臺灣博碩士論文加值系統

同源性排斥(Superinfection exclusion)現象，為當病毒感染一個細胞之後會去排斥具有同源性的病毒重複感染這個細胞，具有加速病毒在細胞間的傳播速度、防止病毒間的資源競爭、保護病毒基因穩定性等功能，此現象廣泛存在於噬菌體、植物病毒及動物病毒中。為分析日本腦炎病毒(Japanese encephalitis virus，簡稱JEV)是否具有同源性排斥的現象，我選用膜蛋白具有差異性的第一型及第三型JEV進行實驗。將小倉鼠腎臟細胞(BHK-21)先以第一型或第三型JEV感染細胞24小時後，則另一基因型JEV就無法順利感染同一細胞；而在蚊子腸道上皮細胞(C6/36)的實驗中也有相同的發現；隨著病毒感染細胞的時間越長，表現出的同源性排斥現象就越強烈，可見病毒在感染細胞後需要一定的時間來建立排斥其它病毒的防衛機制。分析JEV同源性排斥的作用機制，發現JEV的同源性排斥現象不是發生在病毒與宿主細胞膜上受體的結合，而是作用在抑制病毒穿透細胞膜進入細胞的階段。此外，利用螢光染劑將第一型日本腦炎病毒標示上螢光訊號後，再感染已被第三型日本腦炎病毒感染過或未被感染過的細胞，藉由流式細胞儀分析螢光進入細胞的表現量做為同源性排斥現象的定量方法，實驗結果證明在第三型感染後24小時抑制掉65%第一型的螢光病毒的感染。利用分析標定的運鐵蛋白(Transferrin)作為宿主細胞Clathrin-mediated endocytosis pathway的指標，以進一步分析病毒同源性排斥現象抑制進膜的機制，結果顯示BHK-21細胞在感染第三型日本腦炎病毒24小時後，仍然能正常攝入運鐵蛋白，推測JEV的同源性排斥作用並非抑制在Clathrin-mediated endocytosis pathway。本研究首次證明JEV具有同源性排斥現象，並提出此作用的可能機制。

第一章 前言......................................................................................1
1. 日本腦炎病毒與基因體........................................................1
2. 病毒生活史...............................................................................2
3. 同源性排斥......................................................................3
4. 黃質病毒屬病毒外套膜蛋白與進入細胞的路徑.................6
5. 日本腦炎病毒基因型.......................................................7
6. 研究目的……………........................…………………8
第二章 材料與方法...................................................................................9
1. 本研究使用之引子整理與表一...............................................9
2. 細胞株培養................................................................................9
3. 病毒培養.................................................................................9
4. 病毒力價測試..........................................................................10
5. 同源性排斥現象分析...............................................................10
6. 總量RNA萃取……................................................................11
7. 反轉錄作用....................................................................................12
8. 聚合酶鏈鎖反應....................................................................12
9. 病毒吸附分析..........................................................................13
10. 病毒穿膜分析.............................................................................13
11. 病毒穿膜時效分析........................................................................13
12. 濃縮、純化日本腦炎病毒......................................................14
13. 日本腦炎病毒顆粒的螢光標定..............................................15
14. 免疫螢光染色法………………..............................................15
14.1 觀察感染細胞後的螢光標定病毒……………………...........…15
14.2 觀察Alexa 647-transferrin進入細胞的情形…….………….16
15. 流式細胞儀定量病毒感染率.....................................................17
第三章 結果...............................................................................................19
1. 日本腦炎病具有同源性排斥現象........................................19
2. 日本腦炎病毒的同源性排斥現象並非發生於病毒與宿主細胞膜上的受體結合的時候............................................................20
3. 日本腦炎病毒的同源性排斥現象發生在病毒穿透(Penetration)進入細胞膜的階段..................................................................21
4. 當病毒感染細胞的時間越長，對超感染病毒的同源性排斥作用就越強...................................................................................22
5. 同源性排斥現象使超感染病毒穿透細胞膜的能力下降了65%..............................................................................23
6. BHK-21細胞在感染第三型JEV後不影響Clathrin mediated endocytosis pathway的功能…...............................…………23
第四章 討論...............................................................................................25
第五章 參考文獻.......................................................................................31
表圖.............................................................................................................37

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