Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66473
標題: 家禽霍亂巴斯德桿菌絲狀血球凝集素之胜肽酶C58功能區之功能分析
Functional Analysis of the Peptidase C58 Domain Encoded by the Filamentous Haemagglutinin Genes of Pasteurella multocida
作者: 劉姵君
Liu, Pei-Chun
關鍵字: 巴斯德桿菌
Fowl cholera
絲狀血球凝集素
胜肽酶C58功能區
Pasteurella multocida
Filamentous haemagglutinin
pfhB1
pfhB2
peptidase_C58
出版社: 微生物暨公共衛生學研究所
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摘要: 家禽霍亂巴斯德桿菌(Pasteurella multocida) 所引起的家禽霍亂(fowl cholera)為具高度傳染性之疾病,此菌對於多種禽類皆有致病性並可造成嚴重的經濟損失。P. multocida含有多種毒力因子,本篇針對可幫助細菌的散佈及聚生的絲狀血球凝集蛋白(filamentous hemagglutinin, FhaB)之Peptidase_C58功能區進行研究。首先以PCR增幅P. multocida X73、P1059、P1662菌株的pfhB2基因,設計一系列的「walking primers」進行定序,分析不同菌株之絲狀血球凝集素基因(pfhB)之核酸序列。定序結果發現,上述菌株之pfhB基因,皆可轉譯出peptidase C58活性功能區,且此功能區之位置與PfhB1不同,並非位於PfhB2蛋白之C端尾部。接著將P. multocida X73菌株之pfhB2 Peptidase_C58功能區片段構築於PET-23a質體,以E. coli BL21 (DE3) 系統進行重組蛋白表現,將其純化後蛋白對雞隻進行免疫,兩週免疫一次並於兩次免疫前後採血,最後以血清進行western blot分析,發現此血清可辨識P. multocida所表現之PfhB蛋白。另外,針對P. multocida X73菌株pfhB1之Peptidase_C58功能區基因作deletion,將residues 2320-2613與residues 2400-2613區段以PCR增幅並構築於真核細胞表現載體pEGFP-N1,將此質體轉染(transfect)至鴨胚纖維母細胞(DEF),以倒立式螢光顯微鏡觀察,可見轉染成功細胞之型態的不同。帶有完整胜肽酶C58功能區的residues 2320-2613轉染後之組別,可見DEF細胞骨架斷裂而皺縮,而未完整帶有此區的residues 2400-2613轉染後組別,則型態正常,此結果證實胜肽酶C58功能區對細胞具有毒性,可能是P. multocida之重要致病因子。
Pasteurella multocida is the causative agent of fowl cholera (FC), which is a highly contagious disease of the poultry and causes economic losses. The filamentous hemagglutinin (Fha) is one of the virulence factors of Pasteurella multocida, and could mediates bacterial dispersion and colonization. In this study, peptidase C58 domain encoded by the filamentous haemagglutinin genes was characterized. PCR was used to amplify pfhB2 genes from P. multocida X73, P. multocida P1059, and P. multocida P1662, and then walking primers were designed for sequencing analysis of the P. multocida pfhB sequence. Sequence analyses showed that both PfhB1 and PfhB2 contain a hemagglutinin activity domain, peptidase C58 (Yersinia cystein protease_YopT) domain, and a numbers of haemmagglutinin repeats domains. However, in contrast to pfhB1, the peptidase_C58 domain of pfhB2 is not located at the end of the C-terminal of PfhB2 protein. The pfhB2 peptidase_C58 gene was cloned into pET23a vectors and expressed in E .coli, and SPF chickens were immunized subcutaneously with purified recombinant pfhB2 peptidase_C58 proteins. Antisera against this recombinant protein reacted with PfhB expressed by P. multocida. The truncated pfhB1 gene encoding residues 2320-2613 and residues 2400-2613 of pfhB1 was amplified by PCR, and then cloned into the eukaryotic expression vector pEGFP-N1. The recombinant plasmid was transfected into duck embryo fibroblast (DEF) cells. After incubation for 22 hours, expression of the gene was analyzed by using fluorescence microscope. Cells transfected with ΔN1 of residues 2320-2613 which contain full length of peptidase_C58 showed disruption of the actin cytoskeleton and rounding up of the cells, but those transfected with ΔN2 of residues 2400-2613 did not. The result shows that peptidase_C58 domain is toxic to cell and might be an important virulent factor of Pasteurella multocida.
URI: http://hdl.handle.net/11455/66473
其他識別: U0005-1806201311443200
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1806201311443200
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