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研究生:孫世昌
論文名稱:從靈長類長臂猿分離與分析新型B型肝炎病毒
論文名稱(外文):Isolation and Characterization of New Primate Hepatitis B Virus from Gibbon
指導教授:許宗雄許宗雄引用關係張秀鳳張秀鳳引用關係
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:肝炎B型肝炎病毒長臂猿靈長類
外文關鍵詞:HepatitisHepatitis B VirusGibbonPrimate
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中文摘要
利用ELISA之血清學方法對由“屏東科技大學野生動物保育中心”所提供的15個長臂猿、6個紅毛猩猩、及35個台灣彌猴之靈長類血清檢體與4個取自人類之血清檢體,進行血清檢體中所含有的HBsAg測試,來檢測被B型肝炎病毒(HBV) 感染的檢體,並藉由PCR技術放大10個長臂猿血清檢體中所存在的HBV-DNA。經過核甘酸定序與分析得到了病毒之次基因體與完整基因體序列。4個完整長臂猿B型肝炎病毒基因序列總長度皆為3,182個核甘酸,其上含有四個基因,可產生包括聚合酶(polymerase),表面蛋白(surface protein),核酸鞘蛋白(core protein)與X-蛋白;其與哺乳類之B型肝炎病毒基因的組成相同。藉著遺傳演化分析資料的顯示,由長臂猿血清檢體所分離出來的B型肝炎病毒可與其他長臂猿B型肝炎病毒形成一個群集,而且分離出來的長臂猿B型肝炎病毒在遺傳演化關係上也與紅毛猩猩以及黑猩猩B型肝炎病毒相近,但是卻與人類與鳥禽類B型肝炎病毒的遺傳演化關係較為疏遠。經過與標準長臂猿B型肝炎病毒序列進行比對後,所決定的次基因體區域之序列相似度全部皆超過88﹪(而所有次基因體區域之氨基酸序列相似度也都全部超過84﹪)。這個結果為我們所分離出的長臂猿B型肝炎病毒是屬於“長臂猿專一性”的基因型提供了一個非常重要的證據。而在病毒之完整基因體序列與其他HBV序列的比對中,也顯示出了將近有10﹪的序列發散性,也許這顯示我們所分離出來的長臂猿HBV是一種新型基因型的長臂猿B型肝炎病毒(Okamoto et al. 1988, Norder et al.1992, Magnius et al. 1995)。經由前述,我們已成功地由台灣地區靈長類長臂猿的身上檢測並分離出了新型B型肝炎病毒,而且也為日後建立研究HBV新式實驗動物模式提供了一個新的機會。
Abstract
To screen primate hepatitis B virus (HBV), the sera of fifteen gibbons, six orangutans, and thirty-five Taiwan-macaques supported from Recourse Center for Endanger Wild Animal, Pintung Technology University were tested by using ELISA. The human sera were used as the control. ten tested positive sera from gibbons were used as the template to amplify subgenome and complete genome of HBV by polymerase chain reaction (PCR). The PCR products were analyzed by nucleotide sequencing. The complete gibbon HBV genomes has 3,182 nucleotides and encode polymerase, surface protein, core protein and X-protein, which is similar to mammalian HBV in gene organization. The sequences comparison analysis showed that the sequences similarity of viral sub-genomic sequences including pre C, Core, S regions, respectively, are over 88﹪in nucleotide sequence and over 84﹪in protein sequence. These data indicated that the new isolated gibbon HBV is a “gibbon—specific” genotype of HBV. The complete genome sequences has approximately 10﹪nucleotide divergence compared to other giHBV sequences. This suggested that new isolates of gibbon HBV were new genotype (Okamoto et al. 1988, Norder et al.1992, Magnius et al. 1995). Phylogenetic analysis also revealed the sequences between new isolated gibbon HBV and published gibbon HBV are very similar. Furthermore, new isolated gibbon HBV are more related to orangutan HBV and chimpanzee HBV, and they are less close evolutionary relationship with human HBV and Duck HBV. In this report, new primate hepatitis B virus from gibbon has been isolated and characterized. This provides a new opportunity for construction of new experimental animal model for HBV research.
目 錄
名詞縮寫…………………………………………………………………i
英文摘要…………………………………………………………………ii
中文摘要………………………………………………………………...iii
緒論………………………………………………………………………1
實驗材料及方法………………………………………………………..28
實驗結果………………………………………………………………..44
實驗討論………………………………………………………………..56
論文參考資料…………………………………………………………..67
實驗表格與實驗結果附圖……………………………………………..79
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