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研究生:游舒翎
研究生(外文):Shu-Ling Yu
論文名稱:日本腦炎病毒持續性感染之研究
論文名稱(外文):Study of Japanese Encephalitis Virus Persistence
指導教授:林宜玲林宜玲引用關係廖經倫
指導教授(外文):Yi-Ling LinChing-Len Liao
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
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:1999
畢業學年度:87
語文別:中文
論文頁數:65
中文關鍵詞:日本腦炎病毒持續性感染
外文關鍵詞:Japanese Encephalitis Viruspersistent infectionp35
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本論文第一部份是研究p35基因之表現是否可使日本腦炎病毒持續感染幼倉鼠細胞。日本腦炎病毒對細胞的持續性感染,已可被建立於鼠類神經元細胞(N18)、星狀細胞(DBT)中;但卻無法建立於幼倉鼠腎細胞(BHK-21)株和中國倉鼠卵細胞(CHO cell)株。幼倉鼠腎細胞株在感染了日本腦炎病毒後,會引起細胞自裁反應而死亡;若該細胞大量表現Bcl-2,不僅能抑制細胞自裁,並能造成病毒持續性感染的現象。由本實驗結果顯示p35對於幼倉鼠腎細胞亦有相類似的作用。p35原為baculovirus用以對抗細胞自裁之病毒蛋白質。表現p35的BHK-21細胞(B-p35),在感染日本腦炎病毒之後,能稍延遲細胞死亡,並造成部份細胞被病毒持續感染(B-p35/RP9)。由於本實驗室先前的研究發現,若細胞被日本腦炎病毒持續感染時,會表現斷裂的病毒第一非結構性蛋白質(Truncated nonstructural protein-1)。從本實驗我們得知由p35所造成在幼倉鼠腎細胞的持續性感染亦會表現出斷裂的病毒第一非結構性蛋白質。另外,與BHK-21相似,對於原本無法建立日本腦炎病毒持續性感染的CHO細胞,p35的表現可使日本腦炎病毒在細胞中建立持續性感染;並表現出斷裂的病毒第一非結構性蛋白質。我們已証實它是為持續感染。本論文的第二部份是研究日本腦炎病毒持續感染人類骨髓白血球細胞(K562)。對於不同的細胞株,感染日本腦炎病毒多會先造成細胞大量自裁而亡,僅少數存活的細胞能建立起持續性感染。K562細胞為一人類骨髓白血球細胞,由我們先前研究結果顯示:此細胞株能在不造成細胞病變的情況之下,被登革病毒持續感染。同樣的,由本實驗的結果顯示,日本腦炎病毒感染K562後亦無細胞病變之現象,且在長期培養下,接近100%的細胞成為持續性感染。另外,藉由西方墨點法我們觀察到病毒第一非結構性蛋白質也有斷裂型之存在,且細胞內之持續性感染病毒能對後來感染之病毒有干擾之現象。與持續性感染登革病毒相比較下,K562在持續感染日本腦炎病毒後,對低血清培養時生長較好。每個登革病毒持續感染K562細胞約可釋出3個病毒顆粒,而每個日本腦炎病毒持續感染之K562細胞卻能釋出500個病毒顆粒。由此發現K562細胞似乎較利於日本腦炎病毒之感染。

The portion of this research is studying the expression of p35 gene will make Japanese encephalitis virus (JEV) persistently infect the baby hamster kidney fibroblast (BHK-21) cells or not. Persistent JEV-infection can be readily established in murine N18 and astrocytoma DBT cells, but not in BHK-21 and CHO cells. BHK-21 cells were killed upon JEV infection for JEV induced apoptosis. However, over-expression of human bcl-2 in BHK-21 cells delayed JEV-induced apoptosis and rendered cells to be persistently infected with JEV. The results of this study suggest that p35 and bcl-2 have similar fuctions in BHK-21 cells. p35 is an anti-apoptotic viral protein of baculovirus. Expression of p35 in BHK-21 cells (B-p35) delayed JEV-induced apoptosis and rendered a portion of the cells to be persistently infected with virus (B-p35/RP9). The results of the studies above in our laboratory suggested that cells would express the truncated viral nonstructural protein-1 (NS1) upon JEV persistent infection. For the experiments, we got the same suggestion that truncated viral NS1 as well as its derivative NS1' were also detected in B-p35/RP9 cells. Besides, CHO cells are similar to BHK-21 cells that would not be persistently infected with JEV. Expression of p35 in CHO cells also rendered cells to be persistently infected with JEV and to express the truncated viral NS1. We proved that was persistent infection. The other portion of this research is studying JEV persistent infection in human myeloid leukemia cells (K562). The different cell lines were dead upon JEV infection and JEV-induced apoptosis, and little survival cells would be persistently infected with virus. K562 is a human myeloid leukemia cell line. The results of our research suggested that the cell line could be persistently infected with dengue virus and cells had no CPE. The same as dengue virus, the results of this research suggested that the cell line would be infected with JEV and had no CPE. The cells almost be persistently infected under culturing for long time. We could detect the truncated viral NS1 by western blot. And K562/JEV or DEN could interfere K562/DEN or JEV super-infection. K562/JEV grew in serum starvation is better than K562/DEN. Each K562/DEN cell could release 3 virus particles, and the K562/JEV cell could release 500 virus particles. The results suggested that the K562 cells prefer to JEV infect.

