with FAdV-4. The data demonstrate that the Fiber protein of FAdV-4 expressed by baculovirus and the Fiber based indirect ELISA for FAdV-4 antibody detection have fine application prospect in monitoring the immunological state of the vaccination and diagnosing the clinical infection of FAdV-4.