Developing an Influenza Vaccine Based on Genetic Engineering: The Use of Enhanced Green Fluorescent Protein to Increase the Selection of Recombinant Baculovirus with Genes Encoding Influenza Viral-like Particles


Developing vaccines based on viral-like particles (VLPs) can be beneficial. VLPs represent viral antigens that resemble viruses in nature. Unlike viruses that can replicate inside their host causing infection, VLPs have no capability to replicate and cause infection due to the lack of a viral genome. These properties make VLPs a
preferable vaccine rather than inactivated viruses. In this research, VLPs were produced using a baculovirus infection system. Upon infection in SF9 insect cells, recombinant baculovirus produces new baculovirus proteins as well as the influenza proteins hemagglutinin (HA), neuraminidase (NA), and matrix (M1) protein, which can auto assemble into VLPs. VLPs can be separated from baculovirus using an ultracentrifuge. The first step of VLP production is selection of the baculovirus with recombinant genes encoding influenza proteins. The obstacle in selecting recombinant baculovirus is that infected SF9 cells do not produce specific cytopathogenic effects. To assist in the observation of baculovirus production in SF9 cells, we developed a protein reporter system for recombinant baculovirus production. Enhanced green fluorescent protein (eGFP) was used as a reporter protein. The gene encoding eGFP was cloned into the same transient plasmid that also encodes HA, NA, and matrix protein M1. Those proteins were expressed under four different open reading frames. This plasmid was
co-transfected with a linearized baculovirus genome. Structural proteins of baculovirus and VLP forming recombinant proteins as well as the reporter protein were produced. Structural proteins of the baculovirus together with the genome of the baculovirus containing recombinant genes would co-assemble, producing recombinant baculovirus. The recombinant proteins then assembled, producing VLPs, and expression of the
reporter protein eGFP would be visible in the cytoplasm by fluorescence microscopy. Using a TALI cytometer, the percentage of cells that produced GFP was determined to be 3% of the total population, indicating that eGFP can be used to observe the production of recombinant baculovirus in SF9 cells. The infectivity of baculovirus can be improved by passaging baculovirus in SF9 cells and isolating highly infective recombinant baculovirus by plaque assays.

Keywords: VLP, Recombinant baculovirus, eGFP

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