臺灣博碩士論文加值系統

嚴重急性呼吸道症候群(SARS)是一種新興的傳染性疾病，為一種新型的冠狀病毒(SARS-CoV)所引起。嚴重急性呼吸道症候群冠狀病毒已經被發現可以抑制干擾素反應的訊息傳遞路徑。SARS-CoV ORF6 蛋白被報導與第一型干擾素產生拮抗作用，以及細胞內抑制STAT-1 進入細胞核內之作用。本篇論文的主要目的為利用噬菌體呈現技術鑑定與SARS-CoV ORF6 蛋白結合之細胞宿主蛋白及其交互作用與拮抗第一型干擾素訊息傳遞之相關性。本論文首先構築SARS-CoV ORF6 重組載體，藉細菌表現系統，製備ORF6 重組蛋白。噬菌體表現人類肺cDNA 基因庫，經過五次bio-panning 後，找出與ORF6 蛋白結合的肺細胞蛋白。PI-3 kinase-related kinase SMG-1 為其中之一。我們以共軛焦顯微鏡觀察確認病毒蛋白ORF6 與宿主細胞蛋白SMG-1 在細胞中有co-locolization 的現象。之後以ISRE-reporter gene assay 以及Real-time RT-PCR 對表現SARS CoV ORF6 蛋白的HL-CZ 細胞及對照組細胞加入第一型干擾素作用4 小時，發現與對照組相較下， SARS CoV ORF6 蛋白表現細胞的ISRE 啟動子的活性表現量以及ISRE 下游PKR mRNA 有明顯被抑制的現象。而後同樣以ISRE-reporter gene assay 對SARS CoV ORF6 蛋白與SMG-1 蛋白共同表現的細胞同樣加入第一型干擾素作用，相較下發現ISRE 啟動子的活性表現量因有SMG-1 蛋白的存在而明顯有回復升高的現象。接著，我們使用共軛焦顯微鏡觀察發現單獨表現ORF6 蛋白的細胞株加入第一型干擾素作用會抑制STAT-1 進入細胞核內，但是SARS CoV ORF6 蛋白與
SMG-1 蛋白共同表現的細胞株加入第一型干擾素作用後，會有使STAT-1進入細胞核內的現象。同樣地，以西方點墨法來觀察STAT-1 磷酸化情形，發現單獨表現ORF6 蛋白的細胞株加入第一型干擾素作用無STAT-1磷酸化，但是SARS CoV ORF6 蛋白與SMG-1 蛋白共同表現的細胞株加入第一型干擾素作用後，會有使STAT-1 產生磷酸化作用。由上述推測SARS CoV ORF6 蛋白抑制第一型干擾素訊息傳遞之現象會因共同表現宿主細胞SMG-1蛋白而對干擾素反應，如ISRE 啟動子活性以及下游PKR基因表現與單獨表現病毒蛋白ORF6 的細胞株相較之下有回復提升活性的現象。由此我們推測會與病毒蛋白ORF6 作用的宿主細胞蛋白SMG-1在對於嚴重急性呼吸道症候群冠狀病毒拮抗第一型干擾素免疫反應中可能扮演重要腳色。

Abstract
Severe acute respiratory syndrome (SARS) is a newly emerged
infectious disease. The causative agent of SARS has been identified to be a new type of coronavirus, namely SARS coronavirus (SARS-CoV).SARS-CoV proteins including the ORF6 protein have been reported to inhibit interferon signaling response. The goal of this study is to identify human SARS-CoV ORF6-interacting proteins using the phage displayed human lung cDNA libraries and to investigate their interaction on the type I interferon antagonist function. At first, the recombinant ORF6 protein was generated in BL21(DE3) and purified by using IMAC (immobilized mental-affinity chromatography). After five rounds of biopanning, PI-3 kinase-related kinase SMG-1 protein was identified as SARS-CoV ORF6 interacting protein from phage displayed lung cDNA library. Confocal imaging revealed co-localization of SARS-CoV ORF6 protein with SMG-1 protein in HL-CZ cells. In vivo signaling pathway assay and real time RT-PCR showed that single gene expression of SARS-CoV ORF6-expressing cells blocked the INFα/β-induced responses, such as ISRE-responsive firefly luciferase activity and the expression of PKR. However, both gene expression of SARS-CoV
ORF6 and SMG-1 had a significantly higher relative activities of INFα/β-induced ISRE-responsive firefly luciferase than single gene expression of SARS-CoV ORF6 in HL-CZ cells. In addition, confocal imaging and Western blotting assays revealed that the translocation of STAT-1 into nucleus and the STAT-1 phophorylation were found in the transfected cells expressing both genes of ORF6 and SMG-1, but not in the ORF 6-expressing cells in response to INFα/β. Therefore, cell expression of PI-3 kinase-related kinase SMG-1 could restore the INFα /β responses in SARS-CoV ORF6 expressing cells. The interaction of SARS CoV ORF6 and SMG-1 may be responsible for the inhibition of type I IFN response by SARS-CoV.

