Genetic analysis of pCFC2 and studying of virulence associated protein in Riemerella anatipestifer

• Main Authors: Weng, Shu-Chuan, 翁淑娟
• Other Authors: Chao-Fu Chang
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/09189285926010612727

碩士 === 國立臺灣大學 === 獸醫學系 === 86 === Riemerella anatipestifer (RA) 感染症為鴨鵝之重要傳染性疾病，引起 病禽全身性漿膜炎 ；本病普遍發生於世界各地水禽類，造成嚴重經濟損失。由台灣地區罹患 傳染性漿膜炎鴨鵝分 離之 RA 菌，以電泳法檢測分子量大小為 5.6 kb 質體經定序後得知氮鹼 基對數為 5609 bp， 命名該質體為 pCFC2。基因分析結果得知 pCFC2 具三個開放讀碼 (open reading frame, orf)。 第一個 orf 命名為 vapD3 與 Dichelobacter nodosus， Actinobacillus actinomycetemcomitans ，Haemophilus influenzae、Helicobacter pylori 及 Neisseria gonorrhoeae 之 virulence-associated protein gene (vapD) 分別有 83%，75%，80%，69%，67% 的同質性； 此 VapD 在致病機制中 可能扮演重要的角色。為研究 VapD 蛋白質之特性，構築一 vapD1 表現 載體。西方免疫轉漬試 驗結果證明具 vapD 基因者皆可表現此毒力相關蛋白。第二個 orf 命名為 RepA3 則 與 Pseudomonas alcaligenes，Lactococcus lactis，Neisseria gonorrhoeae ，Pseudomonas syringae， Pediococcus pentosaceus及 Campylobacter hyointestinalis 之複製蛋 白 (replication protein) 分別具有 36%，30%，32%，33%，39%，27% 同質性。第三個 orf 命名為 ISRa1 為一推測性插入序列 (putative insertion sequence) 與 Lactococcus lactis 之推測性轉 位 (putative transposase) IS982 及 Bacillus stearothermophilus 之 RSBst-a 有 36% 的同質性 ；以南方雜合法證明此插入序 列可在染色體及質體 DNA 間轉移，國內外菌株可見不同的 ISRa1 patterns，此 ISRa1 可作為流行 病學上之研究工具。 Riemerella anatipestifer (RA) is the principal causative agent of serositis in duck and geese. RA infection occurs in water fowls world wide and accounts major for the economic losses in duck and geese industries. According to gel electrophoresis there was a 6.5 kb plasmid in RA isolates. The 5.6 kb plasmid was sequenced and had 5609 base pairs of nitrogen bases. It was designated as pCFC2. There are three open reading frame (orf) in pCFC2. The first orf is designed as vapD3 and it has 83%，75%，80%，69% ，67% homologous to the virulence-associated protein gene (vapD) in Dichelobacter nodosus, Actinobacillus actinomycetemcomitans, Haemophilus influenzae and Helicobacter pylori, respectively. The VapD may play an important role in the mechanism of pathogenesis. I overexpressed and purified the virulence-associated protein by using an expression vector, pRSETA. The western blotting test showed that all RA strains which have vapD gene could express VapD protein. This protein might be used as a subunit vaccine against R. anatipestifer infection. The second orf is designed as RepA3. It has 36%，30% ，32%，33%，39%，27% homologous Pseudomonas alcaligenes，Lactococcus lactis， Neisseria gonorrhoeae ， Pseudomonas syringae，Pediococcus pentosaceus and Campylobacter hyointestinalis, respectively. The third orf is designed as ISRa1. It is a putative insertion sequence. ISRa1 has 36% homologous to the putative transposase IS982 from Lactococcus lactis and the RSBst-a from Bacillus stearothermophilus. The southern blotting indicated that ISRa1 could transfer between chromosome and plasmid. This ISRa1 might be a useful tool for epidemiological studies.