Figure 5
From: Baculovirus as an efficient vector for gene delivery into mosquitoes
Enzymatic analysis of influenza virus NA expressed in mosquito C6/36 cells by recombinant baculovirus. (a) Schematic representation of a pABb1p10-NA3 transfer vector for generating recombinant baculovirus. The transfer vector contains the EGFP reporter gene driven by the pag1 promoter; NA (H5N3) gene under the control of dual host b1 and p10 promoters for mosquito C6/36 and insect Sf21 cells; FLAG and HIS tags were inserted at c-terminal; AcMNPV lateral ends flanking the expression cassette for homologous recombination. (b) C6/36 cells were transduced with vABb1p10-empty (virus backbone without NA3) and vABb1p10-NA3 baculovirus and the EGFP florescent pictures were taken at 48 h. (c) Western blot analysis. The cell lysates of MOCK, vABb1p10-empty and vABb1p10-NA3 were harvested at 48 h, analyzed by SDS-PAGE, followed by Western detection of NA3 protein in C6/36 cells. (d) Neuraminidase activity and Neuraminidase inhibition assays: The cell lysates of MOCK, vABb1p10-empty and vABb1p10-NA3 were mixed with or without (10 µM) NA inhibitors (Oseltamivir or Zanamivir) in the presence of 4-MUNANA substrate. Purified NA9 protein (H5N9 strain) was used as a positive control in the experiment. The experiment was performed three times and the data shown is the representative of one independent experiment done in triplicate. The error bars indicate the standard deviations.
