| Summary: | 碩士 === 國立中興大學 === 獸醫微生物學研究所 === 94 === Avian influenza viruses are members of the orthomyxoviridae family and are grouped into type A influenza virus according to antigenic characteristics of their core proteins. These viruses can be further classified by their surface proteins hemagglutinin (H1-H16) and neuraminidase (N1-N9). During 1999 to 2000, outbroke of low or high pathogenic H7N1 avian influenza occured in Italy and led to a sobering economic loss. In consequence, the government of Italy developed an effective DIVA methodology as a tool for the eradication of AI. The goal of this study is to establish a DIVA methodology for rapid subtyping of NA-antibodies. The method is to use Bac-to-Bac® baculovirus express system to construct recombinant baculovirus containing N1 and N2 genes. Sf9 and High FiveTM insect cells were then used to produce recombinant NA (rNA) protein by infection with recombinant baculovirus. Possible glycosylations of the full length N1 and N2 recombinant proteins were observed based on the molecular weight of SDS-PAGE. However, the amount of recombinant N1 and N2 proteins expressed in insect cells were low. These two rNA proteins were probed with antibodies of 9 NA subtypes by multi-screen channel Western blot analysis, and the result showed that both N1 and N2 recombinant protein could discriminate the N1 and N2 sera, respectively. Moreover, the two rNA proteins were used as antigens to detect antibodies of 9 NA subtypes by indirect immunofluorescence assay (iIFA) analysis, and the result showed that recombinant N1 can discriminate each subtype sera except for the N2. The recombinant N1 and N2 proteins produced in this study could be used to establish a DIVA method that could help the surveillance and prevention of AIV in Taiwan.
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