臺灣博碩士論文加值系統

親水性產氣單胞菌( Aeromonas hydrophila ) 會引起甲魚 ( 鱉 ) 肝、脾、腎腫大出血，腹水，腸道出血，黏膜下層水腫及體表潮紅出血等病變。本試驗根據其頗具毒力之外毒素hemolysin (hly A) 基因設計一引子對，並以甲魚分離而來之親水性產氣單胞菌之DNA為模板，以聚合酶鏈反應方法增幅出親水性產氣單胞菌A. hydrophila之hemolysin基因之片段，其大小為1866 bp。將此DNA片段及載體 pGEX-6P-1分別以Eco RI及Xho I切割後，再以T4黏接酶黏接至載體中進行重組質體之構築後，轉殖到大腸桿菌DH5α中進行表現。將表現產物以GST親和性管柱純化後，可純化出一種重組融合蛋白，其分子量約為100 kDa。將含有hly A基因所選殖之菌株進行溶血試驗，證實其可對血液培養基產生β-溶血現象。上述表現之重組融合蛋白 ( 100 kDa ) 以西方氏轉漬法分析結果獲知該表現之重組蛋白 ( 100 kDa ) 能與特異性抗體產生強烈之結合反應，足見此重組融合蛋白確具有抗原性 ( antigenicity )。在動物試驗方面，將此純化後表現之重組蛋白以腹腔注射小鼠進行毒性試驗，結果可造成小鼠之死亡，其LD50為320μg/ 22~24 公克體重，經注射後死亡之小鼠剖檢後可見胃及腸管內容有水腫液蓄積、漿膜面潮紅。其主要之組織病理學變化為胃黏膜上皮壞死脫落、腸管黏膜杯狀細胞大量增生，證明此純化表現之重組蛋白不但具有抗原性，而且是一種具有功能性之蛋白質，期能來日經過減毒後用於A. hydrophila基因工程次單位疫苗研發之重要參考。

Aeromonas hydrophila is pathogenic to soft-shell turtle. It could cause hemorrhagic lesions in gastric tract, liver, spleen and kidneys and body surface of the soft-shell turtle as well. A. hydrophila could be isolated from the infected soft-shell turtle. Since hemolysin ( hly ) gene is a distinguished exotoxin of A. hydrophila, we design a pair of specific primers for the amplification of hly gene by using polymerase chain reaction ( PCR ). The amplificon was 1866 bp to be used as an inserted DNA. After the inserted DNA fragment and vector, pGEX6P-1, were digested with Eco RI and Xho I respectively, and then T4 ligase was employed for the ligation and construction of the recombinant plasmid. The recombinant plasmid was transferred to DH5α E.coli for expression. The purified expressed recombinant fusion protein was obtained by using GST affinity column and was strongly reactive to the specific antibody arisen from rabbit by using Western blotting assay in terms of antigenicity. However, in vivo, the purified fusion protein was toxic to BALB/c mice. After intraperitoneal injection, the mice were suffering from congestion and edema in intestines showing intestinal goblet cells massive infiltration and necrosis in the epithelial cells of stomach, multifocal necrosis in liver. In the other hand, this recombinant protein capable of inducing lesions in mice indicates that it is a functional protein and it’s pathogenicity against mice was demonstrated. However, it could be potential used as a candidate antigen for the development of subunit vaccine against A. hydrophila if the recombinant fusion protein could be appropriately attenuated in the near future.

