Rapid construction of EGFP labled recombinant adenovirus containing hVEGF165 and its expression in haematopoietic cells

Abstract

By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labeled recombinant adenovirus vector containing hVEGF165 was constructed quickly and efficiently expressed in mouse haematopoietic cells, First, hVEGF165 coding sequence was subcloned into shuttle plasmid pAdTrack-CMV, then cotransformed with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ5183. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF165. The expression of EGFP could be easily detected. The rate of EGFP positive mouse bone marrow mononuclear cells by flow cytometric analysis was 27.3% (MOI=100), and the expression of hVEGF165 protein in the supernatant was (1385±332) pg/10⁶ cells. These results suggest that the construction of adenovirus vector by homologous recombination in bacteria features high efficiency and simplicity. The prepared high titer Ad-EGFP/hVEGF165 can be used an efficient helpful vector to infect hematopoietic cells.

Keywords

EGFP / vascular endothelial growth factor / adenovirus / homologous recombination / haematopoietic cells

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