- 學位論文
發展典型豬瘟E2結構蛋白之ELISA檢測試劑
Development of E2 Protein ELISA Kit for Monitoring Classical Swine Fever Antibody
摘要
豬瘟（classical swine fever, CSF）是一種高度傳染性與高死亡率之疾病，其病原為豬瘟病毒（classical swine fever virus, CSFV），造成養豬業者重大之經濟威脅，被世界動物衛生組織列為需要通報的疾病之一。豬瘟是屬於黃病毒科中的瘟病毒屬的單鏈核糖核酸病毒。豬瘟病毒基因組長度約為12.3 kb，其外層封套具有三種結構醣蛋白，分別為Erns、E1及E2，而其中Erns和E2已被證實可誘發宿主產生中和抗體。本實驗之目的為利用蛋白表現系統大量製備豬瘟病毒E2蛋白片段，進行抗原性分析並製作CSF檢驗試劑。桿狀病毒表現系統所表現的重組E2醣基化蛋白大小約為55kDa，藉由鎳離子純化管住純化蛋白，並利用抗His血清及抗E2抗體進行西方點墨法，以確認E2重組蛋白抗原性。之後把純化的蛋白塗覆在ELISA盤，以測試最有效檢測抗CSFV抗體的條件。另外本研究中的E2 ELISA Kit的有效性也以市售CSFV ELISA Kit檢測結果相比教，以更進一步確認對豬隻血清樣本的敏感性與特異性。目前E2重組蛋白ELISA Kit成功的分辨出接種疫苗後陰性以及陽性豬血清樣品的抗E2抗體的力價 。未來希望能藉由開發的檢測試劑套組快速檢測受感染的豬隻，以達到防止病毒傳播，監控疾病的目標。
並列摘要
Classical swine fever（CSF）, caused by classical swine fever virus（CSFV）, is a highly contagious fatal disease in pigs, and leads to severe economic losses worldwide. CSFV belongs to the genus Pestivirus within the family Flaviviridae and contains three structural glycoproteins Erns , E1, and E2. The CSFV glycoprotein E2 have been shown to be capable of inducing neutralizing antibodies in host. The purpose of this study is using CSFV E2 protein as an antigen to make an ELISA plate and then to detect the anti-CSFV antibody in the pigs. We have construct the recombinant Bac-E2 baculovirus to efficiently express the E2 protein in Sf-9 cells. The 55-kDa E2 glycosylated proteins were identified by anti-His and anti-E2 western-blot. Then the E2 protein was purified by Ni2+ affinity column with high purity and efficiency. The purified proteins were coating on the ELISA plate with specific conditions for efficiently detecting the anti-CSFV antibody. The effectiveness of the E2 ELISA Kit was analyzed by using the serum specimens of out-bred farm sows that was rested by the commercial CSFV kits. The resulting E2 recombinant proteins ELISA was successfully used to measure anti-E2 antibody titers in vaccinated and field pig sera samples. We hope this rapid detection kit of infected pig is potentially used for preventing the spread of virus and control the disease in the future.