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研究生:劉柔辰
研究生(外文):Rou-Chen Liu
論文名稱:鑑定調控B型肝炎病毒核殼蛋白C端之Serine 170位點磷酸化修飾之激酶
論文名稱(外文):Identification of the Putative Kinase for Serine-170 in C-terminal Domain of HBV Core Protein
指導教授:葉秀慧葉秀慧引用關係
指導教授(外文):Shiou-Hwei Yeh
口試委員:陳培哲鄧述諄
口試委員(外文):Pei-Jer ChenShu-Chun Teng
口試日期:2016-07-29
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:42
中文關鍵詞:B型肝炎病毒核殼蛋白磷酸化激酶
外文關鍵詞:Hepatitis B virus (HBV)core proteinphosphorylationkinase
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B型肝炎病毒(Hepatitis B virus;HBV)所產生之核殼蛋白(HBV core protein;HBc)由183個胺基酸組成,其中1-149胺基酸負責病毒nucleocapsid之組裝;而150-183胺基酸(C-terminal domain;CTD)則負責病毒pregenomic RNA (pgRNA)之包裹(encapsidation)。HBc為一磷酸化蛋白,磷酸化主要發生在CTD中之多個Serine位點,先前研究利用點突變方式發現這些位點的磷酸化可能參與HBV RNA encapsidation以及DNA synthesis等步驟之調控,然這些結果仍有待在wild type HBV replicon中進行確認。為此必須找出負責調控HBc-CTD特定位點磷酸化的激酶 (kinase),利用影響kinase之活性以探討特定位點磷酸化對病毒複製之影響。雖然目前已有數個kinase被報導可能參與HBc-CTD的磷酸化,然而這些kinase的相關研究主要是利用in vitro kinase assay,無法直接證明這些kinase在細胞內(in vivo) 確實參與HBc蛋白的磷酸化,因此負責HBc-CTD磷酸化之kinase仍有待釐清。
本研究提出利用本實驗室所發展之一可區分HBc蛋白Serine-170位點有無磷酸化的專一性抗體,搭配kinase inhibitor處理細胞之策略,來找尋細胞中負責調控HBc蛋白磷酸化的候選kinase(s),結果發現Akt可能參與HBc蛋白的磷酸化修飾。進一步利用phosphatase作用檢測HBc蛋白的磷酸化之程度,發現只有部分的HBc蛋白磷酸化是受到Akt inhibitor影響。有鑒於細胞內活化態的Akt主要是分佈於membrane compartment,實驗利用subcellular fractionation分析亦發現主要是分佈在membrane compartment中的HBc蛋白之磷酸化會受到Akt調控。本論文目前的研究結果初步發現Akt可能參與membrane中調控HBc蛋白serine-170位點的磷酸化修飾,未來將進行探討此Akt調控HBc蛋白磷酸化修飾對HBV複製生活史的影響。

Hepatitis B virus (HBV) core protein (HBc) is a 183-amino acid protein, in which the 1-149 a.a. N-terminal domain is sufficient for nucleocapsid assembly and the 149-183 a.a. C-terminal domain (CTD) is critical for viral RNA encapsidation. The HBc-CTD contains several serine residues, including S155, S162, S170, as the major phosphorylation sites. As revealed by site-directed mutagenesis, the phosphorylation of HBc-CTD might regulate HBV RNA encapsidation and DNA synthesis, which however still awaits to be confirmed in wild-type HBV replicon. Identification of the putative cellular kinase(s) that phosphorylates specific serine residues at HBc-CTD will help address this issue. Though several candidate kinases were identified mainly by in vitro kinase assay, but none of them were proven to occur in cells undergoing active viral replication. The putative kinases are thus still remained to be identified.
The current study proposed to identify the kinase for Serine-170 of HBc-CTD by using the C-S170 antibody, which can specifically recognize the HBc with dephosphorylated S170. The HBc expressing cells were treated with a panel of kinase inhibitors and then processed for the Western blotting by probing with C-S170 antibody. The result pointed out Akt as one candidate in regulating HBc-S170 phosphorylayion. The phosphatase reaction helps illustrate that ~30% of HBc protein in cells is affected by the Akt inhibitor treatment. Regarding to that the Akt is activated at the membrane compartment, the fractionation analysis further demonstrated that the core protein at the membrane compartment is the target HBc to be regulated by Akt. In conclusion, the current study identified that Akt might contribute to the phosphorylation of HBc-S170 at the membrane compartment. This phoshsphorylation in regualating the viral replication is worthy to be further investigated.

口試委員會審定書 i
誌謝 ii
摘要 iii
Abstract iv
圖表目錄 viii
第一章 序論 1
1.1 HBV感染引起的肝臟疾病與目前治療方式 1
1.2 HBV的基因體結構 1
1.3 HBV的生活史 2
1.4 HBV核殼蛋白之結構與功能 3
1.5 HBV核殼蛋白磷酸化修飾對病毒生活史之影響 4
1.6 HBV核殼蛋白與可能參與其磷酸化修飾的激酶 5
1.7 研究目的 6
第二章 實驗材料與方法 8
2.1 質體 8
2.2 激酶抑制劑(protein kinase inhibitors) 8
2.3 細胞培養 (Cell culture) 8
2.4 細胞轉染 (Transfection) 9
2.5 蛋白質抽取 9
2.6 去磷酸化反應 (Lambda Protein Phosphatase reaction) 10
2.7 Sucrose cushion purification 10
2.8 西方墨點法 (Western blot) 10
2.9 HBV nucleocapsid之偵測 11
2.10 Lenti-siRNA病毒感染 11
2.11 RNA之抽取 12
2.12 純化HBV nucleocapsid內之核酸 12
2.13 北方墨點法 (Northern blot) 13
第三章 結果 14
3.1 利用激酶抑制劑(protein kinase inhibitors)篩選可能參與HBV core protein磷酸化修飾的kinase 14
3.2 磷酸化的HBV core protein 可經由autophagy / lysomosome pathway降解 15
3.3 Akt kinase可能參與調控HBV core protein serine-170位點之磷酸化修飾 17
3.4 Akt kinase調控HBV core protein serine-170位點的磷酸化主要發生在membrane compartment 18
3.5 利用Akt inhibitor探討core protein之磷酸化修飾對HBV生活史可能的影響 20
第四章 討論 22
參考文獻 26
圖表 33

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