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研究生： 蕭又嘉
Yu-Chia Hsiao
論文名稱： TAT 融合蛋白質抑制D 型肝炎病毒包裹及分泌之機制
The feasibility of TAT fusion protein in inhibition of hepatitis D virus assembly and secretion
指導教授： 黃琇珍
Hsiu Chen Huang
黃 琤
Cheng Huang
口試委員:
學位類別： 碩士
Master
系所名稱：
論文出版年： 2014
畢業學年度： 102
語文別： 中文
論文頁數： 70
中文關鍵詞： D型肝炎病毒 、大型delta抗原 、核輸出訊號
外文關鍵詞： hepatitis delta virus, HDAg-L, NES
相關次數： 點閱：3  下載：0 
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    • D型肝炎病毒 (hepatitis delta virus, HDV)為一種缺陷型的RNA病毒，必須獲得B型肝炎病毒所提供的的外套膜才具有感染力，因此被歸類為B型肝炎病毒的衛星病毒。D型肝炎病毒顆粒內部含有單股環狀之RNA基因體，並可轉譯出兩種抗原，分別是小型delta抗原 (small delta antigen, HDAg-S)及大型delta抗原 (large delta antigen, HDAg-L)。在功能上，小型delta抗原則為HDV之RNA基因體複製所必需，而大型delta抗原具有CRPQ isoprenylation motif，能夠促使B型肝炎表面抗原共同組成D型肝炎類病毒顆粒之能力。比較胺基酸序列發現，大型delta抗原在C端多了19個胺基酸，此段胺基酸對於病毒顆粒的包裹是重要的，並在第198至210個胺基酸片段(HDAg-L(198-210))，具有核輸出訊號 (nuclear export signal, NES)，藉由此訊號可將細胞核內HDV的核糖核蛋白複合體輸出至細胞質。另外，也已知HDAg-L(198-210)與clathrin蛋白的結合效率會影響病毒顆粒的包裹，更與病毒的致病程度呈正相關。在本研究中，我們想要探討利用TAT蛋白之細胞通透性，觀察TAT-HA-HDAg-L(198-210)融合蛋白質對於HDV的包裹與分泌是否會有影響。
    首先利用大腸桿菌建立大量表現TAT融合蛋白質的系統。以轉導的方式將TAT-HA-HDAg-L(198-210)蛋白質進入細胞內作用，再使用免疫螢光染色和西方墨點法進行確認，發現能夠在細胞內偵測到TAT-HA-HDAg-L(198-210)。使用細胞存活率試驗觀察TAT-HA-HDAg-L(198-210)融合蛋白質對細胞是否具有毒性。為了探討TAT-HA-HDAg-L(198-210)融合蛋白質對於HDV之影響，首先以GST pull-down assay分析，證實TAT-HA-HDAg-L(198-210)融合蛋白質能夠抑制大型delta抗原與細胞因子clathrin heavy chain蛋白質 (CHC)的結合。進一步以類病毒顆粒組裝分析收集類病毒顆粒 (virus-like particles, VLPs)，觀察細胞分泌病毒顆粒的表現情形，證實TAT-HA-HDAg-L(198-210)融合蛋白質能夠抑制HDV病毒顆粒的組裝與分泌。另外，以免疫螢光染色觀察在B型肝炎表面抗原的存在下，其細胞中大型delta抗原的分佈位置，證實TAT-HA-HDAg-L(198-210)蛋白質能夠影響病毒的核輸出功能，並且能抑制病毒顆粒的包裹活性。
    經由以上結果，我們推測TAT-HA-HDAg-L(198-210)融合蛋白質能夠抑制大型delta抗原之核輸出功能，並且可以影響CHC與大型delta抗原C端的專一性結合，而作為一種影響HDV包裹及分泌之抑制劑，未來我們期望能在動物實驗上加以佐證，並且更進一步的研究。

