Construction of eukaryotic expressing plasmids encodingHA andHA 1 of influenza A virus and their transient expression in HEK293 cells
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In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encodingHA andHA 1 of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells.HA andHA 1 genes were amplified by RT-PCR and cloned into pcDNA3. 1(+) to generate pcDNA3. 1(+)/HA and pcDNA3.1(+)/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3. 1(+)/HA and pcDNA3.1(+)/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3.1(+)/HA or pcDNA3.1(+)/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA1, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.
Key wordsinfluenza virus haemagglutinin pcDNA3.1(+) immunofluorescence assay
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