title: [Construction of recombinant adenovirus vector pAdxsi-GFP-HIF containing hypoxia inducible factor gene and its expression in endothelial cells].
abstract: To construct a recombinant adenovirus (pAdxsi-GFP-HIF) encoding human hypoxia inducible factor 1 α gene (HIF-1 α) and to express it in endothelial cells.
  
  
  
  HIF-1 α gene was obtained from human lung cancer cell line A549, which was cultured in hypoxia condition, by RT-PCR. The HIF-1 α gene was subcloned into shuttle vector p Shuttle-CMV-EGFP at KpnI and BamHI sites. After identified with restriction enzymes, plasmid p Shuttle-GFP-HIF was linearized by digestion with restriction endonuclease I-CeuI and I-SceI, and subsequently cotransformed into E.coli DH5a with adenoviral backbone plasmid pAdxsi to make homologous recombination. After linearized by PacI, the homologous recombinant adenovirus plasmid was transfected into 293 cells to package and amplify. The recombinant adenovirus was infected with human umbilical vein endothelial cells (ECV304), and the expression level of HIF-1 α protein was evaluated by ELISA.
  
  
  
  The recombinant adenovirus vector containing HIF-1 α gene (pAdxsi-GFP-HIF) was successfully constructed and amplified with titer of 3.38 X 10(10) pfu/mL. The green fluorescence protein was detected under fluorescent microscope in ECV304 at 24h after transfection and with a stronger degree after 48h. The concentration of HIF-1 protein was (48.93 ±3.86)ng/mL in supernatant at 48 h after transfection.
  
  
  
  A recombinant adenovirus vector pAdxsi-GFP-HIF, encoding human hypoxia inducible factor 1 α gene, has been constructed in vitro and expressed successfully in ECV304 cells.
authors.affiliation: Department Clinical Laboratory, Lanzhou Military Command General Hospital of the People's Liberation Army; Key Laboratory of Stem Cell and Gene Medicine of Gansu Province, Lanzhou 730050, China.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Ha
authors.forename: Donghong
authors.initials: D
authors.lastname: Jiang
authors.forename: Zhiyun
authors.initials: Z
authors.lastname: Deng
authors.forename: Juzi
authors.initials: J
authors.lastname: Dong
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhao
authors.forename: Junhua
authors.initials: J
authors.lastname: Peng
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Yang
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D002478:Cells, Cultured; D005822:Genetic Vectors; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics*; D010957:Plasmids / Q000235:genetics
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit