豬生殖與呼吸綜合症為養殖豬隻的重要疾病，其病源為一種病毒稱為豬生殖與呼吸綜合症病毒(PRRSV)。該病毒感染會引發豬隻食慾不振、高燒不退、腹瀉等臨床症狀，懷孕母豬被感染更會造成流產、產下木乃伊胎等，因此每當爆發疫情大流行時皆造成養殖業大量的損失，且後續養殖業恢復產能相當耗時費力。施打PRRSV疫苗為目前最有效的防疫策略，但是現有疫苗施打小豬身上時，需要四週時間才能引發足夠免疫反應保護小豬，這四週將是很大的免疫空窗期。因此我們希望研發出一種疫苗可以在小豬離乳前施打，能夠具有足夠長的免疫時效而使小豬在離乳後仍能誘發對PRRSV的免疫力。
因此初期本研究方向為利用桿狀病毒作為疫苗載體，結合Cre/LoxP重組系統形成小型環狀游離態抗原表現質體(episome)，施打重組桿狀病毒於豬隻後可在細胞內長期表現PRRSV的抗原核殼蛋白(N protein)。確認了Cre/LoxP系統可經由桿狀病毒轉導在豬腎臟細胞PK(15)中形成episome，但N protein在PK(15)細胞中表現不如預期而更改研究方向。而後研究主題改使用桿狀病毒表現系統在昆蟲細胞(High FiveTM細胞)中生產PRRS病毒的GP5及M蛋白，我們設計了六株的GP5蛋白及一株的M蛋白重組桿狀病毒，原本預期的Bac-GP5組別因其搭載的分泌訊號來自於PRRS病毒而無法在昆蟲細胞作用，GP5蛋白會停留在胞內或分泌效果較差等問題，但結果顯示與Bac64-GP5組別以西方墨點法觀察沒有太大差異，而除了以上兩組外只有Bac64-GP5dN組別能夠分泌出胞外，且有較高的產量，所以後續生產以這三株重組桿狀病毒測試並生產蛋白。在大量生產方面，不同的桿狀病毒劑量感染昆蟲細胞時，高劑量的病毒會使細胞快速凋亡，以致重組蛋白停留在胞內。使用低劑量病毒不只可使重組蛋白分泌到胞外，且可有效增加產量。同時在不同時間收取細胞液以西方墨點法分析，找出後續生產時的最佳收穫時間約為4天。而後驗證突變GP5蛋白上不同數量的醣基化位，確實能夠有效減少醣基化產生。在同時共感染(co-infection) GP5及M重組桿狀病毒時，GP5及M蛋白可以有效形成heterodimer。這些結果顯示其未來應用於疫苗的潛力。

Porcine reproductive and respiratory syndrome (PRRS) is a disease causes by porcine reproductive and respiratory syndrome virus (PRRSV). PRRS clinical symptoms are inappetence, fever, diarrhea and make farrowing sows birth mummified piglets. Developing PRRS vaccine is the best strategy to reduce economic loss. Existing commercial vaccines have problem is using on piglet have window period, so we try to develop a vaccine which have can vaccination on pre-weaning piglet in the long term.
First we try to combine baculovirus-based vector with Cre/LoxP recombination system. Recombination system make our target gene form episome that stable episome can remain in the cell. We confirm that Cre/LoxP system working on the pig kidney cell (PK(15)), but N protein don’t have high exression level. Second we use baculovirus expression system to produce PRRS virus GP5 and M protein in High FiveTM cell as subunit vaccine. We designed gp64 signal peptide on GP5 replace GP5 ownself signal peptide to optimize secretory signal peptides. The result show that no significant difference between Bac-GP5 and Bac64-GP5. We found that only Bac-GP5, Bac64-GP5, Bac64-GP5dN, Bac64-M could secrete GP5 and M in medium, the others couldn’t. Co-infection GP5 and M protein in High FiveTM could form heterodimer that can induce better immunity than monomer. These result show our antigen is a potent vaccine candidate against PRRS virus.

摘要 II
Abstract III
致謝 IV
目錄 V
圖表目錄 VIII
第一章 文獻回顧 1
1-1 豬生殖與呼吸綜合症病毒 1
1-1-1 病毒結構與基因特性 1
1-1-2 豬生殖與呼吸綜合症病毒疫苗發展 2
1-1-3 商業化疫苗 3
1-2 桿狀病毒 4
1-2-1 桿狀病毒/哺乳動物細胞表現系統 5
1-2-2 桿狀病毒疫苗載體 6
1-2-3 桿狀病毒表現系統 10
1-2-4 flashBACTM桿狀病毒表現系統 11
1-3 Cre/LoxP 重組酶系統 12
1-4 Gibson Assembly 13
1-5 研究動機 14
第二章 實驗材料及方法 25
2-1 重組桿狀病毒建構 25
2-1-1 Bac-to-Bac®轉殖載體(Donor Plasmid)之建構 25
2-1-2 重組bacmid之轉位反應 26
2-1-3 重組bacmid之分離 26
2-1-4 重組bacmid之轉染反應 27
2-1-5 Gibson Assembly Reaction 27
2-1-6 Gibson Assembly重組載體的轉染與放大 29
2-1-7 放大重組桿狀病毒 29
2-1-8 重組桿狀病毒之感染效價分析(infectious titer) 30
2-1-9 PRRS病毒膜醣蛋白及膜蛋白生產 30
2-1-10 PNGase F去蛋白醣基化 31
2-2 細胞培養 31
2-2-1 昆蟲細胞培養 31
2-2-2 哺乳動物細胞培養 31
2-3 轉染哺乳動物細胞 32
2-4 桿狀病毒轉導與分析 32
2-4-1 轉導哺乳動物細胞 32
2-4-2 流式細胞儀分析(flow cytometry) 32
2-4-3 Bradford 蛋白質濃度測定 33
2-4-4 SDS-PAGE、西方墨點法(Western blot analysis) 33
2-5 蛋白質純化 35
第三章 結果與討論 44
3-1 Cre/LoxP系統於PK(15)細胞內的重組效率 44
3-2 長效型PRRS病毒載體疫苗開發 45
3-2-1 重組桿狀病毒的建構 46
3-2-2 以Cre/LoxP長效系統於哺乳動物細胞內表現N protein 46
3-3 病毒表現載體測試與生產條件篩選 48
3-4 GP5蛋白及M蛋白的醣基化和二聚體 49
3-5 蛋白質的純化與分析 49
3-6 討論 50

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