Study on the expression of Norovirus (GenegroupII) VP2 protein in insect cells and its subcellular localization

Abstract:

[Objective] This study is to express Norovirus (GenegroupII) VP2 in insect cells, analysis the subcellular localization of VP2 and lay the foundation of the function study of VP2. [Methods] Norovirus (GenegroupII) ORF3 gene was amplified from plasmid pMD-ORF3 using primers P1/P2 (including 6×His tag coding sequence). The product was cloned into the vector pFastBac1 to construct recombinant plasmid pFB-ORF3. pFB-ORF3 was transformed into competent DH10Bac to get recombinant bacmid Bac-ORF3. Recombinant baculovirus, Ac-VP2, was generated for expressing VP2, by transfecting recombinant Bac-ORF3 with LipofactamineTM 2000 into sf9 insect cells. The expression product was testified by the western blot and indirect immunofluorescence assay with monoclonal antibody against 6×His tag. [Results] The result of western blot showed a 29 kD specific bind in sf9 cells infected by Ac-VP2. Indirect immunofluorescence assay showed the specific green fluorescence in sf9 cells infected by Ac-VP2, and the green fluorescence could localize at nuclear and membrane of sf9 cells. [Conclusion] These results demonstrated that the Norovirus (Gengroup II) VP2 was expressed in sf9 cells successfully and could localize at nuclear and membrane of sf9 cells.