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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張麗冠(Li-Kwan Chang) | |
| dc.contributor.author | Ping-Han Huang | en |
| dc.contributor.author | 黃柄翰 | zh_TW |
| dc.date.accessioned | 2021-06-15T02:22:19Z | - |
| dc.date.available | 2012-08-22 | |
| dc.date.copyright | 2011-08-22 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-17 | |
| dc.identifier.citation | Attasart P, Kaewkhaw R, Chimwai C, Kongphom U, Namramoon O, Panyim S (2009) Inhibition of white spot syndrome virus replication in Penaeus monodon by combined silencing of viral rr2 and shrimp PmRab7. Virus Res 145(1): 127-133
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43487 | - |
| dc.description.abstract | Sp1-like proteins /Kruppel-like factors (Sp/KLF)轉錄因子家族，為相似度極高的鋅指蛋白質 (zinc finger protein)，在細胞內調控多種轉錄活性。Sp/KLF轉錄因子於胺基酸序列C端皆具有三組Cys2-His2 (C2H2)鋅指元素 (zinc-finger DNA binding motif)，並且會結合在GC-Box、CACCC-Box以及GT-Box序列上調節轉錄活性。本研究成功地自草蝦 (Penaeus monodon)選殖出Kruppel-like factor (PmKLF)，PmKLF cDNA全長為1702 bps，並且可以轉譯出由360個胺基酸組成的蛋白質產物。胺基酸序列比對後發現，PmKLF和人類 (Homo sapiens)、小鼠(Mus musculus)、斑馬魚 (Danio rerio)以及水蚤的KLF11具有較高的相似度。此外，免疫螢光分析顯示，GFP-KLF融合蛋白質可於秋夜盜蛾細胞 (Spodoptera frugiperda, Sf9)細胞核中形成點狀聚集。冷光報導分析 (reporter assay)也指出 PmKLF促進白點症病毒(White spot syndrome virus, WSSV)啟動子ie1之轉錄活性。接著以電泳流動性轉移分析 (EMSA)發現PmKLF會結合於ie1啟動子GC- box序列形成蛋白質-DNA複合體。另外，若抑制白蝦PmKLF基因表現，能降低白點症病毒感染造成的高死亡率，因此推論PmKLF於白點症病毒感染時應扮演重要角色。而且當PmKLF表現受到抑制時，白點症病毒的複製數以及極早期基因ie1的表現量也會受到抑制，因此PmKLF可能藉由調節ie1基因表現影響白點症病毒的感染。 | zh_TW |
| dc.description.abstract | Sp1-like proteins and Kruppel-like factors (Sp/KLF) are highly related zincfinger proteins that have crucial roles in transcription. The characteristic feature of this fami- ly is the presence of three zinc fingers in the C-terminal domain, which bind to GC- box, CACCC-box and GT-box sequence, to mediate transcription. This study reports the cloning and characteristics of a Kruppel-like factor (PmKLF) from shrimp, Penae- usmonodon (PmKLF). The full-length PmKLF cDNA is 1702 bps, encoding a poly- peptide of 360 amino acids. Sequence analysis revealed that the sequence of PmKLF is similar to that of KLF11 in humans, mice and zebrafish. Additionally, immunofluo- rescence analysis revealed that GFP-fusion KLF protein is located in the nucleus as dots in an insect cell line, Sf9. Reporter assay revealed that PmKLF enhances the ie1 promoter activity. Moreover, a specific protein-DNA complex binds to the GC-box sequences in the ie1 by electrophoresis mobility shift assays (EMSA). Knockdown of the expression of PmKLF transcript in WSSV-infected shrimp resulted in delayed cu- mulative mortalities, suggesting that PmKLF is important to WSSV infection. Additi- onally, inhibition of PmKLF expression reduced the copy number of WSSV and ie1 expression,revealing that PmKLF affects WSSV infection via interfering with ie1 expression. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T02:22:19Z (GMT). No. of bitstreams: 1 ntu-100-R97b47401-1.pdf: 1431809 bytes, checksum: 8e37472dff563284c27318b3fbe1b578 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 目錄
中文摘要 i 英文摘要 ii 一、前言 1 二、材料和方法 9 1. 細胞株、實驗動物、草蝦組織以及白點症病毒液 9 2. 北方墨點分析 (Northern blotting) 9 3. 快速放大cDNA端點-草蝦KLF基因分析(Rapid amplification of cDNA ends, RACE) 10 4. 表現質體的建構 10 5. PmKLF於大腸桿菌 BL21 (DE3)之過量表現 11 6. PmKLF於Sf9昆蟲細胞之暫時性表現 11 7. 草蝦組織蛋白質萃取 11 8. 昆蟲細胞核蛋白質萃取 12 9. 西方點墨法 (Western blotting) 12 10. PmKLF影響白點症病毒ie1啟動子的活性分析 12 11. PmKLF於Sf9昆蟲細胞內之分布型態 13 12. 電泳游動性轉移分析 (Electrophoretic mobility shift assay, EMSA) 13 12. 雙股RNA製備 15 13. 白點症病毒感染、白蝦死亡率觀察以及病毒複製數檢測 15 14. 白蝦RNA萃取以及反轉錄反應(Reverse transcription) 16 三、結果 17 1. 草蝦Kruppel-like factor的選殖 17 2. 偵測草蝦KLF蛋白質以及草蝦KLF重組蛋白質之表現 18 3. 草蝦KLF蛋白質於細胞內之分布狀態 18 4. 草蝦KLF蛋白質調控白點症病毒ie1啟動子之活性 19 5. 草蝦KLF蛋白質以及電泳流動性轉移分析 19 6. 草蝦KLF蛋白質對白點症病毒感染以及蝦體死亡率的影響 20 四、討論 22 五、圖表 26 表1、本研究所使用之引子列表 26 圖1、草蝦KLF的序列以及分析。 27 圖2、以西方墨點分析偵測PmKLF的表現。 30 圖3、PmKLF於細胞內的分布型態觀察。 31 圖4、PmKLF對ie1啟動子轉錄活性的影響。 32 圖5、以電泳流動性轉移分析PmKLF和ie1啟動子的結合。 33 圖6、抑制白蝦KLF基因表現影響對白點症病毒感染造成之影響。 34 圖7、抑制白蝦KLF基因表現影響對白點症病毒ie1基因表現量之影響。 36 六、 參考文獻 37 | |
| dc.language.iso | zh-TW | |
| dc.subject | ie1 | zh_TW |
| dc.subject | Kruppel-like factors | zh_TW |
| dc.subject | GC-box | zh_TW |
| dc.subject | Penaeus monodon | zh_TW |
| dc.subject | WSSV | zh_TW |
| dc.title | 草蝦Kruppel-like factor 在白點症病毒感染時所扮演之角色 | zh_TW |
| dc.title | Role of Penaeus monodon Kruppel-like factor (PmKLF)
in infection by white spot syndrome virus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃偉邦,呂健宏,王涵青 | |
| dc.subject.keyword | Kruppel-like factors,GC-box,Penaeus monodon,WSSV,ie1, | zh_TW |
| dc.relation.page | 48 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-08-17 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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