目錄
中文摘要 ………………………………………………………………ⅤⅠ
英文摘要 ………………………………………………………………ⅤⅢ
謝辭………………………………………………………………………Ⅹ
表目錄 ………………………………………………………………ⅩⅡ
圖目錄 ………………………………………………………………ⅩⅢ
第一章 前言……………………………………………………………… P1
第一節 研究背景與文獻查證…………………………………………… P1
1-1嚴重急性呼吸道症候群………………………………………………P1
1-1-1. 嚴重急性呼吸道症候群…………………………………………P1
1-1-2. 嚴重急性呼吸道症候群冠狀病毒(SARS coronavirus ,SARS CoV)
……………………………………………………………………………P2
1-1-3. 嚴重急性呼吸道症候群冠狀病毒基因組(genome) ……………P3
1-1-4. 嚴重急性呼吸道症候群冠狀病毒ORF 6蛋白…………………P4
1-1-5. 嚴重急性呼吸道症候群的診斷與治療…………………………P5

1-2第一型干擾素之分子機轉……………………………………………P6
1-2-1干擾素系統介紹…………………………………………………P6
1-2-2干擾素……………………………………………………………P7
1-2-3干擾素相關訊息傳遞路徑………………………………………P8
1-2-4. 病毒逃避宿主第一型干擾素免疫機轉之方法………………P9

第二節 研究目的…………………………………………………………P10
第二章 研究方法…………………………………………………………P11
第一節 研究設計…………………………………………………………P11

2-1-1 : 構築表現SARS CoV ORF6重組蛋白之基因載體………P11
2-1-2 : 利用噬菌體篩選法(phage display)找出可能與SARS CoV ORF6重組蛋白有高度結合的宿主蛋白…………………P11
2-1-3 : 確認此宿主蛋白的確與SARS CoV ORF6重組蛋白有高度的結合作用，並鑑別此宿主蛋白為何………………………P12
2-1-4 : 利用ISRE-Luciferase assay system以及Real-time RT- PCR等技術偵測SARS CoV ORF6重組蛋白與第一型干擾素之作用……………………………………………………………P12
2-1-5 : 利用SARS CoV ORF6與宿主蛋白(SMG-1)的結合力來偵測是否會影響第一型干擾素及其下游的訊息傳導路徑……P13

第二節 研究材料…………………………………………………………P14
2-2-1. SARS CoV全長分段基因組cDNA載體……………………P14
2-2-2.細胞株…………………………………………………………P14
2-2-3.大腸桿菌菌株（ Escherichia coli）…………………………P14
2-2-4.質體(Vector) …………………………………………………P15
2-2-5.抗體(Antibody)………………………………………………P16
2-2-6.引子(Primer)…………………………………………………P16
2-2-7.噬菌體呈現人肺cDNA基因庫………………………………P17
2-2-8.實驗試劑及緩衝液……………………………………………P17

