臺灣博碩士論文加值系統

在全世界C型肝炎病毒已是造成輸血後非A非B型肝炎的主要病原。根據世界衛生組織的統計現在全球有3 %的人口感染C型肝炎病毒；感染後大多數呈現慢性肝炎的症狀，最終發展成肝硬化或肝癌。目前，以干擾素合併ribavirin的方法治療較為有效，可是仍有半數以上的病患等待開發新療法來治癒這棘手的疾病。因此，分析研究C型肝炎病毒與尋找新的治療方式或藥物是非常急迫的。
C型肝炎病毒的非結構性蛋白質3（NS3）位於複合蛋白質第1027-1657個氨基酸的位置，是一個設計巧妙、具有多重酵素活性的蛋白質。它的N端1/3部分（第1027-1207個氨基酸）是絲氨酸裂解，作用在非結構性蛋白質3/4A、4A/4B、4B/5A、5A/5B的切點，以NS4A為輔因子；另外C端2/3的部分具有核甘三磷酸及RNA解螺旋的活性。NS3蛋白質的N端及C端在自然狀況下不會再解離成二個次產物，而且NS3會與NS4以非共價鍵鍵結著。本論文以RNA解螺旋活性為主軸，探討NS3與NS4A分子間的交互作用是否會影響解螺旋及核甘三磷酸的活性；又NS3分子內N端和C端的酵素活性是否會彼此干擾或相互協助。
利用昆蟲桿狀病毒表現系統表現NS3- NS4A、全長NS3蛋白質、及只含NS3 C端helicase domain蛋白質。比較這三種蛋白質的RNA解螺旋活性、核甘三磷酸活性與RNA結合能力。結果發現NS4A會增進RNA解螺旋脢活性、RNA結合能力、和核甘三磷酸活性；而N端的裂解雖會抑制RNA解螺旋活性，卻會促進核甘三磷酸的活性。此外，一價鉀離子對這三種蛋白質helicase活性的影響不大相同：鉀離子濃度高過75 mM時，NS3- NS4A helicase活性下降，而在鉀離子75 mM的環境下，NS3蛋白質的helicase活性最大；反之，鉀離子不利NS3 helicase domain之helicase反應。這些初步的實驗結果，將可提供未來更進一步探究其分子間、分子內相互影響的機制，期望從中找出一絲端倪，提供發展抗C型肝炎病毒新藥物的開發。

Hepatitis C virus (HCV) is the major etiological agent of post- transfusion- associated non- A, non- B hepatitis worldwide. An estimated 3 % of the world’s population is infected with HCV, according to the World Health Organization. HCV infection most commonly results in chronic hepatitis that eventually develops into cirrhosis and hepatocellular carcinoma. The current available therapy, using interferon- α and combination with ribavirin, has worked on less than 50 % of the patients. Therefore, there is an urgent need for new therapies.
The non-structure protein 3 (NS3) of HCV ranges from a. a. 1027 to 1657 of the poly-protein and is a well- designed and multi- functional protein. The N terminal one-third (from a. a. 1027 to 1207) of NS3 protein contains a serine protease activity, which catalyzes the cleavage between NS3/ NS4A, NS4A/ NS4B, NS4B/ NS5A and NS5A/ NS5B. The NS4A protein is its functional cofactor. The C terminal two-thirds region of NS3 protein contains an ATPase activity and an RNA helicase activity. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic procession in vivo. The thesis investigates the inter-molecular effects on RNA helicase activity between NS3 and NS4A and the intra-molecular influences between the N terminal domain and the C terminal domain of the NS3 protein.
We used baculovirus expression system to produce NS3- NS4A, full-length NS3 protein, and a truncated NS3 protein that only contains the C terminal helicase domain, and assayed their helicase activity. Our data showed that NS4A up-regulated the RNA helicase activity, RNA binding ability, and the ATPase activity. Unlike the NS4A protein, the N terminal protease domain of the HCV NS3 protein down-regulated the RNA helicase activity, but enhanced the ATPase activity. Besides, the effects of the potassium ion to the helicase activities of three proteins were different. When the concentration of potassium ion was above 75 mM, the helicase activity of the NS3- NS4A protein began to decline. The helicase activity of the NS3 protein reached maximum at 75 mM KCl. But, any the helicase activity of NS3 helicase domain was greatly inhibited by K+, even at low concentration. The preliminary results should provide valuable information to further investigate the mechanisms of intermolecular and intramolecular interactions of the HCV NS3 protein. This may provide a hope to develop new therapeutic agents for hepatitis C.

目錄
中文摘要
英文摘要
壹、導論
一、前言 1
二、C型肝炎病毒的傳播方式及其感染診斷方法 1
三、治療方法 2
四、C型肝炎病毒之分類 2
五、C型肝炎病毒結構及基因體組成 3
六、C型肝炎病毒之各基因產物 5
七、在人工細胞培養環境下複製C型肝炎病毒 11
八、昆蟲桿狀病毒表現系統 12
九、研究方向及目的 13
貳、材料與方法
一、增殖帶有C型肝炎病毒NS3- NS4A之基因重組桿狀病毒 15
二、重組桿狀病毒力價的偵測及免疫螢光染色分析 15
三、利用桿狀病毒表現C型肝炎病毒的NS3- NS4A、NS3及NS3 helicase domain蛋白質及純化 16
四、Helicase受質：雙股RNA的製備 18
五、Helicase活性分析 19
六、RNA結合能力分析 20
七、ATPase活性分析 20
參、結果
一、帶有NS3- NS4A基因之重組桿狀病毒增殖結果 21
二、各蛋白質的表現與純化 21
三、各蛋白質的活性分析及比較 22
四、各蛋白質水解的能力及比較 23
五、各蛋白質與結合的特性及比較 23
六、各蛋白質之活性對一價離子敏感程度不同 24
肆、討論 26
伍、圖與表 31
陸、參考文獻 43

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