Development of flu vaccine by a novel polycistronic baculovirus expression vector

• Main Authors: Ya-Wen Lo, 羅雅雯
• Other Authors: Tzong-Yuan Wu
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/86374289848000199133

碩士 === 中原大學 === 生物科技研究所 === 98 === Since 2003, outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in human and poultry, that is Orthomyxoviridae. H5N1virus lead to generalized infections in chickens with mortality rates close to 100%. At present, there are over hundreds of millions of birds and several hundred people died and cause significantly economic losses in worldwide. Avian influenza virus have two glycoproteins on viral surface, hemagglutinin (HA) and neuraminidase (NA). HA is the major immunogen on the envelope of avian influenza virus, and it is the main target antigen of the humoral immune response in host animals. NA facilitates viral release from cells by removing sialic acid from sialyloligosaccharides on surface of cells and virals. In response to the pandemic disease by H5N1 influenza virus, the flu vaccine development is imperative. Commercial vaccines come from ckicken embryos, which is time-consuming and the cost is too high for poultry. In this study, a novel tricistronic baculovirus expression vector was generated and was used to express NA, HA gene and a convenient traceable fluorescent reporter gene, EGFP. The tricistronic transcript containing ORFs of NA, HA and EGFP is controlled by the polyhedron promoter. To express the three genes simultaneously, NA was translated through a cap-dependent mechanism, while the EGFP and HA were translated by the IRESs derived from Perina nuda virus (PnV) and the 5' UTR of Rhopalosiphum padi virus (RhPV), respectively. We conducted the following experiment to proof the correctly expression of the tricistronic baculovirus vector in Sf21 cells and T. ni larvae. First, the recombinant baculovirus, vAc-NA-EGFP-Rhir-HA, can be identified by the green fluorescence under fluorescence microscope. Second, the vAc-NA-EGFP-Rhir-HA infected Sf21 cells and T. ni larvae revealed the neuraminidase enzyme activity. Finally, Western blot and with anti-HA monoclonal antibody indicate that the third ORF, the HA was expressed. And immune flouracence assay confirned NA and HA expressed in Sf21 cells. Thus, the tricistronic recombinant baculovirus, vAc-NA-EGFP-Rhir-HA, can expressed the NA and HA proteins simultaneously in Sf21 insect cells. We hypothesize that the HA and NA combined vaccine formula produced by this vAc-NA-EGFP-Rhir-HA tricistronic vector can be used as an effective for poultry, the HA will induce the antibodies that against the flu virus entry route and the NA will induce the antibodies that prevent the flu viruses releasing.