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標題: Flagellin gene expression and the low-molecular-weight bacteriocin secretion through the T3SS-like mechansim in Pectobacterium carotovorum
Pectobacterium carotovorum鞭毛基因表現與低分子量細菌素透過類第三型分泌系統機制之研究
作者: 王佩郁
Wang, Pei-Yu
關鍵字: 第三型分泌系統
出版社: 化學系所
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摘要: Pcc是一種會造成植物根莖部腐爛的病原菌。造成植物經濟作物的損害。Pcc會分泌carocin的毒性蛋白,抑制親緣相近的菌種生長,以利於生存競爭。Carocin會藉由第三分泌系統 (T3SS) 送至胞外,因此本論文針對兩者進行更深入的探討。 T3SS分為flagellum和injectisome兩種機制,兩者的基因並不會互相影響彼此的蛋白表現。本研究針對flagellum,純化出其中一個膜外組成蛋白—FliC。將純化並單離後的FliC送入紐西蘭白兔體內,使其產生抗體,並利用此抗體為實驗材料,交叉比對carocin抑制分泌的突變株。 實驗結果發現,△flhA菌株表現了較野生株大量的FliC,而△ysaT菌株的FliC消失表現,並在其基因恢復株 (△ysaT/△ysaT+) 中恢復表現,因此我們推測flhA和ysaT除了影響carocin的分泌,對FliC的組成也有關聯。 P. aeruginosa是一種會分泌毒性蛋白的病原菌,在即少數的P. aeruginosa情況下,仍有可能引發疾病,嚴重甚至會致命。但P. aeruginosa本身即具有多樣的抗藥性和高度環境適應力,因此增加治療上的困難。 本論文針對其Flagellum,將FliC蛋白純化出來,並製備成抗體。利用ELISA實驗測試抗體的力價,並以此抗體檢測P. aeruginosa,確定FliC抗體具有高專一性和敏感性。 有鑑於P. aeruginosa日益嚴重的抗藥性,希望藉由本實驗製備的抗體,提供作為感染P. aeruginosa初期的檢測試劑。
Pcc is a phytopathogenic enterobacterium responsible for the soft-rot disease of many plants species to cause the economic lost. Pcc will inhibit the related strain growth to live by secreting carocin toxin proteins. As known so far, carocin is transported to extracellular by the Type III secretion system (T3SS), and here we study the relationship between T3SS and carocin. T3SS is classified as flagellum and injectisome structures, and the genes from the two systems have no cross-effect on the expression of proteins for each other. FliC is a kind of filament proteins to construct flagellum. FliC protein was isolated and purified from Pcc, and the polycloned antibody was prepared by New Zealand rabbit for the following experiments. The Pcc FilC antibody processed cross-comparison of mutants incapable to delivery carocin to extracellular. The △flhA was mutated gene of flagellum and the western blot experiments of △flhA revealed the FliC expression was large. However, the FliC band disappeared from the western blot of △ysaT mutant, but this band showed again in the western blot of these recovered strains (△ysaT/△ysaT+). This discovery leads to the conclusion that flhA and ysaT have influences on the secretion of carocin and the expression of FliC in T3SS. P. aeruginosa is a kind of bacterium secreting toxin proteins, and pathogenic to die even the amount of bacterium is few. P. aeruginosa has various drug resistance and high resilience to environment, which increase the difficulty of curing the disease. We purified and isolated FliC from the flagellum of P. aeruginosa, and then polycloned to antibody. ELISA experiment was used to evaluate the titer of this antibody, and after the test, the antibody was employed to detect the existence of P. aeruginosa to confirm the sensitivity and selectivity. Our efforts of preparing the antibody of P. aeruginosa could contribute toward the initial discovery of the bacterium.
其他識別: U0005-2208201115592500
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