(3.238.235.155) 您好!臺灣時間:2021/05/16 08:45
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:::

我願授權國圖
: 
twitterline
研究生:林家慧
研究生(外文):Chia-Hui Lin
論文名稱:利用噬菌體展現技術製造蘭花病毒鞘蛋白抗原之雞抗體片段重組基因庫
論文名稱(外文):Chicken single chain variable fragment recognizing orchid virus coat protein antigen using phage display antibody technology
指導教授:楊沂淵楊沂淵引用關係
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:81
中文關鍵詞:噬菌體蘭花病毒:鞘蛋白抗體基因庫
外文關鍵詞:single chainphage displayorchid viruslibrary
相關次數:
  • 被引用被引用:0
  • 點閱點閱:215
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
蘭花是開花植物中最大的一科,估計約有1萬5千至3萬種,遍及全世界。由於極具觀賞價值以及商業性而被大量栽種。相對的在蘭花病蟲害的防治上極其重要,蕙蘭嵌紋病毒(Cymbidium mosaic virus, CyMV)是目前最為普遍造成蘭花傷害的其中一屬。受到病毒感染的蘭花可產生嚴重的系統性黃化病徵,造成蘭花外觀有極大變化與傷害,相關產業也遭受損失,嚴重影響國內經濟。感染途徑主要是經由機械性傷口感染蘭花本身,而本篇的研究目標是利用重組表現CyMV蘭花病毒鞘蛋白片段來免疫雞隻,藉由嗜菌體展現技術(Phage display technique)來建立重組抗體基因庫(antibody library)篩選對於CyMV蘭花病鞘蛋白具有特異性結合力的單株抗體片段(scFv, single chain variable fragment)。重組的CyMV蘭花病毒鞘蛋白基因經酵素作用確認,將重組基因送入大腸桿菌表現並純化重組鞘蛋白,藉由SDS-PAGE染色證實。將純化後的鞘蛋白片段與佐劑混合均勻注射雞隻大腿肌肉部位,連續四周免疫雞隻,收集每次免疫雞蛋,萃取蛋中抗體Poly-IgY,藉由西方墨點法確認。利用西方墨點法(western blot)以及酵素連結免疫分析實驗(enzyme-linked immunosorbent assay;ELISA)偵測經四次免疫後雞蛋中的抗體IgY效價,證實在免疫四次後雞隻體內的確誘發出高劑量的抗體反應並可辨認His-CyMV融合蛋白。將雞隻犧牲,取出脾臟,萃取RNA反轉成cDNA並利用多鏈聚合酶反應(PCR)做出抗體重鏈和輕鏈基因片段,將做出的重鏈與輕鏈接到pComb3X載體中,建構出CyMV為1.1× 103蘭花病毒外鞘蛋白的雞隻抗體片段基因庫,經隨機挑取15個clone,萃取其DNA證實。再利用嗜菌體感染並使其表現出抗體片段基因庫上的scFv。經四次篩選(panning),萃取四次scFv DNA確認。同時表現與重組CyMV鞘蛋白最具有特異性結合力的scFv抗體片段,最後共篩選出14個有表現的抗體片段,將抗體片段基因定序分析其中的相似性,最後共分成三群具有不同序列的片段。將三群抗體片段從中各挑出一個作為代表大量純化,利用純化後可溶性抗體片段以western blot與ELISA方法證實抗體與抗原間結合的能力,發現不同抗體片段具有與抗原間不同的結合能力,對於往後在蘭花病毒的防治及偵測上,期待更有良好的效果。
The Orchidaceae is the largest and most diverse plant family, consisting of more than 25,000 species in approximately 900 genera. It is important to control virus diseases in orchid collections. Orchids are affected by more virus disease problems than others. CyMV (CyMV, family Flexiviridae) has been reported in many cultivated orchid species in many countries, and than is now considered to be spread worldwide. The symptoms of infected orchids were such as system yellowing of leaves, necrotic spot. In this study, we must develop CyMV scFv (single-chain variable fragment) antibody library to produce specific antibody by phage display, and screen the binding ability of CyMV coat protein antibodies. First, we constructed CyMV coat protein genes into pET32a vector and expression the fusion protein; second, we used fusion protein to immunize chicken for IgY production. After four times of boost, the titers of anti CyMV coat protein antibody were increased. And than, identified antibodies with western blot and ELISA analysis. Additionally, we have to analysis the binding ability with total IgY antibody and CyMV coat protein. Finally, We successfully used phage display to generate CyMV scFv library and the size was 1.1× 103. After four times panning amplified the phage, we found 14 scFv clones were expressed antibody protein.After all, we alignmented these 14 scFv clone sequences and evaluated specificity of scFv antibodies with CyMV coat protein for western blot and ELISA analysis. We hope they could use to prevent and detect the orchids virus in the future.
