Construction of recombinant adenovirus vector pAdxsi-GFP-HIF containing hypoxia inducible factor gene and its expression in endothelial cells / 浙江大学学报·医学版
Journal of Zhejiang University. Medical sciences
;
(6): 654-659, 2013.
Artigo
em Chinês
| WPRIM
| ID: wpr-251739
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Plasmídeos
/
Células Cultivadas
/
Adenoviridae
/
Subunidade alfa do Fator 1 Induzível por Hipóxia
/
Células Endoteliais da Veia Umbilical Humana
/
Vetores Genéticos
/
Genética
/
Metabolismo
Tipo de estudo:
Estudo prognóstico
Limite:
Humanos
Idioma:
Chinês
Revista:
Journal of Zhejiang University. Medical sciences
Ano de publicação:
2013
Tipo de documento:
Artigo
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