臺灣博碩士論文加值系統

ORF50是KSHV極早期表現的轉錄活化子，可以誘發潛伏感染的KSHV進入溶裂循環。ORF50由691個胺基酸所組成，包括N端的DNA結合區 (胺基酸1到390)及C端轉錄活化區 (胺基酸486到691)，已知在細胞中ORF50是個高度磷酸化的蛋白質，但並不清楚ORF50是否還有其他轉錄後修飾。在本研究中，藉由西方點墨法分析發現，sodium butyrate (SB)會使Cytomegalovirus immediate-early (CMV-IE)啟動子所調控的ORF50表現量增加，再藉由 ORF50的突變株進一步證實，SB是透過C端357到691胺基酸造成此一現象。冷光酶活性分析也發現，只包含ORF50 C端胺基酸357到691的突變株和全長的ORF50都具有抑制CMV-IE及p21啟動子活性的能力。雖然目前仍然無法證實ORF50是否為乙醯化修飾的蛋白質，但實驗中發現，ORF50 C端胺基酸也是調控其蛋白質磷酸化修飾的重要區域。本研究顯示，ORF50 C端胺基酸不僅僅是ORF50的轉錄活化區，也是影響ORF50在細胞中的含量、轉錄活性及轉譯後修釋重要的調控區域。

The ORF50 protein of Kapsosi’s sarcoma-associated herpesvirus (KSHV) is a multifunctional transcriptional activator that drives the viral lytic cycle. It is a 691-amino acid (aa) polypeptide that is extensively phosphorylated and may be subject to other posttranslational modifications. The DNA binding domain of ORF50 protein is located in the N-terminal portion (aa 1 to 390) and an acidic activation domain is found in the C-terminal portion (aa 486 to 691). This study finds that sodium butyrate treatment markedly enhances the expression level of the full-length ORF50 protein, but not the C-terminal truncated mutants, encoded from the expression vector with a cytomegalovirus immediate-early (CMV-IE) promoter. Extensive deletion analyses showed that the C-terminal region from aa 357 to 691 of the ORF50 protein is sufficient to mediate an abundant enhancement in response to sodium butyrate. Furthermore, transient transfection assay revealed that the C-terminal ORF50 (aa 357-691), similar to its full-length protein, possesses an unexpectedly repressive effect on the CMV-IE promoter and a cellular p21 promoter. Despite the lack of evidence showing that whether ORF50 protein is an acetylated protein, the critical region conferring ORF50 hyperphosphorylation may be also located in the C-terminal region. Taken together, these findings suggest that the C-terminal region of ORF50 protein acts as not only a general transactivation domain, but also an important regulatory motif for protein abundance, transcriptional repression and posttranslational modifications.

目錄

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中文摘要
英文摘要
目錄
第一章、緒論
一、卡波西氏肉瘤疱疹病毒的發現及歷史
二、卡波西氏肉瘤疱疹病毒的型態、遺傳物質與致病性
三、卡波西氏肉瘤疱疹病毒的生活史
四、Sodium butyrate對ORF50 基因調控的影響
五、ORF50的功能與特性
六、ORF50轉譯後修飾的調控
七、ORF50的重要性
八、本研究計畫的目的
第二章、材料與方法
一、質體
二、細胞株
三、細胞轉染作用
四、抗體
五、免疫沈澱分析
六、西方點墨法分析
七、14C同位素標定
八、3H同位素標定
九、冷光酶活性測定
第三章、結果
一、SB對ORF50蛋白質表現的影響
二、不同的ORF50胺基酸片段對CMV-IE啟動子回饋抑制的影響
三、ORF50乙醯化修飾的分析
第四章、討論
一、SB對ORF50在細胞內含量的影響
二、ORF50胺基酸片段對CMV-IE啟動子回饋抑制的影響
三、ORF50回饋抑制p21啟動子在生理上的意義
四、ORF50的轉譯後修飾
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