臺灣博碩士論文加值系統

人類巨細胞病毒〈Human cytomegalovirus (HCMV)〉是一種廣泛存在於人類之間的一種病原菌，不僅是導致胎死腹中的主要病毒，且是免疫不全病人發病及致死的重要因素之一。HCMV的迅早期〈immediate-early (IE)〉基因IE1及IE2是病毒感染細胞後最早表現的基因，是由轉錄活性很強的主要迅早期啟動子〈major IE promoter (MIEP)〉來控制迅早期基因的表現。其中較為重要的是分子量86-kDa的IE2蛋白〈IE2-86〉，被認為在病毒感染過程與致病不可或缺的蛋白。IE2-86的特性是透過位於轉錄起始位置下游的同位抑制訊息〈cis-repression signal (CRS)〉抑制調節MIEP的活性。由於IE2-86在轉錄調節上的特性，因此進一步研究IE2-86在HCMV中是否存在其他可調節的基因或DNA片段，就顯得非常重要。本論文綜合“標的循環增幅與選擇”〈cyclic amplification and selection of targets (CASTing)〉及“結合位置的選擇與增幅”〈selection and amplification of binding sites (SAAB)〉等兩種方式，合併輔以DNA足跡分析〈DNase I footprinting〉及電泳漂移實驗〈electromobility shift assay (EMSA)〉，篩選可和IE2-86結合的HCMV基因體DNA片段。結果顯示—170 box及—240 box不僅存在MIEP中，且具有與CRS類似的DNA序列架構，並且以定位突變反應分析這兩個DNA片段說明功能上亦與CRS類似，當與IE2-86蛋白結合時，MIEP的活性即受到抑制。—170 box及—240 box的中間DNA序列存在AP1轉錄因子的結合序列〈TRE〉，推論IE2-86與—170 box或—240 box結合所產生的抑制機制與CRS並不相同，而是IE2-86與AP1兩種蛋白與新發現的IE2-86結合序列〈—170 box及—240 box〉間的動態調節。

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen that is the leading viral cause of birth defects and also causes significant morbidity and mortality in immunosuppressed individuals. The immediate-early (IE) genes, IE1 and IE2, are the first HCMV genes expressed after infection under the control of a strong transcriptional enhancer-promoter, major IE promoter (MIEP). Gene expression mediated by the predominant HCMV 86-kDa IE2 protein (IE2-86) is believed to be essential for the progression of the viral production, as well as for the development of HCMV-associated pathogenesis. Further, IE2-86 can shut off the activity of MIEP through a specific DNA signal called the cis-repression signal (CRS). It therefore becomes important to identify transcriptional activity of IE2-86 by recognizing downstream targets within HCMV. By a modified approach coupling the CASTing (cyclic amplification and selection of targets) and SAAB (selection and amplification of binding sites), several HCMV genomic fragments were selected based on their ability to interact with IE2-86. Among them, two additional CRS-like DNA sequences, —170 box and —240 box, were confirmed further by DNase I footprinting and electromobility shift assay. Substituted mutation analysis showed that these two sites negatively regulate activity of MIEP with IE2-86. Both the novel boxes contain a consensus-binding site (TRE) for AP1 complex. It shows that the mechanisms of —170 and —240 boxes are different from that of CRS element. The regulated activity of the complicate MIEP is in the dynamic configuration of two proteins (IE2-86 and AP1) and two additional DNA elements (—170 box and —240 box).

目 錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ I
表 目 錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ III
圖 目 錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ IV
中文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ VI
英文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ VII
緒言‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 1
材料與方法‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 16
壹、 實驗材料
一、 化學藥品及酵素 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 16
二、 實驗應用之寡核酸的種類及序列 ‧‧‧‧‧‧‧ 16
貳、 實驗方法
一、 質體的獲得與構築 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 18
二、 質體DNA之抽取與純化 ‧‧‧‧‧‧‧‧‧‧ 20
三、 勝任細胞的製備與質體DNA轉形‧‧‧‧‧‧‧ 23
四、 定位突變法 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 24
五、 西方點墨法 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 26
六、 人類細胞株培養 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 28
七、 質體DNA轉染‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 29
八、 純化大腸桿菌表現的融合蛋白 ‧‧‧‧‧‧‧‧ 31
九、 部分純化細胞萃取液中的IE2-86蛋白 ‧‧‧‧‧ 35
十、 人類巨細胞病毒基因體DNA的純化‧‧‧‧‧‧ 35
十一、 CASTing-SAAB複合步驟篩選人類巨細胞病毒IE2-86蛋白可結合的DNA片段 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 37
十二、 螢火蟲螢光素酶的活性分析 ‧‧‧‧‧‧‧‧‧ 40
十三、 DNA足跡分析‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 41
十四、 電泳流動漂移分析 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 43
十五、 DNA-蛋白免疫共沉澱‧‧‧‧‧‧‧‧‧‧‧‧ 44
結果‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 46
壹、 CASTing-SAAB複合步驟篩選出可與人類巨細胞病毒IE2-86蛋白結合之人類巨細胞病毒基因體DNA ‧‧‧‧‧‧‧‧‧‧‧‧ 46
貳、 DNA足跡分析MIEP中人類巨細胞病毒 IE2-86蛋白結合的位置‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 48
參、 電泳流動漂移分析活體外人類巨細胞病毒 IE2-86蛋 白分別和第—178序列到第—165序列及第—242序列到第—229序列之間結合 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 49
肆、 人類巨細胞病毒 IE2-86蛋白抑制MIEP的活性 ‧‧50
伍、 MIEP內IE2-86和AP1兩者蛋白的DNA結合位置相互重疊‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 52
陸、 在不同細胞株中IE2-86蛋白的存在與否對MIEP活性的影響 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 54
討論 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 56
結論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 73
參考文獻‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 74

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