臺灣博碩士論文加值系統

登革病毒為斑蚊所傳播的蟲媒病毒之一，一旦可能會引起登革熱及嚴重的登革出血熱(Dengue Hemorrhagic Fever；DHF)和登革休克症候群(Dengue Shock Syndrome；DSS)。本實驗室先前曾發現源自於蚊蟲之—C6/36細胞在感染登革病毒後，GRP78/BiP會大量表現，已有研究指出，GRP78/BiP會與登革病毒的外套蛋白產生交互作用；也有報導GRP78/BiP會幫助蛋白摺疊、並且作為內質網(endoplasmic reticulum; ER)壓力的訊息調節者。為了瞭解GRP78/BiP與登革病毒之間的互動關係，本實驗利用反轉錄聚合酶反應(RT-PCR)以及TA選殖(TA cloning)的方式找出C6/36細胞中的GRP78/BiP基因序列，作為後續研究的基礎。此外，我們也利用共同免疫沉澱分析法 (Co-immunoprecipitation assays)分析GRP78/BiP與登革病毒蛋白質之間的交互作用，發現GRP78/BiP與病毒的套膜蛋白有直接之交互作用。同時我們以confocal microscopy觀察GRP78/BiP與病毒蛋白在C6/36細胞內的分布情形，發現兩者有co-localization的現象。另外我們利用mircoRNA的方式來降低GRP78/BiP的表現，並且觀察到病毒的產生會受到影響，因此我們推測在登革病毒感染時，GRP78/BiP增加表現量，可能幫助病毒複製有密切關係。

Dengue virus (DENV), a member of Flavivirus, which is transmitted by the bite of infected mosquito of the genus Aedes between human. Clinical symptoms of the infection can vary from severe to asymptomatic. In addition to Dengue fever, Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS) are severe forms which can threaten the patient’s life. Based on our previous study, BiP/GRP78 gene was identified for its differential expression in DENV-infected mosquito cell line C6/36 compared with mock-infected cells. BiP/GRP78 is a major endoplasmic reticulum (ER) chaperone protein critical for protein quality control of the ER, as well as for the activation of the ER-transmembrane signaling. BiP/GRP78 is also important for assisting protein folding and modulating ER stress. This study is aimed to investigate the response of BiP/GRP78 in mosquito cells with Dengue virus infection. In this study, we have found that GRP78/BiP was up-regulated in infected C6/36 cells; which full-length gene sequence was then determined. In addition, we have used Co-immunoprecipitation assays to investigate the interaction between GRP78/BiP and viral proteins, demonstrateing there are interactions between them. We also observed GRP78/BiP and viral protein were co-localized by confocal microscopy. A knockdown system was established to reduce BiP/GRP78 expression; from which the influence on Dengue virus production was observed. Presumably, BiP/GRP78 may play a role in the replication of DENV in mosquito cells.

指導教授推薦書
口試委員會審定書
授權書…………………………………………………………………iii
誌謝…………………………………………………………………… v
中文摘要………………………………………………………………vi
英文摘要………………………………………………………………vii
目錄………………………………………………………………… viii
圖表目錄……………………………………………………………… x
緒論……………………………………………………………………1
第一節 登革病毒簡介……………………………………………1
第二節 Glucose-regulated protein 78簡介………………………6
研究動機………………………………………………………………13
研究目標………………………………………………………………14
實驗流程………………………………………………………………16
材料與方法……………………………………………………………17
實驗結果………………………………………………………………31
一、 C6/36細胞中GRP78/BiP基因的序列全長……………31
二、 GRP78/BiP在感染及未感染登革病毒的C6/36細胞之表現………………………………………………………32
三、 利用microRNA knock-down的系統使GRP78/BiP的表現量降低……………………………………………………33
四、 在登革病毒感染的C6/36細胞與miBiP細胞及未感染的細胞中，其細胞的存活率…………………………………34
五、 在miBiP細胞中病毒的複製是否會受到GRP78/BiP表現量的降低而影響…………………………………………34
六、 GRP78/BiP與登革病毒在C6/36細胞內分佈的情形……35
七、 以免疫共同沉澱法探討在C6/36細胞表現GRP78/BiP與登革病毒感染細胞後的E蛋白的蛋白質間交互作用…………………………………………………………35
討論…………………………………………………………………… 37
參考文獻……………………………………………………………… 43
附錄…………………………………………………………………… 52


表 目 錄
表一、不同病毒的感染時GPR78/BiP的功能………………………52
表二、 不同的病毒感染細胞會引起ER stress的表現………………53
表三、探討GRP78/BiP全長所用之引子……………………………54
表四、應用於micro RNA設計所用之片段序列……………………55

圖 目 錄
圖一、當細胞產生ER stress或是不正常的蛋白堆積時，BiP/GRP78會藉由PERK、IRE1、ATF6三條路徑調控細胞的存活……56
圖二、白線斑蚊GRP78/BiP的基因序列…………………………… 57
圖三、白線斑蚊的GRP78/BiP胺基酸序列與其他兩種蚊子的比較
圖………………………………………………………………58
圖四、白線斑蚊GRP78/BiP在演化樹位置的關係圖………………59
圖五、登革病毒感染C6/36細胞在不同時期GRP78/BiP的基因表現
量………………………………………………………………61
圖六、轉染microRNA的miBiP細胞中GRP78/BiP的基因表現量…62
圖七、感染登革病毒後的miBiP細胞其GRP78/BiP的基因表現
量……………………………………………………………… 63
圖八、C6/36細胞與miBiP細胞感染及未感染之細胞存活率………64
圖九、登革病毒在C6/36細胞和miBiP細胞中的基因表現量及蛋白表現量……………………………………………………………65
圖十、C6/36細胞轉染pAC5.1-neo(b)-GFP-HA-BiP…………………66
圖十一、C6/36細胞未感染病毒、轉染pAC5.1-neo(b)-GFP-HA-BiP蛋
白以及感染病毒後的位置分布……………………………67
圖十二、感染後18小時，病毒E蛋白與pAC5.1-neo(b)-GFP-HA-BiP
蛋白轉染後的位置分布……………………………………68
圖十三、感染後24小時，病毒E蛋白與pAC5.1-neo(b)-GFP-HA-BiP
蛋白轉染後的位置分布……………………………………69
圖十四、C6/36細胞表現GRP78/BiP與登革病毒感染後利用免疫共同沉澱法分析之結果…………………………………………70

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