https://scholars.lib.ntu.edu.tw/handle/123456789/162627
標題: | 登革病毒中和性抗原決定位之研究及其疫苗研發(2/2) | 作者: | 吳漢忠 | 關鍵字: | 登革病毒;中和性單株抗體;噬 菌體顯現法;抗原決定位;登革疫苗;Dengue;Neutralizing monoclonal antibodies;Phage display;B-cell epitope;dengue vaccine | 公開日期: | 2002 | 出版社: | 臺北市:國立臺灣大學醫學院口腔生物科學研究所 | 摘要: | 本計畫主要是要研究登革病毒中和性 的B 細胞抗原決定位。經由ELISA, 和 immunoblotting 方法,我們確認具專一性的 第二型登革病毒單株抗體(3H5)。3H5 經由 中和力測試實驗,証明具有很高中和登革 病毒感染的能力。動物實驗測試,也發現 此抗體在100 倍LD50 的登革病毒的劑量 下,仍可以保護80﹪乳鼠的存活率。此中 和性抗體之抗原決定位的確認,有助於病 毒感染機制的暸解,疫苗及偵檢試劑的研 發。在此研究中,我們以噬菌體顯現法來 研究第二型登革病毒的中和性抗原決定 位。篩選出的噬菌體可以專一性的與第二 型登革病毒抗體3H5 結合。這些專一性噬 菌體所攜帶的外來peptide , 都含有 XXXXXXSELEXXW。此位置恰好位於第 二型登革病毒套膜蛋白的第51-56 amino acid。我們也更進一步証明篩選出的噬菌體 與3H5 具有很高的專一性。當我們用10 倍稀釋的噬菌體與3H5 反應,發現此噬菌 體與3H5 有專一性結合, 且呈現 dose-dependent 現象,而control phage 則完 全不反應。另外篩選出的噬菌體也証明只 與3H5 抗體結合,而不與其它四種單株抗 體、正常小白鼠血清及純化IgG 結合。中 和性抗原決定位的確認與研究,將有助於 epitope-based peptide vaccine 之研發,及了 解出血性登革之病理機制。 In this report, serotype-specific monoclonal antibody (MAb) against dengue virus type 2 (DEN-2), 3H5 (ATCC HB46), was identified by ELISA and immunoblotting analysis. 3H5 possess highly neutralizing activity against DEN-2 infection using plaque reduction neutralization test. This MAb also has 80% protection activity against 100-fold LD50 DEN-2 challenge. We also identified the neutralizing epitopes of this antibody using phage display method. Eighteen immunopositive phage clones that bound specifically to MAb 3H5 were selected. All of these phage-borne peptides had displayed a peptide sequence of XXXXXXSELEXXW that mimicked the sequence, which corresponded to the amino acid residues 51-56 of the envelope protein (E protein) of DEN-2. For further conformation of the selected phage clone bound 3H5 specifically, the antibodies were incubated with ten-fold serial dilution of phage clone. The result shown that only the selected phage clone bound the antibodies specifically and dose dependently. The selected phage clone only specific interacted with 3H5 and did not react with other four MAbs, normal mouse serum, and IgG. Furthermore, serotype-specific MAb of DEN-2, 3H5, bound its selected phage clone specifically, but had no effect on other five MAbs-selected phage clones. The identification of the epitopes of envelope protein of DEN will permit us to functionally dissect the antibody response and to address the role of anti-E protein antibodies either in the protection from or in the immunopathogenesis of DEN infection. Discovery this neutralizing epitope of DEN-2 may be useful for vaccine development in the future. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/22662 | 其他識別: | 902320B002207 | Rights: | 國立臺灣大學醫學院口腔生物科學研究所 |
顯示於: | 口腔生物科學研究所 |
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902320B002207.pdf | 237.12 kB | Adobe PDF | 檢視/開啟 |
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