臺灣博碩士論文加值系統

中文摘要
本試驗旨在研究D型巴氏桿菌毒素（Pasteurella multocida type D toxin，PMT）基因（tox A），除了對此基因之產物加以定性，並進一步分別求證PMT之毒性及其免疫性是座落在5’端或3’端？PMT毒素基因全長為3.8 kb，其分子量約為143 kDa，惟全段3.8 kb太長，因此，以基因刪除（Gene deletion）方法將全段PMT 3.8 kb分為5’端deletion （Δ5’）及3’端deletion （Δ3’）等兩段進行選殖及大量表現及純化其GST融合蛋白，共選殖有1.5 kbΔ3’ clone 1521（83 kDa）、2.3 kbΔ5’ clone 23XL-17（112kDa） 及連結LT clone 23LTBXL-20（126 kDa）等基因片段，與取自Pasteurella multocida type D 所抽取之PMT（143 kDa）分別進行天竺鼠皮膚毒性試驗結果獲知源自Pasteurella multocida之PMT（143 kDa）及Δ3’ clone1521（83 kDa）對天竺鼠皮膚會產生出血壞死病變，可見PMT之毒性座落在5’端，而Δ5’ clone 23XL-17（112 kDa）對天竺鼠皮膚並無產生病變。以西方氏轉漬法分析結果，上述所選殖之表現蛋白均具有抗原性。至於PMT之免疫性可由對小鼠之保護效力結果得知取自Pasteurella multocida type D之 PMT（143 kDa）為100﹪（8/8），其免疫後母鼠所產下的第一子代保護效力亦為100﹪（8/8），1.5 kbΔ3’ clone 1521（83 kDa）為0﹪（0/8），2.3 kbΔ5’ clone 23XL-17（112kDa）免疫劑量（5μg）免疫組為 25﹪（2/8），免疫劑量（2.5μg）免疫組為 62.5﹪（5/8），免疫劑量（1.25μg）免疫組為 87.5﹪（7/8），至於連結LT clone 23LTBXL-20（126 kDa）免疫劑量（5μg）免疫組為37.5﹪（3/8），免疫劑量（2.5μg）免疫組為12.5﹪（1/8），免疫劑量（1.25μg）免疫組為 25﹪（2/8），由上述免疫保護效力之結果顯示重組PMT之免疫性基因座落在PMT基因之3’端，亦即2.3 kbΔ5’ clone 23XL-17（112kDa）組中免疫劑量（1.25μg）之免疫組具有87.5﹪之保護效力，惟該clone連接LTB後則免疫保護效力下降至12.5﹪至37.5﹪之間，此可能LTB與PMT基因3’端之PMT 2.3 kb之基因融合後，所表現之重組PMT是否在結構上因而有所改變，而導致免疫性亦隨之改變，有待日後進一步之探討。

Abstract
For characterization of Pasteurella multocida toxin （PMT）derived from tox A gene of Pasteurella multocida type D had been cloned. This study was also focusing on the epitopes of the PMT which could be encoded in either 5’ end or 3’ end of tox A gene﹐and then the immunogenic part of PMT will be studied by using an approach of gene deletion and polymerase chain reaction（PCR）cloning technique as well. Two clones‚ 1.5 kb Δ3’ clone 1521 and 2.3 kb Δ5’ clone 23XL-17 were selected since they were harboring the fragment of PMT gene of interest inserted into the pGEX6p-1 in E. coli BL21 ( DE-3 ). After expression‚ the purified GST fusion protein with molecular weight of 83 kDa ( 1.5 kb Δ3’ clone 1521 ) and 112 kDa ( 2.3 kbΔ5’ clone 23XL-17 ) were obtained respectively. The 83 kDa ( 1.5 kb Δ3’ clone 1521 ) other than 112 kDa ( 2.3 kb Δ5’ clone 23XL-17 ) and PMT ( 143 kDa ) originally derived from P. multocida type D demonstrated toxic activity in skin test in guinea pigs. It revealed that the functional toxicity of PMT from tox A gene could be located on 5’ end of PMT gene itself. The purified 83 kDa and 112 kDa GST fusion protein were strongly reactive to the specific antibody against PMT arised from immunized rabbits by using Western blotting assay in terms of antigenicity. Moreover‚ Only the 112 kDa ( 2.3 kb Δ5’ clone 23XL-17 ) had the immunoprotection as high as 87.5﹪( 7/8 ) against‚ ip‚ PMT challenge in BALB/c mice mean while 83 kDa ( 1.5 kb Δ3’ clone 1521 ) and control group of mice had no immunoprotection 0﹪( 0/8 ) at all against the same challenge. However‚ the LTB gene from heat-labile enterotoxin B subunit of E. coli which could be an adjuvant potential fused to 2.3 kb Δ5’ clone 23XL-17 derived from PMT gene and expressed as 126 kDa ( 2.3 kb Δ5’ linked to LTB, clone 23LTBXL-20 ) GST fusion protein. It showed unexpectedly immunoprotection rate as low as 12.5﹪( 1/8 ) to 37.5﹪( 3/8 ). This could be due to the conformational changes of antigen after fusion LTB gene to 2.3 kb of PMT gene ( Δ5’ clone 23XL-17 ). Further research need to be studied in the near future.

