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研究生:龔加吟
研究生(外文):Gong Jia-yn
論文名稱:台灣新分離新城病病毒株之單株抗體製備與特性分析
論文名稱(外文):Preparation and Charaterization of Monoclonal Antibodies Against Recent Newcastle Disease Virus Isolate in Taiwan.
指導教授:林茂勇
指導教授(外文):Lin Mow-yeong
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:94
中文關鍵詞:新城病病毒單株抗體
外文關鍵詞:Newcastle disease virusMonocloanl antibodies
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以基因親源樹狀分型及雞胚平均致死時間測定為第Ⅶ基因型之強毒內臟型新城病病毒(vvNDV)之1999年台灣野外分離株(V157株)濃縮病毒液免疫BALB/c小白鼠以製備單株抗體並分析其特性。利用上述經多次V157株病毒濃縮液免疫之BALB/c小白鼠脾臟細胞在經過三次的細胞融合後,獲得14株抗V157株融合瘤細胞株,經免疫墨點法、間接螢光抗體法和中和抗體檢測皆呈陽性反應,而中和試驗計有1、8、2和2株分別可達4、8、16和512倍之抗體力價;以西方轉漬法檢測計有10株(72%)可辨識V157株之F醣蛋白線性抗原決定位,有4株(28%)可辨識其結構依賴型抗原決定位;在亞型分析方面,共獲得5、3、2和4株分別各屬IgG1、IgG2a、IgG2b和IgG3亞型的單株抗體。利用競爭性ELISA可將上述14株單株抗體分群,共可分為A(6株)、B(5株)、C(2株)、 D(1株)四大群,其中B及D群包含了對抗結構依賴型抗原決定基之單株抗體。NDV的單株抗體之製備雖頗為艱辛,但因其具有高度與NDV抗原表面決定位結合之特異性,為日後探討新野外分離vvNDV株之F醣蛋白抗原表面決定位之變異情形所必備。
Fourteen clones of monoclonal antibody (mAb) production by hybridoma cell were prepared from 3 times fusion of the BALB/c mice spleen cell preimmunized with a 1999 local isolate (V157 strain) of genotype Ⅶ viscerotropic velogenic Newcastle disease virus (vvNDV) and mice myeloma cell. The production of mAbs against V157 strain in the hybridoma cell suspension or mice ascites were screened by enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody assay (IFA), then characterized by immuno dot blotting assay (ID), Western blot (WB) and neutralization test (NT). All clones of mAb were positive in ELISA and IFA. Only 1, 7, 3 and 2 clones of mAb showed positive at 4, 8, 16 and 512 dilution folds level, respectively. Ten (72%) clones of mAb recognized the linear epitope and the other 4 (28%) recognized the conformational dependent epitope on F-glycoprotein. At subtype level, 5, 3, 2 and 4 clones of mAb belonged to IgG1, IgG2a, IgG2b and IgG3, respectively. By competitive ELISA classification, 6, 5, 2 and 1 clones of mAb belong to A, B, C and D group, respectively. Which implys at least 4 antigenic epitopes existing on the F-glycoprotein of V157 strain. The conformation-dependent epitope was found only showing in group B and D. Although the production of NDV mAb is time consuming, the high specificity of mAb in binding with its special NDV antigen make the preparation of NDV mAb is very worthful studing the variation on the antigenic F-glycoprotein epitope in new isolate of vvNDV.
中文摘要--------------------------------------------- Ⅰ
英文摘要--------------------------------------------- Ⅲ
誌謝------------------------------------------------- Ⅴ
目錄------------------------------------------------- Ⅵ
圖次------------------------------------------------- Ⅹ
表次------------------------------------------------- ⅩⅠ
第1章 緒言------------------------------------------- 1
第2章 文獻探討--------------------------------------- 5
2.1. 新城病病毒性狀---------------------------------- 6
2.1.1 病毒的形態與結構------------------------------- 6
2.1.2 病毒基因體------------------------------------- 7
2.1.3 病毒蛋白質------------------------------------- 7
2.1.3.1 核鞘蛋白(NP protein)------------------------- 7
2.1.3.2磷酸化病毒蛋白(P protein)--------------------- 8
2.1.3.3 大蛋白(L protein)---------------------------- 9
2.1.3.4 基質蛋白或膜蛋白(M protein)------------------ 9
2.1.3.5 血球凝集素-神經胺酸酶醣蛋白(HN glycoprotein)- 10
2.1.3.6 融合醣蛋白(F glycoprotein)------------------- 13
2.1.4 新城病病毒之生物合成 (Biosynthesis)------------ 16
2.1.4.1 病毒轉錄與轉譯------------------------------- 17
2.1.4.2 病毒的複製----------------------------------- 17
2.1.4.3 病毒組合及釋放------------------------------- 18
2.2 單株抗體之歷史背景------------------------------- 18
2.2.1 細胞融合的原理--------------------------------- 18
2.2.2 單株抗體的特性與應用--------------------------- 20
2.3 新城病單株抗體的應用----------------------------- 21
第3章 材料與方法------------------------------------- 24
3.1 抗原製備----------------------------------------- 24
3.1.1 病毒------------------------------------------- 24
3.1.2 病毒增殖--------------------------------------- 24
3.1.3 病毒之純化------------------------------------- 25
3.1.4 純化後病毒之確認與定量------------------------- 25
3.1.4.1 反轉錄-聚合酶鏈反應(Reverse transcription -polymerase
chain reaction; RT-PCR)-------------- ------- 26
3.1.4.2 血球凝集試驗(Hemagglutination)--------------- 27
3.1.4.3 病毒斑形成試驗(Plaque formation)------------- 27
3.2 融合瘤細胞之製備--------------------------------- 28
3.2.1 實驗動物--------------------------------------- 28
3.2.2 免疫計劃--------------------------------------- 28
3.2.3 骨髓瘤細胞培養--------------------------------- 29
3.2.4 細胞融合--------------------------------------- 29
3.2.4.1 融合細胞培養--------------------------------- 31
3.2.5 融合瘤細胞之篩選------------------------------- 32
3.2.5.1 酵素連結免疫吸附試驗(Enzyme-linked immunosorbent
assay ; ELISA)------------------------------- 32
3.2.5.2 免疫墨點反應(Immuno-dot assay)--------------- 33
3.2.5.3 間接式免疫螢光抗體試驗(Indirect immuno-fluorescent
antibody test) ------------------------------ 34
3.3 單株抗體之生產及純化----------------------------- 34
3.3.1 腹水之生產------------------------------------- 35
3.3.2 飽和硫酸銨純化抗體----------------------------- 35
3.3.3 Protein A親和性管柱(Protein A affinity column)純
化抗體----------------------------------------- 36
3.4 單株抗體分析------------------------------------- 37
3.4.1 單株抗體亞型分析(Isotyping)-------------------- 37
3.4.2 西方轉漬分析(Western blot)--------------------- 38
3.4.3 血清中和試驗(Serum neutralization; SN)--------- 39
3.5 抗原決定位分群----------------------------------- 40
3.5.1 過氧化酶標定單株抗體(Horseradish-peroxidase conjugated
mAbs)------------------------------------------ 40
3.5.2 己標定過氧化酶之單株抗體親和性分析------------- 41
3.5.3 競爭型酵素連結免疫吸附試驗(Competitive ELISA)-- 41
第4章 結果------------------------------------------- 43
第5章 討論------------------------------------------- 49
第6章 參考文獻--------------------------------------- 79
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