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研究生:昌美惠
研究生(外文):Mei-Hui Chang
論文名稱:C型肝炎NS5A蛋白轉錄活化機轉之研究
論文名稱(外文):Study on transactivation mechanism Hepatitis C Virus NS5A protein
指導教授:陳鴻震葉昭廷葉昭廷引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:39
中文關鍵詞:c型肝炎NS5A蛋白轉錄活化
外文關鍵詞:NS5Atransactivationhepatitis c virus
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C型肝炎是造成非A非B型肝炎的主要原因,持續慢性C型肝炎感染可能導致肝癌。C型肝炎的NS5A詳細功能仍未被完全瞭解,在本研究中我們希望能進一步瞭解NS5A在肝癌演變過程中所扮演的角色。已知NS5A蛋白具有一段可能有功能的核轉位訊號(nuclear localization signal ; NLS),此蛋白之某一區段並具有活化轉錄能力。於是我們從肝癌病人血清中取得HCV基因組,並選取產生NS5A蛋白的基因序列區段來探討轉錄活化之機轉。使用一個NS5A-HCC及二個NS5A突變株質體送到Huh-7細胞表現。NS5A -HCC存在細胞核與細胞質中;核轉位訊號之突變株(NLS mt)和核轉位訊號附近的Serine和Threonine被置換成Alanine之突變株(TS mt),則主要存在細胞質。三種NS5A蛋白都具有轉錄活化SV40T和c-fos promoter的能力,但都不具有轉錄活化c-myc promoter的能力,而且,NS5A-TSmt和NS5A-NLSmt的轉錄活化能力都高於NS5A- HCC。因此,NS5A的轉錄活化能力與進入細胞核的能力無關。在酵母菌雙雜交系統和co-immunoprecipitation實驗證實,NS5A- HCC及NS5A-NLSmt可與Grb2作用,NS5A-TSmt雖有轉錄活化致癌基因promoter能力,但不與Grb2產生交互作用。因此,NS5A- HCC與NS5A-NLSmt的轉錄活化能力需要Grb2參與,但NS5A-TSmt的轉錄活化能力,尚有不需Grb2參與的途徑。
Hepatitis C virus(HCV)is the major cause of non-A, non-B hepatitis. A possible sequel of chronic hepatitis C infection is hepatocellular carcinoma. The amino acid sequence of HCV NS5A protein contains a putative nuclear localization signal(NLS)and this protein possessed a transactivation domain. In this study, we hope to understand the role of NS5A in the development of hepatocellular carcinoma. A HCV clone was isolated from the serum of a patient with hepatocellular carcinoma. The sequence encoding NS5A protein was used for our study. Three NS5A proteins were expressed in Huh-7 cells.NS5A- HCC was localized to both the nucleus and cytoplasm. Two other mutants, NS5A-NLSmt (of which the R/K residues on the NLS were mutated) and NS5A-TSmt (of which the T/S residues near the NLS were mutated)were localized to the cytoplasm. All three NS5A proteins were capable of transactivating the promoters of SV40T and c-fos but not the promoter of c-myc. The transactivation activities of NS5A-TSmt and NS5A-NLSmt were even higher than that of NS5A- HCC. Thus, the transactivation ability of NS5A was not dependent on its capability of nuclear localization. By performing yeast 2-hybrid and co-immunoprecipitation experiments, we further demonstrated that NS5A-TSmt could not interact with Grb2 despite of its capability of transactivating oncogenic promoters. Our data indicated that cellular pathways other than those mediated by Grb2 were involved in the transactivation activity of HCV NS5A.
中文摘要-----------------------------------------------------------1
英文摘要-----------------------------------------------------------2
壹、序論
一、C型肝炎的介紹 -----------------------------------------3
二、C型肝炎與肝癌的關係------------------------------------4
三、C型肝炎的病毒複製機制----------------------------------5
四、C型肝炎的病毒結構----------------------------------------5
五、C型肝炎非結構性蛋白5A的結構及功能---------------------6
貳、研究目的
一、 非結構性蛋白5A的核轉位訊號區段
不同的突變株與轉錄活化能力之關係-----------------------10
三、非結構性蛋白5A的核轉位訊號區段之
不同突變株與Grb2的交互作用-----------------------------10
參、材料與方法
一、 細胞培養(Cell culture)-------------------------------11
二、 質體---------------------------------------------------11
三、 轉染(Transfection)-----------------------------------11
四、轉錄活化分析(Transactivation assay)----------------12
五、酵母菌雜交實驗(Yeast two-hybrid system)--------------13
六、西方墨點轉漬法(Western blot)-----------------------15
肆、結果
一、 C型肝炎非結構性蛋白5A的核轉位訊號區段之
不同的突變株與轉錄活化能力之關係-----------------------17
二、非結構性蛋白5A的核轉位訊號區段之
不同突變株與Grb2的交互作用----------------------------19
伍、討論-----------------------------------------------------------21
陸、附圖-----------------------------------------------------------24
柒、參考資料-------------------------------------------------------36
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