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標題: | D型肝炎病毒於表現FLAG-RPB9細胞系中的複製 Hepatitis D Virus Replication in FLAG-RPB9 Expressing Cell Line |
作者: | 陳思瑜 Szu-Yu Chen |
指導教授: | 陳培哲 Pei-Jer Chen |
關鍵字: | 丁型肝炎病毒,RNA聚合酶Ⅱ,RNA聚合酶Ⅱ次單元RPB9,丁型肝炎病毒RNA複製, Hepatitis D virus,HDV RNA replication,RNA polymerase II,RNA polymerase II subunit RPB9, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | D型肝炎病毒是一種會導致人類D型肝炎的RNA病毒。D型肝炎病毒(HDV)和B型肝炎病毒 (HBV) 同時感染可能會引起急性肝炎或慢性病毒性肝炎,並導致肝損傷,且後續可能會發展為肝癌。根據統計,目前全球約有2.96億人患有慢性B型肝炎,其中近5%的患者合併感染HDV,並發展成更為嚴重的肝炎。在此情況下,有關HDV 分子生物學的研究對於預防和治療HDV感染具有重要的意義。
如今,關於HDV RNA在人體中的複製仍存在爭議。先前的研究分別提出了三種人類RNA聚合酶可能參與HDV生命週期中的HDV RNA複製。 在這項研究中,我們假設 RNA 聚合酶 II 主要負責進行 HDV RNA 的複製。 為了證實我們的假說,我們希望純化含有RNA 聚合酶 II (Pol II) 的HDV RNA複製複合物,並進行核連續測試 (Nuclear run on assay) 和體外轉錄 (In vitro transcription) 。我們選擇Pol II 的次單元RPB9作為純化含有 Pol II 的 HDV 複製複合物之目標,使用帶有FLAG-RPB9的質體和HeLa wild type cell進行DNA轉染 (Transfection) ,經過細胞株篩選後挑選出15支可表達FLAG-RPB9的HeLa穩定細胞株,並進行DNA轉染確認細胞株可以支持HDV RNA的複製。接著,我們使用HDV cDNA轉染後的細胞株進行核質分離 (Nucleus cytoplasm fractionation),發現兩種不同的HDV抗原 (HDAg) 在細胞中位於不同位置。此外,我們還建構了可產生HDV mRNA、基因組或抗基因組串聯二聚體RNA的質體,進行體外轉錄生產這三種HDV RNA。我們將這三種HDV RNA分別和表達S-HDAg的質體於FLAG-RPB9 HeLa細胞中進行共轉染,以確認這些RNA的功能。我們確認了HDV基因組和抗基因組串聯二聚體RNA可以與S-HDAg進行RNA複製,並且HDV mRNA可以在FLAG-RPB9 HeLa細胞中被轉譯成S-HDAg。這些結果為後續研究中HDV複製複合物的純化提供了重要的資訊和材料。 在往後的研究將使用此研究中的HDV RNA 和HeLa-FLAG-RPB9細胞株進行RNA轉染,進行核質分離後進行核連續測試,確認分離的細胞核質是否可以進行HDV RNA 的複製,並使用蔗糖梯度(Sucrose Gradient)以及免疫沉澱(Immunoprecipitation)進一步純化含有Pol II 的HDV RNA複製複合物,最後再以體外轉錄測試是否可以進行HDV RNA 的複製。 Hepatitis D virus is a RNA virus which leads to the Hepatitis D in human. The co-infection of HDV and HBV causes acute hepatitis D or chronic viral hepatitis that may lead to liver damage and may develop into liver cancer. In this day, there have been about 296 million people living with chronic Hepatitis B, and nearly 5% of patients are co-infected with HDV and may develop into more severe form of hepatitis. Hence, the research of HDV molecular biology is important for prevention and treatment of HDV infection. There is a dispute of HDV RNA replication. The three human RNA polymerases may be involved in the replication of HDV RNA in HDV life cycle. In this study, we hypothesize that RNA polymerase II (Pol II) is the major enzyme involving in HDV RNA replication. To confirm our hypothesis, we decided to purify the Pol II-containing HDV replication complex and performed the nuclear run-on assay and in vitro transcription. We selected the RPB9, a subunit of Pol II, as a purification target of Pol II-containing HDV replication complex. We first performed DNA transfection of HeLa wild type cell with the plasmid containing FLAG-RPB9 to establish a HeLa stable cell line expressing FLAG-RPB9 by subsequent single cell selection. We have confirmed three HeLa single cell clones S1, S2, and S3 capable of supporting the HDV RNA replication by subsequent transfection with pCR3.1_HDV-I(I)_221T/219C, a plasmid contains HDV genomic tandem dimer cDNA. Further, we have extracted the nuclei from HeLa single cell clones S1 and S2 both transfected by pCR3.1_HDV-I(I)_221T/219C. We have also observed that the two HDAg located in different subcellular localizations in transfected HeLa cells. Finally, we constructed the plasmid encoding the HDV mRNA, genomic, or anti-genomic tandem dimer RNA to produce the three types of RNAs by in vitro transcription. Also, we performed HDV RNA and DNA expressing S-HDAg co-transfection with FLAG-RPB9 HeLa cell to confirm the function of these RNAs. We confirm that HDV genomic and anti-genomic tandem dimer RNA could undergo the RNA replication with S-HDAg, and HDV mRNA could be translation into S-HDAg in FLAG-RPB9 HeLa cell. These results provide the important information and materials for the purification of HDV replication complex in the following research. For the future work, it is necessary to extract the nuclei from the HeLa cells transfected with HDV RNA. With the extracted nuclei, nuclear run-on assay will be performed to initially confirm if the replication of HDV RNA occurs in the nucleus. After confirming the HDV RNA is produced from the run-on assay, sucrose gradient and immunoprecipitation (IP) would be used to further purify the Pol II-containing HDV replication complex. Finally, the in vitro transcription would be performed to confirm if the pol II-containing complex supports the replication of HDV RNA. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89507 |
DOI: | 10.6342/NTU202302848 |
全文授權: | 同意授權(限校園內公開) |
顯示於系所單位: | 微生物學科所 |
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