Abstract
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.