如何表現蛋白質是生物醫學常見的研究課題。除了高效率之外，蛋白質表達系統的另一個挑戰為重組蛋白的偵測與純化。利用桿狀病毒表現系統生產重組蛋白為目前廣為使用的方法之一，此系統除了具有高產量的優點，也可以生產出具有正確摺疊與轉譯後修飾的蛋白質。Fractalkine蛋白具有許多重要的功能，包含調節細胞生長分化和凋亡的作用，並在心血管疾病過程中扮演重要的角色。Fractalkine蛋白主要藉由其chemokine domain與CX3CR1受體結合產生作用。為了進ㄧ步了解fractalkine的作用， 本研究的目標為利用Bac-to-Bac桿狀病毒表達系統建構fractalkine chemokine domain的表現載體，此載體表達的蛋白質在N端具有一段Igκ signal peptide分泌訊號，藉由這段分泌訊號的幫助，細胞會將製造出的重組蛋白分泌至培養液中。而在重組蛋白的C端具有polyhistidine tag與myc tag兩個親和性標籤以方便進行偵測與純化。實驗結果顯示利用桿狀病毒系統在HEK293 和HeLa cells 可以大量表現fractalkine chemokine domain並且從HEK293細胞內分泌出來，但是在THP-1 monocyte與Jurkat T cells則並沒有表現。在HEK293細胞經病毒感染後60小時即可偵測到重組蛋白的表現，在72小時與96小時後產生更強的效果。這些結果顯示我們已經成功地建立一套高效率的蛋白質表現系統，此系統具有易於偵測與純化的優點，在未來可利用本套系統製造並純化出具有活性的蛋白質以用於蛋白質的結構與功能性的研究。

Baculovirus expression system has often been utilized to express recombinant proteins.The advantages of this system include high efficiency, ability to express proteins in manytypes of cells, and production of proteins with correct conformation and posttranslational modifications. Fractalkine is a membrane protein mainly exerting its effects through binding of extracellular chemokine domain to the CX3CR1 receptor. It regulates proliferation, differentiation, and apoptosis of cells. It also plays an important role in cardiovascular diseases. The goal of this thesis is to construct a baculovirus vector system to express fractalkine chemokine domain for further functional studies. The vector system is engineered to contain a secretion signal peptide sequence and two affinity tags, polyhistidine tag for purification and myc tag for immunoblotting. Using this system, it was found that fractalkine chemokine domain is able to express in HEK293 and HeLa cells, whereas THP-1 monocyte and Jurkat T cells are without effect. The expressed recombinant protein could be detected at 60 h after viral infection and the majority appeared to be produced after 72 h. In addition, the fractalkine chemokine domain produced in HEK293 cells was able to purified from the medium through polyhistidine tag pulldown. These results indicate that a highly efficient protein expression system with advantages of easy detection and purification has been established and this system is a valuable tool in structural and functional studies of proteins.

中文摘要 i
英文摘要 ii
目錄 iii
圖目錄 vi
表目錄 vii
縮寫對照表 viii
第一章 緒論 1
1-1 蛋白質表現系統 1
1-1.1 原核細胞表現系統 1
1-1.2 酵母菌表現系統 2
1-1.3 昆蟲細胞表現系統 3
1-1.4 哺乳動物細胞表現系統 4
1-2 桿狀病毒之簡介 5
1-2.1 桿狀病毒之生活史 5
1-2.2 桿狀病毒進入細胞之路徑 7
1-2.3 昆蟲桿狀病毒載體表現系統 8
1-2.4 桿狀病毒轉導哺乳動物細胞 9
1-3 Fractalkine 之簡介 11
1-3.1 Fractalkine 的結構 11
1-3.2 Fractalkine 的受體 12
1-3.3 Fractalkine 的訊息路徑 13
1-3.4 Fractalkine 的功能 14
1-4 Affinity tag 之簡介 15
第二章 研究動機與目的 18
第三章 材料與方法 19
3-1 材料 19
3-1.1 Bacteria strains 19
3-1.2 細胞株 19
3-1.3 質體 19
3-1.4 藥品試劑 19
3-1.2.1 Bacteria strains 培養試劑 19
3-1.2.2 細胞培養試劑 19
3-1.2.3 ㄧ般藥品與試劑 20
3-2 研究方法 20
3-2.1 質體DNA 純化 20
3-2.2 聚合酶鏈鎖反應 (PCR) 21
3-2.3 限制酶切割反應 21
3-2.4 DNA 接合反應（DNA ligation） 21
3-2.5 Heat shock- transformation 21
3-2.6 Electroporation - transformation 22
3-2.7 Bacmid DNA 純化 22
3-2.8 細胞培養 22
3-2.9 Transfection 至Sf21 細胞 23
3-2.10 病毒的放大 23
3-2.11 桿狀病毒感染使重組蛋白質於真核細胞表現 23
3-2.12 His-tag purification assay 24
第四章 結果與討論 25
4-1 構築帶有Fractalkine chemokine domain 的質體pFastBac-CMV-FKNCDmyc-
His tag 25
4-2 構築bacmid -CMV-FKNCD-myc-His tag DNA 26
4-3 轉染bacmid -CMV-FKNCD-myc-His tag DNA 至昆蟲細胞產生重組病毒
vAc-FKNCD 27
4-4 重組病毒vAc-FKNCD 在哺乳動物黏附型細胞 (HEK293 cells、CHO-K1
cells 以及HeLa cells)的表現 28
4-5 重組病毒vAc-FKNCD 在哺乳動物懸浮型細胞 (THP-1 cells 和 Jurkat cells)
的表現 29
4-6 重組病毒vAc-FKNCD 在哺乳動物細胞HEK293 cells 中的純化 30
4-7 重組病毒vAc-FKNCD 在哺乳動物細胞HEK293 cells 外的分泌以及純化 31
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