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National Chung Hsing University Institutional Repository - NCHUIR > 獸醫學院 > 獸醫病理生物學所 > 依資料類型分類 > 碩博士論文 >  豬瘟病毒Erns封套蛋白之選殖表現與酵素結合免疫吸附法之開發

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/155166

標題: 豬瘟病毒Erns封套蛋白之選殖表現與酵素結合免疫吸附法之開發
Development of enzyme-linked immunosorbent assay with baculovirus-expressed Erns glycoprotein of classical swine fever virus
作者: 王予雯
Wang, Yu-Wen
Contributors: 簡茂盛
獸醫病理生物學研究所
關鍵字: 豬瘟;Erns;ELISA;DIVA
classical swine fever;Erns;ELISA;DIVA
日期: 2012
Issue Date: 2013-11-21 11:42:50 (UTC+8)
Publisher: 獸醫病理生物學研究所
摘要: 豬瘟病毒 (classical swine fever virus) 屬於黃病毒科 (Flaviviridae) 瘟疫病毒屬 (Pestivirus),為豬隻重要法定傳染性病原之一,可導致產業嚴重之經濟損失。酵素連結免疫吸附法 (enzyme-linked immunosorbent assay, ELISA) 為目前現場普遍使用之抗體檢測方法,而針對豬瘟之 ELISA 診斷試劑可區分為檢測 E2 與 Erns 抗體兩種,前者可作為疫苗免疫效力與免疫適期之評估;而後者則可搭配使用 E2 次單位疫苗之豬群進行檢測,一旦豬隻受到野外毒感染時,可利用 Erns 抗體呈現與否,來區別免疫 E2 次單位疫苗或是被感染之豬群,以篩選出野外毒感染個體進行淘汰,而達到清淨之目的。因此本實驗嘗試將豬瘟病毒 LPC strain 之 Erns 序列轉殖入昆蟲桿狀病毒載體,並以懸浮性培養方式將轉殖病毒感染昆蟲細胞大量表現重組蛋白 (BacErns),再經濃縮與純化等步驟,最後以西方墨點法證實其抗原性,並具完整醣基化結構。於間接型 ELISA 的製備上,先以棋盤格 (checkerboard) 模式檢測 ELISA 最佳化條件,經以每孔以加入適當之抗原濃度塗鍍,再以血清稀釋樣本進行檢測,較可得到最精確與一致性之結果。進一步選取 E2 次單位疫苗免疫組、免疫攻毒組及對照組之 SPF 豬隻血清,與免疫 LPC 活毒疫苗之商用豬隻等不同血清樣本進行檢測,結果顯示若以桿狀病毒表現重組 Erns 塗鍍之間接型 ELISA,其敏感性為 85.6% (77/90),特異性為 91.3% (21/23)。此外,本試驗也另嘗試於檢測過程中加入 Erns 特異性單株抗體,並製備間接競爭型 ELISA來增加檢測時之敏感性與特異性。經由最佳化條件測試後,分別以適當濃度之重組蛋白塗鍍,再以適當濃度之單株抗體混合稀釋待測血清製備競爭反應,實驗結果顯示競爭型 ELISA 具備低成本與操作便利等特性,可應用於使用 E2 次單位疫苗免疫豬群,篩選出野外毒感染豬隻,以達到豬瘟清淨的目的。
Classical swine fever (CSF) is a highly contagious disease causing major losses in pig populations almost worldwide. Enzyme-linked immunosorbent assays (ELISA) is one of the most commonly used tests for detection of specific antibody titer in sera. ELISAs for the detection of antibodies against the viral E2 glycoprotein of CSFV are widely used for monitoring of immunized efficacy after vaccination and/or infection. Besides, Erns ELISA can be designated as a companion test to screen either CSFV infected or vaccinated pigs via immunization of E2 subunit marker vaccines in swine population. In this experiment, recombinant subunit Erns protein based on the genome of attenuated CSFV (LPC strain) was cloned and expressed with baculovirus expression system. The baculovirus-expressed Erns (BacErns) indicated to retain the authentic antigenicity of CSFV Erns with appropriate conformation and glycosylation after concentration and purification. In addition, a checkerbroad model of applying with 50 μL of coating antigen and detected sera were assessed to determine the optimum condition for further BacErns-based indirect ELISA. Positive and negative control sera, including samples from mock SPF pigs, SPF pigs immunized with LPC or E2 subunit vaccines, or immunized pigs and challenged with CSFV, were selected to evaluate the sensitivity and specificity. The result showed the sensitivity of 85.6% (77/90) and specificity of 91.3% (21/23) respectively in this established indirect ELISA diagnostic assay. Moreover, monoclonal antibody against yeast-expressed Erns was utilized to perform an indirect competitive ELISA to elevate the sensitivity and specificity for detection. After applying with 100 μL of monoclonal antibody competed with diluted detected sera on an optimal by adding 100 μL of coating BacErns showed the most significant effect in indirect competitive ELISA. The results indicated BacErns-based indirect competitive ELISA may provide a more accurate with low cost and easy manipulation system for massive screening to discriminate vaccinated or CSFV infected pigs in the field.
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