title: Construction of eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus and their transient expression in HEK293 cells.
abstract: 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 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.
authors.affiliation: Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China.
authors.forename: Weidong
authors.initials: W
authors.lastname: Zhang
authors.forename: Mingyuan
authors.initials: M
authors.lastname: Li
authors.forename: Kang
authors.initials: K
authors.lastname: Cao
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.forename: Qiaofa
authors.initials: Q
authors.lastname: Shi
authors.forename: Baoning
authors.initials: B
authors.lastname: Wang
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Jiang
authors.forename: Hong
authors.initials: H
authors.lastname: Li
mesh_terms: D002460:Cell Line; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000276:immunology*; D007252:Influenza Vaccines / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D019444:Vaccines, DNA / Q000096:biosynthesis / Q000235:genetics
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA