臺灣博碩士論文加值系統

登革病毒(dengue viruses)感染人體之後會產生登革熱(dengue fever, DF)、出血性登革熱(dengue hemorrhagic fever, DHF)和登革休克症候群(dengue shock syndrome, DSS)等症狀。登革感染的病人血清會出現高量的病毒非結構蛋白1(NS1)，而人體免疫系統產生的抗NS1抗體會導致人體產生病理反應。人體在二次感染不同型登革病毒後產生的非中和性抗體會產生不正常的免疫反應，這不正常的免疫反應可能是導致出血性登革熱及登革休克群的原因。但是，登革病毒如何讓人體產生不正常免疫反應的機轉仍需進一步研究。我們之前的研究顯示出血性登革熱病人血清含抗NS1抗體會與人體內TNF receptor 家族中的TRAIL-R1 以及B細胞活化因子的接受器( transmembrane activator and CAML interactor ,TACI )產生交叉反應，而目前已知TACI可由B細胞活化因子(B cell-activating factor, BAFF)調控B細胞的存活、成熟以及耐受性。為了探討這些因登革感染產生的B細胞活化因子接受器抗體(TACI cross-reactive Igs)是否會造成B細胞不正常發育及之後產生出血性登革熱病理現象，我們製備了TACI重組蛋白並且以酵素連結免疫沉澱方法(ELISA)篩選登革熱病人及登革出血熱病人血清中抗TACI抗體的效價。雖然我們尚未證明登革出血熱病人血清中抗TACI IgM抗體效價比登革熱病人高，但初步結果顯示登革出血熱病人血清中含有高效價的抗TACI IgG抗體及抗NS1 IgG抗體，且經NS1免疫的兔子其抗NS1抗體亦會隨著免疫次數的增加而增加與TACI產生交叉反映的效價。在體外細胞研究中，抗NS1抗體可以增加B淋巴瘤細胞的存活但降低T淋巴瘤細胞的生存，而抗NS1抗體中所含的抗TACI、DR4抗體也會影響淋巴瘤細胞的存活率。另外，利用登革病毒膜蛋白（Dengue-2 virus envelope protein domain III, Den2E-D3）免疫已事先對NS1及GST產生抗體的老鼠，再偵測其產生的抗Den2E-D3抗體的IgG 效價，實驗結果發現小鼠在接受NS1的前置免疫注射後約有50 %會對後續Den2E-D3的免疫注射產生較低下的抗Den2E-D3 IgG效價，且這些小鼠血清對病毒的中和能力亦較低落。

Dengue viruses, arthropod-borne flaviviruses, cause dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In dengue viral infections, high level nonstructural protein 1(NS1) was found in patients’ sera, and the antibodies against the NS1 protein was proposed to induce serious abnormal immune response result in autoimmune disease, however, the mechanism that how dengue viral infections elicited autoimmune response remains to be elucidated. According to our preliminary data, it showed that anti-NS1 Immunoglobulin (Ig) present in dengue hemorrhagic fever patients’ sera could cross-react with TNF receptor superfamily members, TRAIL-R1 (DR4) and TACI which is the receptor of B cell-activating factor (BAFF). To investigate whether such cross-reactive Igs could cause B cell abnormalities and later DHF pathogenesis, we prepared the recombinant TACI protein and screen the anti-TACI antibody titers of DF and DHF/DSS sera samples by ELISA. The results indicated that the IgG titers of anti-TACI in DHF patients’ sera are significant higher than DF patients’ sera, but the specific anti-TACI IgM activities are not. The anti-NS1 polyclonal antibodies were also found to enhance and reduce the cell viability of human B lymphoma cell line and T lymphoma cell line, respectively. The viability of lymphoma cells were also influence by anti-NS1 antibodies that cross-reactive with TACI and DR4. Furthermore, we determined the IgG titer of anti- Den2E-D3 in Den2E-D3 immunized mice that pre-immunized with rNS1 and GST. Approximately 50 % of mice with prior immunization of rNS1 have lower IgG titers to Den2E-D3 following subsequent immunization of Den2E-D3 than the naïve mice, and the viral neutralization capability also decreased in this group of mice.

