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研究生:吳偉嘉
研究生(外文):Wei-Jia Wu
論文名稱:腦膜炎雙球菌疫苗的研發:疫苗抗原之鑑定
論文名稱(外文):Development of meningococcal vaccines:Identification of vaccine antigens
指導教授:楊秋英
指導教授(外文):Chiou-Ying Yang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:49
中文關鍵詞:腦膜炎雙球菌疫苗反向疫苗學流式細胞儀
外文關鍵詞:meningococcal vaccinesreverse vaccinologyflow cytometry
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腦膜炎雙球菌疫苗的研發主要是針對其莢膜多醣 (capsular polysaccharides, CPS) 來作為疫苗開發的成分,但是由於多醣類疫苗 (polysaccharide vaccines) 對於血清群 B 群之 NM 無法產生有效的保護力,因此目前在 NM 的 B 群疫苗研發上主要是針對外膜蛋白質 (outer membrane proteins, OMPs) 及外膜囊泡 (outer membrane vesicles, OMVs) 進行研究。本研究利用反向疫苗學 (reverse vaccinology) 的研究策略,鑑定具有疫苗潛力之目標抗原。本論文研究選擇 NMA1697、NMB2095以及 NMB2141進行分析,其原因為:NMA1697預測為一血清群 A 群菌 Z2491的假設性脂蛋白,但是在 B 群菌 MC58序列中則沒有相關的註解;NMB2095預測為一假設性附著蛋白;NMB2141預測為一假設性非典型分泌的蛋白。此三個基因以血清群 B 群菌 Nm22209之 genomic DNA 為模板進行 PCR 的增幅,經核酸定序分析結果證明各增幅之 DNA 片段均為預期之產物。再利用大腸桿菌生產重組蛋白質 (rNMA1697、rNMB2095以及 rNMB2141),並以其免疫小鼠。為了偵測目標蛋白是否確實會表現於 NM 菌株中,以各抗血清為探針對十八株不同的 NM 菌株進行西方墨點法分析。結果發現各抗血清辨認之目標蛋白均表現於所有測試的 NM 菌株中,然而蛋白的表現量在不同菌株中有差異,其中 NMA1697分子量大小亦出現明顯的差異。因此推測 NMA1697、NMB2095與 NMB2141均為具有高度保存性之蛋白。進一步以 whole-cell ELISA、流式細胞儀與免疫螢光顯微鏡觀察 NMA1697在 Nm22209菌體表面的表現情形。雖然 whole-cell ELISA 分析結果顯示所有測試的 NM 菌株都有明顯的吸光讀值,但是流式細胞儀及免疫螢光顯微鏡觀察結果則只有少部分的菌能被 anti-rNMA1697所辨認。有趣的是流式細胞儀分析結果顯示 NM 菌體經由 sodium azide 處理後 NMA1697的表現量是活菌狀態時的2-3倍,但是在 whole-cell ELISA 或西方墨點法分析結果顯示則無明顯差異。根據實驗結果推測經由 sodium azide 處理後可能改變 NMA1697在細胞內表現的位置。最後,進一步利用 in vitro 血清殺菌力測試與 in vivo 保護力分析來評估 rNMA1697能否引起有效的保護能力以對抗 NM 的感染。實驗結果顯示 anti-rNMA1697無法產生具殺菌力或保護力的作用,表示 NMA1697無法單獨作為 NM 疫苗的成分。

