- 學位論文
鞭毛蛋白之佐劑效應分析與其於次單位疫苗設計之應用
Analysis and application of flagellin as a vaccine adjuvant when fused to protein antigens in subunit vaccines
摘要
鞭毛蛋白是細菌鞭毛的主要成分，它激活Toll樣受體5（TLR5）以啟動促炎性免疫反應。然而，全長鞭毛蛋白難以生產高質量且不穩定。因此，我們的研究側重於評估鞭毛蛋白中可能充當蛋白質亞基做為疫苗及佐劑的域。將鑑定出的鞭毛蛋白結構域與細菌和病毒抗原融合，並在激發模型中測試鞭毛蛋白的疫苗增強作用。 對於細菌抗原工作，將多殺巴斯德氏菌脂蛋白E（tplpE）的免疫原性結構域克隆為抗原，並將鞭毛蛋白（nFliC）的N端結構域（1-99個殘基）作為融合蛋白疫苗與tplpE連接。與純抗原組相比，用這種重組蛋白質免疫雞後，觀察到抗體水平，CD4和CD8種群以及細胞因子mRNA水平升高。當使用多殺性巴氏桿菌攻擊已免疫重組蛋白的雞隻時，鞭毛蛋白的N-末端結構域將存活率提高到75％，而僅抗原組則為25％。作為一個額外的發現，在設計我們的抗原tplpE時，我們發現天然信號肽的加入增強了疫苗效力。 為了進行病毒抗原工作，將鴨產蛋下降綜合徵病毒（DEDSV）的包膜（E）蛋白克隆為抗原，並選擇和表達E蛋白內的結構域。為了將來的工作，這些E蛋白構建體將與鞭毛蛋白結構域融合以增強免疫力，並將評估疫苗的功效。
並列摘要
Flagellin, a major component of bacterial flagella, activates Toll-like receptor 5 (TLR5) to initiate pro-inflammatory immune responses. However, the full-length flagellin is challenging to produce with high quality and is unstable. Therefore, our studies focused on evaluating domains within flagellin that may act as a vaccine adjuvant for protein subunit vaccines. Identified flagellin domains were fused to bacterial and viral antigens and the vaccine enhancement effect of flagellin was tested in challenge models. For bacterial antigen work, the immunogenic domain of Pasteurella multocida Lipoprotein E (tplpE) was cloned as the antigen and the N-terminal domain (1-99 residues) of flagellin (nFliC) was linked to tplpE as a fusion protein vaccine. When chickens were immunized with this protein construct, elevated antibody levels, CD4 and CD8 populations, and cytokine mRNA levels were observed when compared to the antigen-only group. When the immunized chickens were challenged with P. multocida, the N-terminal domain of flagellin boosted survival rate to 75%, compared to 25% for the antigen-only group. As a side- discovery, while designing our antigen tplpE, we have found that the inclusion of the native signal peptide enhanced vaccine efficacy. For viral antigen work, the Envelope (E) proteins of Duck Egg-Drop Syndrome Virus (DEDSV) were cloned as the antigen and domains within the E protein were also selected and expressed. For future work, these E protein constructs will be fused with flagellin domains for immune enhancement, and the vaccine efficacy will be evaluated.