臺灣博碩士論文加值系統

本論文以分離自罹病草蝦( Penaeus monodon)之病原弧菌Vibrio harveyi，探討其菌體、細胞外產物及部分純化蛋白分解酵素對蝦類血漿中凝血蛋白(coagulogen)之影響，分別進行體內及體外的試驗，以進一步了解本弧菌對蝦類感染之致病機制及疾病生理現象。
本研究先製備V. harveyi細胞外產物，並部分純化出具致死性之蛋白分解酵素。此部份純化之蛋白分解酵素，以SDS-PAGE鑑定分子量，在未加熱處理時，其具有酵素活性的蛋白帶分子量為22 kDa，但如加入sample buffer (含2-mercaptoethanol)再予以加熱處理，會使分子量轉變為38 kDa。
觀察南美白蝦(Litopenaeus vannamei)與草蝦凝血蛋白與血藍素在SDS-PAGE的位置皆很相近，以交叉免疫電泳與西方點墨法可觀察到南美白蝦與草蝦的凝血蛋白在電泳位移、分子量與免疫性方面具極高的相似性。
以交叉免疫電泳，SDS-PAGE及Western blotting等方法探討V. harveyi細菌、細胞外產物、部分純化蛋白分解酵素三者對白蝦血漿之影響。結果發現V. harveyi部分純化蛋白分解酵素在體外會使白蝦血漿中的凝血蛋白發生電泳移動較快的現象，而細胞外產物主要除了影響凝血蛋白之外也影響白蝦血漿中血藍素之電用位移。使用西方點墨法，可證明凝血蛋白被作用後會降解成較小分子量的蛋白質。活菌在體外的試驗條件下對白蝦血漿並無顯著的影響，但在體內試驗，活菌、細胞外產物與部分純化的蛋白分解酵素皆會讓蝦體中的血漿蛋白量減少，亦使血漿中凝血蛋白的含量降低。就影響的程度而言則為細胞外產物最大，其次為部分純化的蛋白分解酵素。以西方點墨法測試也初步證明在白蝦蝦體受感染後凝血蛋白會降解成主要約89kDa次要約43 kDa的小片段蛋白質。草蝦凝血蛋白被降解成大小約80 kDa的小片段蛋白質
本研究結果顯示弧菌V. harveyi菌體、細胞外產物及部分純化致死蛋白分解酵素會影響蝦血漿中成分，尤其是蝦體的凝血機制，特別是造成凝血蛋白的降解。此為首次證明蝦類弧菌對血漿凝血蛋白的影響為使其降解，造成血淋巴無法凝結，且致死蛋白分解酵素可能扮演了本弧菌感染蝦類致病機制之主要角色。

This thesis investigates the effects of bacterial cells, extracellular products (ECP) and partially purified protease of Vibrio harveyi, originally isolated from diseased tiger shrimp (Penaeus monodon), on plasma coagulogen of shrimps in vitro and in vivo, in order to further understand the pathogenesis and pathophysiological phenomenon caused by the Vibrio species.
The ECP of V. harveyi was prepared and the toxic protease was partially purified. This partially-purified protease was a 22 kDa protein, determined by SDS-PAGE, exhibiting caseinolytic activity without boiling while it was a 38 kDa protein after boiling in sample buffer (contained 2-mercaptoethanol).
On SDS-PAGE, the molecular weights of coagulogen and hemocyanin of white shrimp (Litopenaeus vannamei) and tiger shrimp found similar. In addition, the migration, molecular weights and immune cross-reactivity of coagulogen of both shrimp species were also found were similar.
The effects of bacterial cells, ECP and partially purified protease of V. harveyi were investigated using crossed immunoelectrophoresis (CIE), SDS-PAGE and Western blotting. The results revealed that a fast- migrating phenomenon of plasma coagulogen in CIE gels could be observed after pre-incubation with partially purified protease. In addition to fast-migrating of plasma coagulogen, the hemocyanin was also affected after pre-incubation with the ECP. Plasma coagulogen was proven to be degraded into smaller molecular-weight proteins on Western blot, plasma coagulogen was not remarkably affected after pre-incubation with bacterial cells. However, the reduction of plasma protein content and coagulogen amount was determined when the shrimps were pre-injected with bacterial cells, ECP and partially purified protease, separately. As for the degree of reduction, the group pre-injected with ECP was the highest while that pre-injected partially purified protease was the second. The plasma coagulogen was degraded into 89 kDa and 43 kDa, and 80 kDa protein in white shrimp and tiger shrimp, respectively.
The present results revealed that plasma components and blood clotting mechanism of shrimp could be affected by bacterial cells, ECP and partially purified protease. In particular, the plasma coagulogen was degraded. This is the first report which demonstrates that plasma coagulogen of shrimp is degraded when the animal is infected with V. harveyi leading to occurrence of unclottabe hemolymph. Meanwhile, the toxic protease may play a major role in the pathogenesis of V. harveyi infection.

