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標題: 豬萎縮性鼻炎巴氏桿菌毒素之純化與其分子性狀之探討
Purification and molecular characteristics of Pasteurella multocida toxin ( PMT ) association with atrophic rhinitis of swine
作者: 胡秋蘭
關鍵字: Pasteurella multocida toxin
genomic DNA
染色體 DNA
出版社: 獸醫學系
摘要: 自野外罹患豬萎縮性鼻炎豬隻所分離之 D 型巴氏桿菌產毒株,製備成粗 製毒素並進行毒素純化。 粗製毒素在經過膠體層析及離子交換樹脂層析 並經電泳回收後, 於 144 kDa 處可見有單一 peptide 。毒性試驗顯示 ,引致天竺鼠皮膚壞死的最小劑量粗製毒素為 0.84 ug ,純化毒素為 0.105 ug ; 在細胞株的毒性試驗方面引致 Vero 細胞發生 CPE 粗製毒 素為 35 ug ,純化毒素為 0.02 ug ;而 BEK 細胞則粗製毒素為 35 ug , 純化毒素為 0.006 ug 。在抗原性試驗方面,粗製毒素與純化毒素經 福馬林不活化後進行動物接種, 經由免疫轉印法顯示所產生的抗血清對 144 kDa 處的蛋白質仍具有辨識能力。 在中和試驗方面,利用細胞株測 定中和抗體力價顯示粗製毒素的抗體力價為 2 倍,而純化毒素的力價為 16 倍;但利用免疫連結酵素吸附法 ( ELISA ) 則粗製毒素的力價為 32倍, 而純化毒素的力價為 1024 倍。 依據已知的毒素基因序列, 依 sense 方向及 anti- sense 方向共製備成 8 個引子 ( primers ) , 並配對組成 5 組引子, 利用聚合脢連鎖反應 ( PCR ) 進行 30 週期的 增幅後於 1 % agarose 進行電泳, 結果可知在 D 型菌株之間不論是產 毒株或非產毒株,在經 PCR 反應後均有相同大小的產物產生。
Crude and partial purifided Pasteurella multocida toxin (PMT ) , derived from a toxigenic strain of P. multocida type D originally isolated from a pig with atorphic rhinitis , were prepared to study their biological properties. The protein toxin apparently composed of one peptide with 144 kDa was purified by using the methods of gel - filtration , ion - exchange chromatography and sodium dodecyl sulphate polyacrylamide gel. The results indicated that the partial purified toxin had the same biological properites as crude extract to product dermonecrotic change in guinea pigs and cytopathic effect in both Vero cells and embryonic bovine kidney cells. Animals vaccinated with formaldehyde - detoxified preparations of either crude or partial purified PMT revealed the same antigenicity demonstrated by immunoblotting analysis , and produced the neutralizing antibodies determined by cytopathic effect of PMT on Vero cells and indirect enzyme - linked immunosorbent assay. According to the sense strand and antisense strand , eight oligonucleotide primers were prepared and grouped as 5 sets of synthesize primers. The PCR targeted partial of toxA gene encoding a 144 kDa was developed for the differentiation of toxigenic and nontoxigenic P. multocida type D and toxigenic P.multocida type A. The results indicated that either toxigenic or nontoxigenic P.multocida type D had the same PCR - amplified fragments which suggested that the major immunologic determinants would be the same among P.multocida type D.
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