Functional characterization and epitope mapping of meningococcal lipoprotein Ag473

Ag473, a vaccine candidate for the protection against Neisseria meningitidis infection, is a highly conserved lipoprotein of unknown function and predicted to form an extremely high degree of α-helical structure. The specific aims of this study were of two folds: to investigate the possible role of Ag473 plays in inflammation and to define the surface accessible region(s) of the Ag473 protein. To achieve the first goal, the expression profiles of proinflammatory cytokines in the human cell lines nasopharyngeal carcinoma NPC076, neuroblastoma SK-N-SH, and glioblastoma G5T/VGH after being co-cultured with the wild type (WT) meningococci or the ag473-knockout mutant (MT473) were examined by RT-PCR. In NPC076 cell line, IL-1α, IL-1β, IL-6 and IL-8 were attenuated after coculturing with the WT for 24 h; similar effect was observed when co-cultured with MT473 in the presence of recombinant Ag473 (rAg473) but not with either one alone. In SK-N-SH, only expression of IL-1αand IL-1β was down regulated after co-culturing with the WT for 24 h. In G5T/VGH, the expression of IL-1α, IL-1β, IL-6, and TNF-α was stimulated by both WT and MT473 strains. However, a delayed cytokine expression in response to MT473 was observed compared to that of WT. Together, these results implicate that Ag473 may participate in the modulation of host immune response during infection. To achieve the second goal, monoclonal antibodies (mAb) against the native Ag473 (4-7-3) and rAg473 (α-rAg473) were used as the primary antibody to probe the Western blots of E. coli lysates each containing an Ag473 variant deleted at different region. The results showed that the functional epitope of 4-7-3 lies in aa 48-60. Surprisingly, the fourα-rAg473 mAbs analyzed all recognized the aa 55-60 regions. Sequence analysis of the variable region genes cloned from the corresponding hybridomas confirmed that all antibodies analyzed were independent clones. These results indicate that aa 55-60 of Ag473 is an immunodominant region in both native and purified recombinant Ag473 proteins although subtle differences may exist.Ag473 為一具有開發為腦膜炎疫苗潛力之 Neisseria meningitidis 外膜脂蛋白。此蛋白質具有高度保留性，經程式分析預估其二級結構主要由α-helix 組成；但目前尚未明瞭其功能。本研究之目的著重於兩部分，分別探討其在發炎反應中扮演的角色及定出具備 immuno-dominant 性質之區域。 首先利用野生株 (WT) 及不表現 Ag473 的突變株 (MT473) 感染人類鼻咽癌上皮細胞株 NPC076、神經原母細胞瘤 SK-N-SH 細胞株及神經膠母細胞瘤 G5T/VGH 細胞株後，利用 RT-PCR 觀察其 pro-inflammatory cytokines 的表現情形。在 NPC076 細胞株部分，實驗結果顯示以 WT 感染 NPC076 細胞株 24 小時後，會導致 IL-1α、IL-1β、IL-6 及 IL-8 表現量的降低；此外以重組蛋白 rAg473 及 MT473 共同感染 NPC076 細胞株後亦可重現此一趨勢，但若以 MT473 或重組蛋白質 rAg473 單獨感染時卻不會有此現象出現。在 SK-N-SH 細胞株的部分，WT 持續 24 小時的感染會降低 IL-1α 及 IL-1β 的表現量。在 G5T/VGH 細胞株的實驗顯示，WT 及 MT473 的感染均會刺激 IL-1α、IL-1β、IL-6 及 TNF-α 的表現，但 MT473 所引起的趨勢較 WT 來得緩慢。綜合上述實驗結果，推測 Ag473 或許在感染時期參與了對宿主免疫反應的調控。 至於另一部分的研究，利用辨識 native Ag473 的單株抗體 4-7-3 及由重組蛋白質 rAg473 製備的單株抗體 (α-rAg473) 作為西方墨點法的一級抗體，偵測帶有不同 Ag473 片段的 Escherichia coli lysate，來定義出 Ag473 上具備 immuno-dominant 區域的胺基酸片段。實驗結果顯示單株抗體 4-7-3 的抗原決定位落在 Ag473 第 48 個到第 60 個胺基酸的片段。至於 4 株α-rAg473 的單株抗體，其抗原決定位則位於第 55 個到第 60 個胺基酸的區域。藉由抗體重、輕鏈變異區的序列比對顯示，這 4 株單株抗體均來自不同的融合瘤細胞。雖然上述各單株抗體其抗原決定位的界定有些微差異，但仍可作出 Ag473 第 55 個到第 60 個胺基酸片段在 native Ag473 及 rAg473 均具備 immuno-dominant 性質的結論。中文摘要………………………………………………………………………………3 英文摘要………………………………………………………………………………4 壹、前言……………………………………………………………………………….. 一、緒論……………………………………………………………………….6 二、致病機制………………………………………………………………….6 三、疫苗研發………………………………………………………………….7 四、immuno-dominant 區域之應用……………………………………….....8 五、脂蛋白 Ag473……………………………………………………………9 六、研究目的………………………………………………………………….9 貳、材料與方法…………..………………………………………………………….… I. 實驗材料………………………………………………………………..………. 一、菌株、細胞株及其培養條件.…………………..……..……………….11 二、質體及引子對………………………………………..…………………11 三、培養基與溶液配方…………………………………..…………………11 II. 實驗方法………………………………………………………………….......... 一、感染細胞實驗…………………………………..………………………12 二、萃取 total RNA…………………………………………………………12 三、反轉錄酶酵素作用及聚合酶鏈鎖反應………..……………………….12 四、DNA 洋菜膠電泳分析…………………………………………………13 五、DNA 之黏接反應………………………………………………………13 六、製備 E. coli 勝任細胞及熱休克轉型作用……………………………13 七、小量質體 DNA 之萃取………………………………………………..13 八、重組蛋白質之表現及純化……………………………………………...14 九、重組蛋白質之透析及濃縮…………………………………………...…14 十、重組蛋白質之濃度測定………………………………………………...15 十一、重組蛋白質膠體電泳分析…………………………………………...15 十二、西方墨點法…………………………………………………………...15 十三、融合瘤細胞之製備………………………………………………...…16 十四、酵素連結免疫呈色反應分析………………………………………...16 十五、單株抗體之單株化…………………………………………………...17 十六、單株抗體之抗體亞群鑑定…………………………………………...17 参、結果……………………………………………………………………………….. 一、探討 Nm 膜外蛋白 Ag473 與發炎反應之關係………………………....18 二、以免疫學方法搜尋 Ag473 具 immunodominant 區域…………………..20 肆、討論………………………………………………………………………………... 一、探討 Nm 膜外蛋白 Ag473 與發炎反應之關係………………………....23 二、以免疫學方法搜尋 Ag473 具 immunodominant 區域…………………..26 參考文獻 ……………………………………………………………………………30 圖表..…………………………………………………………………………………36 附錄…………………………………………………………………………………..55 論文口試記錄………………………………………………………………………..6