以豬丁型冠狀病毒棘狀蛋白研發酵素免疫吸附套組

Abstract

豬丁型冠狀病毒（porcine deltacoronavirus; PDCoV）屬於冠狀病毒屬中的丁型冠狀病毒，受其感染之仔豬會出現下痢、嘔吐及脫水，甚至導致死亡，是造成台灣田間仔豬下痢的病原之一，目前尚無商業化的酵素結合免疫吸附分析法（enzyme-linked immunosorbent assay, ELISA）套組可使用於偵測抗體，因此，本研究將利用不同長度之棘狀蛋白（Spike, S）研發 ELISA 套組。除了應用於未來調查台灣田間豬丁型冠狀病毒流行率之外，亦可以被使用於評估疫苗效力，對於未來開發相關疫苗相當重要。本研究使用人類胚胎腎細胞 293（human embryonic kidney 293, HEK 293）表現 N 端蛋白域（N-terminal domain, NTD）、NTD 到 C 端蛋白域（C-terminal domain, CTD）、S1 及膜外全長 S 蛋白三聚體為 ELISA 套組的披覆抗原 （coating antigen）。我們成功建立了能夠穩定表現不同長度Ｓ蛋白的細胞株，並利用親和性樹脂進行純化，接著利用免疫細胞化學染色（immunocytochemistry; ICC）和西方墨點法進行確認，最後再將純化蛋白作為 ELISA 的披覆抗原。為了評估 ELISA 的敏感性（sensitivity）和特異性（specificity），本研究使用 408 個來自 26 個不同田間豬場之血清樣本，計算不同 ELISA 的 ROC 曲線（receiver operating characteristic curve; ROC curve），分別得出了各 ELISA 的臨界值（cut-off value）、敏感性和特異性。實驗結果顯示，相較於膜外全長 S 蛋白，以 NTD、NTD-CTD 或 S1 為披覆抗原的 ELISA 會有較佳的敏感度和特異性。

Porcine Deltacoronavirus (PDCoV) belongs to the genus Deltacoronavirus and the family Coronavirus, is one of the enteric pathogens occurring in Taiwanese pig farms. Piglets infected with PDCoV may suffer from diarrhea, vomiting, dehydration, and death. Since there is no available commercial enzyme-linked immunosorbent assay (ELISA) kit so far, the aim of this study is to develop a PDCoV spike (S) protein-based ELISA kit for not only conducting serological surveys for PDCoV prevalence in Taiwan in the future, but evaluating the efficacy of PDCoV S-based vaccines. In the present study, we successfully established human embryonic kidney cells 293 (HEK 293) cell lines stably expressing PDCoV S truncates, such as N-terminal domain (NTD), NTD to C-terminal domain (CTD), S1, and full-length ectodomain. After purification by affinity resins, the proteins were confirmed by performing immunohistochemistry (ICC) and western blot, and respectively used as coating antigen for different ELISAs. In order to evaluate the sensitivity and specificity of each ELISA, 408 pig sera from 26 different pig farms were used to calculate the receiver operating characteristic curve (ROC curve) in this study. Finally, we obtained the cut-off value, sensitivity, and specificity of each ELISA. Based on the results, NTD, NTD-CTD, and S1 served as a better coating antigen than full-length ectodomain.