跳到主要內容

臺灣博碩士論文加值系統

(3.236.110.106) 您好!臺灣時間:2021/07/29 17:04
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:林雅葶
研究生(外文):Ya-tin Lin
論文名稱:以抗登革病毒前趨膜蛋白單株抗體結合磁珠研究未成熟的登革病毒
論文名稱(外文):Anti-prM antibody-conjugated magnetic bead as a probe to study the immature dengue virus
指導教授:黎煥耀黎煥耀引用關係
指導教授(外文):Huan-Yao Lei
學位類別:碩士
校院名稱:國立成功大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:48
中文關鍵詞:前趨膜蛋白磁珠未成熟型登革病毒
外文關鍵詞:immaturedengue virusantibodyprM protein
相關次數:
  • 被引用被引用:1
  • 點閱點閱:173
  • 評分評分:
  • 下載下載:40
  • 收藏至我的研究室書目清單書目收藏:0
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
Alcon, S., Talarmin, A., Debruyne, M., Falconar, A., Deubel, V., and Flamand, M. (2002). Enzyme-linked immunosorbent assay specific to Dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections. J Clin Microbiol 40, 376-381.
Bhamarapravati, N. (1989). Hemostatic defects in dengue hemorrhagic fever. Rev Infect Dis 11 Suppl 4, S826-829.
Chang, C.J., Luh, H.W., Wang, S.H., Lin, H.J., Lee, S.C., and Hu, S.T. (2001). The heterogeneous nuclear ribonucleoprotein K (hnRNP K) interacts with dengue virus core protein. DNA Cell Biol 20, 569-577.
Chao, D.Y., Davis, B.S., and Chang, G.J. (2007). Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important flaviviruses in mosquitoes. J Clin Microbiol 45, 584-589.
Deubel, V., Laille, M., Hugnot, J.P., Chungue, E., Guesdon, J.L., Drouet, M.T., Bassot, S., and Chevrier, D. (1990). Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood. J Virol Methods 30, 41-54.
Drosten, C., Panning, M., Guenther, S., and Schmitz, H. (2002). False-negative results of PCR assay with plasma of patients with severe viral hemorrhagic fever. J Clin Microbiol 40, 4394-4395.
Falgout, B., Chanock, R., and Lai, C.J. (1989). Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a. J Virol 63, 1852-1860.
Falgout, B., Pethel, M., Zhang, Y.M., and Lai, C.J. (1991). Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol 65, 2467-2475.
Halstead, S.B. (1970). Observations related to pathogensis of dengue hemorrhagic fever. VI. Hypotheses and discussion. Yale J Biol Med 42, 350-362.
Halstead, S.B. (2007). Dengue. Lancet 370, 1644-1652.
Hathirat, P., Isarangkura, P., Srichaikul, T., Suvatte, V., and Mitrakul, C. (1993). Abnormal hemostasis in dengue hemorrhagic fever. Southeast Asian J Trop Med Public Health 24 Suppl 1, 80-85.
Henchal, E.A., and Putnak, J.R. (1990). The dengue viruses. Clin Microbiol Rev 3, 376-396.
Huang, J.H., Liao, T.L., Chang, S.F., Su, C.L., Chien, L.J., Kuo, Y.C., Yang, C.F., Lin, C.C., and Shu, P.Y. (2007). Laboratory-based dengue surveillance in Taiwan, 2005: a molecular epidemiologic study. Am J Trop Med Hyg 77, 903-909.
Huang, K.J., Yang, Y.C., Lin, Y.S., Huang, J.H., Liu, H.S., Yeh, T.M., Chen, S.H., Liu, C.C., and Lei, H.Y. (2006). The dual-specific binding of dengue virus and target cells for the antibody-dependent enhancement of dengue virus infection. J Immunol 176, 2825-2832.
Kittigul, L., Pitakarnjanakul, P., Sujirarat, D., and Siripanichgon, K. (2007). The differences of clinical manifestations and laboratory findings in children and adults with dengue virus infection. J Clin Virol 39, 76-81.
Kurane, I., and Ennis, F.E. (1992). Immunity and immunopathology in dengue virus infections. Semin Immunol 4, 121-127.
Lanciotti, R.S., Calisher, C.H., Gubler, D.J., Chang, G.J., and Vorndam, A.V. (1992). Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 30, 545-551.
