| Summary: | 碩士 === 台北醫學院 === 細胞及分子生物研究所 === 87 === Hepatitis C virus (HCV) is one of the major pathogens of chronic hepatitis, which may lead to liver cirrhosis and hepatoma. Nonstructural protein 3 (NS3) of HCV is a highly conserved and multi-functional protein with protease and helicase activity, which plays an important role in HCV replication. To further study HCV NS3 and the possible applications of monoclonal antibody, phage display system offers an alternative method for the generation of large numbers of anti-NS3 mono-specific antibody molecules. In the present study, two phage display antibody libraries were established by combining PCR products of heavy chain genes with those of either kappa or lambda light chain genes, resulting in 4.65x10*4 and 2.5x10*5 clones in size, respectively. After 4th panning against the NS3 of HCV, the results of restriction analysis on randomly selected clones showed that both heavy and light chain gene inserts increased from 46.6% to 93.3% (kappa library) and 71.4% to 100% (lambda library), respectively. Furthermore, the detection of a 50 kD protein molecule using western blotting technique suggested the heavy and light chain polypeptides have been expressed and associated into the correct configuration. The preliminary ELISA data suggested that 4 clones may be specific for NS3, but not for BSA. DNA sequences of 4 clones indicated that 2 clones are identical but different from the other 2 identical clones. Whether or not these Fab fragments are specific for NS3 antigen remains to be determined. Taken together, our data suggested phage display system could offer an efficient way in the generation of NS3-specific clones.
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