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標題: | 探討核酸內切酶FEN1在B型肝炎病毒中環狀共價鍵結去氧核醣酸形成過程所扮演的角色 The role of FEN1 in formation of hepadnavirus covalently-closed circular DNA |
作者: | Kung-Yu Lin 林冠佑 |
指導教授: | 楊宏志(Hung-Chih Yang) |
關鍵字: | B 型肝炎病毒,cccDNA,rcDNA,flap endonuclease 1(FEN1),FEN1 inhibitor,PTPD, Hepatitis B virus,cccDNA,rcDNA,flap endonuclease 1(FEN1),FEN1 inhibitor,PTPD, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | B型肝炎病毒Hepatitis B virus屬於hepadnavridae的一員,病毒顆粒具有外套膜,而病毒基因體本身相當的精小,只有3.2 kb左右,以不完全雙股DNA存在於病毒顆粒中。當B型肝炎病毒感染肝臟細胞後,會脫去外殼將病毒基因體 (relaxed-circular DNA )釋放出來,並且在細胞核中被修補成為環狀共價鍵結DNA的型態(cccDNA),這個cccDNA會當作模板轉錄出大小不同的mRNA ( 3.5, 2.4, 2.1, 0.6 kb ) 來合成各種不同的病毒蛋白。其中3.5 kb mRNA ( pregenomic RNA )會透過本身所轉譯出來的反轉錄酶來合成病毒的子代基因體,包裹入新的病毒顆粒,緩和的釋放到血液裡進行下一波的感染。在B型肝炎的治療上,疫苗在台灣已獲得不錯的成果,但仍有為數不少的病人必須面臨終身服用抗病毒藥物的情況,一旦停藥後,血清中的病毒量又便會上升,造成治療上的問題。而問題的根源在於細胞核中的cccDNA並沒有辦法對目前的治療產生有效反應,同時cccDNA降解速率相當的漫長,無法被清除的cccDNA就會造成病人產生持續性的感染。因此,若能夠解破cccDNA是如何從rcDNA形成的,就是提供另一種抗病毒藥物的選擇策略。而從rcDNA的結構中可以觀察到具有單股 DNA的存在,這是類似於一個DNA的損傷情況。在閱覽Base-excision repair (BER)中我們發現到了一個相當有趣的宿主蛋白, FEN1, (flap endonuclease 1)。 FEN1 是一個核酸內切酶參與在解除Okazaki fragment的RNA primer和BER的5’flap結構。我們觀察到在rcDNA的結構上也具有RNA primer和5’flap結構的存在,而在將FEN1 knockdown之後,我們發現到cccDNA的形成有20 % 顯著意義的下降。而在我們以FEN1 inhibitor, PTPD,抑制FEN1功能的情況下,我們觀察到高濃度的PTPD同時抑制了rcDNA和cccDNA的形成,同時出現一個未知的DHBV band存在;另外,在低濃度的情況下,我們觀察到cccDNA形成也有一個下降的趨勢。總結,從我們的實驗裡可以發現FEN1 確實顯著的影響cccDNA的形成,同時在inhibitor的結果可以得知,在特定的濃度下,rcDNA的形成也受到了影響而消失。 Hepatitis B virus (HBV) is an enveloped dsDNA virus belongs to hepadnaviridae. The viral genome is a partially double strand DNA form or called the relaxed circular DNA form (rcDNA), in the mature virion. After HBV entry the hepatocyte, the rcDNA will be transported into nucleus and converted to the covalently close circular DNA (cccDNA). The cccDNA persists in the nucleus and serves as template to transcript four viral mRNA, 3.5 kb pregenomicRNA (pgRNA), 2.4 kb preS RNA, 2.1 kb S RNA and 0.7 kb X RNA. The longest transcript, pgRNA, also serves as a template for viral replication and will be converted into the rcDNA by the unique reverse transcription in the nucleocpasid. In the current therapy of chronic HBV infection, the RT inhibitors can success to reduce the viral titer in the serum. However, most of the patients must take the RT inhibitor for life-long due to the persistent cccDNA in the nucleus. This cccDNA was from its precursor rcDNA with the help of some host factors. The host factors involve in the cccDNA are still unidentified now. We found a host factor, FEN1, a crucial endonuclease involved in Okazaki fragment maturation and Base-excision repair might play a role in removal of the negative-strand 5’ redundancy. In this study, we found the significant reduction of cccDNA formation by knockdown of FEN1. And when we treated with FEN1 specific inhibitor, PTPD, the cccDNA is reduced in low concentration inhibitor. However, in high concentration of inhibitor, both rcDNA and cccDNA formation were blocked. Meanwhile, an unidentified band was accumulated in high concentration of inhibitor treatement. According to our results, we found that the FEN1 significantly influent the cccDNA formation and the inhibitor data also consist with this result. And we also observe that the high concentration inhibitor block rcDNA formation. However, the detail blocking level still needs further identifications. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64345 |
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顯示於系所單位: | 微生物學科所 |
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