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研究生:譚美珍
研究生(外文):Mei-Chan Tan
論文名稱:腦膜炎雙球菌脂蛋白Ag473功能之初步探討
論文名稱(外文):Characterization of meningococcal lipoprotein Ag473
指導教授:楊秋英
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:55
中文關鍵詞:腦膜炎雙球菌Ag473
外文關鍵詞:Neisseria meningitidisAg473
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Neisseria meningitidis (NM) 具有多醣莢膜的革蘭氏陰性菌,為人類致病菌會引起敗血症與腦膜炎。Ag473 為本實驗室發現的外膜脂蛋白可能與 NM 致病力有關。利用免疫螢光染色比較 NM 野生株 (WT) 及 Ag473 缺失株 (MT473) 與人類細胞之交互作用,結果發現 WT 與 MT473 都有絲狀物的形成,但 WT 與 MT473 絲狀物的抗原性卻不同,而且 MT473 與宿主細胞的結合能力卻比 WT 好,顯示 Ag473 蛋白可能影響 NM 與宿主細胞交互作用。已知 type Ⅳ pilus 與多醣莢膜是 NM 的主要致病因子,有莢膜 NM 必須透過 type Ⅳ pilus 才能與宿主細胞結合,本研究將探討 (1) 此絲狀物是否為 type Ⅳ pilus (2) 分析 Ag473 是否會影響 type Ⅳ pilus 與宿主細胞的作用,(3) 分析 NM 感染宿主細胞時 Ag473 的表現情形。為了探討第一個問題,分別增幅WT 與 MT473-1 的 pilE 基因,以 anti-PilE serum 進行西方墨點法分析發現 MT473-1 的 PilE 蛋白 (PilE-MT) 比 WT 的 PilE 蛋白 (PilE-WT) 大。比對 PilE-WT 與 PilE-MT 的胺基酸序列,發現在PilE-MT 之 C 端有 11 個胺基酸與 PilE-WT 不同。為了要探討 Ag473 的功能,必須排除 PilE 蛋白的突變所造成的干擾,所以再重新篩選出 PilE 沒有突變且 Ag473 缺失的菌株稱為 MT473-5。以 anti-MT473-PilE serum 為探針進行免疫螢光染色觀察發現只有 MT473 偵測到絲狀物,顯示此絲狀物為 type Ⅳ pilus,當 PilE 蛋白的氨基酸組成發生改變時會改變 type Ⅳ pilus 的抗原性,而使anti-MT473-PilE serum 無法辨識 WT 及 MT473-5 的 type Ⅳ pilus。NM 對細胞貼附能力的分析,發現 MT473 貼附在宿主細胞的能力比 WT 及 MT473-5 貼附在宿主細胞的能力好,顯示貼附在宿主細胞的能力之差異不是透過 Ag473 但可能與 PilE 蛋白有關。FACS、西方墨點法及免疫螢光染色觀察 NM 感染宿主細胞,發現 Ag473 蛋白的表現菌會增加,顯示 Ag473 蛋白在 in vivo 下的量會增多。這個特性顯示以 Ag473 作為疫苗的潛力很大。
Neisseria meningitidis (NM) is an encapsulated Gram-negative diplococcus causing meningitis and septicemia in human. Ag473 is an outer membrane lipoprotein newly identified in our laboratory which may involve in pathogenicity of NM. It has previously been shown during infection of human cells that both the wild-type (WT) and the Ag473 deficient strain (MT473-1) could form filamentous structure; however, these structures manifested different antigenicities. In addition, MT473-1 exhibited better ability in adhesion with host cell than WT. These observations suggested that Ag473 may affect the interaction between NM and host cell. Since the initial steps of attachment of encapsulated NM to the epithelial cells is mediated by type IV pilus, this study was aimed to understand 1) whether the filamentous structures consist of type IV pilus, 2) whether deficiency in Ag473 affects the interactions between type IV pilus and host, and 3) the expression of Ag473 upon infecting the host cells. To address the first question, pilE genes (coding for prepilin protein) were amplified from WT and MT473-1. Amplicons with expected size but different in sequences, pilE-wt and pilE-mt from the wild-type and the mutant, respectively, were obtained. Antisera against the resulted recombinant PilEs (designated PilE-WT and PilE-MT) were obtained by immunizing mice. Western blotting showed that PilE-mt in the whole cell extracts prepared from MT473-1 had an apparent molecular size larger than that from WT. Sequencing indicated that the pilE gene of MT473-1 had 11 amino acid residues (near the C-terminus) different from that of WT. Furthermore, filamentous structures were detected in WT and MT by using the respective anti-PilE antisera. These results confirmed the filamentous structure to be type IV pilus. To address the second question, a new Ag473 deficient strain without pilE mutation was then screened from the stock in our laboratory and designated as MT473-5. MT473-5 exhibited similar adhesion activity to that of WT, indicating that Ag473 is not involved in the interactions between NM and human cells. To address the third question, Ag473 expression in NM adhered to host cells were monitored by FACS, Western blot and immunofluorescence. The results showed that Ag473 expression was upregulated during infection, suggesting that Ag473 may be more abundant in vivo. This feature strengthens the vaccine potential of Ag473.
中文摘要…………………………………………………………………i
英文摘要………………………………………………………………ii
目次……………………………………………………………………iii
圖表目次…………………………………………………………………v
壹、前言……………………………………………………………
一、緒論……………………………………………………………1
二、NM 的外膜蛋白………………………………………………1
三、致病機制……………………………………………………2
四、脂蛋白 Ag473…………………………………………………4
五、研究目的………………………………………………………5
貳、材料與方法……………………………………………………
Ⅰ. 實驗材料……………………………………………………………
一、菌株及其培養條件……………………………………………6
二、質體與引子對…………………………………………………6
三、培養基與溶液配方……………………………………………6
Ⅱ. 實驗方法…………………………………………………………
一、製備 E. coli 勝任細胞 (competent cell) 及熱休克轉形作用 (heat shock transformation)………………………………7
二、小量質體 DNA 之萃取………………………………………7
三、重組蛋白質之表現及純化……………………………………7
四、重組蛋白質之透析 (dialysis) 及濃縮…………………8
五、蛋白質膠體電泳分析 (SDS-PAGE)…………………………8
六、西方墨點法 (Western blotting)…………………………9
七、抗體血清之製備……………………………………………10
八、貼附力分析 (Adhesion assay)…………………………10
九、免疫沉澱……………………………………………………10
十、生長曲線……………………………………………………11
十一、免疫螢光染色 (Immunofluorescence)………………………11
十二、流式細胞儀分析 (FACS analysis)…………………………12
參、結果……………………………………………………………
一、探討絲狀物是否為 type Ⅳ pilus 及 Ag473 如何影響 NM 與宿主細胞的交互作用……………………………………………14
二、Ag473 蛋白對多醣莢膜 (CPS) 的影響…………………16
三、免疫沉澱分析 RmpM 蛋白與 Ag473 的交互作用………16
四、比較不同環境下 WT 與 MT473-5 的生長情形…………17
五、Ag473 在 NM 感染宿主細胞後的表現情形………………18
肆、討論…………………………………………………………20
參考文獻………………………………………………………………23
圖表……………………………………………………………………29
附錄……………………………………………………………………51
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劉依倫 (2006) 腦膜炎雙球菌表面抗原 Ag473 之功能及免疫特性分析。國立中興大學分子生物學研究所,碩士論文。

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