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中文摘要
EB病毒開放譯讀架BGLF4，經氨基酸序列比對發現具有ser/thr蛋白質激坆功能區保守的氨基酸，因此推測BGLF4可能具有蛋白質激坆的活性。之前的研究可在BGLF4轉染及vTF 7-3病毒感染的細胞中表現BGLF4，以免疫沉澱部分純化後進行蛋白質激坆實驗，觀察到BGLF4的自我磷酸化，能夠磷酸化casein、histone、及EB病毒的early-antigen diffuse type (EA-D)，並測定其基本特性。本論文利用此系統進一步分析BGLF4上對自我磷酸化的重要區域，並研究BGLF4相關蛋白質激坆活性的特性。以突變株分析對BGLF4自我磷酸化重要的區域中，定位出BGLF4氨基酸1至26對BGLF4磷酸化是重要的，刪除原推測對蛋白質激坆重要的區域，對BGLF4自我磷酸化影響不大。heparin及okadaic acid對BGLF4的磷酸化沒有影響，但heparin可抑制對casein的磷酸化。以抗磷酸化氨基酸抗體觀察到BGLF4自我磷酸化的位置是在serine及threonine上，表示BGLF4上的磷酸化由ser/thr蛋白質激坆活性造成。在BGLF4轉染293T細胞實驗中，利用293T細胞所帶的SV40 T Ag啟動BGLF4前SV40啟動子的作用使之表現，在此系統中BGLF4表現量比vTF7-3病毒感染較低，但亦可進行蛋白質激坆實驗。利用免疫螢光實驗觀察到BGLF4表現位置在細胞質。

Abstract
BGLF4 of Epstrin-Barr virus (EBV) was identified as a possible protein kinase based on the homology alignment to the conserved motifs of known protein kinases. In a previous study, the expression of BGLF4 product was observed in a transient transfection following by the recombinant vaccinia virus vTF7-3 which carrying a T7 RNA polymerase. By using an EBNA-1 tag, the immunoprecipitates BGLF4 was demonstrated for its ability of autophosphorylation and phosphorylating casein, histone, and early-antigen diffuse type (EA-D) of EBV. This study was therefore aimed to identify the important functional domains of BGLF4 and further characterize the BGLF4-associated protein kinase activity. A panel of deletion mutants of BGLF4 were generated and analyzed for their kinase activities. Sequence between amino acid (aa) 1-26 but not the predicted conserved catalytic domain of BGLF4 was found essential for the BGLF4 kinase activity. The kinase activity was not affected in the presence of heparin or okadaic acid. Anti-phosphoamino acid monoclonal antibodies were used to demonstrated that the phosphorylation sites of BGLF4 were on serine and threonine. However, the BGLF4-associated protein kinase activity was sensitive to heparin in the transphosphorylation of casein. It's possible that there is more than one protein kinase appeared in the autophosphorylation and transphosphorylation of BGLF4. Finally, the BGLF4 product was found to express in the cytoplasm of 293T cells by immunofluorescence staining.

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