The formation and activation of influenza virus-like particles with Drosophila S2 expression system

Abstract

Objective To produce influenza virus-like particles(VLPs) with drosophila S2 cell line and identify its bioactivity. Methods The persistent expression vector sof pAc-V5-HA, NA and M1 suitable for drosophila S2 cell line were constructed at the earlier stage, which were co-transfected into S2 cell with pCoblast vector. Then we screened monoclonal S2 cell lines which integrated HA, NA and M1 genes through blasticindin combined with limited dilution methods. We used PCR assay to identify whether HA, NA and M1 genes were successfully integrated into genome, and used Western-Blot assay to identify whether there was VLPs in the supernatant. The HA activity was evaluated with hemagglutination test, and NA activity was reflected with the ability to hydrolyze sialic acid. Results The HA, NA and M1 genes were successfully amplified from the monoclonal S2 cell using PCR assay. The VLPs integrated HA, NA and M1 protein was identified with Western-Blot assay. The hemagglutination test confirmed influenza VLPs could agglutinate chicken red blood cells with titer of 1∶160. The NA could dose-dependent hydrolyze sialic acid, and no significance was observed at the high dose(250 ng, 500 ng)between wild type virus and VLPs. Conclusion We successfully established the monoclonal S2 cell line which integrated influenza HA, NA and M1 genes, and it could persistently produce influenza VLPs with perfect HA and NA activity. VLPs could be considered as effective influenza vaccine candidate in the future. Key words: Drosophila; Influenza; Virus-like particles; Cell line