第三節 實驗方法…………………………………………………………P18
2-3-1 質體純化(plasmid purification)……………………………P18
2-3-2 聚合酵素連鎖反應（polymerase chain reaction，PCR）…P18
2-3-3 限制?﹞蘢恁K………………………………………………P19
2-3-4 質體構築：DNA 接合ligation（DNA ligation）…………P19
2-3-5 勝任細胞（competent cell）製備……………………………P19
2-3-6 大腸桿菌的轉型（transformation）…………………………P20
2-3-7 誘導大腸桿菌上乳醣啟動子（lac promoter）表現蛋白…P20
2-3-8 IMAC純化重組病毒蛋白質（FPLC）……………………P21
2-3-9 蛋白質濃度測定……………………………………………P21
2-3-10 蛋白質之電泳分析(SDS-PAGE)…………………………P22
2-3-11 西方墨點法（Western bloting）…………………………P23
2-3-12 噬菌體的生長與儲存（Growth and storage of Bacteriophage T7）…………………………………………………………P23
2-3-13 測定噬菌體的效價（phage titer determination）…………P24
2-3-14 噬菌體篩選（biopanning）…………………………………P24
2-3-15 噬菌體酵素連結免疫吸附分析（phage enzyme-linked
immunosorbent assay）……………………………………P25
2-3-16 噬菌體DNA製備（ Rapid purification of phage sequencing
templates）……………………………………………………P26
2-3-17 共同免疫沉澱法(Co-immunoprecipitation) ………………P26
2-3-18 DNA轉染（DNA transfection）…………………………P27
2-3-19細胞內分子之免疫螢光染色 (Immunofluorescence staining)
………………………………………………………………………P27
2-3-20冷光測試ISRE啟動子的活性（measure ISRE activity by luciferase assay）……………………………………………P28
2-3-21細胞RNA 抽取(purification of cell RNA) …………………P28
2-3-22反轉錄酵素-聚合酵素連鎖反應……………………………P29
2-3-23即時定量聚合??連鎖反應…………………………………P30
第三章 研究結果…………………………………………………………P32
第一節 鑑定與急性呼吸道症候群冠狀病毒非結構蛋白ORF6作用的相關細胞因子………………………………………………………P32
3-1-1. 構築表現SARS CoV ORF6重組蛋白之基因載體…………P32
3-1-2. 以西方墨點法鑑定經FPLC純化後之急性呼吸道症候群冠狀 病毒ORF6重組蛋白表現……………………………………P32
3-1-3. 以急性呼吸道症候群冠狀病毒ORF6重組蛋白進行噬菌體親和性篩選……………………………………………………P33
3-1-4. 以共同免疫沉澱法證明急性呼吸道症候群冠狀病毒ORF6重組蛋白與SMG-1蛋白有結合力……………………………P34
3-1-5. 共同轉染質體觀察細胞內表現重組蛋白質交互作用……P35

第二節 急性呼吸道症候群冠狀病毒ORF6蛋白抵抗第一型干擾素反應訊息傳遞的機制探討…………………………………………P35
3-2-1. 細胞內表現急性呼吸道症候群冠狀病毒ORF6重組蛋白…P35
3-2-2. 在第一型干擾素作用下偵測急性呼吸道症候群冠狀病毒ORF6重組蛋白在細胞中表現對ISRE啟動子的效能影響……………………………………………………………P36
3-2-3. 以Real-time PCR偵測在第一型干擾素作用下急性呼吸道症候群冠狀病毒ORF6重組蛋白在細胞中所影響ISRE下游基因表現……………………………………………………………P37

第三節 探討SARS CoV ORF6與宿主蛋白(SMG-1)的作用是否會影響第一型干擾素及其下游的訊息傳導路徑…………………………P37
3-3-1. 表現SMG-1與ORF6重組蛋白之細胞，對於第一型干擾素反應之影響……………………………………………………P37
3-3-2. 表現SMG-1與ORF6重組蛋白之細胞，對於STAT-1訊息傳遞之影響……………………………………………………P38
3-3-3. 表現病毒蛋白ORF6與SMG-1的細胞，抑制細胞內STAT-1蛋白的磷酸化………………………………………………P39

第四章討論………………………………………………………………P40
第一節 急性呼吸道症候群冠狀病毒ORF6蛋白與細胞內宿主蛋白的交互作用……………………………………………………………P40
第二節 急性呼吸道症候群冠狀病毒ORF6蛋白抵抗干擾素反應即訊息 傳遞的機制………………………………………………………P41
第三節 探討SARS CoV ORF6與宿主蛋白(SMG-1)的作用是否會影響第一型干擾素及其下游的訊息傳導路徑…………………………P42

第五章結論與建議………………………………………………………P43
第一節 結論………………………………………………………P43
第二節 建議………………………………………………………P43
參考文獻…………………………………………………………………P44
表…………………………………………………………………………P51
圖…………………………………………………………………………P56

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