Browsing by Subject "Sf9"

Great concern is to be addressed to safety measures in order to guarantee work safety when studying novel, possibly pandemic influenza A viruses. These safety measures slow down the research process and their upkeep is expensive. To overcome these hindrances a virus-like-particle (VLP) can be used as a model to replace the need for a live virus. Because VLPs are non-infectious, they are suitable for being used in research where experiments are done with slighter safety precautions. In addition, VLPs are usually highly immunogenic and thus influenza A VLP may function as a model system for further vaccine development. This research was done in a research group, in which a VLP had previously been made with cloning the genes of hemagglutinin (HA), neuraminidase (NA) and matrix 1 (M1) proteins from the year 2009 pandemic Influenza A virus (H1N1)v to a single baculovirus protein expression vector. During this earlier research project it was found problematic that the expression level of a particular gene could not be controlled. In this research, HA, NA and M1 genes were cloned to different baculovirus protein expression vectors so that the expression level of individual genes could be enhanzed with plaque purification and the multiplicity of infection (MOI) adjusted individually for each vector. It was hypothesized that an optimal configuration of MOI rates between vectors could be found in order to maximise VLP production in Spodoptera frugiperda 9 (Sf9) cells. Baculovirus protein expression vectors were made via traditional cloning of the HA, NA and M1 genes into three pAcYM1 baculovirus transfer vectors under polyhedrin promoter, which has been shown to be a strong promoter. Transfer vectors were used to transfer the genes into linearised baculovirus’ genomes by homologous recombination and the genomes were transfected into Sf9 cells to produce recombinant baculoviruses. These expression vectors were plaque-purified and their titers were amplified. Their expression level was studied using SDS-PAGE and coomassie blue analysis and with metabolic labeling using [S35]-labeled methionine. The formation of VLPs was measured with hemagglutination assay when Sf9 cells were co-infected with all three protein expression vectors. It was found that changing expression vectors MOI between 1, 3 and 5 did not have a great impact on protein expression from individual vectors. The presence of NA protein was found to be necessary for the formation of influenza A virus VLPs with a detectable hemagglutination activity. Differences between VLP formations were obtained when MOI rate compositions were changed, but further study is needed to find the significance of this result. The research to find an optimal configuration of MOI rates between vectors is still to be continued.