嗜水性產氣單胞桿菌Aeromonas hydrophila質體之探討與 表現載體之建構

Abstract

嗜水性產氣單胞桿菌為革蘭氏陰性、兼性厭氧性菌，廣泛的存在於各類水域之中，一般被視為伺機性病原菌，對於人類及動物均有致病力。由於目前為止仍然缺乏適當的選殖系統可供研究其基因功能，所以本研究的目標是利用 Aeromonas 屬內生質體與大腸桿菌之載體融合，建構成複製區域選殖載體。爲得到 Aeromonas 屬內生質體，因此我們檢測 Aeromonas 菌株之臨床及環境分離株共70株之質體存在，發現有 4 株臨床株及 4 株環境株帶有質體。所有質體以限制酶進行切割，由其切割後之片段與標準品比較計算質體的大小，分別為：臨床株中 pAH 32為17 Kb、pAH 35為15kb；環境株中pAH 132、pAH 169、pAH191均為2.4kb。再選取其中限制酶切位較多之質體pAH 35為研究對象，將其切割成適當之片段與載體融合，製作成找尋複製區域之質體。我們將質體送入大腸桿菌複製放大數量後，再利用電穿孔法轉形入嗜水性產氣單胞桿菌，利用抗生素作為標記篩選出帶有嗜水性產氣單胞桿菌質體中複製區域的重組質體。利用網路核酸資料庫 NCBI 比對其序列，發現其與 Aeromonas salmonicida subsp. salmonicida strain A449.質體 pAsa2有極高之相似性。該質體為ColE1-type plasmid ，因此本質體亦可能為 ColE1-type plasmid，我們針對此質體探討其複製起始點，並製作穿梭載體。本實驗中，質體pAH35的複製區域尚未得到證實。

Aeromonas hydrophila is a gram negative, facultatively anaerobic freshwater bacterium, occurring widely in aquatic environment. It has been indicated as a factor causing diseases in humans and animals. As there was no suitable system to study the gene function in Aeromonas spp. The purpose of this work is to construct vectors which contain replication origin by fuse the vector of E.coli with indigenous plasmids of Aeromonas hydrophila. In order to get indigenous plasmids of Aeromonas, we examined the plasmid carriage rate of a total 70 clinical and environmental Aeromonas isolates. Four clinical Aeromonas species, AH25, AH32, AH35, AH69 and four environmental Aeromonas species carry plasmids. All plasmids were examined after digestion with restriction endonucleases. Restriction endonucleases fragments were sized by comparison with standard marker. Plasmids of clinical Aeromonas species, pAH 32、pAH 35, and environmental Aeromonas species, pAH 132、pAH 169、pAH191, could be digested by restriction endonucleases. The size of these plasmids are 17 Kb、15kb、2.4kb、2.4kb、2.4kb respectively. We had interest in Plasmid, pAH35, which had more restriction endonucleases digestive site than others. For this purpose, we fused vector of E.coli with fragment of indigenous plasmids in Aeromonas hydrophila to construct a replication origin-screening vector. We transformed these plasmids to E.coli to amplify them then transferred these vector into Aeromonas spp by electroporation. Once the recombinant contains a replication origin from plasmid of Aeromonas hydrophila, antibitic trait is used as a selection marker as it is transformed into Aeromonas hydrophila. The plasmid which contains replication origin could be constructed an expressional vector. On the basis of similarity to other genes in the Gene Bank Database, NCBI, we found a fragment named pAH35-3 which similarity to a plasmid pAsa2 of Aeromonas salmonicida subsp. salmonicida strain A449. Plasmid pAsa2 is the type of ColE1-type. We suggest plasmid pAH35 is a ColE1-type plasmid, too. In this work, we investigate the replication origin of this plasmid and try to construct a shuttle vector for this genus. In this work, the replication origin of plasmid pAH35 has not been confirmed.