目錄
誌謝 I
中文摘要 II
英文摘要 III
目錄 IV
圖目錄 VI
緒論 1
材料與方法 8
(一) 材料
1. 細胞株 8
2. 病毒株 9
3. 實驗動物 9
4. 實驗使用之抗體及重組蛋白 9
5. 實驗使用之相關試劑 10
6. 實驗相關藥品 11
(二) 方法
1. 載體構築 13
2. 蛋白質的表現與純化 18
3. 抗體的製備 20
4. SDS-聚丙烯醯電泳 21
5. 西方墨點法 22
6. 酵素連結免疫沈澱法 23
7. 流式細胞分析 23
8. 細胞存活實驗 24
9. 病毒株的感染與繁殖 24
10.病毒效價的測定 25
11.病毒中和試驗 25
結果 27
討論 35
參考文獻 38
圖 41
附錄 56

Baker, K. P. (2004). BLyS--an essential survival factor for B cells: basic biology, links to pathology and therapeutic target. Autoimmun Rev 3, 368-375.
Bielefeldt-Ohmann, H. (1997). Pathogenesis of dengue virus diseases: missing pieces in the jigsaw. Trends Microbiol 5, 409-413.
Boonpucknavig, S., Vuttiviroj, O., Bunnag, C., Bhamarapravati, N., and Nimmanitya, S. (1979). Demonstration of dengue antibody complexes on the surface of platelets from patients with dengue hemorrhagic fever. Am J Trop Med Hyg 28, 881-884.
Do, R. K., and Chen-Kiang, S. (2002). Mechanism of BLyS action in B cell immunity. Cytokine Growth Factor Rev 13, 19-25.
Falconar, A. K. (1997). The dengue virus nonstructural-1 protein (NS1) generates antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells: potential implications in haemorrhagic fever pathogenesis. Arch Virol 142, 897-916.
Gross, J. A., Johnston, J., Mudri, S., Enselman, R., Dillon, S. R., Madden, K., Xu, W., Parrish-Novak, J., Foster, D., Lofton-Day, C., et al. (2000). TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature 404, 995-999.
Guzman, M. G., and Kouri, G. (1996). Advances in dengue diagnosis. Clin Diagn Lab Immunol 3, 621-627.
38
Guzman, M. G., and Kouri, G. (2002). Dengue: an update. Lancet Infect Dis 2, 33-42.
Hales, S., de Wet, N., Maindonald, J., and Woodward, A. (2002). Potential effect of population and climate changes on global distribution of dengue fever: an empirical model. Lancet 360, 830-834.
Henchal, E. A., and Putnak, J. R. (1990). The dengue viruses. Clin Microbiol Rev 3, 376-396.
Kautner, I., Robinson, M. J., and Kuhnle, U. (1997). Dengue virus infection: epidemiology, pathogenesis, clinical presentation, diagnosis, and prevention. J Pediatr 131, 516-524.
Kimberley, F. C., and Screaton, G. R. (2004). Following a TRAIL: update on a ligand and its five receptors. Cell Res 14, 359-372.
Lin, C. F., Chiu, S. C., Hsiao, Y. L., Wan, S. W., Lei, H. Y., Shiau, A. L., Liu, H. S., Yeh, T. M., Chen, S. H., Liu, C. C., and Lin, Y. S. (2005). Expression of cytokine, chemokine, and adhesion molecules during endothelial cell activation induced by antibodies against dengue virus nonstructural protein 1. J Immunol 174, 395-403.
Lin, C. F., Lei, H. Y., Shiau, A. L., Liu, C. C., Liu, H. S., Yeh, T. M., Chen, S. H., and Lin, Y. S. (2003). Antibodies from dengue patient sera cross-react with endothelial cells and induce damage. J Med Virol 69, 82-90.
Lin, C. F., Lei, H. Y., Shiau, A. L., Liu, H. S., Yeh, T. M., Chen, S. H., Liu, C. C., Chiu, S. C., and Lin, Y. S. (2002). Endothelial cell apoptosis induced by antibodies against dengue virus nonstructural protein 1 via production of nitric oxide. J Immunol 169, 657-664.
39
Morens, D. M. (1994). Antibody-dependent enhancement of infection and the pathogenesis of viral disease. Clin Infect Dis 19, 500-512.
Ranga, S., Prakash, I., Choudhry, Y., Thergaonkar, A., Khurana, S. K., and Talib, V. H. (1997). Dengue haemorrhagic fever: an overview. Indian J Pathol Microbiol 40, 103-117.
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.
Seshasayee, D., Valdez, P., Yan, M., Dixit, V. M., Tumas, D., and Grewal, I. S. (2003). Loss of TACI causes fatal lymphoproliferation and autoimmunity, establishing TACI as an inhibitory BLyS receptor. Immunity 18, 279-288.
Chang, Y. C. (2003). Roles of TRAIL pathway in dengue viruses mediated pathogenesis.