臺灣博碩士論文加值系統

嗜水性產氣單胞桿菌（Aeromonas hydrophila）為革蘭氏陰性，通性厭氣性桿菌，屬於化學異營菌，廣泛存於各水域環境中，被認為動物的病原菌
，對人類也具有致病性。
產氣單胞桿菌的致病機制至今未明，在眾多可能的致病因子中，我們選擇蛋白質分解來研究。
為比較產氣單胞桿菌環境株與致病株之間的差異，我們自各水域環境中篩選環境株，利用SA培養基初步篩選後，經過oxidase活性與O/129耐性測試後，以AH培養基測試菌株的生理、生化性質，最後篩選得到環境株共85株。
我們由各產氣單胞桿菌致病株中，選擇蛋白質分解活性高的A.
hydrophila CKH-29菌株為材料，製作基因庫以進行蛋白質分解基因選殖，利用含2﹪skim milk的LA培養基篩選E. coli轉形株，篩選到一帶有10.2 kb外來DNA片段重組質體pPS1的轉形株，具有蛋白質分解活性。
由重組質體pPS1進行次選殖，利用限制Kpn I切割，回收一5.5 kb大小的DNA片段，並發現其重組質體pPS155具有蛋白質分解活性。重組質體pPS155經定序分析後，發現一段ORF，共有1,362 bp可以轉譯出453個胺基酸，將此基因命名為prtS1。
prtS1基因經比對後，發現有部分序列與E. coli htrA基因有相似性，進一步分析胺基酸序列得知，PrtS1上209-215位置上有serine protease HtrA的催化區GNSGGAL motif。
以osmotic shock區分細胞，分析蛋白質分解活性，得知prtS1基因產物主要位於E. coli轉形株之胞內部分。

Aeromonas hydrophila and related aeromonads are gram negative
, facultatively anaerobic freshwater bacteria, many strains of which are pathogens of humans and animals. In humans they cause gastroenteritisin healthy individuals or septicemia in
individuals with impaired immune systems or various
malignancies. The pathogenicity of the microorganism may involve several extracellular enzymes including two hemolysins, lipase, and proteases. Some of the toxins have been isolated and characterized biochemically , but their roles in the pathogenesis of A. hydrophila is still unknown. It has been suggested that proteolytic enzymes excreted by Aeromonas spp. play an important role in invasiveness and establishment of
infection by overcoming initial host defenses and by providing nutrients for cell proliferation. Although all these exoproteins are secreted extracellularly by A. hydrophila , they can only be exported to periplasm in E. coli. In this study we report the molecular cloning and expression in E.coli
of the protease gene prtS1 from A. hydrophila CKH-29. We have
sequenced prtS1 gene and found an open reading frame (ORF) of 1,362 bp coding for a protein of 453 amino acids. The predicted primary structure of PrtS1 displayed similarity to HtrA, a serine protease involved in proteolysis of abnormal proteins and required for resistance to oxidative and heat stress in enteric bacteria. Towards the middle of the deduced protein sequence between residues 209 and 215 has a GNSGGAL motif, which is the conserved serine protease catalytic domain in many bacteria.

壹.前言........................................................1
一.Aeromonas hydrophila的基本性質..............................1
二.A. hydrophila的危險性.......................................4
三.A. hydrophila的致病因子.....................................6
貳.研究材料...................................................12
一.菌株.......................................................12
二.質體.......................................................12
三.培養基.....................................................16
四.藥品與儀器.................................................18
參.研究方法...................................................19
肆.結果與討論.................................................37
一.A. hydrophila菌株的篩選....................................37
二.A. hydrophila蛋白質分解基因之選殖........................42
三.重組質體pPS1的次選殖與定序.................................48
四.分析prtS1基因..............................................52
五.prtS1基因在E. coli宿主中的表現.............................60
伍.結論.......................................................66

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