| Summary: | 碩士 === 中原大學 === 生物科技研究所 === 101 === Highly pathogenic avian influenza viruses of subtype H5N1 not only cause serious economic loss in poultry cultivation but also pose a continuous threat to public health and life. The development of vaccines is one of the most viable means to against a pandemic threat. In our previous works, we aimed to develop vaccine candidates by using Baculovirus Expression Vector System (BEVS) as a platform. In this study, we utilized dual promoter which were combined the CMV promoter with polyhedron and chimeric IRES derived from parts of IRESs of Enterovirus 71 and Rhopalosiphum padi virus to simultaneously express HA and NA protein of H5N1 in both insect cells and mammalian cells. First of all, we successfully generated vAc-CMVph-HA-Lir-NA, vAc-ph-HA-Lir-NA and vAcCMV-HA-Lir-NA viruses. By western blotting analysis, we could detect HA and NA proteins in infected Sf21cells or tranduced U2OS cells. We further confirmed the expression of HA and NA proteins on cell membrane by immunofluorescence assay. Meanwhile, we could also detect the NA activity in the infected cell lysates. Comparing the three viruses, vAc-CMVph-HA-Lir-NA might produce the higher level of HA or NA proteins in insect cells or mammalian cells. We finally validated the glycosylation of HA and NA protein produced in BEVS through de-glycosylase treatment. In sum, we expected the vAc-CMVph-HA-Lir-NA would be used as DNA or subunit vaccine candiates to elicit a better immune response against H5N1 virus infection.
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