臺灣博碩士論文加值系統

犬冠狀病毒 (canine coronavirus, CCoV) 、犬小病毒 (canine parvovirus, CPV) 及鉤端螺旋體菌 (Leptospira interrogans) 皆為常見之犬類病原，其中CCoV及CPV皆能引起犬隻之腸胃疾病，CPV另會導致犬類呼吸道之疾病，而鉤端螺旋體根據其血清型不同可影響肝及腎功能及造成內臟出血及腸胃道疾病。CCoV表面突刺 (spike) 蛋白與病毒進入細胞有關。而VP2蛋白也和CPV進入宿主細胞扮演重要角色；鉤端螺旋體則利用Lig.A及Lig.B蛋白進入宿主細胞。本研究主要目的為生產單株抗體。由於目前市售抗體較昂貴，若可生產專一性及表現量皆高之單株抗體，用於後續犬隻次單位疫苗之抗原鑑定及快速篩檢之用途，可有效降低實驗及後續應用之成本，以期得到較大利潤。本研究經由確認後，共篩選出2株CCoV S1蛋白之單株抗體，分別命名為S1-35及S1-38。而在鉤端螺旋體Lig.B蛋白也得到兩株單株抗體，分別命名為Lig.B-1及Lig.B-2。利用西方墨點法和免疫墨點法分析得知S1-35及S1-38其辨認抗原皆為非構型依賴 (線性抗原)；Lig.B-1及Lig.B-2其辨認同樣為非構型依賴之抗原決定位。進一步以套組分析抗體之亞型，S1-35及S1-38兩株單株抗體其抗體亞型皆為 IgG1，抗體輕鏈同樣為κ，而Lig.B-1及Lig.B-2兩株單株抗體其抗體亞型亦為IgG1，抗體輕鏈同樣亦為κ。利用刪除突變 (deletion mutation) 方式進行抗原決定位之定位分析，其中單株抗體S1-35辨認S1 170-262胺基酸，單株抗體S1-38則辨認S1 262-353胺基酸，單株抗體Lig.B-1辨認Lig.B 301-598胺基酸，而單株抗體Lig.B-2辨認Lig.B 1-300胺基酸。另外定位分析過去本實驗室鄭年倫學長製備出CPV-VP2之抗原決定位，結果顯示CPV-VP2兩株單株抗體所辨認抗原決定位之位置分別為VP2蛋白第52-62胺基酸及第85-97胺基酸。綜合以上結果，本研究所製備之單株抗體已可應用於實驗中犬隻次單位疫苗之抗原鑑定，未來更可進一步可作為於免疫色層分析法 (Immunochromatographic assay, ICT) 之材料，應用於快速檢測犬隻病原。

Canine coronavirus (CCoV), Canine parvovirus (CPV) and Leptospira interrogans, All are common pathogens for canine. CCoV　and CPV can cause gastrointestinal disease of dogs. CPV will lead to canine respiratory disease and the Leptospira can affect liver and kidney function and cause internal bleeding and gastrointestinal diseases according to their different serotypes. CCoV enters the cell utilize spike protein. CPV VP2 protein is important role into host cells. Leptospirosis is use Lig.A and Lig.B proteins into host cells. In this study, we product protein by BL21 (DE3), After the fusion protein was purified immunized BALB / c mice, Whichever spleen cells with myeloma cells (NS1) for cell fusion, use enzyme-linked immunosorbent assay (ELISA) Preliminary screening antibody secreting tumor cell fusion, And TrxA-His and GST negative control group was further confirmation may identify specific antigen positive. After confirming via we were selected from two CCoV S1 protein of the monoclonal antibody, we named S1-35 and S1-38. In Lig.B Leptospira proteins have also been two monoclonal antibodies, named Lig.B-1 and Lig.B-2. Use of western blot and immune blot analysis showed that the S1-35 and S1-38 which identify antigens are non-configuration-dependent (linear antigen)；Lig.B-1 and Lig.B-2 which recognize the same antigen non-conformation-dependent decision bits. In Pierce ™ Rapid Antibody Isotyping Kit-Mouse (Thermo) antibody subtype analysis kit, S1-35 and S1-38 two monoclonal antibodies are all of its antibodies in IgG1, the same as the antibody light chain κ, The Lig.B-1 and Lig.B-2 two monoclonal antibodies for antibody subtype IgG1, the same as the antibody light chain κ. Similarly, to remove the mutation (deletion mutation) positioning mode antigenic determinant bit of analysis, Monoclonal antibodies S1-35 recognize S1 170-262 amino acids and monoclonal antibodies S1-38 recognize S1 262-353 amino acids, Lig.B-1 monoclonal antibody identified LigB 301-598 amino acids and monoclonal antibodies identify Lig.B 1-300 amino acids. Over the past years our laboratory Nian Lun Zheng locate CPV-VP2 epitopes bit of analysis prepared, The results showed that two strains of CPV-VP2 monoclonal antibody to identify the location of epitopes bit of VP2 protein were first and second amino acids 85-97 amino acids 52-62. Preparation of monoclonal antibodies identified in this study can be used in addition to antigen subunit vaccine experiments dogs, the future may further be used as in Immunochromatographic assay (ICT) of the material, for rapid pathogen detection.

