EB病毒感染已被證實與許多人類細胞癌化有相關，其中包括B細胞淋巴癌及鼻咽癌。EB病毒的第一型潛伏膜蛋白(LMP1)曾被發現能啟動數種細胞訊息傳導途徑，並且是病毒引發B細胞永生化的主要蛋白質。第一型潛伏膜蛋白亦被發現會降低上皮細胞中細胞黏合蛋白的表現，並且會增加細胞的轉移能力。由於第一型潛伏膜蛋白經常在很多的EB病毒相關的癌症細胞中被發現，並且跟細胞癌化有相當密切的關係。因此，第一型潛伏膜蛋白可以作為臨床診斷以及治療EB病毒感染非常好的目標。本論文的主要研究目標即為偵測第一型潛伏膜蛋白，並且嘗試抑制此蛋白質在引起細胞癌化的相關功能，希望能解決EB病毒有關腫瘤的問題。為了偵測與估計第一型潛伏膜蛋白在細胞內的表現量，實驗中選殖了5個對第一型潛伏膜蛋白專一的單株抗體。這些單株抗體的抗原決定位位於第一型潛伏膜蛋白的CTAR3內的重複序列區，對應到氨基酸254-319之間的位置。這些單株抗體可以辨識不同來源的第一型潛伏膜蛋白，包括淋巴以及表皮組織起源，並且可以應用在免疫墨點法，酵素連結吸附免疫分析法(ELISA)以及免疫細胞螢光染色等偵測上。本研究亦利用這些單株抗體建構了夾層式ELISA (sandwich ELISA)分析法，以用來定量第一型潛伏膜蛋白。由此結果顯示這些單株抗體可以應用在基礎研究或臨床上對於第一型潛伏膜蛋白的偵測及定量。第二部分的研究則是利用噬菌體呈現系統進行對抗第一型潛伏膜蛋白C端的專一單鏈抗體篩檢。經過4次篩檢後得到對第一型潛伏膜蛋白C端的CTAR1，CTAR2及CTAR2區域具有專一性的單鏈抗體。其中一個單鏈抗體scH3，在293細胞中可以抑制第一型潛伏膜蛋白所提昇的NF-kB活性。利用transwell來分析細胞轉移程度的實驗中亦發現，將單鏈抗體scH3表現於具有第一型潛伏膜蛋白的MDCK-LMP1細胞中亦可以抑制細胞的轉移能力。由此得知單鏈抗體scH3在細胞內可以結合至第一型潛伏膜蛋白的C端CTAR1的位置，並且降低其所引發的細胞轉移的能力。這些抗體或許可以提供未來在臨床上治療EB病毒相關的腫瘤上的應用。

Epstein-Barr virus (EBV) has been implicated in the development of many human neoplasias including B lymphomas and nasopharyngeal carcinoma. The EBV latent membrane protein 1 (LMP1) has been found to participate in diverse cellular signaling pathways and is essential for virus-induced B-cell immortalization. LMP1 also down-regulated cell adhesion molecules expression and increase cell motility in epithelial cells. Since LMP1 can be found in many EBV-associated tumors and its role in carcinogenesis, it can be served as a good target for clinical diagnosis and therapy of LMP1-positive tumors. In this study, I focused on the detection of LMP1 and impairment of its functions in vivo. In order to determine quantitatively the amount of LMP1 in cells, five monoclonal antibodies (Mabs) specific to LMP-1 were generated. The epitopes recognized by these Mabs were found to cluster within the repeat region of CTAR3 domains, corresponding to amino acid positions 254-319 of LMP1. These Mabs were capable of recognizing LMP1 proteins of both lymphoid and epithelial origin as revealed by immunoblot, ELISA and immunocytofluorescence analysis. A sandwich ELISA for the quantification of LMP1 has been established using these Mabs. These results indicate that the Mabs generated in this study are suitable for the detection of LMP1 in biomedical research. In the secondary study, a phage display library carrying the human synthetic single-chain Fv (scFv) antibodies was used for screening of recombinant antibody clones that specific to the LMP1 C-terminal. After 4 times of panning, individual clones that react to the CTAR1, CTAR2 and CTAR3 region of LMP1 C-terminal were obtained. These LMP1 C-terminal targeted scFvs were cloned as intrabodies and transfected into LMP1-positive cells. The scFv H3 clone, when co-transfected with LMP1, were found capable of decreasing the NF-kB activity which was upregulated by LMP1 in HEK293 cell. Expression of scFv H3 also reduced cell motility in MDCK-LMP1 cells, as demonstrated by the transwell cell migration assay. These data indicates that the anti-LMP1 C-terminal scFv H3 intrabody has the ability to bind to the LMP1 C-terminal in vivo and can inhibit LMP1 functions in epithelial cells. This scFv antibodies may be useful in attenuating the LMP1 function in LMP1-positive tumors.

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