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系統識別號 U0026-0812200912010073
論文名稱(中文) 利用噬菌體展示系統鑑定登革病毒免疫增強性抗體所辨識之抗原決定位
論文名稱(英文) Identification of the epitope recognized by enhancing monoclonal antibody against dengue virus using phage-displayed library
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 94
學期 2
出版年 95
研究生(中文) 雷仲益
研究生(英文) Chung-I Rai
電子信箱 barry@ms29.url.com.tw
學號 t3692105
學位類別 碩士
語文別 英文
論文頁數 65頁
口試委員 口試委員-蔡淦仁
指導教授-葉才明
口試委員-黎煥耀
中文關鍵字 登革  登革出血熱  抗體依靠性增強 
英文關鍵字 Dengue  dengue hemorrhagic fever  antibody-dependent enhancement 
學科別分類
中文摘要 登革病毒分成四種血清型態,是引起較輕微的登革熱及致命的登革出血熱和登革休克症候群的病原體。學者所提出的抗體依靠性增強假說,被用來解釋為何在二次感染不同血清型會明顯地造成較嚴重的登革出血熱和登革休克症的表現,然而,至今對於這些增強性抗體所辨識的抗原決定位仍不清楚。近年來,抗前膜蛋白的單株抗體 (mAb 70-21) 已被證實是ㄧ增強性抗體且能夠辨識所有的登革病毒血清型而不具有中和作用活性。在本次研究中,我們利用了噬菌體展示隨機胜肽庫來鑑定mAb 70-21辨識的抗原決定位,在經過三次的生物淘選 (biopanning) 之後,利用酵素免疫分析結果得知,篩選所得陽性反應之噬菌體 (immunopositive phage clones) 能夠和mAb 70-21有特異性的結合,而不會和正常老鼠血清或純化的正常老鼠免疫球蛋白結合。陽性的噬菌體於DNA定序後並配合GCG軟體分析得知,這些噬菌體有著相同的胺基酸序列QNNLGPR,且和登革前膜蛋白的胺基酸序列58-61及熱休克蛋白60的N端序列有部份相似性。再者,這些陽性的噬菌體經免疫老鼠所得的抗血清 (phage-induced antisera) 不僅能辨認所對應的phage clone,亦能夠和所合成的胺基酸序列QNNLGPR結合。除此之外,由間接免疫螢光染色法證實免疫噬菌體的抗血清能夠和正常的Vero和RD細胞結合,而在西方點墨法及免疫沉澱法的結果得知,噬菌體的抗血清能夠辨認BHK-21上的熱休克蛋白60,並且由plaque assay證實,噬菌體抗血清和mAb 70-21ㄧ樣能夠增強登革病毒感染BHK-21細胞。此外,在嚴重的登革病人的血清中發現對抗合成的胺基酸序列QNNLGPR之抗體有顯著增加。總之,由我們的實驗結果顯示,增強性抗體與登革前膜蛋白及熱休克蛋白60的結合,可能會促進登革病毒感染宿主細胞,並可能在發展成登革出血熱及登革休克症候群的致病機轉中扮演重要角色。

英文摘要 Dengue virus (DV), consists of four serotypes (DEN1-4), is the causative agent of mild dengue fever and lift-threatening dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). The antibody dependent enhancement (ADE) hypothesis has been proposed to explain why more severe manifestations of DHF/DSS occur predominantly during secondary infection with different serotypes. However, the epitopes recognized by these enhancing antibodies are unclear. Recently, anti-prM monoclonal antibody (mAb 70-21) which recognized all DV serotypes without neutralizing activity were generated and demonstrated as an enhancing antibody for DV infection. In this study, the epitope recognized by mAb 70-21 was identified using a phage-displayed random peptide library. After three rounds of biopanning, immunopositive phage clones showed specific binding to mAb 70-21 but not to normal mouse serum or purified normal mouse IgG by ELISA. DNA sequencing and GCG analysis of these phage clones showed a consensus sequence, QNNLGPR, which shared sequence homology to the amino acid 58-61 of dengue prM protein and N-terminus of heat shock protein 60. Moreover, phage clones induced-antisera recognized not only these clones but also synthetic peptide of the consensus sequence. In addition, phage-induced antisera also bound to normal Vero or RD cells as demonstrated by indirect fluorescent assay. Western blot analysis and immunoprecipitation showed heat shock protein 60 in BHK-21 cell lysate was recognized by phage clones induced-antisera. Like mAb70-21, these phage-induced antisera also could enhance dengue virus infection of BHK-21 cells. Moreover, antibodies against QNNLGPR synthetic peptide were significantly increased in dengue patients with severe syndrome. Taken together, our results suggest that antibodies which recognized the epitope shared by prM of DV and heat shock protein 60 may enhance DV infection of host cells, which may play a role in the development of DHF or DSS.

