Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13152
標題: 豬萎縮性鼻炎巴氏桿菌之分子分型及毒素基因之選殖
Molecular characterization and cloning of the Pasteurella
作者: 周育賢
Chou, Yuh-Shyen
關鍵字: Pasteurella multocida
巴氏桿菌
Atrophic rhinitis
PMT gene
Clone
RAPD-PCR
萎縮性鼻炎
毒素基因
選殖
隨機引子聚合酵素連鎖反應
出版社: 獸醫學系
摘要: 摘 要D型Pasteurella multocida產 毒株為豬萎縮性鼻炎(atrophic rhinitis;AR)之主要病原,其所產生的 P. multocida toxin (PMT)亦被証實為其主要毒力因子。本實驗選取P. multocida D型、A型菌株及國外標準菌株進行實驗,以探討P. multocida 產毒株與非產毒株的各種檢測方法之特異性,結果顯示小白鼠致死試驗與 天竺鼠皮膚壞死試驗會有偽陽性或偽陰性發生,而以vero cell 細胞毒性 試驗及利用PCR檢測PMT基因,兩者皆具特異性但以PCR檢測毒素基因方式 最為快速與簡便。此外,A型P. multocida產毒株亦會導致AR臨床病徵, 但傳統方法於鑑定A型及D型菌株時常有誤判之情形,因此嘗試利用RAPD- PCR (Random Amplified Polymorphic DNA; RAPD-PCR)研究其分子分型, 結果發現以10-mer random primer進行RAPD-PCR進行分析時,D型與A型菌 株之PCR產物於2.8 kb位置處具明顯差異性,可用於快速鑑別A型及D型P. multocida。選取P. multocida type D產毒株Liu-48,利用PCR增幅完整 PMT基因後,經限制酵素切割將各不同基因片段分別選殖至適當的表現載 體pET system vector,以進行完整及部份PMT基因的選殖及表現。結果共 構築了Tox1 (2~486 a.a.)、Tox2 (486~987 a.a.)、Tox3 (987~1282 a. a.)、Tox4 (2~987 a.a.)、Tox5 (486~1282 a.a.)、Tox6 (2~1282 a.a.) 及Tox7 (987~1282 a.a.)等七個選殖株,並且可成功地誘導其表現。所表 現產物除Tox1外,都可被swine anti-PMT polyclonal Ab所辯識,且各個 選殖株之表現產物對vero cell皆不具有毒性。而選取Tox4及Tox5進行豬 隻免疫攻毒試驗以旍銣K疫原性,結果顯示以此兩個選殖株之表現產物免 疫豬隻後,可使豬隻耐過高劑量PMT攻毒(>20mg),且其血清在vero cell 中和抗體力價亦上升至>256倍,顯示具有良好之免疫保護效力。
AbstractThe toxin-producing strain of Pasteurella multocida (type D) is characterized to be the major causative agent of atrophic rhinitis (AR) in swine. The P. multocida toxin (PMT) with a calculated molecular weight of 146 kDa has been identified to cause the atrophy of nasal turbinate bones. Both type D and type A of P. multocida including several wild isolates were selected to study the specificity for their toxin-producing activity. The results suggested the vero cell toxicity test and the PCR methods for screening PMT gene were more specificity if in comparison with both mouse lethality test and guinea pig skin test. The random amplification polymorphic DNA (RAPD) PCR method was also applied and successfully identified the type A and type D of toxin or non-toxin producing strains of P. multocida. The highly homogeneity of PMT genes between type D and type A were analyzed using PCR automated sequencing method and suggested the PMT gene was very conserved. Moreover, cloning full length and partial of PMT gene in pET- system expression vectors were applied for investigation the PMT biological!activity. Several clones contained partial or complete open reading frame of 3.8kb in size of PMT gene were successfully constructed into different vectors. Total four clones, Tox3, Tox5, Tox6 and Tox7 respectively, could be identified with both polyclonal and monoclonal antibody by Western blotting assay. Furthermore, the expression products of Tox4 and Tox5 can mount good protective immunity in experimental swine when they were challenged with high dose of PMT (>20 mg) and the neutralizing antibody titer was markedly increased up to 256X with vero cell toxicity test.
URI: http://hdl.handle.net/11455/13152
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