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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳美如(Mei-Ru Chen) | |
dc.contributor.author | Hsiu-Yu He | en |
dc.contributor.author | 何修宇 | zh_TW |
dc.date.accessioned | 2021-06-17T08:27:23Z | - |
dc.date.available | 2024-08-27 | |
dc.date.copyright | 2019-08-27 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74273 | - |
dc.description.abstract | EB病毒屬於人類皰疹病毒的一員,感染了全球約95%的人口,不僅可能造成感染性單核球增多症,更與許多惡性腫瘤疾病擁有高度相關性。當受EB病毒感染時,大部分病毒會潛伏於體內,然而當病毒受到刺激時便會從潛伏期轉為溶裂期。此時EB病毒會開始複製DNA並表現大量的病毒基因。BGLF4是EB病毒唯一的蛋白激酶,先前已有許多研究指出BGLF4不只透過磷酸化不同的宿主以及病毒蛋白進而協助EB病毒的感染與複製,也促進數種本身不具有入核序列的病毒蛋白進入細胞核中協助病毒的複製,包括EB病毒引子酶BSLF1及DNA螺旋酶 (BBLF4) 等DNA複製複合體中的酵素。此外近來研究指出,分子伴護蛋白DNAJB6除了負責蛋白的摺疊以及運輸外,也被報導參與在數種病毒的複製過程中。因此我們在本篇研究中探討DNAJB6是否參與在BGLF4所調控的引子酶入核過程中,以及DNAJB6是否參與在EBV的複製過程。結果顯示在HeLa細胞中DNAJB6a與BGLF4皆可促進引子酶的進核,然而將在三者共同表現的系統中並未看見更加顯著的入核現象,而DNAJB6b則是會降低BGLF4將引子酶帶入細胞核的能力。在帶有EB病毒的上皮細胞株NA或淋巴球細胞株Akata中我們藉由即時定量聚合酶連鎖反應來檢測病毒DNA的表現,我們發現在shRNA剔弱DNAJB6a表現時會使EB病毒的複製效率以及病毒顆粒產生的總量下降。除此之外,我們也利用經過設計的嗎啉寡核苷酸,一種透過與特定RNA序列結合進而抑制基因表現的分子來抑制DNAJB6a的表現並觀察其對EB病毒的影響。結果顯示嗎啉寡核苷酸能確實剔弱DNAJB6a蛋白在Akata 細胞中的表現,也一同抑制了其餘EB病毒溶裂期蛋白的表現,進而抑制EB病毒的DNA複製效率以及病毒顆粒的產生。進一步我們想透過帶有EB病毒及綠色螢光蛋白的細胞設計能快速篩選對抗EB病毒或者對抗DNAJB6a的系統,初步結果顯示嗎啉寡核苷酸能降低綠色螢光蛋白的表現。綜合以上實驗結果,我們發現DNAJB6不只參與在引子酶的進核過程中,DNAJB6a更影響EB病毒DNA的複製以及病毒顆粒的產生。 | zh_TW |
dc.description.abstract | Epstein-Barr virus, which is the first identified oncogenic virus, infects 95 % of human populations and is highly associated with several malignancies. During EBV lytic replication, EBV synthesizes viral DNA in the nucleus and expresses many viral genes for replication. BGLF4, as the only protein kinase encoded by EBV, phosphorylates several cellular and viral proteins to facilitate virus replication and maturation process. Previous studies showed that BGLF4 translocates into nucleus through interaction with nucleoporins; and BGLF4 induces the nuclear import of several non-NLS-containing EBV proteins, including EBV primase BSLF1 and DNA helicase BBLF4 within the DNA replication complex. Recent studies reported that DNAJB6, a member of Heat shock protein 40 (Hsp40), regulates the nuclear import of several viral proteins and facilitates viral replication. To reveal whether BGLF4 promotes the nuclear targeting of BSLF1 in a DNAJB6-dependent pathway, we did a transfection and knockdown assay in immunofluorescence assay and we found that the nuclear import ratio of BSLF1 was not significantly changed in DNAJB6a overexpression group, however the nuclear import of BSLF1 was decreased in DNAJB6b overexpression group. We then observed the viral DNA replication and found that knockdown of DNAJB6a decreased EBV replication and virion secretion in EBV-positive NA cells and Akata cells. In addition, we used Morpholino Mo-MRJ to target DNAJB6a and found Mopholino treatment reduced the cell growth and decreased EBV intracellular DNA and released virion DNA copy numbers. Moreover, EBV lytic protein expression was also suppressed, suggested that this molecule might exhibit anti-EBV potential. We also aimed to establish an anti-EBV drug screening system by AGS-BX1 and Akata-BX1 cells. We found that Morpholino treatment slightly decreased the GFP value in both cell lines. Taken together, DNAJB6a is involved in EBV primase nuclear import and EBV lytic replication. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:27:23Z (GMT). No. of bitstreams: 1 ntu-108-R06445103-1.pdf: 5416289 bytes, checksum: f8b46414592d82457a6dbe61efae5827 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 口試委員審定書 II
