In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus （A/PR/8/ 34） and studied their expressio...In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus （A/PR/8/ 34） and studied their expression in HEK293 cells. HA and HA1 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 HAl , 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.展开更多
基金This project was supported by a grant from the NationalNatural Sciences Foundation of China (No .39970830)
文摘In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus （A/PR/8/ 34） and studied their expression in HEK293 cells. HA and HA1 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 HAl , 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.
