臺灣博碩士論文加值系統

傳染性胰臟壞死病毒( infectious pancreatic necrosis virus, IPNV )，屬於兩段雙股核醣核酸病毒科，是養殖漁業的重要病原體。在台灣IPNV也曾經造成吳郭魚、鰻魚、虱目魚及文蛤等養殖魚類的重大損失。因此，如何有效抑制IPNV的感染乃當今一大課題。今欲研究基因功能及發展DNA 疫苗，故先構築表現載體，將IPNV的VP2 和VP3的基因插入可在哺乳類細胞表現的載體pCDNA3裡，它包含有CMV(cytomegalovirus) 立即早期啟動子。確定VP2和VP3的基因是正確的插入pCDNA3，經由定序，得知T42G之VP2和Jasper 株之基因序列有99.08%之同質性，而氨基酸序列則有99.17%相同。VP3之基因序列和Jasper株有99.05%之同質性，而氨基酸序列則有99.18%相同。所構築的pCDNA3-VP2和pCDNA3-VP3質體轉染進CHSE-214細胞裡，再以西方墨點法來分析重組蛋白質在細胞中的表現，結果顯示VP3在細胞中有兩個分子，分別是34.5kDa及31kDa。而VP2以西方墨點法來分析，並不能明顯的證實VP2在細胞中表現，故用體外轉譯的方式來確定VP2是可以表現的，大小是54kDa，可能是VP2的前趨蛋白(pre-VP2)。先前本實驗室的證據顯示，VP3可和雙股核糖核酸結合，故以pCDNA3-VP3與pAcUW21-Luc質體共同轉染TO-2細胞，檢測VP3是否對轉譯有關係，發現VP3可以增加luciferase 蛋白質的表現。進一步，將pCDNA3-VP2與pCDNA3-VP3分別轉染CHSE-214細胞，挑選穩定表現之細胞株，穫得10個含VP2的細胞群落及12個含VP3細胞群落，現進行細胞放大中。鱒魚(0.3g w.t.) 以傳染性胰臟壞死病毒之VP2的cDNA質體進行DNA疫苗注射，免疫5個星期後，再以傳染性胰臟壞死病毒的致病株來進行攻擊試驗，兩星期後，發現魚體研磨液有高的抗體力價，但沒有中和病毒感染的抗體存在。

Infectious pancreatic necrosis virus(IPNV), a birnavirus, is an important pathogen in fish farms. In Taiwan, IPNV has caused great damages and loss in the aquacultures of tilapia, eel, milk fish and clam. Therefore, how to effectively control the IPNV infection is an important issue to the aquacultures.To order to study the gene function and develop of DNA vaccine. So construction of the expression vector is the first, the VP2 and VP3 cDNA clones of IPNV were cloned into the pCDNA3 expression vector for mammalian cell. It contains cytomegalovirus (CMV) immediate early promoter. Those plasmid with virus gene (VP2 or VP3) have been confirm of the insert was in frame by sequence. The DNA sequence of T42G strain VP2 gene is very similar to that of the known Jasper strain, with 99.08% identity, while the homology of amine acid sequence between two strain is 99.17%. The homology between T42G and Jasper strain VP3 is 99.05% for DNA sequence, and 99.18% for the amine acid sequence. The construction pCDNA3-VP2 and pCDNA3-VP3 was transient transfected into to CHSE-214 cell and detected by Western blotting analysis. The data showed VP3 have two size(34.5kDa and 31kDa). The VP2 can't detected by Western blotting, so the in vitro translation of pCDNA3-VP2 was conducted. The data showed a major protein expressed and the size was 54kDa. This protein maybe the precursor of VP2. VP3 has been confirmed to have dsRNA binding activity. Therefore, the effect of VP3 on the translation has to be mesured. The pCDNA3-VP3 and pAcUW21-Luc plasmid was cotransfected into TO-2 cells. The VP3 increase the luciferase translation in vivo. Furthermore, the pCDNA3-VP2 and pCDNA3-VP3 was transfected into CHSE-214 cells and stable clones was screened. Now 10 clones of VP2 and 12 clones of VP3 were detained and the cells amplify. Rainbow trout (0.3g w.t) were injected with plasmid pCDNA3-VP2 of IPNV e as a DNA vaccine. Five weeks after immunization, fish were challenged with IPNV (Buhl strain ) and of anti-IPNV antibody of fish were measures by ELISA. The immunized fish had a good antibody titer in against IPNV but not the neutralizing antibody of virus.

英文摘要 1中文摘要 2前言 3文獻整理 5 一、感染性胰臟壞死病毒 5 (一) IPNV 之發現、分類、分佈及其引起之病症 5 1. IPNV 之發現 5 2. IPNV 之分佈 6 3. IPNV 之分類 6 4. IPNV 在血清型上之分類 7 5. IPNV 感染之病症 8(二) IPNV 之特性 8 1. IPNV 之外部形態 8 2. IPNV 之生長 9 3. IPNV 之遺傳物質 10 4. 病毒的轉錄與複製 10 5. 病毒之轉譯 11 6. 病毒蛋白質及其功能 11 二、雙股核糖核酸結合蛋白質 16 三、有關DNA疫苗方面的研究 191. 疫苗的發展的由來 192. 各種疫苗之缺點 203. DNA疫苗 214. DNA疫苗傳送方式 225. 人類DNA疫苗之發展 236. 其他動物及魚類之DNA 疫苗發展 257. DNA疫苗之免疫增強法 26材料與方法 28 一、材料 28 1. 細胞株 28 2. IPNV 病毒 28 3.鱒魚 28 4.菌種與質體 28 5.培養基 28 6. 反應試劑 29 二、實驗方法 301. 反應溶液或緩衝液 30 2. 細胞之培養 33 3. 病毒之增殖及感染力價的測定 33 4. 病毒斑分析法 34 5. 病毒 ( virion ) 純化 35 6. 不連續 SDS-PAGE 平板電泳膠體之製備 35 7. 轉印及西方點墨雜交法 36 8. 蛋白質 Coomassie blue 染色法 36 9. 蛋白質定量 37 10. 構築VP2和VP3表現載體 37 11. 製備大腸桿菌勝任細胞及轉型 37 12. 小量及大量質體 DNA 置備 38 13. 質體DNA以酵素及聚合酵素連鎖反應鑑定 39 14. 穩定轉殖CHSE-214細胞珠之建立 40 15. 體外轉錄轉譯質體蛋白 41 16. 鱒魚之免疫與攻擊試驗 41 17. 酵素免疫法(ELISA) 41 18.VP3對轉譯的影響 42結果 43 (一)、質體之構築 43 (二)、篩選構築成功之載體 43 (三)、質體方向之鑑定 44 (四)、嵌入體質體序列分析 45 (五)、轉染細胞之病毒蛋白表現 45 (六)、體外轉錄及轉譯質體蛋白質 46 (七)、穩定轉殖之單一細胞群落(Colony)篩選 46 (八)、VP3對轉錄轉譯之影響 46討論 48文獻整理 53附圖

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