目錄……………………………………………………………………………………1
中文摘要………………………………………………………………………………5
英文摘要………………………………………………………………………………8
正文
第一章、前言………………………………………………………………………11
第二章、材料與方法………………………………………………………………17
材料與方法(一)……………………………………………………………18
(一)實驗用之細胞………………………………………………………18
(二)實驗用之菌株………………………………………………………18
(三)實驗用之病毒株……………………………………………………18
材料與方法(二)………………………………………………………………19
(一)Competent cells的製備…………………………………………19
(二)菌株及菌種之儲存…………………………………………………19
(三)日本腦炎病毒之增殖………………………………………………19
(四)病毒效價之測試……………………………………………………20
(五)大腸桿菌的轉形作用………………………………………………20
(六)微量質體DNA之抽取……………………………………………20
(七)間接免疫螢光法(IFA)…………………………………………21
(八)西方墨點法………………………………………………………22
(九)細胞轉染(Transfection)………………………………………23
(十)體外轉譯轉錄法(in vitro transcription and translation;TNT)
………………………………………………………………23
(十一)放射免疫沉澱法(RIP)………………………………………24
(十二)增幅聚合酵素鏈結反應(PCR)………………………………24
(十三)斷裂DNA的分析………………………………………………25
(十四)計算活細胞數……………………………………………………25
第三章、結果………………………………………………………………………26
(一) Baculovirus之p35的表現對日本腦炎病毒感染BHK-21與
CHO-k1細胞的影響………………………………………………………27
1. 以TA-cloning之方式於p35的C端接上V5 epitope…………………27
2. 以TNT和RIP確定p35/V5之表現……………………………………27
3. 以細胞生長情況觀察p35/V5對BHK-21與CHO-k1細胞之
影響……………………………………………………………………28
4. p35之表現對JEV誘發細胞死亡的影響………………………………28
5. 以測試DNA階梯式片段(DNA Ladder)來研究p35/V5的
表現是否能抑制JEV所引發之細胞自裁……………………………29
6. 利用間接免疫螢光法(IFA)來測定持續感染細胞內JEV蛋
白質的表現……………………………………………………………29
7. 利用西方墨點法偵試上述持續性感染細胞中是否有斷裂的
JEV第一非結構性蛋白質……………………………………………30
(二) JEV感染對K562細胞之影響……………………………………………31
(三) 黃質病毒在K562細胞中持續感染的生物特性…………………………32
1. Serum starvation對於持續感染JEV或DEN之K562細胞的
影響………………………………………………………………………32
2. Serum starvation對於持續感染JEV或DEN之K562細胞
在病毒產量上的影響……………………………………………………33
(四) 黃質病毒持續感染K562細胞後對再次感染之病毒複製有干擾
現象…………………………………………………………………………34
第四章、討論………………………………………………………………………35
第五章、參考文獻…………………………………………………………………40
第六章、圖…………………………………………………………………………45
圖1、以in vitro transcription and translation(簡稱TNT)表現
p35/V5,並以RIP加以確認
圖2、表現p35/V5對BHK-21及CHO-k1細胞生長的影響
圖3、p35/V5對JEV誘發細胞病變之影響
圖4、p35/V5對JEV誘發細胞自裁作用之影響
圖5、以間接免疫螢光法(IFA)測定B-p35/RP9與C-p35/RP9
內有JEV蛋白質的表現
圖6、以西方墨點法測試斷裂之JEV第一非結構性蛋白質
(Truncated-NS1;簡稱:tNS1)
圖7、JEV感染對K562細胞生長的影響
圖8、以間接免疫螢光法(IFA)偵測K562細胞內JEV之NS1
的表現
圖9、以西方墨點法偵測JEV持續感染K562細胞內JEV之
tNS1的存在
圖10、Maintenance media之血清濃度測試
圖11、Serum starvation對K562/JEV及K562/DEN細胞生長之
影響
圖12、Serum starvation對於在K562細胞持續感染之病毒於產
量上的影響
圖13-1、觀察JEV(RP9)在持續感染DEN之K562細胞內複
製之情形(感染JEV後第一天)
圖13-2、觀察JEV(RP9)在持續感染DEN之K562細胞內複
製之情形(感染JEV後第三天)
圖13-3、觀察JEV(RP9)在持續感染DEN之K562細胞內複
製之情形(感染JEV後第六天)
圖13-4、觀察JEV(RP9)在持續感染DEN之K562細胞內複
製之情形(感染JEV後第九天)
圖14-1、觀察DEN在持續感染JEV之K562細胞內複製之情形
(感染DEN後第一天)
圖14-2、觀察DEN在持續感染JEV之K562細胞內複製之情形
(感染DEN後第三天)
圖14-3、觀察DEN在持續感染JEV之K562細胞內複製之情形
(感染DEN後第六天)
圖14-4、觀察DEN在持續感染JEV之K562細胞內複製之情形
(感染DEN後第九天)

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