臺灣博碩士論文加值系統

由琵琶鼠(Corydoras aeneus) 病魚感染肝臟分離到的一株嗜水性產氣單胞菌(Aeromonas hydrophila) AH20病原菌株，於添加羊血的TSB培養基上形成一混濁溶血環(α-hemolysin)，顯示該菌株能分泌溶血性的酵素。以嗜水性產氣單胞菌的溶血毒素aerolysin當探針( probe )來檢測AH20菌株的體染色體，AH20菌株的溶血基因無法以南方氏核甘酸雜合法檢測到。AH20菌株釋出溶血毒素的特性在TSB培養液中生長，於進入對數後期時溶血力價達到高峰，之後急速下降；而磷脂脢C (phospholipase C)在溶血毒素下降後即進入細胞生長的靜止期時才開始分泌。利用此菌株之溶血毒素釋出特性，將AH20菌株培養在TSB培養液於37 oC生長至對數後期後，離心收集的細胞外產物，含有高量的溶血毒素但不含有磷脂脢C，經過過濾和濃縮，再經由Sephacryl S-100 HR色層分析依分子量大小部份純化， 依序分作80管收集液，其中會造成溶血的收集液和離心純化的吳郭魚紅血球細胞膜於37 oC反應30分鐘，比對SDS-PAGE電泳解析檢測吳郭魚紅血球的膜蛋白組成，結果可觀察到四條主要紅血球膜蛋白染色帶被分解掉。由於嗜水性產氣單胞菌會釋出多種細胞外產物，其中溶血毒素應包含可將細胞膜分解的蛋白脢和磷脂脢C。利用AH20菌株在TSB培養液於37 oC下生長，成長期與溶血毒素釋出時機之對應關係，可證實該菌株的溶血酵素功能應為蛋白脢而非磷脂脢C。 由SDS-PAGE染色解析此溶血酵素的分子量應小於39 kDa。若以AH20菌株的細胞培養液，於離心過濾去除細胞後將含溶血毒素的上清液作用於吳郭魚血、羊血及人血，其溶血力價都不相同；作用於吳郭魚的紅血球時溶血力價256最高，作用於人的紅血球用時其溶血力價為128，作用於羊的紅血球用時溶血力價最低為32。

Aeromonas hydrophila strain AH20 was isolated from Corydoras aeneus which shown the symptom of hemorrhage. A plasmid, pPH4, which carry a unique aerolysin sequence was labeled by DIG and used as a probe to hybridize strain AH20 chromosomal DNA. Homologous DNA residing in the AH20 genome could not be detected. Enzymatic activity of either protease or phospholipase C, both are postulated to be virulent factors to destroy red blood cell (RBC) membrane and consequently resulting in hemolysis. An interesting characteristic of this strain AH20 is observed as that the secretion of hemolysin is growth-phase dependent. The highest hemolytic unit was monitored in the late log-growth phase, then decline soon. On the other hand, while entering into the stage of stationary-phase, AH20 began to export phospholipase C only in the stationary phase and sustain 30-40 hours. Yields of concentrated extracellular products (ECP) from the culture in the state of late log phase were resolved by Sephacryal S-100 HR chromatography. The partial purified ECP from the collected eluents were mixed with tilapias'' erythocyte''s membrane for enzymatic digestion. Function of degrading RBC membrane proteins which determined by SDS-PAGE resulted in the conclusion of that the hemolysin exporting by strain AH20 is due to the activity of protease not phospholipase C.

目錄壹、中文摘要………………………………………………………..1英文摘要………………………………………………………..3貳、前言……………………………………………………………..4參、材料與方法…………………………………………………….11一. 測AH20菌株之特性………………………………………..11（i）溶血力價……………………………………………..11(ii)脂肪脢活性測定……………………………………….12二. 大量質體的萃取…………………………………………….13三. 大腸桿菌DH5α勝任細胞之製備…………………………. 14四. 電穿孔法…………………………………………………….15五. 三層包埋…………………………………………………….16六. 嗜水性產氣單胞菌體染色體的製備……………………….16七. 南方氏點墨法雜交試驗…………………………………….17（i）DNA的酵素剪切…………………………………………17（ii）製作南方氏點墨法探針pPH4…………………………18(iii)Acrylamide gel的作法……………………………….18（iv）分離DNA片段…………………………………………18(v)純化pPH4 DNA…………………………………………..18(vi)標幟反應…………………….……………………………19(vii)洋菜膠之配置……………………………………….….19(viii)轉移洋菜膠之DNA至Nylon member………………….20(ix)Hybridization…………………………………………….20(x)Post hybridization and detection product…………..20八.吳郭魚紅血球之處理…………….……………………………..21紅血球細胞膜與菌上清液之作用……………………………….22九.SDS聚丙醯胺膠之配製………………………………………….22SDS-PAGE之染色………………………………………………..23SDS-PAGE之退染…………………………………………………23十.Native-PAGE的配製…………………………………………….23Native-PAGE的清洗和壓片….………………………………..24十一.色層分析Sephacryal S-100 HR管柱的設立……………….24Sephacryal S-100 HR分離AH20之細胞外粗產物…………..25Sephacryal S-100 HR的清洗………….……………………..25蛋白量的測定………………….……………………………….25肆、結果……………………………………………………………….26一、吳郭魚紅血球細胞膜蛋白質電泳解析…….……………..26二、AH20菌株特性之探討……………………………………….26三、電泳及色層分析AH20菌株培養液細胞外產物…………….28四、南方氏核甘酸雜合…………………………………………..29五、aerolysin和AH20溶血毒素的比較……..………………..29伍.討論…………………………………………………………………31陸.附圖………………………………………………………………..35柒.參考文獻…………………………………………………………..46捌.謝辭………………………………………………………………..50玖.作者簡歷…………………………………………………………..51

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