目 錄
目錄...............................................................................................................................i
圖表目錄.......................................................................................................................iii
中文摘要.......................................................................................................................􀒊
英文摘要.......................................................................................................................􀒌
壹、緒論.......................................................................................................................1
一、CyMV蘭花病毒簡介.........................................................................................1
二、蛋黃免疫球蛋白簡介.....................................................................................2
三、單株抗體簡介及應用.....................................................................................3
四、嗜菌體技術重組基因庫簡介及應用.............................................................5
貳、研究目的...............................................................................................................9
叁、實驗材料與方法...................................................................................................10
一、實驗材料.........................................................................................................10
1、化學藥品.....................................................................................................10
2、酵素類.........................................................................................................12
3、抗體類.........................................................................................................12
4、其他.............................................................................................................12
5、各式溶液.....................................................................................................12
二、實驗方法.........................................................................................................16
1、建立一個重組CyMV病毒鞘蛋白序列.....................................................16
2、表現及純化重組CyMV病毒鞘蛋白.............................................................17
3、聚丙烯氨膠體(SDS-PAGE)電泳法.............................................................17
4、西方墨點法(Western blot) ....................................................................18
5、免疫注射雞隻.............................................................................................18
6、純化雞蛋之抗體(IgY)................................................................................19
7、檢測純化出的抗體IgY與蘭花病毒鞘蛋白結合反應..............................20
8、Chicken cDNA 資料庫建構.......................................................................20
9、抗CyMV蘭花病毒鞘蛋白的scFv基因庫的建立.........................................21
10、抗CyMV 蘭花病毒鞘蛋白重組基因庫篩選.........................................24
11、表現經過篩選的CyMV 重組鞘蛋白抗體scFv 片段...............................25
12、CyMV scFv 抗體片段序列分析................................................................26
13、利用西方墨點法分析抗體片段與CyMV 病毒鞘蛋白抗原之結合力.....26
14、酵素結合免疫分析法(ELISA)分析scFv 抗體片段與CyMV 蘭花病毒
組織的結合反應........................................................................................26
肆、實驗結果................................................................................................................28
1、重組CyMV蘭花病毒鞘蛋白基因....................................................................28
2、重組CyMV融合鞘蛋白表現以及純化............................................................28
3、純化免疫前後蘭花病毒IgY 抗體............................................................... 28
4、利用西方墨點法分析並偵測雞隻雞蛋中抗體(poly-IgY)與特異性結合反應
..........................................................................................................................29
5、ELISA 偵測IgY 與CyMV 蘭花病毒鞘蛋白特異性結合反應.......................29
6、分離脾臟並抽取RNA.....................................................................................29
ii
7、抗CyMV 蘭花病毒鞘蛋白的scFv 基因庫的建立........................................30
8、確認重組抗體DNA送入XL-1 E.coli 中......................................................30
9、利用限制酶sfi 1 確認重組抗體DNA 後送入的基因片段........................30
10、評估利用嗜菌體展現技術篩選CyMV 重組蛋白抗體基因庫片段效價..... 31
11、評估利用噬菌體展現技術後篩選出與CyMV 鞘蛋白具特異性結合
的重組抗體基因片段(scFv)的一致性........................................................31
12、表現經過篩選的CyMV 重組鞘蛋白抗體scFv 片段....................................31
13、序列分析其CyMV 重組鞘蛋白scFv 抗體片段............................................32
14、西方墨點法分析純化CyMV 重組scFv 抗體片段........................................32
15、西方墨點法分析CyMV重組scFv抗體片斷與CyMV鞘蛋白蛋白抗原的結合
反應................................................................................................................32
16、酵素連結免疫反應分析CyMV 重組scFv 抗體片斷與感染CyMV 的組
織結合反應....................................................................................................33
伍、討論........................................................................................................................53
陸、未來展望................................................................................................................57
柒、附錄一....................................................................................................................58
附錄二....................................................................................................................65
捌、參考文獻................................................................................................................79
iii
1.Wong, S.M., et al., Cymbidium mosaic potexvirus RNA: complete nucleotide sequence and phylogenetic analysis. Arch Virol, 1997. 142(2): p. 383-91.
2.Goodman, R.M. and A.F. Ross, Enhancement of potato virus X synthesis in doubly infected tobacco occurs in doubly infected cells. Virology, 1974. 58(1): p. 16-24.
