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Aeromonas hydrophila 是一株具有菌體外良好分泌能力的革蘭氏陰性、
兼性厭氣菌，遍存於流水、潮溼土壤以及未處理的廢水中。此外，也被認
為是一種人類與魚類的伺機性病原菌，其分泌的許多菌體外蛋白質如溶血
素、(hemolysins)、腸毒素(enterotoxins)、與蛋白質分解酵素(
proteases)一般咸認為與該菌的致病性有關，然而這些毒素在
Aeromonashydrophila致病過程中真正扮演的角色至今仍不清楚。藉由研
究從 Aeromonas hydrophila 基因庫中篩選的蛋白質分解酵素基因，我們
試圖去探究蛋白質外分解酵素在A. hydrophila致病性方面的貢獻以及革
蘭氏陰性菌分泌菌體 A. hydrophila 致病性方面的貢獻以及革蘭氏陰性
菌分泌菌體外蛋白質的可能機制。首先我們針對帶有蛋白質分解酵素基因
的 4.3kb DNA 片段，進行一連串的次選殖及限制圖譜分析，而後以
Sanger 的雙去氧鏈終止法 (dideoxy chain termination method) 進
行核■酸序列分析的結果，推測一個由 1038bps 組成的可能開放閱讀架
(open reading frame)，可解譯出由 346 個氨基酸組成、分子量約
為37.5kD 的多胜■；此與經 minicell analysis 與 SDS-PAGE 分析鑑
定蛋白質分解酵素的分子量為 37kD 相符。將此蛋白質分解酵素在 56
0C 加熱 30 分鐘後活性便告喪失；且經由 minicell fractionation發現
其主要集中在細胞膜部份。因此，我們認為此蛋白質分解酵素是一種熱不
安定性的外膜蛋白質。此外，以A.hydrophila 的蛋白質分解酵素基因作
為探針，由臨床病人不同檢體培養所得菌體的染色體 DNA 進行南方吸漬
法 (Southern blotting) 分析，發現絕大多數都有雜交帶出現；且這些
有雜交帶的菌種經鑑定大都屬於Aeromonas species。因此，蛋白質分解
酵素 (protease)在 A.hydrophila的致病性方面可能扮演某種重要的角色
肌氨酸分解酵素 (creatinase)臨床上被用來偵測血清與尿液中肌氨酸■(
creatinine)與肌氨酸(creatine)量；另外，罹患肌肉萎縮症的病人，其
酵素之酵素動力學與正常人有很大不同。因此，利用本實驗室已由
Pseudomonas putida的基因庫中篩選的肌氨酸分解酵素基因作探針，我
們試圖選殖人類骨骼肌 cDNA 基因庫中的肌氨酸分解酵素基因。目前初步
得到五個可能的 positive clones ， 而關於 creatinase 的活性分析則
仍在進行當中。
Aeromonas hydrophila is a gram-negative , facultatively
anaerobic bacterium. This organism has been recognized as an
opportunistic pathogen of humans and fish . The pathogenicity
of A. hydrophila may involve several extracellular proteins
including hemolysins , enterotoxins, and proteases.We cloned
the protease gene from genomic library of A. hydrophila and
investigated the role of protease in the pathogenicity of this
organism and the extracellular protein secretion mechanisms of
gram-negative bacteria. A 4.3 kb DNA fragment containing
protease-encoding gene was cloned from genomic library of A.
hydrophilia . For nucleotide sequence determination , we
obtained several subclones and each was sequenced by Sanger's
dideoxy chain termination method, nucleotide sequence analysis
predicted a possible open reading frame composed of 1038bps
encodes a 346 amino acids polypeptide and the predicted
molecular weight is appromixmately 37.5kD , which is in
reasonable agreement with the size derived from minicell
analysis. The cloned protease was unstable by heating at 56C
for 30 min and detected in the membrane fraction . It was
proposed that the protease is a heat-labile and outer membrane
protein . In addition , many bacteria were cultured from
different samples of different clinical patients . When the
protease gene was used as a a probe , Southern blotting
analysis and bacteria species determination revealed that
protease may play an important role in the pathogenesis of A.
hydrophila. Creatinase is used in clinics to determine the
level of creatinine and creatine in serum and urine. We
attempted to clone the creatinase gene from human skeletal
muscle cDNA library by using P. putida creatinase gene as a
probe . Five positive clones have been obtained and the
creatinase activity assay is currently undergoing.
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