目錄
頁次
中文摘要 Ⅰ
英文摘要 Ⅲ
誌謝 Ⅴ
目錄 Ⅶ
圖次 Ⅹ
表次 ⅩⅢ
第一章 前言 1
第二章 文獻探討 4
2. 1 巴氏桿菌 4
2. 2 巴氏桿菌之型態及生化性狀 5
2. 3 巴氏桿菌之分離及鑑定 6
2. 4 豬萎縮性鼻炎之臨床性狀及組織病理學變化 7
2. 5 豬萎縮性鼻炎之致病機轉 9
2. 6 巴氏桿菌毒素 12
2. 6. 1 巴氏桿菌毒素純化及其特性 12
2. 6. 2 巴氏桿菌毒素之生物活免疫學特性 16
2. 6. 3 巴氏桿菌毒素之毒素基因 20
2. 7 豬萎縮性鼻炎之疫苗 23
第三章 材料及方法 25
3. 1 菌株分離及鑑定 25
3. 2 巴氏桿菌毒素之製備 25
3. 3 引子設計 26
3. 4 模板 ( Template ) DNA之製備 27
3. 5 聚合酶鏈反應 ( Polymerase chain reaction; PCR ) 29
3. 6 瓊膠（agarose）電泳 30
3. 7 PCR產物及載體Vector DNA之處理 30
3. 8 連結（Ligation） 31
3. 9 轉型（Transformation） 31
3. 10 選殖（Cloning） 32
3. 11 次選殖（Subcloning） 33
3. 12 表現（Expression） 33
3. 13 DNA序列之測定 34
3. 14 DNA序列之比對分析 34
3. 15 GST（Glutathione S-transferase）融合蛋白純化 35
3. 16 聚丙烯醯胺膠片（Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis，SDS-PAGE ）
電泳分析 36
3. 17 蛋白質濃度測定（Protein assay） 36
3. 18 兔子抗PMT毒素抗體之製備 37
3. 19 西方氏轉漬法（Western blotting） 37
3. 20 動物毒性試驗 38
3. 20. 1 PMT最小致死量（Minimum lethal dose）測定
38
3. 20. 2 肉眼及組織病理學檢查 39
3. 20. 3 天竺鼠皮膚壞死試驗 40
3. 21. 疫苗效力試驗 40
3. 21. 1 攻擊（Challenge） 40
3. 21. 2 幼鼠移行抗體保護效力試驗 41
第四章 結果 42
4. 1 全段3.8 kb之巴氏桿菌毒素基因選殖 42
4. 2 Δ3’ 1.5 kb之巴氏桿菌毒素基因選殖 43
4. 3 Δ5’ 2.3 kb之巴氏桿菌毒素基因選殖 43
4. 4 Δ5’ 2.3 kb之巴氏桿菌毒素基因連結E.coli忌熱性腸毒素B次單位（LTB）基因融合之選殖
44
4. 5 重組蛋白質之表現及純化 45
4. 6 重組蛋白質之DNA及胺基酸序列之比對及分析 46
4. 7 巴氏桿菌毒素及表現純化之重組蛋白之毒性試驗 46
4. 8 組織病理學變化 47
4. 9 PMT重組蛋白之抗原性 48
4. 10 PMT重組蛋白之免疫性 48
第五章 討論 50
參考文獻 81
附錄 107
附錄一 DNA之純化 107
附錄二 質體之抽取 109
附錄三 Glutathione Sepharose 4B再生及保存 111
附錄四 蛋白質濃度測定之標準曲線 113
作者簡介 114
圖次
頁次
圖4-1. 以Api 20E快速鑑定Pasteurella multocida（本試驗所分離之菌株編號為Pm-5） 56
圖4-2. 載體pGEX6p-1之圖式 57
圖4-3. PMT △3’ 1.5 kb clone 1521之Insert DNA之確認1.2％ agarose電泳圖：M：λⅡ DNA marker (以Hind III切割)，列1：100 bp DNA marker，列2、3：PMT 1.5 kb clone 1521。抽取PMT △3’ 1.5 kb clone 1521重組質體之DNA，以BamH I與XhoI限制酶切割後，確有Insert DNA 1.5 kb與載體pGEX6P-1DNA為4.9 kb 58
圖4-4. PMT △5’ 2.3 kb PCR產物之0.9％ agaorse電泳圖：M：λⅡ DNA marker (以Hind III切割)，列1-2：增幅PMT 基因之PCR product 2.3 kb 59
圖4-5. PMT △5’ 2.3 kb clone 23XL-17之Insert DNA之確認1.2％ agarose電泳圖：M：λⅡ DNA marker (以Hind III切割)，列1-14為抽取PMT △5’ 2.3 kb 之14個選殖菌株之重組質體DNA並以BamH I及Sma I限制酶切割後，其中列2確有PMT 2.3 kb之 Insert DNA存在，列6為pGEX6P-1（4.9 kb） 60
