| Summary: | 碩士 === 中原大學 === 生物科技研究所 === 100 === The H5N1 avian influenza is a kind of high pathogenic avian influenza that usually spread in poultry. It always brings significant economical loss on agriculture. Since 1997, the H5N1 avian influenza had caused 367 fatalities in humans and the fatality rate is about 60%. These serious cases suggest the necessity to develop vaccines or neutralizing antibodies for the control of H5N1 influenza virus infection and spreading. In this study, we tried to express H5N1 monoclonal antibody by baculovirus expression vector system, which has been used to produce antibodies for other infectious disease. To produce a functional antibody, it is essential to express heavy chain and light chain simultaneously. Here, we used polycistronic expression vector which combined a polyhedrin promoter and several IRESs (internal ribosome entry sites) to co-express two or more genes simultaneously. We cloned the genes of heavy chain variable region and light chain variable region from H5N1 monoclonal antibody YY1 into expression vector. After isolating the recombinant virus, we expressed these subunits in insect cells. By western blot analysis, we could detect the signals for heavy chain in both infected cell lysate and supernatant. Besides, we also fused the secreted alkaline phosphatase (SEAP) gene and enhanced cyan fluorescent protein (ECFP) gene with C-terminal domain of heavy chain variable region respectively to make a fusion antibody. We hope to develop a novel approach for diagnostic and therapeutic applications.
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