3.Ajjikuttira, P., C.S. Loh, and S.M. Wong, Reciprocal function of movement proteins and complementation of long-distance movement of Cymbidium mosaic virus RNA by Odontoglossum ringspot virus coat protein. J Gen Virol, 2005. 86(Pt 5): p. 1543-53.
4.Seoh, M.L., S.M. Wong, and L. Zhang, Simultaneous TD/RT-PCR detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus with a single pair of primers. J Virol Methods, 1998. 72(2): p. 197-204.
5.Verchot-Lubicz, J., A new cell-to-cell transport model for Potexviruses. Mol Plant Microbe Interact, 2005. 18(4): p. 283-90.
6.Yu, H.H. and S.M. Wong, A DNA clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene. Arch Virol, 1998. 143(1): p. 163-71.
7.Meng, C.M., et al., [Production of monoclonal antibodies to Cymbidium mosaic virus and application in orchids virus detection]. Wei Sheng Wu Xue Bao, 2007. 47(5): p. 928-31.
8.Chen, C.L., J.E. Lehmeyer, and M.D. Cooper, Evidence for an IgD homologue on chicken lymphocytes. J Immunol, 1982. 129(6): p. 2580-5.
9.Burns, R.B. and M.H. Maxwell, Probable occurrence of IgE in the adult domestic fowl (Gallus domesticus) after horse serum stimulation. Vet Res Commun, 1981. 5(1): p. 67-72.
10.Williams, J.W. and D.D. Donermeyer, The interaction of bovine serum albumin and its chicken antibodies. J Biol Chem, 1962. 237: p. 2123-30.
11.Leslie, G.A. and L.W. Clem, Phylogen of immunoglobulin structure and function. 3. Immunoglobulins of the chicken. J Exp Med, 1969. 130(6): p. 1337-52.
12.Latreille, J., et al., Dexamethasone improves the efficacy of granisetron in the first 24 h following high-dose cisplatin chemotherapy. Support Care Cancer, 1995. 3(5): p. 307-12.
13.Gassmann, M., et al., Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. Faseb J, 1990. 4(8): p. 2528-32.
14.Hadge, D. and H. Ambrosius, Evolution of low molecular weight immunoglobulins--IV. IgY-like immunoglobulins of birds, reptiles and amphibians, precursors of mammalian IgA. Mol Immunol, 1984. 21(8): p. 699-707.
15.Carlander, D., et al., Peroral immunotherapy with yolk antibodies for the prevention and treatment of enteric infections. Immunol Res, 2000. 21(1): p. 1-6.
16.Fryer, J., et al., IgY antiporcine endothelial cell antibodies effectively block human antiporcine xenoantibody binding. Xenotransplantation, 1999. 6(2): p. 98-109.
17.Erhard, M.H., et al., Adjuvant effects of various lipopeptides and interferon-gamma on the humoral immune response of chickens. Poult Sci, 2000. 79(9): p. 1264-70.
18.Rietschel, E.T., et al., Bacterial endotoxin: molecular relationships of structure to activity and function. Faseb J, 1994. 8(2): p. 217-25.
19.Woof, J.M. and D.R. Burton, Human antibody-Fc receptor interactions illuminated by crystal structures. Nat Rev Immunol, 2004. 4(2): p. 89-99.
20.Schwaber, J. and E.P. Cohen, Human x mouse somatic cell hybrid clone secreting immunoglobulins of both parental types. Nature, 1973. 244(5416): p. 444-7.
21.Kohler, G. and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975. 256(5517): p. 495-7.
22.Kipps, T.J., et al., Importance of immunoglobulin isotype in human antibody-dependent, cell-mediated cytotoxicity directed by murine monoclonal antibodies. J Exp Med, 1985. 161(1): p. 1-17.
23.Khazaeli, M.B., R.M. Conry, and A.F. LoBuglio, Human immune response to monoclonal antibodies. J Immunother Emphasis Tumor Immunol, 1994. 15(1): p. 42-52.
24.Jaenisch, R. and B. Mintz, Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA. Proc Natl Acad Sci U S A, 1974. 71(4): p. 1250-4.
25.Bruggemann, M., et al., A repertoire of monoclonal antibodies with human heavy chains from transgenic mice. Proc Natl Acad Sci U S A, 1989. 86(17): p. 6709-13.
26.Holliger, P. and P.J. Hudson, Engineered antibody fragments and the rise of single domains. Nat Biotechnol, 2005. 23(9): p. 1126-36.
27.Smith, G.P., Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 1985. 228(4705): p. 1315-7.