誌謝 III 中文摘要 IV Abstract V Contents 1 Chapter 1 Introduction 4 1.1 Epstein-Barr virus 4 1.1.1. The classification, characterization and associated diseases of EBV 4 1.1.2. The life cycle of EBV 4 1.2 Nuclear and cytoplasmic transport 5 1.3 EBV protein kinase BGLF4 7 1.3.1. Conserved herpesviruses protein kinase (CHPK) 7 1.3.2. Characteristics of EBV BGLF4 protein kinase 7 1.3.3. Functions of BGLF4 in EBV infection 8 1.3.4 The nuclear targeting of BGLF4 9 1.4 Molecular chaperones 9 1.4.1. The chaperone-dependent machinery 9 1.4.2. Characteristics of heat shock protein 40s (Hsp40s) 10 1.4.3. Characterization and functions of DNAJB6 11 1.5 Specific aim of this study 12 Chapter 2 Materials and Methods 13 2.1 Cell culture 13 2.2 Transfection 13 2.3 Plasmid construction 14 2.4 Western blot analysis 15 2.5 Co-immunoprecipitation assay 15 2.6 Immunofluorescence assay 16 2.7 shRNA lentivirus production and transduction 17 2.8 Extraction of intracellular EBV DNA 18 2.9 Isolation of the secreted EBV virions and DNA extraction 18 2.10 Quantitative real-time PCR (qPCR) analysis 19 2.11 Anti-sense oligonucleotides Morpholino treatment 19 2.12 GFP quantitation assay 20 Chapter 3 Results 21 3.1 GFP-BGLF4 and HA-DNAJB6a promote the nuclear targeting of BSLF1, but overexpression of HA-DNAJB6b decreases BGLF4-mediated nuclear import of BSLF1. 21 3.2 HA-DNAJB6a and HA-DNAJB6b are co-immunoprecipitated with GFP-BGLF4. 22 3.3 BGLF4 changes the pattern expression of DNAJB6 and stabilizes the protein expression of BSLF1-myc and DNAJB6. 23 3.4 Knockdown of DNAJB6a and DNAJB6b does not affect BGLF4-mediated BSLF1 nuclear import significantly. 23 3.5 Knockdown of DNAJB6a attenuates EBV replication and virion secretion in EBV-positive NA cells and EBV-positive Akata cells. 24 3.6 Morpholino-MRJ treatment targets DNAJB6a and downregulates DNAJB6a expression, EBV DNA replication and lytic protein expression in EBV-positive Akata cells. 24 3.7 Enhanced GFP signal is correlated with EBV lytic protein expression in reactivated AGS-BX1 cells and Akata-BX1 cells. 25 3.8 Morpholino treatment slightly decreases the GFP intensities of AGX-BX1 cells and Akata-BX1 cells. 26 Chapter 4 Discussion 27 4.1 The mechanisms of nuclear targeting of EBV primase BSLF1 27 4.2 The function of DNAJB6 on EBV lytic replication 27 4.3 The roles of anti-EBV drug screening system 28 4.4 Evaluation of GFP-tagged BSLF1 constructions in screening anti-DNAJB6a drug 29 4.5 The roles of DNAJB6 in multiple diseases 29 Figure 1. GFP-BGLF4 and HA-DNAJB6a promote the nuclear targeting of BSLF1, but overexpression of HA-DNAJB6b decreases BGLF4-mediated nuclear import of BSLF1. 31 Figure 2. HA-DNAJB6a and HA-DNAJB6b are co-immunoprecipitated with GFP-BGLF4. 33 Figure 3. BGLF4 changes the pattern of DNAJB6 and stabilizes the protein expression of BSLF1-myc and DNAJB6. 34 Figure 4. Knockdown of DNAJB6a and DNAJB6b does not affect BGLF4-mediated BSLF1 nuclear import significantly. 36 Figure 5. Knockdown of DNAJB6a attenuates EBV replication and virion secretion in TSA reactivated EBV positive NA cells. 38 Figure 6. Knockdown of DNAJB6a attenuates EBV replication and virion secretion in anti-IgG reactivated EBV positive Akata cells. 39 Figure 7. Morpholino-MRJ reduces the cell growth and attenuates EBV DNA replication in EBV-positive Akata cells. 40 Figure 8. Morpholino-MRJ targets DNAJB6a and attenuates EBV lytic protein expression. 41 Figure 9. Enhanced GFP signal is correlated with EBV lytic protein expression in reactivated AGS-BX1 cells and Akata-BX1 cells. 42 Figure 10. Morpholino treatment slightly decreases the GFP value of AGX-BX1 cells and Akata-BX1 cells. 43 Supplementary Figure 1. Nuclear transport cycles. 44 Supplementary Figure 2. Gene structure and functional domains of DNAJB6 (MRJ) isoforms. 45 Supplementary Figure 3. Zta lentivirus transduction had the better effect for mild EBV reactivation. 46 Supplementary Figure 4. Constructions of GFP-tagged BSLF1 protein show diffuse pattern under microscopy. 47 Reference 48 | |
dc.language.iso | zh-TW | |
dc.title | EB病毒BGLF4蛋白激酶及分子伴護蛋白DNAJB6對於病毒DNA引子酶進核調控及病毒溶裂期複製之探討 | zh_TW |
dc.title | Regulation of nuclear targeting of EBV primase and viral lytic replication by EBV BGLF4 kinase and chaperone DNAJB6 | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃立民(Li-Min Huang),張明富(Ming-Fu Chang),蔡明翰(Ming-Han Tsai) | |
dc.subject.keyword | BGLF4蛋白激?,分子伴護蛋白DNAJB6,引子?,溶裂期複製,嗎?, | zh_TW |
dc.subject.keyword | BGLF4 protein kinase,chaperone DNAJB6,primase,lytic replication,Morpholino, | en |
dc.relation.page | 53 | |
dc.identifier.doi | 10.6342/NTU201903118 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-13 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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