|摘要: ||雞傳染性華氏囊病(infectious bursal disease, IBD)為一具有高度傳染性並在年幼雞隻造成死亡及免疫抑制的疾病，主要由雞傳染性華氏囊病病毒(infectious bursal disease virus, IBDV)所引起，而VP2為IBDV之結構蛋白並能夠誘發宿主產生中和性抗體。Pichia pastoris為真核表現系統，能高度表現外源性蛋白。本實驗以真核表現系統Pichia pastoris表現IBDV VP2重組蛋白(Y-VP2)當作抗原應用於IBD疫苗及ELISA檢測之研發。在ELISA檢測方面，本實驗檢測141隻田間雞隻血清，並以商品化之IDEXX IBD ELISA檢測結果當作黃金標準，自製之Y-VP2-ELISA主要分為間接型(indirect) ELISA、三明治型(sandwich) ELISA、阻斷型(blocking) ELISA三種，其中間接型 ELISA是以經由蔗糖密度梯度離心方法純化後之Y-VP2蛋白當作抗原；而三明治型與阻斷型ELISA則以未純化的Y-VP2蛋白當作抗原，並配合市售Anti-VP2單株抗體之使用。本實驗選擇兩種截切點(cut-off)判定方式，分別為傳統上以無特定病原之雞隻血清平均值加上標準差當作截切點，和以Two-graph receiver operating characteristic (TG-ROC)公式計算出之截切點，並比較其ELISA之結果。結果顯示Y-VP2 indirect ELISA以血清讀值平均值加上三個標準差當做截切點時，其敏感性、特異性及準確率分別為95.6%、85.7%及92.2%；而以TG-ROC判定截切點時其敏感性、特異性及準確率分別為90.27%、90.1%及90.32%，且ROC曲線下面積 (area under the ROC curve; AUC) 達0.96表示其為判別力良好的檢測方式，其中Y-VP2 indirect ELSIA比起其他兩種ELISA具有較高之敏感性、特異性、準確率。然而在疫苗保護力之探討，發現於施打Y-VP2重組蛋白之動物實驗中，對於雞隻並無法產生有效之保護力。綜合以上結果，實驗中以酵母菌系統表現之VP2蛋白，並無法引起具保護力之免疫反應，但Y-VP2 indirect ELISA經實驗證明其仍然不失為一種有效且成本低廉、操作簡便之血清檢測方法，可應用於未來IBD的診斷。|
Infectious bursal disease (IBD) is a highly contagious, immunosuppressive disease of young chickens caused by infectious bursal disease virus (IBDV). The VP2 of IBDV is a structural protein and relates to the host-protective immune response. The Pichia pastoris is a methylotrophic yeast, which can express heterologous proteins with high efficiency. The objectives of this study were to develop a subunit vaccine against IBD and ELISAs for detecting IBDV infection using IBDV VP2 expressed by Pichia pastoris as antigen. The In-house Y-VP2-ELISAs including indirect ELISA, sandwich ELISA, and blocking ELISA were developed and compared with commercial IDEXX IBD ELISA. Total 141 chicken sera from field were used for the ELISA evaluations and the commercial IDEXX ELISA was used as a gold standard method. Amoung these three ELISAs, the best results were obtained by Y-VP2 indirect ELISA. When the cut-off value was set on that mean plus three standard deviations, the sensitivity, specificity and accuracy of the Y-VP2 indirect ELISA were 95.6%, 85.7% and 92.2%, respectively. When the cut-off value was determined by the two-graph receiver operation characteristics (TG-ROC), the sensitivity, specificity, and accuracy were 90.27%, 90.1% and 90.32%, respectively. Moreover, the area under the ROC curve (AUC) of the Y-VP2 indirect ELISA was 0.96. Although the results of animal study showed that the yeast expressed VP2 protein couldn’t induce protective immune response against IBD, the results of Y-VP2-ELISA indicated that the Y-VP2 indirect ELISA is an effective method for the detection of IBDV infection.