臺灣博碩士論文加值系統

細胞素為天然衍生蛋白且主要作用為調節、提升免疫反應，故有潛力成為好的疫苗佐劑。雞傳染性支氣管炎( infectious bronchitis, IB)是家禽之重要疾病，其病原為雞傳染性支氣管炎病毒 (infectious bronchitis virus, IBV)，此病毒血清型眾多，但是他們的交叉保護力甚低，故導致免疫成效不佳引發重大的經濟損失。本研究目的是表現融合細胞素與IBV S1次單位蛋白供疫苗組合使用。在細胞素方面則以10個胺基酸(GGGGS)2組成的彈性胜肽連接子(S2 linker)接合A蛋白及C蛋白或B蛋白，採用pET32表現質體，構築形成A-S2-C及A-S2-B序列表現質體，而後表現及純化重組蛋白，並與先前本研究室以15個胺基酸(EAAAK)3的螺旋胜肽連接子(αH linker)接合之衍生蛋白比較，分別進行試驗評估重組細胞素之活性。結果顯示，在淋巴球增生或一氧化氮生成試驗結果以αH連接子連接蛋白效果較好，但在淋巴球試驗中顯示以S2 linker連接蛋白可於不同濃度間維持穩定活性。IBV S1次單位蛋白表現方面，則分別以IBV H120疫苗株及野外分離TW1病毒株之RNA為模板，增幅出全長或部分的S1基因片段，構築6個原核表現質體，並誘導重組蛋白表現，其中只有表現部分的S1基因能獲得預期大小的蛋白產物，本研究表現的IBV S1重組蛋白及融合細胞素將來可以分別提供做為疫苗之免疫原與佐劑進行評估。

The cytokines are naturally derived proteins that play important roles in controlling and promoting immune responses, and therefore may have potential to serve as good vaccine adjuvants. Avian infectious bronchitis (IB) is an important avian disease and its causative agent is avian infectious bronchitis virus (IBV). Many different IBV serotypes have been recognized with little cross protection, rendering great economic losses due to vaccination failure. In this study, we expressed fusion cytokines and S1 subunit of IBV for the future vaccine usage. To produce fusion cytokines, we linked A protein with C or B protein by a 10 amino acid (flexible peptide linker (S2 linker). Using pET32a vector, we constructed expression plasmids for A-S2-C and A-S2-B. After protein induction and purification, both S2 linker-derived recombinant proteins were compared in parallel with αH linker (15-amino acid helix forming linker) derived proteins, which were developed earlier in our laboratory. Activity assays were conducted to evaluate the activities of recombinant cytokines. The results showed that αH linker derived cytokines were better than S2 linker-derived cytokines in stimulating lymphocyte proliferation or inducing nitric oxide production from macrophages. S2 linked cytokine showed a stable lymphocyte proliferation stimulating activity in a wide range of protein concentration. For the expression of IBV S1 subunit proteins, we amplified the entire or partial S1 gene fragments from a vaccine strain (H120) and a field strain (TW1), and constructed 6 prokaryotic expression plasmids. After induction, bacteria carrying partial S1 gene fragments expressed recombinant proteins with the expected molecular weights. These IBV S1-derived proteins will be evaluated together with fusion cytokines as immunogen and adjuvant for the vaccine usage.

摘要 I
Abstract II
目次 III
表次 VI
圖次 VII
第一章 序論 1
第二章 文獻探討 2
第一節 細胞素(cytokine) 2
第二節 A 家族(A family) 2
2-1 A蛋白 3
第三節 B家族 (family) 3
3-1 B蛋白 4
第四節 C家族(family) 5
4-1 C 蛋白 5
第五節 細胞素佐劑 (cytokine adjuvants) 6
第六節 胜肽連接子 (peptide linker) 7
第七節 雞傳染性支氣管炎 (avian infectious bronchitis, IB) 7
7-1 雞傳染性支氣管炎簡介 7
7-2 病毒介紹 8
7-3 病毒分類依據及S1高度變異區介紹 9
7-4 台灣目前IBV現況及分群情形 9
7-5 臨床症狀、肉眼及組織學病變 9
7-6 防疫計畫 10
第三章 材料與方法 11
第一節 原核表現質體之構築與重組蛋白表現及純化 11
1-1 增幅A-S2-B基因序列及構築原核表現質體 11
1-1.1誘導表現A-S2-B蛋白及所需時間 11
1-1.2大量表現A-S2-B蛋白及純化 12
1-2 利用限制酵素Pst I置換pET32A-S2-C質體錯誤序列及構築原核表現質體 12
1-2.1誘導表現A-S2-C蛋白及所需時間 13
1-2.2大量表現A-S2-C蛋白及純化 13
1-3 大量表現及純化細胞素重組蛋白 13
1-4 增幅IBV H120 病毒株之S1基因序列及構築原核表現質體 13
1-4.1誘導表現IBV S1衍生蛋白及所需時間 14
1-4.2大量表現IBV S1衍生重組蛋白及純化 15
1-5 增幅IBV TW1 病毒株之S1 基因序列及構築原核表現質體 15
1-5.1誘導表現IBV S1衍生蛋白及所需時間 15
1-5.2 大量表現IBV S1衍生重組蛋白及純化 15
第二節 重組細胞素活性分析試驗 16
2-1 分離周邊血液單核球 (peripheral blood mononuclear cell, PBMC) 16
2-2 A蛋白活性試驗 16
2-3 細胞免疫染色 (immunocytochemistry) 16
2-4 B蛋白活性試驗 17
2-5 C蛋白活性試驗 17
2-6 統計分析及軟體 18
第四章 結果 19
第一節 構築細胞素表現質體、基因序列比對及表現重組蛋白 19
1-1 A-S2-B表現質體之構築序列比對及蛋白表現 19
1-2 A-S2-C表現質體之構築、序列比對及蛋白表現 20
1-3 其他細胞素重組蛋白之大量表現及純化 20
第二節 IBV之S1次單位表現質體、基因序列比對及重組蛋白表現 21
2-1 IBV H120 疫苗株之S1次單位表現質體、基因序列比對及重組蛋白現 21
2-2 IBV TW1病毒株之S1次單位表現質體、基因序列比對及重組蛋白表現 22
第三節 重組細胞素蛋白活性測試 23
3-1 A 蛋白活性試驗 23
3-2 細胞免疫染色 (immunocytochemistry) 24
3-3 B蛋白活性試驗 24
3-4 C蛋白活性試驗 24
第五章 討論 26
表次 31
參考文獻 57

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