HIV融合抑制物之作用機制研究

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許長勝(Chang-Sheng Hsu) 查詢紙本館藏

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化學學系

論文名稱

HIV融合抑制物之作用機制研究

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摘要(中)

人類免疫缺乏病毒入侵人體的過程中，病毒的套膜醣蛋白扮演一個很重要的角色，而這個醣蛋白是由gp41和gp120這兩個具有非共價性連結的子蛋白所組成。在表面子蛋白gp120與輔助T細胞上的CD4受器和一些輔助受器結合後，導致穿膜子蛋白gp41構形上的重大改變，而造成gp41形成一三髮夾的結構核心，進而達到病毒與目標細胞的融合。當gp120分離且gp41並未摺合成三髮夾結構核心時的中間狀態，是為愛滋病毒融合抑制藥物的作用目標。目標是和gp41之CHR區域，或是NHR之三寡聚合物螺旋coiled-coil核心結構發生結合反應，以阻斷可造成細胞融合之三寡聚合物髮夾結構的形成。
從gp41裸露於細胞膜外之胺基酸序列所得到一些胜?，能夠有效的抑制愛滋病毒的融合，其中的一些胜?：T-20 (aa. 638-673)、T-649 (aa. 628-663)，被發現於某一條件下具有較低的溶解度且於溶液中呈均勻渾濁高光散射度的狀態。但當加入包含gp41N-terminal區域(NHR)某ㄧ胺基酸序列時可造成T-20、T-649渾濁度的減少而變得澄清。利用此簡單方法與渾濁度的變化可找出gp41N-terminal區域重要胺基酸序列為何。由一系列選自gp41N-terminal區域且部份胺基酸序列重疊的方式，發現胺基酸片段LLSGIV是與取自於CHR的T-20、T-649的主要作用區域，而在NHR胺基酸序列567-573為一次要作用區域。
另外由表面電漿子共振所得到的結合能力數據，NHR的LLSGIV區段對T-20、T-649具有最大的結合能力；此外尚有一接近T-649疏水性口袋的次要的作用區域，這些數據與渾濁度實驗的結論相符合。因此，由以上實驗所得到的結果，我們可以推論出NHR的LLSGIV及QLTVWGI區段對於與CHR摺合成三寡聚合物髮夾結構是很重要的；同時可以知道T-649較T-20對gp41N-terminal區域擁有較強之反應能力，這也暗示T-649具有較佳的抗HIV能力。

摘要(英)

The envelope glycoprotein of human immunodeficiency virus type I (HIV-1) plays an important role in viral entry and consists of two non-covalently associated subunits, gp120 and gp41. The surface subunit gp120 is responsible for virus attachment and it binds to CD4 and a chemokine coreceptor. Consequently, gp41 undergoes conformational changes, leading to trimeric hairpin structure core, and achieves fusion between virus and target cell. The pre-hairpin intermediate of gp41 is the target for fusion inhibitors that bind to the C-terminal heptad repeat region or the trimeric coiled-coil of NHR helices and prevent formation of the fusogenic trimeric hairpin structure.
A number of peptides derived from the membrane proximal region of gp41 ectodomain are found to be effective fusion inhibitors. Two of them, T-20 (aa. 638-673) and T-649 (aa. 628-663), were found disordered and sparingly soluble in water but became clear upon mixing with structured peptides from the N-terminal heptad repeat (NHR) region using a simple method of reduction in the scattering of T-20 and T-649 suspension. From the results on mapping the locus of interaction with T-20 and T-649 by using partially overlapping peptides derived from NHR, it was included that the LLSGIV region was the major docking site and a minor docking site around aa. 567-573 was found.
The binding affinity data measured by surface plasmon resonance (SPR) also showed a maximum around the LLSGIV segment for the two fusion inhibitors and a less prominent binding region near the hydrophobic pocket for T-649. These results imply that the two segments of NHR are important to the formation of trimeric hairpin structure and T-649 has better antiviral ability than T-20.

論文目次

章節目錄
中文摘要 i
英文摘要 iii
章節目錄 iv
圖表目錄
vi
名詞縮寫及胺基酸序列對照表 viii
第一章序論
1.1愛滋病的歷史 1
1.2 HIV的生命週期 2
1.3愛滋病的治療 3
1.4 gp41構形之改變及其抑制 5
第二章原理簡介
2.1 胜?合成 15
2.2 表面電漿子共振原理 16
2.3 Biacore之光學原理 19
2.4 Biacore感應圖(sensorgram)之介紹 19
2.5 Biacore之基本組成 20
第三章實驗方法
3.1胜?樣品製備 29
3.2 Resin的Cleavage 31
3.3 HPLC純化 32
3.4渾濁度澄清分析(Turbidity clearance assay) 33
3.5表面電漿子共振分析(SPR assay) 34
第四章實驗結果
4.1渾濁度澄清分析(Turbidity clearance assay) 36
4.2表面電漿子共振分析(SPR assay) 38
第五章討論
5.1 HR1之重要作用位置尋找 47
5.2 HR1-HR2的bi-model binding model作用機制 49
參考文獻 54
附錄 62

參考文獻

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指導教授

張定國、蔡惠旭
(Ding-Kwo Chang、Hui-Hsu Gavin Tsai)

審核日期

2006-7-20

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