臺灣博碩士論文加值系統

卡波西氏肉瘤疱疹病毒（人類第八疱疹病毒）為造成卡波西氏肉瘤的病原體，並且與兩種B淋巴球不正常增生疾病有關。K15開放性閱讀框架位於病毒基因體的最右端，已知有兩種高變異度類型：P類型及M類型；由不同的病毒株中分離。在前人研究中指出，此蛋白會與HAX-1蛋白質結合，然而生理意義不明。我們使用互補去氧核糖核酸微陣列比較表現K15P之內皮細胞株（ECV304）與對照組之基因表現量，找出有差異者。分析後，發現其中包含與機動蛋白改變、細胞移動、細胞轉移等相關的基因。以傷痕試驗法確定K15P蛋白具有促進細胞移動的能力。為了深入了解機制，以慢病毒為載體建立K15P HAX-1結合區刪除突變細胞株。在傷痕試驗及Transwell細胞移動試驗中，均發現K15P野生型細胞株擁有較好的移動能力，突變株略遜，但依然高於母細胞株。因此，我們認為：K15P蛋白可以透過與HAX-1的作用而促進細胞移動能力。

Kaposis’s sarcoma-associated herpesvirus (KSHV/HHV8) is the infectious cause of Kaposis’s sarcoma and also link to two kinds of lymphoproliferation, primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). Open reading frame K15 is at the extreme right-hand side of the KSHV genome, and two highly divergent forms, the predominant (P) and minor (M) forms, have been identified from different strains. A previous report showed that K15P interacts with HAX1 (HS-1 associated protein X-1). By oligonucleotide microarray, we compared the gene expression profile of K15P-ECV304 stable cell line to those of control cell lines. Several genes, which are involved in the regulation of actin filaments, cellular motility and adhesion, were regulated by K15P. Using wound assay, we showed that K15P increased cell motility. We further established a cell line expessing a K15P mutant, whose HAX1-binding site is deleted, by a lentiviral vector system. Wound assay and transwell cell migration assay indicated that the migration ability of K15P mutant cell line is weaker than that of K15P cell line, but is still stronger than that of parental cell line. We conclude that K15P can induce cell migration via a HAX1-dependent pathway.

中文摘要 4
Abstract 5
壹、緒論 6
一、 卡波西氏肉瘤疱疹病毒（KSHV）概論 6
I. 卡波西氏肉瘤疱疹病毒的發現與簡介 6
II. KSHV相關疾病 7
III. 卡波西氏肉瘤疱疹病毒基因體（KSHV Genome） 9
二、 K15蛋白概論 10
I. K15蛋白簡介 10
II. K15蛋白功能研究 11
三、 實驗目的 13
貳、材料與方法 14
一、 材料（Materials） 14
二、 大腸桿菌勝任細胞製備（E. coli competent cell production） 14
三、 大腸桿菌轉型（Transformation） 14
四、 質體DNA製備（Plasmid DNA extraction） 14
I. 微量質體DNA製備（Mini-preparation of plasmid DNA） 14
II. 中量質體DNA製備（Midi-preparation of plasmid DNA） 15
五、 細胞培養（Cell Culture） 15
六、 細胞轉染（Cell transfection） 15
七、 慢病毒載體製備（Producing lentiviral vectors） 15
八、 細胞感染（Cell infection） 16
九、 病毒感染單位測定 17
十、 聚丙醯氨膠體電泳及西方墨點法（Western Blot） 17
I. 蛋白質樣品製備及聚丙醯氨膠體電泳 17
II. Commassie Blue染色 18
III. 半乾式轉漬及西方墨點法 18
十一、 間接免疫螢光染色法（Indirect immunofluorescence staining） 19
十二、 傷痕試驗（Wound Assay） 20
十三、 Transwell cell migration assay 20
十四、 反轉錄聚合酶連鎖反應（RT-PCR） 21
I. RNA分離純化 21
II. 反轉錄反應 21
III. 聚合酶連鎖反應 21
十五、 肌動蛋白染色（Actin staining） 22
十六、 微陣列分析（Microarray analysis） 22
I. Microarray analysis 22
II. Computational analysis 22
參、結果 23
一、 確認K15P（UK）細胞株中K15P蛋白表現 23
二、 K15蛋白促進細胞移動 24
三、 HAX-1結合區刪除突變之K15P細胞株製備及確認 25
四、 HAX-1結合區刪除突變造成K15P促進細胞移動之能力下降 27
肆、討論 29
伍、參考文獻 33
陸、附圖 39
圖一、KSHV基因體 40
圖二、 42
（A）K15P預測之結構及已知功能 42
（B）K15P及K15M C端胺基酸序列比對 42
（C）Gα13－HAX-1調控細胞移動之推測模式 42
圖三、K15P西方墨點法偵測 43
圖四、K15P免疫螢光染色法及流式細胞儀分析結果 44
圖五、兩組細胞間表現量差異之基因表熱圖（Heat map） 45
圖六、GeneOntology Website分析結果 46
圖七、傷痕試驗與肌動蛋白染色 48
圖八、細胞株中K15P基因表現量之比較 49
圖九、間接免疫螢光染色法 50
圖十、Actin染色結果 51
圖十一、傷痕試驗結果 52
圖十二、Transwell Cell Migration Assay結果 53
圖十三、K15P蛋白促進細胞移動力之可能機制 54
柒、附錄 55
一、 材料 55
二、 菌株 57
三、 細胞株 58
四、 抗體 58
五、 Lysis Buffer 比較 59

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