目錄
致謝.................................................................................................................................i
中文摘要........................................................................................................................ii
英文摘要.......................................................................................................................iii
目錄...............................................................................................................................iv
圖目錄............................................................................................................................v
表目錄.........................................................................................................................vii
第一章 前言...................................................................................................................1
第二章 文獻探討...........................................................................................................2
2.1 犬小病毒之歷史背景.....................................................................................2
2.2 犬小病毒之特性.............................................................................................2
2.3 犬小病毒之傳播方式.....................................................................................3
2.4 犬小病毒之臨床症狀.....................................................................................3
2.5犬小病毒之治療..............................................................................................3
2.6 犬冠狀病毒之歷史背景.................................................................................3
2.7 犬冠狀病毒之特性.........................................................................................4
2.8 犬冠狀病毒之傳播方式.................................................................................5
2.9 犬冠狀病毒之致病機制.................................................................................5
2.10 犬冠狀病毒之臨床症狀...............................................................................6
2.11 犬冠狀病毒之治療.......................................................................................6
2.12 鉤端螺旋體之歷史背景...............................................................................7
2.13 鉤端螺旋體之特性.......................................................................................7
2.14 鉤端螺旋體之傳播方式...............................................................................8
2.15 鉤端螺旋體之臨床症狀...............................................................................9
2.16 鉤端螺旋體之治療.......................................................................................9
2.17 酵素連結免疫分析法 (enzyme-linked immunosorbent assay, ELISA).....9
2.18 單株抗體之種類介紹.................................................................................10
2.19 單株抗體之結構介紹.................................................................................10
2.20 單株抗體製備之發展過程.........................................................................10
第三章 材料與方法....................................................................................................11
3.1犬冠狀病毒及鉤端螺旋體基因之建構........................................................11
3.1.1 引子 (primer) 設計...........................................................................11
3.1.2 聚合酶連鎖反應 (polymerase chain reaction；PCR) .....................11
3.1.3 PCR產物純化.....................................................................................11
3.1.4 pET21a-CCoV S1之載體建構...........................................................11
3.1.5 勝任細胞製備....................................................................................11
3.1.6 轉型作用 (transform) .......................................................................12
3.1.7 重組質體之抽取與鑑定....................................................................12
3.2 小量抗原製備及確認...................................................................................12
3.2.1十二烷基硫酸鈉聚丙烯酰胺凝膠電泳.............................................12
3.2.2 西方墨點法 (Western blot) ..............................................................12
3.3 CCoV S1及 Leptospira interrogans Lig.B 融合蛋白之製備.....................13
3.3.1 CCoV S1及 Leptospira interrogans Lig.B 融合蛋白之大量表現...13
3.3.2 CCoV S1及 Leptospira interrogans Lig.B融合蛋白之純化及確認13
3.4 融合瘤細胞之製備.......................................................................................14
3.4.1實驗動物.............................................................................................14
3.4.2 免疫實驗............................................................................................14
3.4.3 棋盤式力價測定法 (Box titration) ..................................................14
3.4.4 間接型酵素免疫分析法 (indirect ELISA) ......................................15
3.4.5 骨髓瘤細胞培養................................................................................15
3.4.6 細胞融合............................................................................................15
3.4.7 HAT 培養........................................................................................16
3.5初步篩選可分泌目標抗體之融合瘤細胞....................................................16
3.5.1 酵素連結免疫分析法 (ELISA) .......................................................16
3.5.2 極限稀釋法 (limit dilution) .............................................................16
3.6 融合瘤細胞篩選...........................................................................................16