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中文摘要……………………………………………………………… I
英文摘要………………………………………………………………III
誌謝……………………………………………………………………IV
目錄……………………………………………………………………V
表次……………………………………………………………………IX
圖次……………………………………………………………………X
第1章 前言……………………………………………………………1
第2章 文獻探討………………………………………………………3
2.1自然界中之親水性產氣單胞菌（Aeromonas hydrophila）… 3
　 2.1.1 親水性產氣單胞菌之型態及性狀…………………… 3
2.1.2 親水性產氣單胞菌之生長條件………………………… 5
2.1.3 親水性產氣單胞菌之產毒條件………………………… 5
2.1.4 親水性產氣單胞菌對環境之耐受性…………………… 6
2.2 親水性產氣單胞菌之分類特性……………………………… 7
2.2.1 親水性產氣單胞菌之命名……………………………… 7
2.2.2 親水性產氣單胞菌之分類特徵………………………… 8
2.3 親水性產氣單胞菌之病害與治療…………………………… 12
　 2.3.1 親水性產氣單胞菌之感染範圍………………………12
2.3.2 親水性產氣單胞菌對養殖漁業之危害………………13
2.3.3 親水性產氣單胞菌對人類之致病性…………………15
2.3.4 親水性產氣單胞菌之抗藥性…………………………16
2.4 親水性產氣單胞菌之致病因子……………………………… 17
2.4.1 親水性產氣單胞菌致病因子之種類..……………… 17
2.4.2 親水性產氣單胞菌之內毒素致病因子………………20
2.4.3 親水性產氣單胞菌之外毒素致病因子………………21
第3章 材料及方法………………………………………………… 26
3.1 菌株來源……………………………………………… 26
3.1.1 親水性產氣單胞菌之來源與鑑定………………………. 26
3.2製備不活化A. hydrophila及其抗體……………………… 26
3.2.1 親水性產氣單胞菌A. hydrophila不活化菌株之製備26
3.2.2 親水性產氣單胞菌多株抗體之製備………………… 27
3.3 重組質體之構築與製備………………………………... 27
3.3.1 模板 (template) DNA之製備……………………… 27
3.3.2 親水性產氣單胞菌A. hydrophila引子之設計.…… 28
3.3.3 以PCR增幅A. hydrophila hemolysin gene ( hly A )序列.. 29
3.3.4 表現載體pGEX6P-1…………………………………… 30
3.3.5 PCR產物及載體 (vector) DNA之限制酶處理…… 30
3.3.6 標的序列與載體之接合反應 (ligation).………… 32
3.3.7 勝任細胞 (competent cell)之製備.……………… 32
3.3.8 轉型反應 (transformation)……………………… 33
3.3.9 單一菌落之溶血性狀試驗…………………………… 34
3.3.10 篩選 (screening)………………………………… 34
3.3.11 重組DNA之定序 (sequencing)…………………… 35
3.4 重組蛋白之表現與分析……………………………… 36
3.4.1 重組蛋白之表現與萃取……….…………………… 36
3.4.2 重組蛋白之純化……………………………………… 37
3.4.3 重組蛋白之分子量確認……………………………… 37
3.4.4 重組蛋白之抗原性分析……………………………… 38
3.5 重組蛋白之動物試驗……………………………....... 39
3.5.1 大量hly A基因融合蛋白之製備…………………… 39
3.5.2 hly A基因融合蛋白標籤蛋白( tag protein)之切割..…….. 40
3.5.3 重組蛋白濃度之測定………………………………… 40
3.5.4 重組蛋白之毒性試驗………………………………… 41
3.5.5 組織病理切片之製作及判讀…………………………… 41
第4章 結果……………………………………………………………43
4.1 親水性產氣單胞菌不活化菌株之結果確認……………… 43
4.1.1 不活化菌株之菌落觀察……………………………… 43
4.2 重組質體之構築…………………………………………… 43
4.2.1 聚合酶鏈反應之結果………………………………… 43
4.2.2 單一菌落之溶血性狀觀察結果……………………… 43
4.2.3 A. hydrophila hemolysin ( hly A ) gene 之選殖結果…….. 45
4.2.4 核苷酸及胺基酸序列之比對及分析………………… 45
4.3 重組蛋白之表現及分析……………………….……………50
4.3.1 hly A融合基因之表現結果……………………………50
4.3.2 hly A基因融合蛋白純化之結果………………………50
4.3.3 重組蛋白之抗原性分析……………………………… 52
4.4 重組蛋白之動物試驗結果…………………….……………52
4.4.1 重組蛋白濃度測定結果……………………………… 52
4.4.2 重組蛋白於小鼠致死劑量試驗之結果……………… 54
4.4.3 組織病理學變化之結果……………………………… 59
第5章 討論……………………………………………………………63
5.1 hly A基因之選殖………………………….….……………63
5.2 hly A基因重組蛋白之抗原性與活性之分析………………66
5.3 未來展望………………………………….…………………69
參考文獻………………………………………………………………70
作者簡介………………………………………………………………83

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