臺灣博碩士論文加值系統

新穎的串接親合純化技術加上質譜分析與資料庫搜尋的結合使得辨識與NC有交互作用的蛋白質變得可行。我們的最初始目標是辨認及定性在HIV Gag輸送及組裝RNA時與NC有關之細胞蛋白質。NC蛋白參與了許多病毒生活史中的重要過程，包含與病毒RNA結合，病毒組裝，反轉錄過程中的伴護蛋白活性，病毒感染後的cDNA合成，以及Gag組裝過程中將RNA包裝入病毒顆粒中。在細胞內HIV NC會專一的結合在病毒RNA上的Ψ序列，並將病毒RNA經由NC包裝入病毒中，但是在胞外實驗時卻沒有看見這現象。因此推測可能有細胞或是其他病毒因子參與這項工作。我們建立包含TAP純化標籤的HIV NC或突變型NC(在NC的鹼性片段中存在10個氨基酸的變異)重組質體，此標籤經文獻證實能夠更有效率的從哺乳類細胞中回收具有交互作作用的蛋白質。這些重組質體經過核酸定序後，轉染至293T細胞中進行表現。表現出的融合蛋白也藉由標定CBP與蛋白質A等標籤蛋白而證實其表現。我們也已經最佳化TAP純化程序，並用以純化與NC有交互作用的細胞蛋白。考馬斯藍染色的1D SDS-PAGE以及西方氏墨點法成功顯示出純化出的蛋白質圖譜。這些蛋白質經過多次實驗的比對，我們依據蛋白質含量以及再現的一致性選出30個蛋白質利用LC/MS/MS進行蛋白身份辨識。辨識結果包含Topoisomersase I，Nuclear DNA helicase II，tubulin，heterogeneous nuclear ribonuclear protein U(HNRPU)，suppressor of cyclic-AMP receptor (SCAR)，以及ribosomal protein subunits等會與cDNA合成、Gag前驅蛋白運送，或是HIV RNA包裝等工作有關的蛋白。我們未來將比較病毒RNA上的包裝訊號(Ψ)存在與否是否會造成NC結合蛋白圖譜的改變。並嘗試抑制會與NC結合的細胞蛋白的表現，以瞭解其在HIV生活史中的功能。本研究建立了一個初步的純化以及鑑定NC結合蛋白成員的蛋白質體學方法，有助於未來提供重要的細胞內NC蛋白交互作用網路機制的研究，特別是NC在早期病毒生活史的角色，病毒Gag的運送，或是RNA的包裝過程。

A novel Tandem Affinity Purification (TAP) method combined with mass spectrometry (MALDI-TOF, MS) and database search algorithms are adapted to allow identification of HIV NC protein interacting partners. Our preliminary goal is to identify and characterize cellular proteins associated with HIV nucleocapsid (NC) or during precursor Gag trafficking. NC participates in many steps in virus lifecycle, including (1) viral RNA binding and chaperone activity during reverse transcription and cDNA synthesis in the early stages of viral infection and (2) precursor Gag assembly and HIV RNA packaging process in the late stage of viral particle biosynthesis. The packaging process is highly selective in vivo; however, the affinity and specificity of NC for Ψ (packaging signal) containing viral genomic RNA sequences in vitro is modest at best, suggesting that other viral or cellular factors contribute to the selectivity process. We constructed plasmids and viral vectors harboring HIV NC gene sequences (including mutants with 10 mutations in the NC basic region) with aligned TAP sequence to allow sufficient expression in mammalian cell cultures. In addition to the confirmed in-frame sequences, transfection of these constructs into 293 T cells resulted in the expression of the NC and mNC fusion proteins in the cytosol and can be efficiently identified by specific antibodies against the Tag proteins CBP and proteinA. An optimized Tandem affinity purification (TAP) protocol has been established to compare and isolate candidate proteins that are physically associated with NC. Many NC but not mNC interacting proteins in the final affinity elutents were evident on one-dimension SDS-PAGE gradient gel via comassive blue staining and western blot analysis. Repetitive comparison of the staining patterns and analysis of the protein identities through LC/MS/MS and data mining revealed 30 consistent NC-specific binding proteins. Topoisomersase I, Nuclear DNA helicase II, tubulin, heterogeneous nuclear ribonuclear protein U(HNRPU), suppressor of cyclic-AMP receptor (SCAR), and ribosomal protein subunits are predominant species that may play important roles for NC during cDNA synthesis or precursor Gag trafficking and genomic viral RNA incorporation. Further verification of these NC interaction proteins may require the comparison between the presence and absence of the packaging signal (Ψ) containing viral genomic RNA. Inhibition of the NC binding proteins would assist to reveal their identities and functional relevance in HIV life cycle. This study established a preliminary purification and identification protocols for the proteomic analysis of HIV NC binding proteins and may provide in the future significant insights into the mechanisms of HIV NC-cellular protein interaction networks during initial HIV life cycle, precursor Gag trafficking, and RNA packaging process.