Lee, W.C., Lien, K.Y., Lee, G.B., and Lei, H.Y. (2008). An integrated microfluidic system using magnetic beads for virus detection. Diagn Microbiol Infect Dis 60, 51-58.
Lei, H.Y., Yeh, T.M., Liu, H.S., Lin, Y.S., Chen, S.H., and Liu, C.C. (2001). Immunopathogenesis of dengue virus infection. J Biomed Sci 8, 377-388.
Lin, C.F., Wan, S.W., Cheng, H.J., Lei, H.Y., and Lin, Y.S. (2006). Autoimmune pathogenesis in dengue virus infection. Viral Immunol 19, 127-132.
Mady, B.J., Erbe, D.V., Kurane, I., Fanger, M.W., and Ennis, F.A. (1991). Antibody-dependent enhancement of dengue virus infection mediated by bispecific antibodies against cell surface molecules other than Fc gamma receptors. J Immunol 147, 3139-3144.
Randolph, V.B., and Stollar, V. (1990). Low pH-induced cell fusion in flavivirus-infected Aedes albopictus cell cultures. J Gen Virol 71 ( Pt 8), 1845-1850.
Randolph, V.B., Winkler, G., and Stollar, V. (1990). Acidotropic amines inhibit proteolytic processing of flavivirus prM protein. Virology 174, 450-458.
Rico-Hesse, R., Harrison, L.M., Salas, R.A., Tovar, D., Nisalak, A., Ramos, C., Boshell, J., de Mesa, M.T., Nogueira, R.M., and da Rosa, A.T. (1997). Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas. Virology 230, 244-251.
Rigau-Perez, J.G., Clark, G.G., Gubler, D.J., Reiter, P., Sanders, E.J., and Vorndam, A.V. (1998). Dengue and dengue haemorrhagic fever. Lancet 352, 971-977.
Rothman, A.L., Kurane, I., Lai, C.J., Bray, M., Falgout, B., Men, R., and Ennis, F.A. (1993). Dengue virus protein recognition by virus-specific murine CD8+ cytotoxic T lymphocytes. J Virol 67, 801-806.
Shu, P.Y., Chen, L.K., Chang, S.F., Yueh, Y.Y., Chow, L., Chien, L.J., Chin, C., Lin, T.H., and Huang, J.H. (2003). Comparison of capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) and nonstructural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections. Clin Diagn Lab Immunol 10, 622-630.
Shu, P.Y., and Huang, J.H. (2004). Current advances in dengue diagnosis. Clin Diagn Lab Immunol 11, 642-650.
Vaughn, D.W., Green, S., Kalayanarooj, S., Innis, B.L., Nimmannitya, S., Suntayakorn, S., Endy, T.P., Raengsakulrach, B., Rothman, A.L., Ennis, F.A., et al. (2000). Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 181, 2-9.
Winkler, G., Maxwell, S.E., Ruemmler, C., and Stollar, V. (1989). Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization. Virology 171, 302-305.
Winkler, G., Randolph, V.B., Cleaves, G.R., Ryan, T.E., and Stollar, V. (1988). Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer. Virology 162, 187-196.
Wu, P.C., Guo, H.R., Lung, S.C., Lin, C.Y., and Su, H.J. (2007). Weather as an effective predictor for occurrence of dengue fever in Taiwan. Acta Trop 103, 50-57.
Yu, I.M., Zhang, W., Holdaway, H.A., Li, L., Kostyuchenko, V.A., Chipman, P.R., Kuhn, R.J., Rossmann, M.G., and Chen, J. (2008). Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 319, 1834-1837.
Zhang, Y., Corver, J., Chipman, P.R., Zhang, W., Pletnev, S.V., Sedlak, D., Baker, T.S., Strauss, J.H., Kuhn, R.J., and Rossmann, M.G. (2003). Structures of immature flavivirus particles. EMBO J 22, 2604-2613.
Zhang, Y., Kaufmann, B., Chipman, P.R., Kuhn, R.J., and Rossmann, M.G. (2007). Structure of immature West Nile virus. J Virol 81, 6141-6145.
Zhang, Y., Zhang, W., Ogata, S., Clements, D., Strauss, J.H., Baker, T.S., Kuhn, R.J., and Rossmann, M.G. (2004). Conformational changes of the flavivirus E glycoprotein. Structure 12, 1607-1618.
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