EB病毒和一些人類的惡性疾病具有高度相關性，它感染宿主後會進入潛伏期，也能經由刺激再度活化而進入溶裂期。EB病毒於溶裂期時會利用自己所製造的複製相關蛋白質有效率地複製病毒的DNA。病毒所製造的單股DNA結合蛋白質(ssDNA-binding protein)，BALF2，即為DNA複製所需的蛋白質之一。單股DNA結合蛋白質能夠經由結合單股DNA使其不被核酸酶所分解，並且防止其形成二級結構，同時也可能會透過與其他蛋白質形成一個複製複合體(replication complex)以輔助DNA進行複製。本研究的目的是分析BALF2的功能區域(functional domain)，並且探討BALF2可能的生物功能。首先，為了定位BALF2的ssDNA-binding domain和nuclear localization signal (NLS)，根據一些生物資訊以及和其他單股DNA結合蛋白質的氨基酸序列比對，建構表現不同片段BALF2的質體，並以ssDNA-cellulose層析法來測試利用試管內轉錄及轉譯系統所表現的不同片段BALF2與單股DNA的親和力。實驗結果確認利用氨基酸序列比對所發現的單股DNA結合蛋白質保守結構ssDNA-binding motif (BALF2 a.a. 741-785)的確對於BALF2結合單股DNA是重要的。其他的BALF2 deletion mutant與單股DNA之間的親和力也觀察到有下降的情形，推測可能是蛋白質結構的改變進而造成與單股DNA之間的親和力下降。此外，利用免疫螢光實驗發現缺少N端、C端或是保守結構ssDNA-binding motif的BALF2都會影響進核的比例。另外，為了確認能夠與BALF2有交互作用的病毒蛋白質，本研究以共免疫沉澱實驗進一步驗證BALF2與其他病毒蛋白質之間的交互作用，結果證實BALF2能夠和BGLF5 (核酸酶)以及BVRF1 (病毒外殼相關披膜蛋白質)產生交互作用。此外，為了探討BALF2在溶裂期DNA複製時的生物功能，透過PCR-targeting的方式，建構了一個將EB病毒B95-8 strain bacmid (p2089)的BALF2 ORF剔除的bacmid，簡稱為p2089ΔBALF2。目前已經利用PCR，BamHI切割及定序這三種方式確認p2089ΔBALF2的正確性。綜合實驗的觀察，對於BALF2的ssDNA-binding domain、進核機制以及BALF2和BGLF5 (核酸酶)及BVRF1 (病毒外殼相關披膜蛋白質)之間交互作用的生物意義仍然需要進一步的研究。

Epstein-Barr virus (EBV) is highly associated with several human malignancies. After infection, EBV can become latent and be reactivated into lytic replication. EBV is equipped with lytic viral proteins to efficiently replicate viral DNA. EBV-encoded ssDNA-binding protein, BALF2, is one of the essential viral replication proteins. The functions of ssDNA-binding proteins are to protect ssDNA from nucleases digestion and prevent ssDNA from forming secondary structure. It may also help DNA replication through forming a replication complex with other proteins. The specific aim of this study is to dissect the functional domain of BALF2, and to explore possible biological functions of BALF2. First, different deletion mutants were generated according to bioinformatics and sequence alignment with other ssDNA-binding proteins for mapping the ssDNA-binding domain and nuclear localization signal (NLS) of BALF2. The DNA binding affinities of different in vitro translated BALF2 deletion constructs were examined by ssDNA-cellulose chromatography. The result indicated that the conserved structure ssDNA-binding motif (BALF2 a.a. 741-785), identified by amino acid alignment, is important for BALF2 to bind ssDNA. Moreover, the decrease of ssDNA-affinity was also observed in other BALF2 deletion mutants, which may result from abnormal protein structure. In addition, the immunofluorescence assay revealed that the deletion of N-terminus or C-terminus or ssDNA-binding motif regions affected the nuclear targeting of BALF2. To search for BALF2 interacting viral protein, co-immunoprecipitation assay was used to confirm the interaction between BALF2 and other viral proteins, which showed that BALF2 interacts with BGLF5 (nuclease) and BVRF1 (capsid-associated tegument protein). To explore the biological function of BALF2 on EBV lytic replication, a BALF2 knock-out B95-8 bacmid (p2089ΔBALF2) was generated through PCR-targeting protocol, and its correctness was confirmed by PCR, BamHI-digestion, and sequencing. Overall, the ssDNA-binding domain of BALF2, the mechanism of BALF2 nuclear localization, and the biological significances of the interaction with BGLF5 (nuclease) and BVRF1 (capsid-associated tegument protein) all need to be further studied.

口試委員審定書
碩博士論文授權書
誌謝...............................................I
中文摘要..........................................II
英文摘要.........................................III
序論...............................................1
實驗材料與方法.....................................10
實驗結果...........................................21
討論...............................................25
圖表...............................................31
參考文獻...........................................46

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