英文摘要
中文摘要
導論： 1
1. 後天性免疫不全症候群之流行病學 1
2. 人類免疫不全病毒的分類 1
3. 人類免疫不全病毒簡介 2
4. 人類免疫不全病毒之生活史 3
5. 人類免疫不全病毒Gag蛋白質之結構與功能 4
6. 人類免疫不全病毒NC蛋白質之結構與功能 5
7. 人類免疫不全病毒之成熟化作用 7
8. 人類免疫不全病毒蛋白與其他因子之交互作用 7
9. 串聯親和性純化標籤純化技術(Tandem Affinity Purification) 9
實驗目的 10
實驗材料與方法： 12
實驗材料： 12
實驗方法： 14
A. 質體之製備(Plasmid extraction)： 14
A-1. 小量的製備(mini preparation) 14
A-2. 中量的製備(midi preparation) 16
B. 聚合酶鏈鎖反應(Polymerase chain reaction, PCR)：17
B-1. DNA擴增(DNA amplification) 17
B-2. 菌落聚合酶鏈鎖反應(Colony PCR) 18
C. 重組質體建構之策略(Cloning strategy)： 19
C-1. BT系列質體建構 (construction of BT series constructs) 19
C-2. pNTAP系列質體建構(construction of pNTAP series constructs) 20
C-3. pCTAP系列質體建構(construction of pCTAP series constructs) 20
D. 酵素之作用(Enzyme treatment)： 21
D-1. 限制酶作用實驗(Restriction enzyme digestion) 21
D-2. DNA去磷酸化(CIP)處理 22
E. DNA電泳分析(DNA electrophoresis analysis)實驗： 22
E-1. 瓊酯膠體電泳(Agarose gel electrophoresis) 22
E-2. DNA片段的純化(DNA fragment purification) 23
F. 細胞的轉型(Transformation)： 24
F-1. 化學勝任細胞的製備(Competent cell preparation)24
F-2. 轉型作用(Transformation) 25
F-3. 菌種的保存(Bacteria culture preservation) 26
G. 真核細胞之轉染(Transfection)： 26
G-1. 磷酸鈣轉染法(Calcium phosphate transfection) 26
G-2. 轉染細胞蛋白質之收集(Cell lysis protocol) 27
G-3. 蛋白質定量方法(Protein quantification assay) 28
H. 西方氏轉漬法(Western blotting)： 29
H-1. 西方氏轉漬法(Western blotting) 29
I. 串接親和標籤純化技術 (Tandem Affinity Purification)： 30
I-1. 串接親和標籤純化 (TAP purification) 30
J. 蛋白質鑑定(Protein Identification with LC/MS/MS)： 33
J-1. 蛋白質膠內酵素切割(Proteolytic Digestion of Protein: In- Gel Digestion) 33
結果： 35
1. 質體之構築與表達： 35
1-1. BT重組質體中人類免疫不全病毒Gag或NC與串接親和標籤融合蛋白之表達 35
2. 串接親和標籤純化與蛋白分析實驗： 36
2-1. BT-NC和BT-mNC串接親和標籤重組蛋白在可溶解之蛋白溶解液(S27)與非溶解沉澱物(P27)之分佈 36
2-2. BT-NC和BT-mNC串接親和標籤重組蛋白與IgG 微珠之結合效率 37
2-3. 利用BT-NC串接親和標籤重組蛋白對串接親和標籤純化過程進行逐步分析 39
2-4. BT-NC和BT-mNC串接親和標籤重組蛋白與細胞蛋白進行比對分析 40
3. 蛋白質體學分析實驗： 42
3-1. 以LC/MS/MS對BT-NC和BT-mNC串接親和標籤所純化之蛋白質樣本進行分析 42
討論： 44
1. BTNC與BTmNC重組質體之建構與表現 44
2. NC及mNC融合蛋白僅能在細胞核外被偵測到 47
3. 與HIV Nucleocapsid有交互作用的細胞蛋白及可能作用47
4. Nucleocapsid的RNA結合能力對串接親和純化結果應再進一步測試 54
5. 嘗試利用HIV Gag進行串接親和純化實驗測試 55
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