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研究生:康上恩
研究生(外文):Shang-En Kang
論文名稱:尋找影響日本腦炎病毒套膜蛋白第138位置E-E138K突變株釋放之細胞因子
論文名稱(外文):Searching for the cellular factor that influenced the release of Japanese encephalitis virus E-E138K mutant
指導教授:陳世順李珮瑜李珮瑜引用關係
指導教授(外文):Shih-Shun ChenPei-Yu Lee
學位類別:碩士
校院名稱:中臺科技大學
系所名稱:醫學生物科技研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:50
中文關鍵詞:日本腦炎病毒套膜蛋白病毒釋放硫酸乙醯肝素
外文關鍵詞:heparan sulfateenvelopevirus releaseJapanese encephalitis virus
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日本腦炎病毒(Japanese encephalitis virus;JEV)套膜蛋白(envelope protein;E protein)E138胺基酸由glutamic acid(E)替換成lysine(K)會使JEV感染細胞之病毒斑變小、降低吸附細胞能力、毒力減弱、對heparin有較的高親和力、感染器官趨向肝臟及部分病毒被累積在細胞內。為了進一步探討影響E-E138K突變病毒釋放的細胞因子及病毒被累積在細胞內的分子機制。我們利用反轉遺傳學(reverse genetics)及infectious cDNA clone,產生野生型的RP9X病毒株及經單點突變獲得E-E138K突變株。實驗結果發現,E-E138K突變株感染細胞後的確影響部分病毒被累積在細胞內,而RP9X卻能正常釋放病毒顆粒。已知細胞膜上膽固醇會影響某些具套膜病毒釋放,於是藉由膽固醇抑制劑methyl-β-cyclodextrin(MβCD)處理受到病毒感染的細胞,觀察缺少細胞膜上的膽固醇是否影響E-E138K突變株之釋放,結果發現E-E138K突變株與野生型RP9X的病毒顆粒釋放不受細胞膜上之膽固醇去除而影響。然而,利用6.25 mM硫化抑制物sodium chlorate(NaClO3)抑制細胞內硫酸乙醯肝素(heparan sulfate)合成,初步發現BHK-21及NT-2細胞內硫化被抑制後能增加E-E138K突變株感染後被累積在細胞內的病毒釋放到細胞外,然而,卻不影響RP9X病毒的釋放。此外,在含較多heparan sulfate的肝臟細胞株Hep-G2及BNL-CL2,細胞內硫化被抑制也能增加E-E138K突變株在感染細胞外的病毒顆粒數目,卻都不影響病毒在細胞內的複製。然而,為何細胞內硫化被抑制後能增加E-E138K在感染細胞外之病毒釋放量之分子機制,仍有待深入釐清。為了進一步探討,我們將以次細胞分層(subcellular fraction)蔗糖密度梯度離心及西方墨點法分別探討病毒累積的部位及細胞內病毒結構蛋白及複製相關蛋白的表現,來回答heparan sulfate影響病毒釋放的那個階段。
Japanese encephalitis virus containing a single Glu-to-Lys mutation at amino acid 138 of the envelope (E) protein, displayed a smaller-plaque morphology, reduced binding efficiency to cells, lower neurovirulence than wild-type virus, increased affinity to heparan sulfate, liver tissue tropism and more likely to be retained in infected cells. However, the mechanism of E-E138K mutant accumulated in infected cells has not been explored. We used reverse genetics and infectious cDNA clone, generation wild-type strain RP9X and a single point mutation E-E138K mutated virus. This study shows that E-E138K mutation affected infectious virions released from the infected cells, although did not influence JEV replication. Known cholesterol on cellular membrane is a critical factor for flavivirus release from infected cells. Treatment virus-infected cells with cholesterol depletion agent methyl-β-cyclodextrin (MβCD) did not influence the release of E-E138K mutant and wild type virus particles. Compared with chlorate analogue nitrate and salicylic acid-treated cell, sulfation inhibitor sodium chlorate (NaClO3) significantly increased the E-E138K mutant release from infected BHK-21, N18, HepG2, and BNL.CL2 cells, but did not affect the release of RP9X. However, the mechanism of inhibition cellular sulfation in increased release of E-E138K mutant from virus infected cells has remained to be investigated. In the following experiment, we mwill examine the localization of accumulated-viruses in infected cells by subcellular fraction sucrose density gradient centrifugation and Western blot analysis to understand the role of heparan sulfate on viral release stage.
中文摘要………………………………………………………… 3
英文摘要………………………………………………………… 4
目錄……………………………………………………………… 5
圖目錄…………………………………………………………… 6
第一章、前言…………………………………………………… 7
第二章、材料與方法…………………………………………… 11
第三章、結果與討論…………………………………………… 23
第四章、結論…………………………………………………… 31
第五章、參考文獻……………………………………………… 33
第六章、圖表…………………………………………………… 40
第七章、附錄…………………………………………………… 47
圖一、比較野生型RP9X及E-E138K突變株的差異…………………… 39
圖二、以MβCD去除膜上膽固醇對病毒釋放的影響…………………… 40
圖三、不同濃度NaClO3對於RP9X及E-E138K釋放的影響………… 41
圖四、以NaClO3抑制硫化反應對於RP9X、E-E138K及E-D389G病毒釋放的影響……………………………………………………… 42
圖五、以NaClO3抑制硫化反應對於RP9X、E-E138K病毒蛋白表現的影響………………………………………………………………… 43
圖六、NaClO3類似物對於日本腦炎病毒釋放的影響…………………… 44
附錄一、黃質病毒RNA基因體及轉譯出的多蛋白質………………… 46
附錄二、黃質病毒感染細胞之生活史…………………………………… 47
附錄三、Heparan sulfate 硫化反應及 NaClO3 作用機制示意圖……… 48
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