臺灣博碩士論文加值系統

　　登革熱仍是目前非常重要的新興病毒感染症，每年都會發生千萬以上的病例。登革病毒感染除了造成類似感冒症狀的登革熱之外，嚴重者也會形成登革出血熱，甚至變成登革休克症候群，死亡率極高。但是由於登革病毒的致病機轉仍是眾說紛紜，莫衷一是，因此對於登革熱的防治也就困難重重。然而最近已有許多研究報告顯示，登革病毒進入標的細胞與硫酸乙醯肝素受體有很大的關係，登革病毒有可能與硫酸乙醯肝素蛋白吸附後，再經由內噬作用進入標的細胞。CD164分子是一種最近才被確認之唾液酸黏蛋白，除了在所有的組織或細胞都會表現外，結構上CD164具有與硫酸乙醯肝素類似之葡萄糖胺聚合醣結合位，功能上CD164也參與了細胞的內噬作用，因此CD164可能在登革病毒入侵標的細胞的機制中扮演著某種角色。本研究初步結果證明，CD164分子重組鑲嵌蛋白可能會和登革病毒套膜蛋白鑲嵌蛋白接合，然而它們之間確切的關係仍需更多的實驗數據方能證明。

　　Dengue virus (DV) is an arthropod-borne human pathogen that represents a serious public health threat. There are at least 20 million infections in the world each year and several hundred thousand cases of a severe, life-threatening syndrome known as dengue hemorrhage fever/dengue shock syndrome (DHF/DSS). However，the mechanism of pathogenesis of DV is still not clear. Recent studies showed that DV serotype-2 binds selectively to highly-sulfated heparan sulfates, a member of glycosaminoglycans (GAG), on target cells via the viral E glycoprotein. The viral envelope E protein of dengue virus, may mediate virus attachment and entry to target cells by penetration or endocytosis. CD164 is a newly identified 80-110 kD sialomucin with a potential GAG attachment site. CD164 is localized on both the cell surface and in the recycling endosomes and lysosomes, and it was more prominently expressed at the cell surface and in recycling endosome in some cells. Therefore, CD164 molecule may play a very important role on the pathogenesis of dengue virus. To further our understanding of the molecular mechanisms that govern dengue virus infection of target cells and the functions of CD164, we tried to investigate the relationship between dengue virus infection and CD164 molecule. Our preliminary results showed that CD164 recombinant construct may bound to envelop protein of DV. However, it still needs more experiments to prove the relationship between CD164 and dengue virus envelope glycoprotein .

正文目錄
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　頁
第一章、 緒言……………………………………………………………1
第二章、 材料與方法
壹、勝任細胞（competent cells）之製備……………………………11
貳、細胞基因轉型（transformation）………………………………11
參、DNA之微量純化（minipreps）……………………………………12
肆、DNA之大量純化（maxipreps）……………………………………13
伍、限制酶（restriction enzymes）反應與瓊脂膠體電泳（agarose gel electrophoresis）……………………………………14
陸、聚合酶連鎖反應（polymerase chain reaction；PCR）…15
柒、Syndecan-4 RNA之純化……………………………………………15
捌、反轉錄酶─聚合酶連鎖反應（reverse transcriptase polymerase chain reaction；RT-PCR…………………………………16
玖、分離與純化syndecan-4 cDNA………………………………………17
拾、人類syndecan-4 cDNA之選殖（cloning）………………………17
拾壹、序列分析（sequencing）………………………………………18
拾貳、細胞培養（cell culture）……………………………………19
拾參、細胞基因轉染（transfection）………………………………19
拾肆、純化可溶性重組鑲嵌蛋白（recombinant chimeric DNA constructs）……………………………………………………………19
拾伍、以DTT（dithiothreitol）處理重組鑲嵌蛋白…………………20
拾陸、以第一型肝素酶（heparainase I）處理syndecan-4重組鑲嵌蛋白………………………………………………………………………20
拾柒、SDS-PAGE電泳（sodium dodecyl sulphate-polyacryamide
gel electrophoresis）…………………………………………………21
拾捌、轉漬（electrotransfer）………………………………………22
拾玖、西方點墨法（Western blot）…………………………………23
第三章、 結果
壹、人類syndecan-4 cDNA質體之製備…………………………………24
貳、選殖之syndecan-4 cDNA之微量純化………………………………24
參、選殖之人類syndecan-4 cDNA之序列………………………………25
肆、各重組DNA之製備……………………………………………………25
伍、各重組鑲嵌蛋白的製備與西方點墨試驗測試……………………26
陸、以DTT處理重組鑲嵌蛋白……………………………………………26
柒、CD164(E6a)與CD164(DE5)重組鑲嵌蛋白之西方點墨試驗………27
捌、登革病毒套膜重組鑲嵌蛋白之西方點墨試驗……………………28
第四章、 討論……………………………………………………………29
參考文獻…………………………………………………………………66
表目錄
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表一、各種不同葡萄糖胺聚合醣之不同結構…………………………60
表二、本研究所使用的引子（primers）………………………………61
表三、本研究中西方點墨法（Western blot）所使用的第一抗體（primary antibodies）………………………………………………62
表四、套膜病毒的細胞表面受體………………………………………63
表五、與肝素或硫酸乙醯肝素結合的蛋白質…………………………64
表六、與細胞表面硫酸乙醯蛋白醣結合的微生物致病原……………65
圖目錄
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　頁
圖一、登革病毒的基因圖………………………………………………41
圖二、硫酸乙醯蛋白醣之預期結構卡通圖……………………………42
圖三、以接受器為媒介的內噬作用（receptor-mediated endocytosis）
……………………………………………………………………………43
圖四、人類、小鼠、大鼠和果蠅之CD164之胺基酸序列………………44
圖五、CD164(E6a)與CD164(DE5)之cDNA序列…………………………45
圖六、CD164(E6a)與CD164(DE5)之預期結構卡通圖…………………46
圖七、本研究中syndecan-4重組DNA所使用的yT&A選殖載體…………47
圖八、人類syndecan-4之預期結構卡通圖……………………………48
圖九、以反轉錄酶聚合酶連鎖反應選殖人類syndecan-4 ……………49
圖十、人類syndecan-4 DNA之微量純化………………………………50
圖十一、人類syndecan-4之cDNA序列…………………………………51
圖十二、本研究中CD164 (E6a)、CD164(DE5)、syndecan-4與L-selection
重組DNA所使用的IgMu/pEF-BOS表現載體………………………………52
圖十三、本研究中DEN2 E重組DNA所使用的pcDNA表現載體…………53
圖十四、本研究所用來製備重組鑲嵌蛋白之重組DNA…………………54
圖十五、U937細胞株之CD164與α-tubulin西方點墨試驗……………55
圖十六、CD164(E6a)重組鑲嵌蛋白之西方點墨試驗…………………56
圖十七、CD164(E6a)與CD164(DE5)重組鑲嵌蛋白之西方點墨試驗…57
圖十八、CD164(DE5)與CD164(E6a)和syndecan-4重組鑲嵌蛋白之西方點墨試驗………………………………………………………………………58

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