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研究生:王佑君
研究生(外文):Yu-Chun Wang
論文名稱:利用新的途徑擴大對登革病毒中和能力
論文名稱(外文):A novel approach to broaden antibody neutralizing activity against dengue viruses
指導教授:趙黛瑜趙黛瑜引用關係
指導教授(外文):Day-Yu Chao
口試委員:劉俊宏吳尚蓉
口試委員(外文):Shang-Rung Wu
口試日期:2016-07-06
學位類別:碩士
校院名稱:國立中興大學
系所名稱:微生物暨公共衛生學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:44
中文關鍵詞:登革病毒中和能力類病毒顆粒
外文關鍵詞:dengue virusneutralizevirus like particle
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登革熱是一種新興起的流行疾病並且威脅著全球人民的健康,因此急需發展有效對抗四種血清病毒的疫苗。登革病毒的特色是釋放到細胞外的過程中會產生不同成熟度的病毒顆粒。過去的血清學研究表示,登革熱患者的血液中擁有來自具有對不同血清交叉反應及中和效力差的辨識前體膜蛋白抗體,以及來自外套膜蛋區域一、二辨識fusion loop的抗體。在這項研究中,嘗試使用含有不同前體膜蛋白含量的登革病毒二型類病毒顆粒,評估免疫小鼠後產生血清中的抗體反應。免疫五組不同前體膜蛋白含量類病毒顆粒及一組在WT上突變2H2抗原決定位類病毒顆粒的DNA載體,結果顯示分泌程度與前體膜蛋白含量會影響後續的免疫反應。為了避免分泌程度與前體膜蛋白含量產生的影響,使用等量的登革二型類病毒顆粒並免疫小鼠,檢測針對登革二型的抗體總量、中和50%病毒能力、前體膜蛋白抗體和fusion loop抗體。我們的數據顯示,切除最多前體膜蛋白含量的登革二型類病毒顆粒產生最少的辨識前體膜蛋白和辨識fusion loop的抗體,分別為7.2%和幾乎為0,並且對四型登革病毒都有很高的中和能力。結果顯示,修飾furin切割位置是拓展抗體反應的新方法。

Dengue virus is an emerging global health threat and development of dengue vaccine requires the induction of neutralizing antibodies against four serotypes. Dengue virus presents as a heterogeneity in maturity of virion particles. Previous studies suggested that dengue-infected patient serum contained cross-reactive and poorly neutralizing antibody recognizing prM and fusion loop (fl) on domain I/II of envelop (E) protein. In this study, we attempted to construct dengue virus serotype 2 virus-like particles (D2VLP) with different proportion of pr peptide cleavage and assess the antibody response after immunization in mice. Both DNA and VLP immunization were performed. After three doses DNA vector (five groups different prM proportion, one group WT construction mutation 2H2 epitope) of intra-muscular injection, the result suggested that both secretion level and proportion of prM might affect immunogenicity of DNA vaccine and hence antibody titer.In order to compare different proportions of prM on the effect of immunigenicity, purified D2VLP with the equal concentration were used to immunize the mice by two doses of intra-muscular injection, the sera of individual mice were subjected to measure total dengue-specific IgG antibody, neutralizing antibody titers and epitope-specific antibody. Our data suggested that D2VLP with maximum pr-cleavage induced lower anti prM and anti-fl antibody proportion (7.2% and 0, respectively) and the highest neutralizing activity against all four serotypes of dengue viruses. Out study concluded that modulating furin cleavage site is a novel approach to broaden antibody response.

第一章、文獻回顧 1
(一)登革病毒簡介 1
(二)登革病毒結構 1
(三)登革病毒生活史 2
(四)抗體依賴性增強作用antibody-dependent enhancement (ADE) 2
(五)血清學研究 3
(六)類病毒顆粒(Virus-like particles, VLPs) 3
(七)各種不同前體膜蛋白含量類病毒顆粒及分泌程度 4
(八)實驗假說 4
第二章、材料與方法 5
(一)DNA質體 5
(二)類病毒顆粒 6
(三)免疫老鼠 8
(四)抗原抓取酵素結合免疫吸附分析法(antigen capture ELISA) 8
(五)病毒斑減少中和試驗(Plaque reduction neutralization test) 9
(六)單株抗體155-49競爭 10
(七)辨識fusion peptide抗體比例 11
(八)區域三抗體檢測 12
第三章、結果 13
(一)DNA質體免疫 13
(二)類病毒顆粒免疫 13
(三)病毒斑減少中和試驗 14
(四)辨識前體膜蛋白抗體比例 15
(五)辨識fusion peptide抗體比例 15
(六)區域三抗體 16
第四章、討論 17
第五章、參考文獻 20
第六章、附錄 40
(一)製備DNA質體試劑 40
(二)製備類病毒顆粒試劑 41
(三)抗原抓取酵素結合免疫吸附分析法試劑 42
(四)病毒斑減少中和試驗(Plaque reduction neutralization test) 43
(五)單株抗體155-49競爭 44


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