目 錄
中文摘要 ------------------------------------------------- V
英文摘要 ------------------------------------------------- VII
圖目錄 --------------------------------------------------- IX
一、前言 ------------------------------------------------- 1
二、文獻整理 --------------------------------------------- 4
三、材料方法---------------------------------------------- 15
3.1 弧菌及其細胞外產物 ----------------------------------- 15
3.1.1 菌株的活化、毒力強化、再鑑定及保存------------------ 15
3.1.2 V. harveyi菌體的生產 ------------------------------ 15
3.1.3 V. harveyi細胞外產物的取得 ----------------------- 16
3.2 V. harveyi細胞外致死蛋白分解酵素的部分純化及其特性---- 16
3.3 蛋白質含量的測定 ------------------------------------- 17
3.4 蛋白分解酵素活性的測定 ------------------------------- 17
3.5 電泳及染色 ------------------------------------------- 17
3.5.1 PhastSystem SDS-PAGE ------------------------------ 17
3.5.2 蛋白質膠體電泳（sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, SDS-PAGE）------------------------ 18
3.6 Western Blotting-------------------------------------- 18
3.7電泳、交叉免疫電泳膠片之蛋白分解酵素活性測定 ---------- 19
3.8白蝦血漿、血清草蝦血漿的取得及保存 -------------------- 20
3.8.1 實驗用白蝦與草蝦 ----------------------------------- 20
3.8.2 血漿的取得 ----------------------------------------- 20
3.8.3 血清的取得 ----------------------------------------- 21
3.8.4 血漿、血清的保存 ----------------------------------- 21
3.9兔子抗白蝦血漿(plasma)抗血清的製備---------------------- 21
3.10 交叉免疫電泳(Crossed Immunoelectrophoresis, CIE) ---- 21
3.11抗血清中抗體含量分析---------------------------------- 22
3. 12 V. harveyi影響白蝦血淋巴成分之體外實驗 ------------- 23
3.12.1 白蝦血漿凝結蛋白物質於交叉免疫電泳中的相對位置及確認 23
3.12.2 V. harveyi 細菌於體外對白蝦血漿的影響-------------- 23
3.12.3 V. harveyi細胞外產物於體外對白蝦血漿不同時間下的影響 23
3.12.4 V. harveyi部分純化致死蛋白分解酵素於體外對白蝦血漿不同時間下的影響 ----------------------------------------------- 24
3.13、V. harveyi影響草蝦血淋巴成分之體外組實驗---------- 24
3.13.1 V. harveyi細胞外產物對體外白蝦血漿不同時間下的影響24
3.13.2 V. harveyi部分純化致死蛋白分解酵素對草蝦血漿不同時間
下的影響--------------------------------------------------- 25
3.14 V. harveyi 細菌、細胞外產物、部分純化致死蛋白分解酵素
於體內對白蝦與草蝦血漿的影響------------------------------- 25
四、結果
4.1 V. harveyi 部分純化之致死蛋白分解酵素------------------ 27
4.2抗血清中抗體力價之測定---------------------------------- 27
4.3 白蝦血漿凝血蛋白於交叉免疫電泳中相對位置之確認 -------- 28
4.4 V. harveyi細菌於體外對白蝦血漿的影響------------------- 29
4.5 V. harveyi 細胞外產物於不同時間下對白蝦血漿的影響 (in vitro) ---- 29
4.6 V. harveyi 部分純化致死蛋白分解酵素於不同時間下對白蝦血漿
的影響(in vitro)------------------------------------------- 30
4.7 V. harveyi 細胞外產物於不同時間下對草蝦血漿的影響
(in vitro) ------------------------------------------------- 31
4.8 V. harveyi 部分純化致死蛋白分解酵素於不同時間下對
草蝦血漿的影響(in vitro) ----------------------------------- 32
4.9 V. harveyi細菌、細胞外產物、部分純化致死蛋白分解酵素於體內
對白蝦血漿的影響-------------------------------------------- 32
4.9.1 V. harveyi細菌於體內(in vivo)對白蝦血漿的影響--------- 33
4.9.2 V. harveyi 細胞外產物於體內(in vivo)對白蝦血漿的影響-- 33
4.9.3 V. harveyi 部分純化致死蛋白分解酵素於體內(in vivo)對
白蝦血漿的影響---------------------------------------------- 34
4.10 V. harveyi細菌、細胞外產物及部分純化致死蛋白分解酵素
於體內對草蝦血漿的影響-------------------------------------- 34
五、討論
5.1 V. harveyi 部分純化致死蛋白分解酵素--------------------- 36
5.2 白蝦血漿凝結蛋白物質於交叉免疫電泳中相對位置之確認------ 36
5.3 V. harveyi細菌、細胞外產物及部分純化致死蛋白分解酵素
於體外(in vitro)、體內(in vivo)對白蝦血漿的影響 ------------ 37
5.4 V. harveyi細菌、細胞外產物及部分純化致死蛋白分酵素
於體外(in vitro)、體內(in vivo) 對草蝦血漿的影響------------ 41
六、結論---------------------------------------------------- 44
七、參考文獻 ----------------------------------------------- 45

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