| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 93 === Epstein-Barr virus (EBV), a human herpesvirus that establishes a life-long persistent infection in more than 90% of adults worldwide, is highly associated with many tumors and lymphoid disease. Latent membrane protein 1 (LMP1), a versatile protein, not only involves in type II and type III EBV latency but also regards as an oncoprotein in rodent fibroblast transformation and EBV-induced B cell immortalization. LMP1 functions as a constitutively activated member of the tumor necrosis factor receptor (TNFR) superfamily and activates several signaling pathways in a ligand-independent manner, which induces various genes that encode anti-apoptotic proteins and cytokines. The distribution of LMP1 was shown to be heterogeneous among individual cells. To identify its cellular localization, we have expressed LMP1 fused with enhanced green fluorescence protein (EGFP), and in cell membrane and the most condensed in perinuclear compartments were found in three epithelial cell lines. Subcellular localizations of the LMP1 were determined with a series of organelle-specific markers for endoplasmic reticulum, Golgi apparatus and cell membrane. By construction, those organelle targeting sequences were reported with red fluorescence protein (DsRed2). We found that a proportion of LMP1 was co-localized with ER, Golgi apparatus and cell membrane. Besides, we had identified a potent siRNA molecule by using two efficient siRNA screening strategy. One is fused LMP1 cDNA with reporter genes, and the other is fused only siRNA target sequence with reporter genes. Using western blot, immunofluorescence staining and northern blot, the potent siRNA can inhibit LMP1 protein and mRNA production specifically. Furthermore, the potent siRNA reduced LMP1 expression in LMP1 transfected stable cell lines. These results suggested the potent siRNA molecular could be a power tool to study LMP1 functions.
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