論文目次 I.Abstract................................................................Ⅰ
A.中文摘要................................................................Ⅰ
B.English abstract........................................................Ⅱ
誌謝..................................................................... Ⅳ

II.Introduction............................................................1
A.Dengue virus.............................................................1
B.Characteristics of dengue virus..........................................2
C.Clinical manifestations of dengue virus infection........................4
D.The mechanism of DHF/DSS.................................................5
E.The role of antibody in antibody dependent enhancement...................7
F.Phage display technology.................................................8
G.Phage peptide library....................................................8

III.Experimental design....................................................10

IV.Materials and Methods...................................................11
A.Materials................................................................11
A.1.Reagents...............................................................11
A.2.Instruments............................................................14
A.3.Antibody list..........................................................15
A.4.Cell lines, Bacteria strains, and kits.................................16
A.5.The compositions of stocks, buffers, and broths........................16
B.Methods..................................................................17
B.1.Virus stocks...........................................................17
B.2.Freezing Cells.........................................................17
B.3.Thawing Cells..........................................................17
B.4.Dengue patients and serum samples......................................17
B.5.Hybridoma production and purification of anti-prM mAb..................18
B.6.Biopanning against anti-prM mAb 70-21 with phage displayed random peptide library....................................................................18
B.7.Phage titration........................................................19
B.8.Detection of positive phage clones by ELISA............................20
B.9.Competitive ELISA......................................................20
B.10.Phage DNA extraction and sequencing...................................20
B.11.Phage immunization of BALB/c mice.....................................21
B.12.Analysis of immune response to phage clones by ELISA..................21
B.13.Evaluation of phage-induced antiserum binding to DV...................21
B.14.Peptide inhibition experiments........................................22
B.15.Flow cytometry........................................................22
B.16.Indirect immunofluorescence assay.....................................22
B.17.Searching for potential amino acid residues of prM & Hsp60......................................................................23
B.18.Western blot analysis.................................................23
B.19.Evaluation of the phagotope binding to hsp60..........................23
B.20.Immunoprecipitation detection of cellular protein binding to phage-induced antisera...........................................................24
B.21.Plaque assay for antibody-dependent enhancement.......................24
B.22.Antibodies against phagotope in dengue patients’sera.................25
V.Results..................................................................26
A. Identification of epitopes recognized by enhancing monoclonal antibody against dengue virus.......................................................26
A.1.Screening of phage-displayed peptide library with enhancing mAb 70-21.........................................................................26
A.2.Analysis of phages recognized by enhancing mAb 70-21...................26
B. Study on the immunogenicity of dengue virus mimicking phagotope..................................................................27
C. Comparison of prM epitope and phagotope recognized by mAb 70-21.........27
D. Analysis of putative epitope on cell surface recognized by mAb 70-21 and phage-induced antisera.....................................................28
E. Analysis of cellular protein recognized by phagotope-induced antisera...28
F. Assessment of the effect on phage-induced antisera on DV infection of cells......................................................................28
G. Antibodies against phagotope in dengue patients’sera...................29
VI.Discussion..............................................................30
A. The application of phage display library in DV infection................30
B. The proposed mechanism of ADE in DV infection...........................30
C. Antibodies induced by positive selected phage clone bound to host cells.31
D. The significance of enhancing antibodies in clinical DV infection.......31
E.Summary..................................................................32

VII.Reference..............................................................33

VIII.Figure List...........................................................42
Figure 1. Analysis of monoclonal antibody 70-21 from mouse ascites and protein G affinity column by 10% SDS-PAGE..........................................42
Figure 2. Scheme for using phage display 7-mer library to screen epitope recognized by monoclonal antibody 70-21....................................43
Figure 3. Positive phage clone bound specific to mAb 70-21.................44
Figure 4. Positive phage clones competitively inhibit mAb 70-21 binding to DV coated plate...............................................................45
Figure 5. (a) Agarose gel electrophoretic analysis of purified phage single strand DNA.................................................................46
(b) Sequence of phage displayed peptide obtained from 3 rounds of biopanning against mAb 70-21...............................................46
Figure 6. Sequence analysis of selected phage clones by automated DNA sequencer..................................................................47
Figure 7. Generation of phage antibody in phage immunized mice.............48
Figure 8. The reactivity of phage-induced anti-sera to dengue viral particles..................................................................49
Figure 9. Competitive inhibition of phage-induced antisera binding to dengue viral particles by synthetic peptide.......................................50
Figure 10. Comparison of epitope of prM protein and phagotope recognized by mAb 70-21..................................................................52
Figure 11. The cross-reactive binding of mAb 70-21 to BHK cells as determined by flow cytometry..........................................................53
Figure 12. Binding of phage-induced antisera to cells without Fc receptor as shown by immunofluorescence staining.......................................54
Figure 13. Sequence alignment of the mimotope of QNNLGPR with hsp60 by GCG tools......................................................................55
Figure 14. Western blot analysis of cell antigen recognized by phage-induced antisera...................................................................56
Figure 15. Binding activity of phagotope (QNNLGPR) to hsp60 antibody.......57
Figure 16. Identification of hsp60 as an autoantigen candidate of DV-infected BHK-21 protein extracts....................................................58
Figure 17. Monoclonal antibody 70-21 and phage induced antisera C5S-mediated antibody-dependent enhancement of DV infection.............................59
Figure 18. Detection of antibody against phagotope peptide in dengue patients’sera by ELISA..............................................................60
Figure 19. The hypothetic diagram showing the possible mechanism of mAb 70-21 to cause ADE...............................................................61
Figure 20. The proposed models for ADE.....................................62

IX.Table List..............................................................63
Table 1. Summary of anti-dengue monoclonal antibody........................63
Table 2. Affinity selection of random peptide library with mAb 70-21.......64
Table 3. Summary of 32 dengue patients in this study.......................65
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