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研究生:陳秋錦
研究生(外文):chun-chin chen
論文名稱:日本腦炎病毒基因體3'端未轉譯保留區衍生之小片段RNA與複製功能分析
論文名稱(外文):Functional analysis of the small RNA derived from the highly conserved 3’-untranslated region of Japanese encephalitis virus in infected mammalian cells
指導教授:張瑞宜張瑞宜引用關係
指導教授(外文):R.Y. Chang
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:59
中文關鍵詞:病毒複製日本腦炎小片段RNA
外文關鍵詞:viral replicationsmall RNAJEV
相關次數:
  • 被引用被引用:0
  • 點閱點閱:133
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  • 下載下載:9
  • 收藏至我的研究室書目清單書目收藏:1
中文摘要
日本腦炎病毒 (Japanese encephalitis virus,簡稱JEV)基因體為一
條正股全長10,976 nts 的RNA 序列,在其複製過程中不會產生任何
次基因體RNA (subgenomic RNA)。本實驗室之前有研究報導指出:
在日本腦炎病毒RNA 複製的過程中,會產生一個比基因體小的有趣
分子,稱此RNA 為small RNA,其大小為521-523 nts 之正股RNA,
序列為病毒基因體3’-端未轉譯區 (3’-untranslated region)中高度保留
區段 (Journal of Virology 78:5133,2004)。為研究small RNA 之弁鄐尷R病毒複製的關係,本研究分別利用Northern hybridization、RNase
protection assay 和RT-real-time PCR 等實驗方法定量日本腦炎病毒正
股和負股RNA 的合成量,結果顯示small RNA 可能扮演抑制負股合
成的角色。在Northern 分析病毒負股的結果中沒有發現負股small
RNA 的存在,只能發現2X 大小的負股基因體。為了進一步說明small
RNA 可能的弁?轉染plus-sense small RNA 或minus-sense small
RNA 於已感染JEV 的細胞中並利用Northern hybridization 和
RT-real-time PCR 的方法分析病毒正負股RNA 的合成有無受到影
響,結果指出當轉染plus-sense small RNA 不影響病毒正股RNA 生合
成,但轉染minus-sense small RNA 卻會造成1X 病毒負股基因體的
產生。顯示 small RNA 可能扮演調節病毒正負股RNA 合成之弁遄C
英文摘要
Japanese encephalitis virus (JEV) contains a single positive-strand
RNA genome of 10,976 nucleotides in length and is not formally thought
to generate subgenomic RNA molecules during replication. Previous
studies in our lab have reported the abundant accumulation of a
3’-terminal 521- to 523-nucleotide genome fragment, representing the
highly conserved region of the 3’-untranslated region, in JEV-infected
cells (Journal of Virology 78:5133, 2004). To address a possible
function of the small RNA during viral replication, several approaches
were carried out. Systematic quantification of plus- and minus-stand
viral RNA synthesis using Northern hybridization, RNase protection, and
RT-Real-time PCR assays suggested that the presence of the small RNA
may play a role in the limitation of minus-stand RNA synthesis. Results
from Northern analysis reveals that a minus-strand complement of the
small RNA is not found, but rather only a minus-strand RNA that is 2X
genome size is found. To elucidate a possible function of the small
RNA and its complementary sequence during viral replication,
unit-length (i.e., 523-nt) plus- and minus-strand forms of the small RNA
were separately transfected in virus-infected cells and the effects on plusand
minus-strand accumulation were measured. By strand-specific
Northern hybridization and RT-real-time PCR assays. Transfection of
the plus-sense small RNA appeared not to affect plus-strand viral RNA
accumulation. However, transfection of the minus-sense small RNA
caused a change in the migration pattern of the normally observed 2X
minus-strand RNA in that nearly equal amounts of 1X-sized minus-strand
3
RNA are now found. The effect of transfection of the small plus-strand
RNA on minus-strand accumulation remains to be determined. These
results suggest that features of the minus-strand RNA may play a
regulatory role during RNA synthesis in vivo.
目次
中文摘要………………………………………………………….1
英文摘要………………………………………………………….2
第一章 簡介………………………………………………………...4
一. 日本腦炎…………………………………………..4
二. 日本腦炎病毒結構及基因體…………………..5
三. 3’-UTR 的保守序列區域(conserved sequence)及其重要性…….6
四. 黃質病毒RNA 的複製………..………………………8
五. Small RNA 的發現………………….…………....10
六. 研究目的……………..……………………......10
第二章 研究材料與方法………………………….…..12
第三章 結果…………………………………….……. 27
一. 分析JEV 病毒感染哺乳類動物細胞後正負股基因體的合成
量……….....27
I. Northern hybridization 分析結果............................................27
II. RNase protection assay 分析結果………....29
III.RT-Real-time PCR 分析結果……………..…...30
二. 轉染plus-或minus-sense small RNA 對病毒複製的影響……...31
I. 轉染plus-sense Small RNA 對於病毒的正股基因體複製沒有顯著
的影響………………………………………….…………31
II. 轉染minus-sense Small RNA 影響病毒負股基因體的結構和合
成量………………………………….……………………32
III. 轉染antisense NS2a RNA 不會影響病毒負股基因體的結
構…………………………………………….……………34
第四章 討.論……………………………………………………….35
一. Small RNA 可能扮演調節負股RNA 合成速率…..35
二. Small RNA 協助維持2X 病毒負股基因體結構的存在,以調節
病毒負股基因體之合成速度…..................…37
三. 轉染活體外製備之minus-sense small RNA 於已感染細胞中,似
乎有複製情況產生………………………………….….39
第五章 參考文獻………………………………………………….…...41
圖表………………………………………………………….45
第五章 參 考 文 獻
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