臺灣博碩士論文加值系統

中 文 摘 要
鼠肝炎病毒(MHV) 的Spike (S)蛋白質, 在病毒的發病原理(pathogenesis)扮演很重要的角色. S蛋白質在宿主或培養細胞內, 受蛋白質酵素切割成含N端(N terminal half) 的S1 subunit 和含C端(C terminal half) 的S2 subunit二段大致等長的多胜(polypeptide). S1多胜是引導病毒和細胞受體(receptor)結合的重要蛋白質; 而S2多胜則是誘發細胞與細胞間融合(cell-cell fusion)的要素. 大多數的鼠肝炎病毒株感染細胞後, 會使細胞與細胞間發生細胞融合, 形成多核巨細胞(multinuclear giant cell), 唯有MHV-2病毒株例外, 不具有誘發細胞間融合的能力. 本研究即利用MHV-2未能誘發細胞間融合的性質來研究可能的融合機轉. 將MHV-2 S蛋白質基因做分子選殖(molecular cloning), 分析其核酸序列, 其結果與會引起細胞間融合的MHV-JHM病毒株之S蛋白質基因比對, 發現MHV-2 S蛋白質基因除少了位於S1超變化區(hypervariable region)的12個基酸(amino acid residues 446至457)外, 在S2發生3個基酸取代(amino acid substitution). 將選殖的MHV-2 S蛋白質基因質體轉染DBT細胞, 發現和利用病毒顆粒感染相同, 皆不誘發細胞間的融合. 此外, 利用MHV-2病毒顆粒感染, 或含S蛋白質基因質體轉染DBT細胞時,都發現MHV-2 S蛋白質有proteolytic cleavage反應, 被切割為S1和S2二個subunits. 由domain swapping實驗結果, 發現當MHV-2 S蛋白質基因引入原來缺失的12個基酸後, 即具有細胞間融合功能. 因此, 推論此12個基酸片段的缺失, 而不是S蛋白質的proteolytic cleavage site, 對於MHV感染細胞後發生的細胞間融合現象是重要的.

Abstract
The spike (S) glycoprotein of mouse hepatitis virus (MHV) plays a major role in the viral pathogenesis. It is often processed into the N-terminal S1 and the C-terminal S2 subunits that were evident to be important for binding to cell receptor and inducing cell-cell fusion, respectively. As a consequence of cell-cell fusion, most of naturally occurring infections of MHV are associated with syncytia formation. So far, only MHV-2 was identified to be fusion-negative. In this study, the S gene of MHV-2 was molecularly cloned and the nucleotide sequence was determined. The MHV-2 S protein lacks a 12-amino-acid stretch in the S1 hypervariable region from amino acid residues 446 to 457 when compared to the fusion-positive strain MHV-JHM. In addition, there are three amino acid substitutions in the S2 subunit, Tyr-1144 to Asp, Glu-1165 to Asp and Arg-1209 to Lys. The cloned MHV-2 S protein exhibited fusion-negative property in DBT cells as the intrinsic viral protein. Furthermore, similar to the fusion-positive MHV-JHM strain, proteolytic cleavage activity was detected both in DBT cells infected with the fusion-negative MHV-2 and in the transfected cells that expressed the cloned MHV-2 S protein. Domain swapping experiments demonstrated that the 12-amino-acid stretch missing in the MHV-2 S1 subunit, but not the proteolytic cleavage site, was critical for the cell-fusion activity of MHV.

目 錄
中 文 摘 要................................................I
英 文 摘 要..............................................III
緒 論................................................1
Part I MHV S蛋白質基因選殖....................................8
前 言................................................9
實驗材料與方法...............................................10
實驗結果與討論...............................................15
Part II MHV-2 S蛋白質生化性質之探討..........................17
前 言...............................................18
實驗材料與方法...............................................20
實 驗 結 果...............................................30
討 論...............................................34
圖 表...............................................39
參 考 資 料...............................................54

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