圖4-6. PMT △5’ 2.3 kb clone 連結LTB之Insert DNA之確認1.2％ agarose電泳圖：M：λⅡ DNA marker (以Hind III切割)，列1：100 bp DNA ladder，列2-7為所篩之菌株，其中列3、5、6確有LTB 372 bp之 Insert DNA存在。篩選PMT △5’ 2.3 kb 與連結LTB基因之菌株抽取其重組質體DNA，並以clone 19-24重組質體之DNA。以BamH I限制酶切割 61
圖4-7. PMT △3’ 1.5 kb clone 1519-1522純化表現蛋白之10％ SDS-PAGE：M：蛋白分子量之marker，列1-4為所篩選之菌株（菌株編號為PMT △3’ 1.5 kb clone 1519-1522）所表現之純化的GST融合蛋（83 kDa） 62
圖4-8. PMT △5’ 2.3 kb clone 23XL-17純化表現蛋白之10％ SDS- PAGE：M：蛋白分子量之marker，列1：PMT △5’ 2.3 kb clone 23XL-17 所表現之粗製蛋白，列2：PMT △5’ 2.3 kb clone 23XL-17 所表現之純化的GST融合蛋白（112 kDa） 63
圖4-9. PMT △5’ 2.3 kb連結LTB clone 23LTBXL-20及23純化表現蛋白之10％ SDS-PAGE：M：蛋白分子量之marker，列1：PMT △5’ 2.3 kb連結LTB clone 20所表現之純化的GST融合蛋白（126 kDa），列2：PMT △5’ 2.3 kb連結LTB clone 23LTBXL-23所表現之純化的GST融合蛋白（126 kDa） 64
圖4-10. PMT △5’ 2.3 kb clone 23XL-17之DNA序列圖 65
圖4-11. PMT △5’ 2.3 kb clone 23XL-17之胺基酸序列圖 69
圖4-12. 天竺鼠皮膚毒性試驗之結果：1~3呈現出血壞死之病灶，4及5無任何病變。1：取自Pasteurella multocida type D之PMT，2：PMT 3.8 kb clone 90表現純化之GST融合蛋白（170 kDa），3：PMT △3’ 1.5 kb clone 1521表現純化之GST融合蛋白（83 kDa），4：PMT △5’ 2.3 kb clone 23XL-17表現純化之GST融合蛋白（112 kDa），5：陰性對照組（0.85﹪NaCl） 71
圖4-13. PMT（陽性對照組）之組織學變化：於天竺鼠皮膚接種之部位其鏡下可見皮膚之真皮層呈廣泛性嚴重充、出血。H&E，A：100倍，B：400倍 72
圖4-14. PMT 3.8 kb clone 90之組織學變化：於天竺鼠皮膚接種之部位其鏡下亦可見皮膚之真皮層呈廣泛性嚴重充、出血。H&E，A：100倍，B：400倍 73
圖4-15. PMT △3’ 1.5 kb clone 1521之組織學變化：於天竺鼠皮膚接種之部位其鏡下可見皮膚之真皮層充、出血。H&E，A：100倍，B：400倍 74
圖4-16. PMT △5’ 2.3 kb clone 23XL-17之組織學變化：於天竺鼠皮膚接種之部位其鏡下可見皮膚之真皮層無任何病變。H&E，A：100倍，B：400倍 75
圖4-17. 生理食鹽水（陰性對照組）之組織學變化：於天竺鼠皮膚接種之部位其鏡下可見皮膚之真皮層為正常皮膚構造。H&E，A：100倍，B：400倍 76
圖4-18. PMT △3’ 1.5 kb clone 1521之西方氏轉漬法：列1：PMT △3’ 1.5 kb clone 1521所表現及純化之GST融合蛋白（83 kDa），列2：該純化之GST融合蛋白能與特異性抗體（兔子抗PMT）產生特異性的結合反應 77
圖4-19. PMT △5’ 2.3 kb clone 23XL-17之西方氏轉漬法：M：蛋白分子量之marker，列1：PMT △5’ 2.3 kb clone 23XL-17所表現之純化的GST融合蛋白（112 kDa）（轉印自SDS-PAGE），列2：PMT △5’ 2.3 kb clone 23XL-17所表現之純化的GST融合蛋白（112 kDa）能與特異性抗體（兔子抗PMT）產生特異性的結合反應 78
表次
頁次
表 4-1. PMT重組蛋白之天竺鼠皮膚毒性試驗結果 79
表 4-2. PMT重組蛋白之BALB/c鼠免疫保護效力試驗結果 80

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