Abstract
Background: Secreted alkaline phosphatase (SEAP) is a reporter assay which has been widely used to study the promoter activity or gene expression. Currently, the most common method to titrate dengue virus is plaque assay, which is very time-consuming. We tried to use SEAP reporter assay as substitution for dengue virus titration in this study. Methods: We first construct a SEAP gene sequence which contains SEAP, dengue NS2bNS3 and EGFP peptide. We then transferred this sequence into XL1-Blue competent cells to produce plasmid DNA, and made it into lentivirus in 293T cell line. The SEAP-lentivirus was collected from the supernatant and transfected into different kinds of cell lines to choose stable clone. Fluorescent microscopy and western blot analysis were used to confirm the viruses in-deed enter into host cells. Subsequently, different multiplicity of infection (MOI) of DENV was used to infect these stable clones and the concentration of SEAP was detected by colorimetric method after different periods of infection. Results: By detecting the concentration of SEAP in supernatants of stable clones after different infection time and MOI, a dose-dependent and time-dependent secretion of SEAP was found. Conclusion: These results indicate the SEAP reporter assay can be used to substitute the plaque assay for DENVs titration, which can be a platform for antibody or drug selection against DENV infection.
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