臺灣博碩士論文加值系統

登革病毒感染為一種每年威脅著亞熱帶及熱帶國家人民的健康問題，主要是經由攜帶登革病毒的病媒蚊叮咬而遭受感染，引發的疾病分成輕微的登革熱以及嚴重的登革出血熱/登革休克症候群，甚至有致死的危險。登革病毒在型態上可分成未成熟型與成熟型兩種，差異在於未成熟型登革病毒顆粒的表面結構具有前趨膜蛋白，而在病毒成熟過程中，特異性酵素會在酸性環境下切割前趨膜蛋白，使前趨胜肽從前趨膜蛋白上脫落而形成膜蛋白，此時的病毒顆粒則為成熟型病毒。在遭受登革病毒感染時若無及時接受治療，引發登革出血熱/登革休克症後群的致死率極高，所以早期的診斷及實驗室檢測則顯得非常的重要，以往的偵測方式主要分成兩個方向，一為偵測血液中登革病毒體RNA的基因組，二為偵測血液中專一性抗登革病毒IgM及IgG抗體，來確知病患有無遭受感染。但是現階段仍沒有好的方法可以瞭解病患體內的登革病毒是成熟型或未成熟型，以及不同型態的登革病毒對病患的影響。於是我們運用能辨識未成熟型登革病毒前趨膜蛋白的單株抗體，及結合具有磁性的磁珠載體，單獨分離出未成熟型的登革病毒，研究未成熟型登革病毒的生物特性。我們發現一般細胞株所繁殖出來的登革病毒大部分都是以未成熟型登革病毒為主，可以被抗前趨膜蛋白單株抗體抓住，而且具有感染力；而人體單核球細胞中繁殖出來的登革病毒則是以含套膜蛋白的成熟型登革病毒為主。我們更進一步利用單株抗體-磁珠去抓取臨床檢體中的登革病毒，發現臨床檢體含有未成熟及成熟型兩種登革病毒。

Dengue virus infection is an important infectious disease that affects many people in the subtropic and tropic country, which is an arthropod born virus transmitted by Ades egypti and Ades albopictus. The disease spectrum ranges from self-limited dengue fever (DF) to life-threatening dengue hemorrhagic fever or dengue shock syndrome (DHF/DSS). Dengue virus has two forms: immature and mature form, the difference being that the immature dengue virus contains the prM protein. During virus maturation, the furin cleavage of prM results in pr-peptide and M protein in low pH, causing the rearrangement of E to form the mature dengue virus. The diagnosis of dengue virus infection can be molecular or serological, detecting the viral RNA or detecting the host specific anti-dengue IgM and IgG antibody. However, there is no simple way to separate the immature dengue virus from mature form and study the characteristics of immature dengue virus. We generate monoclonal anti-prM Ab that can bind the prM of the dengue virus specifically. The anti-prM Ab was conjugated with magnetic beads and was used to isolate the immature form of dengue virus. We found that the anti-prM Ab-conjugated beads can capture the immature dengue virus propagated in different cell lines. The majority of dengue viruses derived from various cell lines are immature one except that from human primary monocytes. The antibody-conjugated beads were further used to capture dengue virus in the clinical samples, and found that the dengue patient sera contain mature dengue virus as well as immature one.

總目錄 …………………………………………………………………I
考試合格證明
中文摘要 ………………………………………………………………IV
英文摘要 ………………………………………………………………V
誌謝 ……………………………………………………………………VI
表目錄 ………………………………………………………………VII
圖目錄 ………………………………………………………………VIII
縮寫索引 ………………………………………………………………IX
緒論 ……………………………………………………………………1
材料與方法
一、 材料
1. 細胞株 ……………………………………………………………8
2. 病毒株 ……………………………………………………………8
3. 登革病人血清 ……………………………………………………8
4. 實驗動物 …………………………………………………………8
5. 抗體…………………………………………………………………9
6. 試劑藥品 …………………………………………………………10
7. 實驗材料 …………………………………………………………13
8. 儀器 ………………………………………………………………14
二、 方法
1. 單株抗體製備與特性分析 ………………………………………16
2. 帶有專一性抗體的磁珠製備 ……………………………………17
3. 專一性抗體磁珠抓取登革病毒 …………………………………18
4. 專一性抗體磁珠抓取不同型態登革病毒 ………………………18
5. 試管內登革病毒 core RNA 的製備 ……………………………20
6. 經專一性抗體磁珠抓取後的登革病毒進行感染實驗 …………22
結果
一、 建立抗登革病毒單株抗體偵測登革病毒的平台……………25
抗登革病毒單株抗體可偵測到登革病毒顆粒 ……………………………25
抗登革病毒單株抗體結合磁珠可以分離出登革病毒 ……………………25
由不同細胞株繁殖出的登革病毒以未成熟型病毒為主 …………………26
由人類初級單核球(primary monocyte)中繁殖出的登革病毒以成熟型病毒為主 …………………………………………………………………………27
二、 抗登革病毒單株抗體-磁珠分離出來的病毒具有感染力 …27
三、 利用單株抗體-磁珠分離臨床檢體中的登革病毒 …………28
討論
抗登革病毒單株抗體-磁珠偵測登革病毒平台的應用………………29
抗登革病毒單株抗體-磁珠所抓取分離到的未成熟型登革病毒……30
單株抗體-磁珠分離出的不同型態病毒具有不同的感染能力………31
登革病毒型態與抗體依靠性增強作用的關係 ………………………32
登革病患血清中的登革病毒 …………………………………………32

參考文獻 ………………………………………………………………34
附錄 ……………………………………………………………………45
作者簡歷 ………………………………………………………………48

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