28.Griffiths, A.D., et al., Human anti-self antibodies with high specificity from phage display libraries. EMBO J, 1993. 12(2): p. 725-34.
29.Huse, W.D., et al., Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science, 1989. 246(4935): p. 1275-81.
30.Fernandez-Gacio, A., M. Uguen, and J. Fastrez, Phage display as a tool for the directed evolution of enzymes. Trends Biotechnol, 2003. 21(9): p. 408-14.
31.Scott, J.K. and G.P. Smith, Searching for peptide ligands with an epitope library. Science, 1990. 249(4967): p. 386-90.
32.Jacobsson, K. and L. Frykberg, Cloning of ligand-binding domains of bacterial receptors by phage display. Biotechniques, 1995. 18(5): p. 878-85.
33.Ajjikuttira, P.A., et al., Genetic variability in the coat protein genes of two orchid viruses: Cymbidium mosaic virus and Odontoglossum ringspot virus. Arch Virol, 2002. 147(10): p. 1943-54.
34.Barbas, S.M. and C.F. Barbas, Filamentous phage display. Fibrinolysis, 1994. 8(Supplement 1): p. 245-252.
35.Polson, A., et al., Improvements in the isolation of IgY from the yolks of eggs laid by immunized hens. Immunol Invest, 1985. 14(4): p. 323-7.
36.Polson, A., Isolation of IgY from the yolks of eggs by a chloroform polyethylene glycol procedure. Immunol Invest, 1990. 19(3): p. 253-8.
37.Manoutcharian, K., et al., Phage-displayed T-cell epitope grafted into immunoglobulin heavy-chain complementarity-determining regions: an effective vaccine design tested in murine cysticercosis. Infect Immun, 1999. 67(9): p. 4764-70.

34. Dexter, J.S., Mosquitoes Pollinating Orchids. Science, 1913. 37(962): p. 867.
35.Sweet, H.R., Orchids of Venezuela. Science, 1966. 152(3724): p. 943-944.
36. Tsen, Y.C., et al., Evaluation and validation of a duck IgY antibody-based immunoassay for high-sensitivity C-reactive protein: avian antibody application in clinical diagnostics. Clin Chem, 2003. 49(5): p. 810-3.
37. Yin, X., et al., Screening of a phage display library of exendin-4 mutants with the extracellular domain of rat GLP-1 receptor. Protein Pept Lett, 2007. 14(8): p. 816-21.
38. Carlander, D. and A. Larsson, Avian antibodies can eliminate interference due to complement activation in ELISA. Ups J Med Sci, 2001. 106(3): p. 189-95.
39. Sherpa, A.R., et al., Complete nucleotide sequence analysis of Cymbidium mosaic virus Indian isolate: further evidence for natural recombination among potexviruses. J Biosci, 2007. 32(4): p. 663-9.
40. Sheets, M.D., et al., Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci U S A, 1998. 95(11): p. 6157-62.
41. Vejaratpimol, R., et al., Detection and serological relationships of cymbidium mosaic potexvirus isolates. J Biosci Bioeng, 1999. 87(2): p. 161-8.
42. Usha, R., et al., Expression of an Animal Virus Antigenic Site on the Surface of a Plant Virus Particle. Virology, 1993. 197(1): p. 366-374.
43. Rasmussen, J.E., Contact dermatitis from orchids. Clin Dermatol, 1986. 4(2): p. 31-5.
44. Rajkumari, J.D. and R.S. Longjam, Orchid flower evolution. J Genet, 2005.584(1): p. 81-4.
45. Martelli, G.P., et al., Family Flexiviridae: a case study in virion and genome plasticity. Annu Rev Phytopathol, 2007. 45: p. 73-100.
46. Vaccaro, P., et al., Efficient display of scFv antibodies on bacteriophage lambda. J Immunol Methods, 2006. 310(1-2): p. 149-58.
47. Carlander, D., J. Stalberg, and A. Larsson, Chicken antibodies: a clinical chemistry perspective. Ups J Med Sci, 1999. 104(3): p. 179-89.
48. Carlander, D., H. Kollberg, and A. Larsson, Retention of specific yolk IgY in the human oral cavity. BioDrugs, 2002. 16(6): p. 433-7.
49. Rose ME, Orlans E, Buttres N. Immunoglobulin classes in the hen''s egg:
their segregation in yolk and white. Eur J Immunology 1974; 4:521-523.
50. Akita EM, Nakai S. Immunoglobulins from egg yolk: Isolation and
purification. Journal of food science 1992; 57(32):629-634.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top