    Hepatitis delta virus (HDV) is a defective RNA virus, which is a satellite virus of hepatitis B virus (HBV) and requires HBV envelope proteins for packaging and infection. HDV contains a single-stranded circular RNA genome that encodes two forms of hepatitis delta antigen (HDAg), the small delta antigen (HDAg-S) and the large delta antigen (HDAg-L). The HDAg-S is necessary for the HDV RNA replication; while the HDAg-L contains CRPQ isoprenylation motif that is critical for the viral assembly with hepatitis B surface antigen (HBsAg). The two HDAgs are identical in the amino acid sequence, except that the HDAg-L possesses an extra 19-amino-acid extension at the C terminus. The domain spanning amino acid residues 198-210 of the HDAg-L (HDAg-L(198-210)) contains a nuclear export signal (NES). This signal is important for the nuclear export of HDV ribonucleoprotein to the cytoplasm. Further, the binding of HDAg-L(198-210) and clathrin is essential for the viral assembly. In this study, we investigated the use of cell permeable TAT-HA-HDAg-L(198-210) protein in inhibition of HDV assembly and secretion.
    Expression and purification of TAT fusion proteins were performed by utilizing gene recombinant method in E. coli protein expression system. To determine whether the purified TAT-HA-HDAg-L(198-210) fusion protein was functional in the cells, the immunofluorescence assay and Western blotting were performed. After transduction, the TAT-HA-HDAg-L(198-210) fusion protein can be detected in the cell. Further, cytotoxicity of TAT-HA-HDAg-L(198-210) fusion protein was investigated by MTT assay. By performing GST pull-down assay, the TAT-HA-HDAg-L(198-210) fusion protein interfered with the interaction between HDAg-L and clathrin heavy chain (CHC). In virus-like particles assembly assay, HDV virus-like particles (VLPs) decreased by TAT-HDAg-L(198-210) fusion protein treatment. Therefore,TAT-HA-HDAg-L (198-210) fusion protein was capable of inhibiting viral particles assembly and secretion of HDV. In addition, in the presence of HBsAg, the cellular distribution of HDAg-L was observed by immunofluorescence staining. The TAT-HA-HDAg-L (198-210) fusion protein had an effect on the nuclear export function of the HDAg-L.
    The results suggest that the TAT-HDAg-L(198-210) fusion protein inhibits nuclear export function with the transport of HDAg-L. TAT-HDAg-L (198-210) fusion protein also affects the interaction between CHC and the C terminus of the HDAg-L, and acts as a HDV assembly and secretion inhibitior.

    中文摘要 … … … … … … … … … … … … … … … … … … … … … … I 英文摘要 … … … … … … … … … … … … … … … … … … … … … … III 縮寫表 … … … … … … … … … … … … … … … … … … … … … … V 第一章 前言 … … … … … … … … … … … … … … … … … … … … … 1 1.1 病毒性肝炎 … … … … … … … … … … … … … … … … … 2 1.2 D型肝炎病毒之發現 … … … … … … … … … … … … … … 2 1.3 D型肝炎之流行病學 … … … … … … … … … … … … … … 3 1.4 D型肝炎病毒結構組成 … … … … … … … … … … … … … 3 1.5 D型肝炎delta抗原 …… … … … … … … … … … … … … … 4 1.6 D型肝炎delta抗原之結構與功能 … … … … … … … … … … 5 1.7 D型肝炎大型delta抗原之病毒顆粒包裹能力 … … … … … … 7 1.8 D型肝炎大型delta抗原之核輸出功能 … … … … … … … … 8 1.9 大型delta抗原和CHC蛋白質之交互作用…… … … … … … … 9 本論文研究主題與實驗目的 … … … … … … … … … … … … …10 第二章 實驗材料 …… … … … … … … … … … … … … … … … … … 12 第三章 實驗方法 …… … … … … … … … … … … … … … … … … … 18 3.1 實驗室提供之質體… … … … … … … … … … … … … … … 19 3.2 聚合酶連鎖反應…… … … … … … … … … … … … … … … 20 3.3 DNA瓊脂膠體電泳 …… … … … … … … … … … … … … 20 3.4 細菌轉型作用 … … … … … … … … … … … … … … … … 21 3.5 質體之微量製備 …… … … … … … … … … … … … … … 22 3.6 質體之大量製備 …… … … … … … … … … … … … … … 23 3.7 細胞株繼代培養 …… … … … … … … … … … … … … … 23 - 2 - 3.8 DNA轉染 … … … … … … … … … … … … … … … … … 24 3.9 細胞內蛋白質之收集 ……… … … … … … … … … … … … 24 3.10 蛋白質定量 …… … … … … … … … … … … … … … … … 25 3.11 正十二烷硫酸鈉-聚丙烯醯胺膠體電泳 …… … … … … … … 25 3.12 Coomassie brilliant blue染色法… … … … … … … … … … … 27 3.13 西方墨點法 …… … … … … … … … … … … … … … … … 28 3.14 TAT融合蛋白質之表現與純化 … … … … … … … … … … 29 3.15 細胞存活率試驗 …… … … … … … … … … … … … … … 30 3.16 免疫螢光染色 … … … … … … … … … … … … … … … … 31 3.17 GST pull-down assay … … … … … … … … … … … … … … 31 3.18 類病毒顆粒組裝分析 ……… … … … … … … … … … … … 32 3.19 酵素免疫分析法 …… … … … … … … … … … … … … … 32 3.20 動物實驗 ……… … … … … … … … … … … … … … … … 33 第四章 實驗結果 … …… … … … … … … … … … … … … … … … … 34 第五章 討論 …… … … … … … … … … … … … … … … … … … … … 41 第六章 圖表 …… … … … … … … … … … … … … … … … … … … … 46 參考文獻 ……… … … … … … … … … … … … … … … … … … … … 66

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