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研究生:陳俊延
研究生(外文):Jyun-Yan Chen
論文名稱:依賴Src的Caspase活化在牛流行熱病毒引起細胞凋亡之研究
論文名稱(外文):Src-Dependent Caspase Activation in Bovine Ephemeral Fever Virus Induced Apoptosis
指導教授:施玟玲
指導教授(外文):Wen-Ling Shih
學位類別:碩士
校院名稱:慈濟大學
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:51
中文關鍵詞:牛流行熱病毒細胞凋亡CaspaseSrcSAPK/JNK
外文關鍵詞:BEFVapoptosiscaspaseSrcSAPK/JNK
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牛流行熱病毒(BEFV)是屬於彈狀病毒科(Rhabdoviridae)中的流行熱病毒屬(Ephemerovirus),會導致感染動物急性高熱、跛腳、漿液性口鼻分泌物、反芻停止等症狀。先前研究證實在細胞培養系統中BEFV會誘導Caspase依賴性的細胞凋亡,但其中的訊息傳遞路徑仍是未知。為了探討MAPK及Src訊息分子是否媒介BEFV對Caspase之活化及細胞凋亡的誘導,利用抑制訊息分子活性之策略進行研究。以SU6656與Csk抑制Src或利用SP600125抑制SAPK/JNK分子的活性,能減緩由BEFV所誘發的細胞病變現象。西方墨點法證實在 Vero與MDBK細胞中,BEFV的感染會增加Src tyrosine-418的磷酸化及Caspase-3, -6, -7, -9與PARP的切割,此磷酸化現象及Caspase切割效應能被SU6656阻斷。本研究證實BEFV所誘導Caspase活化的細胞凋亡是藉由Src分子所傳遞的。另以大腸桿菌系統表現及純化BEFV Matrix(M)蛋白質,免疫動物後獲得之抗血清可專一性辨認BEFV感染細胞後之M蛋白質,未來可作為研究M蛋白質生物功能之工具。
The Bovine ephemeral fever virus (BEFV) belongs to the Ephemerovirus of Rhabdoviridae family. It causes the disease with the sudden onset of fever, lameness, serous oral and nasal discharges and cessation of rumination. Previous reports demonstrated that the BEFV induces a caspase-dependent apoptosis in cultured cells, but the involved signaling pathways are still unknown. To investigate the roles of MAPK and Src signaling in mediating BEFV-induced caspase activation and subsequent apoptosis induction, specific inhibitors were utilized BEFV-infected cells. Inhibition of Src by SU6656 and Csk and inhibition of SAPK/JNK by SP600125 reduced the BEFV-induced CPE. Western blotting analysis demonstrated that BEFV stimulated Src tyrosine-418 phosphorylation and caspase-3, -6, -7, -9 and PARP cleavage. These effects were blocked by SU6656. Thus, these data suggested that Src-dependent caspase activation in BEFV-infected cells. In addition, expression and purification of BEFV Matrix (M) protein in E. coli system, then collection of anti-serum from immunized animal. The anti-serum could detect M protein in BEFV- infected cells, which provide a useful tool for further study of biological function of M protein.
中文摘要………………………………………………………………………2
英文摘要………………………………………………………………………3
1. 文獻探討…………………………………………………………7
1.1. 牛流行熱病毒……………………………………………………7
1.1.1. 病毒簡介…………………………………………………………7
1.1.2. 臨床上的重要性…………………………………………………8
1.2. 細胞凋亡(Apoptosis)現象與機制……………………………9
1.2.1. 病毒與細胞凋亡相關性…………………………………………11
1.2.2. Src與細胞凋亡相關性…………………………………………11
1.2.3. MAPK與細胞凋亡相關性…………………………………………13
2. 研究目的…………………………………………………………16
3. 材料與方法………………………………………………………17
3.1. 化學藥品與試劑(Chemicals and reagents)………………17
3.2. 抗體(Antibody)………………………………………………20
3.3. 緩衝溶液(Buffer)……………………………………………21
3.3.1. PBS………………………………………………………………21
3.3.2. RIPA buffer……………………………………………………21
3.3.3. SDS running buffer……………………………………………22
3.3.4. TBST………………………………………………………………22
3.3.5. Transfer buffer………………………………………………22
3.3.6. Binding buffer…………………………………………………22
3.3.7. Elution buffer…………………………………………………23
3.4. 細胞………………………………………………………………23
3.4.1. Vero、MDBK細胞培養(Cell culture)………………………23
3.4.2. 冷凍細胞(Frozen cells)……………………………………24
3.4.3. 解凍細胞(Cells reconstitution)…………………………24
3.5. 病毒………………………………………………………………24
3.5.1. 病毒增殖…………………………………………………………24
3.5.2. 病毒定量(TCID50 assay)……………………………………25
3.6. 蛋白質……………………………………………………………26
3.6.1. 蛋白質萃取(Protein extraction)…………………………26
3.6.2. 蛋白質定量(Protein concentration assay)……………26
3.7. 蛋白質電泳(SDS-PAGE)………………………………………27
3.8. 西方點墨法(Western Blot)…………………………………28
3.9. 細胞轉染(Transfection)……………………………………29
3.10. 利用E. coli系統大量表現蛋白質……………………………30
3.11. 純化蛋白質………………………………………………………31
3.12. 其他………………………………………………………………32
4. 結果………………………………………………………………33
4.1. 牛流行熱病毒的增殖與定量……………………………………33
4.2. SU6656、Csk、SP600125減緩牛流行熱病毒導致的Vero細胞病變效應(CPE)………………………………………………………………………………33
4.3. 牛流行熱病毒活化Src分子……………………………………36
4.4. 牛流行熱病毒活化Caspase分子………………………………38
4.5. 牛流行熱病毒誘導依賴Src而活化的Caspase………………40
4.6. 牛流行熱病毒Matrix(M)蛋白質表現、純化與免疫動物…41
5. 討論………………………………………………………………45
6. 參考文獻…………………………………………………………48
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