Expression and Identification of Dengue 2 Virus Nonstructural Protein 4B and its Mutant in Mammalian Cells

Objective:To construct the recombinant enkaryotic plasmid expressing the nonstructural protein 4B(NS4B) of dengue 2 virus and its mutant Δ2K-NS4B lacking the 2K segment,and to obverse the location of both the recombinant proteins in the mammalian cells.Methods:The gene fragments of NS4B and Δ2K-NS4B were amplified by PCR from full length cDNA cloned vector of dengue 2 virus 43 strain,and then were cloned into enkaryotic expression vector pcDNA6/V5-HisA to generate the desired recombinant plasmid pc/D2-NS4B and pc/D2-Δ2K-NS4B.After transfected into the mammalian BHK-21 cells by lipofectamine,the expression of recombinant proteins were identified by RT-PCR,indirect immunofluorescence and Western blot.Results:Both the recombinant proteins D2-NS4B and D2-Δ2K-NS4B exist mainly in the cytoplasm,and they could react with the polyclonal antibody again the protein NS4B of dengue 2 virus.Conclusion:The correct expression of both the recombinant proteins in the cytoplasm of the mammalian cells has laid the foundation of interests on the biological function of the protein NS4B during the life cycle of dengue virus.