Vaccine development against meningococci has mostly been focused on the capsular polysaccharides (CPS). However, CPS-based vaccines are not suitable for serogroup B meningococci. Hence, present study for serogroup B meningococcal vaccines have focused mostly on outer membrane proteins and outer membrane vesicles. In this study, reverse vaccinology was employed to identify potential vaccine candidates. NMA1697, NMB2095 and NMB2141 were chosen for the following reasons: NMA1697 is a putative lipoprotein annotated in the genome of serogroup A strain Z2491 but not that of serogroup B strain MC58, NMB2095 is a putative adhesion protein, and NMB2141 is a hypothetical, non-classical secreted protein. The three genes were PCR amplified from serogroup B strain Nm22209, verified by nucleotide sequencing, and expressed in E. coli. The recombinant NMA1697, NMB2095 and NMB2141 were purified and used to immunize mice. To examine whether the target protein is indeed expressed by meningococci, bacterial lysates of 18 meningococcal strains were subjected to Western blot analysis. All three proteins were detected in the tested strains, however, the expression levels were varied among strains, particularly the NMA1697 protein which also exhibits size variation. Accordingly, NMA1697, NMB2095 and NMB2141 are considered meningococcal conserved proteins. The surface expression of NMA1697 in Nm22209 was further characterized by whole-cell ELISA, flow cytometry, and immunofluorescence microscopy. Although significant binding activities were obtained by whole-cell ELISA, flow cytometry and immunofluorescence microscopy revealed that only a small portion of viable meningococci are anti-rNMA1697 positive. Interestingly, the numbers of anti-rNMA1697 positive meningococcus treated with sodium azide was 2-3 folds of that viable meningococcus examined by flow cytometry but no difference was obtained when evaluated by whole-cell ELISA or Western blot analysis. Together, the data suggest that azide treatment alters the subcellular localization of NMA1697. Finally, in vitro bactericidal assay and in vivo active protection assay were performed to evaluate whether rNMA1697 can elicit protective immunity against meningococcal infection. Both assays failed to demonstrate any protective activity indicating that NMA1697 cannot be used as a sole vaccine component.

中文摘要………………………………………………………………………………1
英文摘要………………………………………………………….…………………2
壹、前言
一、奈瑟氏腦膜炎雙球菌之特性………………………………………………..3
二、奈瑟氏腦膜炎雙球菌之致病機轉…………………….……….……………3
三、奈瑟氏腦膜炎雙球菌疫苗之研發…………………………………………..5
四、研究目的……………………………………………………………..………6
貳、材料與方法
I. 實驗材料
一、菌株及其培養條件………………………….…………………………….…7
二、實驗動物……………………………………………………………………..7
三、核酸引子 (primers)…………………………………………….……………7
四、培養基與溶液配方…………………………………………………………..7
II. 實驗方法
一、聚合酶連鎖反應 (polymerase chain reaction, PCR)…….……….…………8
二、DNA 洋菜膠體電泳分析 (DNA agarose electrophoresis)…………………8
三、DNA 之連接反應 (ligation)……………………………………………...…8
四、轉形作用 (transformation)………………………………………..…………8
五、重組蛋白質之表現與純化………………………………………………..…9
六、重組蛋白質之透析 (dialysis) 及濃縮………………………...…..……....10
七、蛋白質濃度之測定…………………………………………………………10
八、蛋白質膠體電泳分析 (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) ……………………….…………………..…10
九、西方墨點法 (Western blotting).……………………. ………….……….....11
十、抗血清之製備………………………………………………………………12
十一、抗體血清力價之測試……………………………………………………13
十二、免疫螢光染色 (Immunofluorescence staining)…………………………13
十三、血清殺菌力測試 (serum bactericidal assay)……………………………14
十四、保護力分析 (protection assay)…………………………………………14
参、結果與討論
一、NM 基因之選殖、序列分析與表現載體之構築…………………….……16
二、重組蛋白質之表現與純化及其抗體之製備………………………………16
三、以西方墨點法及 whole cell ELISA 分析抗血清與 NM 之作用情形….17
四、NMA1697在 NM 個別菌體之表現情形…………………………………18
五、NMA1697疫苗潛力之評估………………………………………………...20
(一) 補體殺菌力分析……………………………………………………….20
(二) 保護力分析…………………………………………………………….21
參考文獻……………………………………………………………………………..23
圖表.………………………………………….………………………………………28
附錄…………………………………………………………………………………..47
論文口試紀錄……………………………………………………………….……….49


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