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研究生:李恆煥
研究生(外文):Heng-Huan Lee
論文名稱:探討EB病毒溶裂期中Rta對於LMP1表現之調控
論文名稱(外文):Rta regulates LMP1 expression during EBV lytic cycle
指導教授:蔡錦華蔡錦華引用關係
指導教授(外文):Ching-Hwa Tsai
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:61
中文關鍵詞:溶裂期潛伏期膜蛋白質病毒
外文關鍵詞:LMP1EBVlytic cycleRta
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LMP1為EB病毒潛伏期蛋白質,主要表現於細胞膜上。弁鄐W類似於持續活化之CD40。其為腫瘤壞死因子受體群成員之一,而LMP1之致癌性可由其能轉形囓齒類纖維母細胞株而印證。身為病毒潛伏期表現之蛋白質,其基因調控的相關研究皆於病毒潛伏期時進行。然而少數報導亦指出LMP1之表現量可隨病毒進入溶裂期而增加,惟對其調控機制所知甚少。
本實驗室利用基因重組之Akata株EB病毒感染293細胞,並經由挑選而得到十種分株細胞。此十種分株細胞可概分為兩型:其中一型僅表現EBNA1蛋白質而稱作潛伏期分株細胞;另一型因表現溶裂期蛋白質而稱作溶裂期分株細胞。有趣的是LMP1蛋白質於溶裂期分株細胞中有很明顯的表現量但卻無法在潛伏期分株細胞中被偵測到。利用對Zta mRNA具專一性之siRNA(si-Z1)抑制此自發性溶裂期進行後,證實病毒溶裂期之進行乃導致LMP1表現量增加之必要因素。比較帶有EB病毒之不同上皮細胞株,發現因Rta所引發病毒溶裂期進行而促進之LMP1表現量遠較Zta明顯。進一步利用si-Z1阻斷病毒溶裂期進行,發現此Rta誘導之LMP1表現量並不需Zta蛋白質的存在。藉由北方墨點法偵測可知LMP1於病毒溶裂期之轉錄產物可來自啟動子ED-L1或TR-L1之轉錄,且於不同上皮細胞有不同之偏好性。此外,利用luciferase reporter assay證實Rta可以直接促進LMP1啟動子ED-L1與TR-L1之活化,並不需要其它病毒基因產物的存在。利用TR-L1不同刪減片段突變株進行的實驗,結果顯示DNA序列170164至170108間之區域對於Rta調控啟動子TR-L1是必要的。另一方面對Rta蛋白質進行不同片段之刪減與突變,經西方墨點法與luciferase reporter assay的探查,暗示Rta蛋白質的N端、C端或核定位訊號序列對於其調控LMP1表現皆具必要性。最後我們亦於B細胞株中證實Rta引發病毒溶裂期所促進之LMP1表現量較Zta顯著,暗示Rta在B細胞中亦具有調控LMP1表現的能力。在本論文中證實了EB病毒可藉由Rta調控LMP1基因的表現,為LMP1在病毒溶裂期中所受到基因調控的機制提供了一個新的觀點,而其於病毒溶裂期中所扮演的角色及弁鄍蝑�得後續的研究與探討。
Latent membrane protein (LMP)-1 is an Epstein-Barr virus latent protein and expressed dominantly in cytoplasmic membrane. Functionally, LMP1 is like a constitutively active CD40, which is a member of tumor necrosis factor receptor family. The oncogenic ability of LMP1 has been demonstrated by rodent fibroblasts transformation. As an EBV latent gene, all the studies about LMP1 gene regulation have been concentrated during the viral latency. However, little is known about the LMP1 gene regulation during the lytic cycle even though few studies indicated that the expression of LMP1 can be up regulated when EBV is entering into the lytic cycle.
In our lab, ten sub clones have been obtained from 293A cells, which are 293 cells infected with a recombinant Akata strain of EBV. They can be classified into two groups: one is latent type, which only expresses EBNA1 protein, and the other is lytic type, which expresses lytic proteins. It is of interest that LMP1 are profoundly detected in five lytic clones but not in latent clones. We have further confirmed that lytic cycle progression is required for LMP1 expression by the Zta-targeted siRNA (si-Z1) blockage approach. Using different EBV-carrying epithelial cell lines, it is found that the induction amounts of LMP1 are more abundant in Rta-triggered lytic cycle than those in Zta-triggered lytic cycle. Furthermore, the results from transfection assay and si-Z1 blockage approach demonstrate that Zta is not required for Rta-mediated LMP1 induction, in our assay system. Data from Northern blotting assay demonstrate that LMP1 products can transcribe either from ED-L1 or from TR-L1 promoter in different epithelial cell lines. In addition, the results of luciferase reporter assay tell us that Rta can activate both LMP1 promoters in the absence of other viral genes. Experiments using a series of deletions on TR-L1 promoter indicate that the nucleotide sequences 170108 to170164 in TR-L1 are required for Rta activation. Also, the Western blot and luciferase assay using various of N-terminal, C-terminal deleted and nuclear localization signal (NLS)- mutant Rta constructs hint that transactivation domain, dimerization and NLS of Rta protein are all required for Rta-mediated LMP1 promoter activation. Finally, we also demonstrate Rta-initiated lytic cycle induces more abundant LMP1 expression than Zta in B cell lines, suggesting that Rta may also regulate LMP1 expression in B lymphocytes system. In this study, the new view for LMP1 regulation is explored, and the possible function of LMP1 expression during the lytic cycle will be discussed.
摘要 ……………………………………………………………..1
Abstract………………………………………………………..3
序論………………………………………………………..5
一、EB病毒與疾病…………………………………………………..5
二、EB病毒生活史…………………………………………………..5
三、Rta……………………………………………………………..8
四、LMP1………………………………………………………..9
實驗目的………………………………………………………..15
實驗材料………………………………………………………..16
一、藥品………………………………………………………..16
二、溶液…………………………………………………………..17
三、套組試劑……………………………………………………..18
四、抗體…………………………………………………………..19
五、質體…………………………………………………………..19
六、細胞株………………………………………………………..22
七、儀器…………………………………………………………..23
方法………………………………………………………………..24
一、細胞培養……………………………………………………..24
二、質體大量製備………………………………………………..24
三、細胞轉染……………………………………………………..25
四、西方墨點分析法……………………………………………..25
五、北方墨點分析法……………………………………………..26
六、螢光酵素-報導基因檢測…………………………………….28
結果………………………………………………………………..29
討論………………………………………………………………..34
圖表………………………………………………………………..38
參考文獻…………………………………………………………..55
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