3.6.1 酵素連結免疫分析法 (ELISA) .......................................................16
3.6.2 免疫墨點法 (Immuno-dot assay) .....................................................16
3.6.3 西方墨點法 (Western blot) ..............................................................17
3.7 單株抗體之生產...........................................................................................17
3.7.1 BALB/c 小鼠腹水生產法.................................................................17
3.7.2 抗體的純化........................................................................................18
3.8 單株抗體之亞型分析 (isotyping) ..............................................................18
3.9 抗原決定位之定位 (epitope mapping) ......................................................18
3.9.1以CPV VP2、CCoV S1及Leptospira interrogans Lig.B之不同缺
損蛋白為材料進行抗原決定位之定位..............................................18
3.9.2 西方轉漬法 (Western blot) ..............................................................19
第四章　結果..............................................................................................................20
4.1 CCoV 之 S1、 Leptospira interrogans 之 Lig.B, 及 CPV 之 VP2 基
因選殖及定序分析........................................................................................20
4.2 CCoV S1、Leptospira interrogans Lig.B融合蛋白表現及純化....................20
4.3 CCoV S1及Leptospira interrogans Lig.B免疫效果之確認........................20
4.4 融合瘤細胞之製備.......................................................................................20
4.5 融合瘤細胞篩選及單株化...........................................................................21
4.6 融合瘤細胞單株化後之抗體篩選及確認...................................................21
4.7 Epitope mapping試驗....................................................................................21
4.8 單株抗體之生產...........................................................................................22
4.9 單株抗體之亞型分析...................................................................................22
第五章　討論.............................................................................................................23
參考文獻.....................................................................................................................59
圖目錄
Figure 4-1. 電泳分析S1、Lig.B及VP2之PCR產物................................................27
Figure 4-2. S1蛋白之製備及純化..............................................................................28
Figure 4-3. Lig.B蛋白利用pGEX4T2表現載體生產目標蛋白................................29
Figure 4-4. 純化Lig.B蛋白並去除GST蛋白...........................................................30
Figure 4-5. ELISA檢測免疫His-S1融合蛋白後之小鼠其血清之抗體力價...........31
Figure 4-6. ELISA 檢測免疫Lig.B-GST融合蛋白後之小鼠其血清之抗體力價...32
Figure 4-7. NS-1及分泌S1抗體之融合瘤細胞. .......................................................33
Figure 4-8. NS-1及分泌Lig.B抗體之融合瘤細胞....................................................34
Figure 4-9. His-S1及Lig.B-GST融合瘤細胞進行單株化細胞後利用ELISA進
行初步篩選................................................................................................35
Figure 4-10. S1及Lig.B單株抗體經由ELISA排除陰性抗原辨認之可能性..........36
Figure 4-11. S1 protein及pET21a only (His) 之coomassie blue染色圖及利用
S1-35、S1-38單株抗體之西方墨點法圖................................................37
Figure 4-12. 胺基酸親水性及抗原性示意圖............................................................38
Figure 4-13. 電泳分析CCoV S1基因之PCR產物...................................................39
Figure 4-14. 以西方墨點法定位CCoV S1之單株抗體S1-35以及S1-38之抗原
決定位............... ....................................................................................40
Figure 4-15. 電泳分析CCoV S1 170-262、262-353基因片段之PCR產物.............41
Figure 4-16. 以西方墨點法測試CCoV S1之單株抗體S1-35以及S1-38...............42
Figure 4-17. CCoV單株抗體S1-35及S1-38辨認S1蛋白抗原決定位之總整理.. 43
Figure 4-18. 電泳分析Leptospira interrogans Lig.B 1-300及 301-598基因片段
之 PCR產物..........................................................................................44
Figure 4-19. 以西方墨點法測試Lig.B之單株抗體Lig.B-1以及Lig.B-2...............45
Figure 4-20. 電泳分析CPV-VP2基因之PCR產物...................................................46
Figure 4-21. 以西方墨點法測試TrxA-His-VP2之單株抗體VP25以及VP28之
抗原決定位之定位................................................................................47
Figure 4-22. 電泳分析CPV-VP2基因之PCR產物...................................................48
Figure 4-23. 以西方墨點法測試CPV VP2之單株抗體VP25以及VP28之抗原
決定位之定位........................................................................................49
Figure 4-24. 以西方墨點法測試CPV VP2之單株抗體VP25以及VP28之抗原
決定位之定位... ....................................................................................50
Figure 4-25. CPV單株抗體VP-25及VP-28辨認VP2蛋白抗原決定位之總
整理..........................................................................................................51
Figure 4-26. 利用抗原亞型分析套組分析S1單株抗體之亞型...............................52
Figure 4-27. 利用抗原亞型分析套組分析 Lig.B單株抗體之亞型........................53
Figure 4-28. 利用腹水生產單株抗體之小鼠與一般小鼠之對照圖........................54

表目錄
表1. 構築CCoV S1所使用之primers.......................................................................55
表2. 本實驗所使用模板來源....................................................................................56
表3. CPV-VP2、CCoV-S1及 Leptospira interrogans-Lig.B抗原決定位之定位所利 用之primer...........................................................................................................57

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