Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98199
標題: 桿狀病毒系統表現豬生殖道與呼吸道綜合症病毒N蛋白在ELISA之應用
Development of ELISA coated with PRRS virus N protein expressed by baculovirus expression system
作者: 沈昌鋒
Chang-Feng Shen
關鍵字: 豬生殖道與呼吸道綜合症病毒;ELISA;桿狀病毒表現系統;ORF7;Porcine reproductive and respiratory syndrome virus;ELISA;Baculovirus expression system;ORF7
引用: 黃儀婷 (2015)。 豬生殖與呼吸綜合症抗體檢測試劑之研發與應用。The Development of ELISA for the Detection of the Antibody against Porcine Reproductive and Respiratory Syndrome Virus. 碩士論文,國立中興大學微生物暨公共衛生學研究所,台中市 Albina, E., 1997. Epidemiology of porcine reproductive and respiratory syndrome (PRRS): an overview. Vet. Microbiol. 55, 309-316. Allende, R., Lewis, T.L., Lu, Z., Rock, D.L., Kutish, G.F., Ali, A., Doster, A.R., Osorio, F.A., 1999. North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions. J. Gen. Virol. 80, 307-315. Benfield, D.A., Nelson, E., Collins, J.E., Harris, L., Goyal, S.M., Robison, D., Christianson, W.T., Morrison, R.B., Gorcyca, D., Chladek, D., 1992. Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332). J. Vet. Diagn. Invest. 4, 127-133. Bierk, M.D., Dee SA, Rossow, K.D., Otake, S., Collins, J.E., Molitor T.W., 2001. 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摘要: 
豬生殖道與呼吸道綜合症 (Porcine Reproductive and Respiratory Syndrome, PRRS)是由豬生殖道與呼吸道綜合症病毒 (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV)感染所引起的,此疾病對於養豬業者造成嚴重的經濟損失。N (nucleocapsid)蛋白為PRRSV的結構蛋白,佔病毒結構蛋白的40%,且N蛋白上富含B細胞及T細胞的抗原決定位,能在病毒感染早期時,產生大量的抗N蛋白之抗體。因此抗N蛋白之抗體經常被作為PRRSV感染之檢測標的。桿狀病毒表現系統,為真核生物表現系統,具有與動物細胞相似之轉譯後修飾作用。因此生產出來的蛋白,與原核表現系統相比具有較正確的蛋白質構型及較高的生物活性,常被應用於真核生物蛋白的表達。本實驗的目的是以昆蟲桿狀病毒表現系統生產PRRSV的N蛋白作為抗原,研發檢測N蛋白抗體之ELISA檢測套組。構築含有N蛋白基因之重組桿狀病毒,使其感染SF9細胞,表現之N蛋白經免疫螢光染色及西方墨點法確認且分子量大小為14.5 kDa。桿狀病毒與大腸桿菌(實驗室先前已構築)產生之重組N蛋白,經金屬螯合親和層析膠體純化後,作為研發之ELISA套組的抗原。利用商品化之PRRS ELISA套組 (IDEXX PRRS X3 Ab Test) 所測得的已知抗體力價之血清作為標準血清,建立與評估間接型與三明治型及阻斷型之ELISA套組。結果指出,桿狀病毒與大腸桿菌表現之N 蛋白間接型ELISA,其ELISA的敏感性與專一性及準確度皆有90 %以上,且ROC曲線下面積皆大於0.9,代表具有良好判別力。而桿狀病毒表現之N蛋白三明治型與阻斷型ELISA,經評估後發現,此兩種ELISA皆無法有效的區分出陰性血清與陽性血清。將建立之桿狀病毒與大腸桿菌表現之N 蛋白間接型ELISA檢測大量血清樣本,其ELISA的敏感性與專一性及準確度皆依然可達90 %以上。結果顯示,桿狀病毒與大腸桿菌表現系統所生產之重組N蛋白可應用於間接型ELISA上,且具有良好之檢測效率。
URI: http://hdl.handle.net/11455/98199
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