Construction Of Nucleoprotein Fusion Gene And Inhibition Of SiRNA On Expression Of NP Gene In Influenza A Virus

Objective:To develop a best method of constructing influenza NP fusion gene containing enhanced green fluorescent protein (EGFP) and to identify common anti-flu vaccine by using siRNA technology in MDCK cells.Methods:The full-length NP gene of influenza A was amplified by RT-PCR and was inserted into an eukaryotic expression vector pEGFP-N1in order to construct a fusion gene of pEGFP-N1-NP using three different methods. The sequencing confirmed constructed pEGFP-N1-NP fusion gene was transfected into MDCK cells by Lipofectamine2000, then the expression of NP gene observed and the function of NP detected by using Fluorescence microscopy, Immunofluorescence, Western blot, and siRNA technology in MDCK cells.Results:The fusion gene of recombinant eukaryotic expression vector pEGFP-Nl-NP was successfully constructed, expressed and effectively inhibitted by three pairs of specific siRNA separately by using Fluorescence microscopy, Immunofluorescence and Western blot in MDCK cells.Conclusion:The successfully constructed NP gene and inhibition of siRNA on expression of NP gene in Influenza A Virus set up a groundwork for understanding the pathogenesis of influenza virus and provide laboratory evidence of developing universal anti-flu vaccines and drugs when multi-influenza virus strains epidemic.