Construction and identification of recombinant adenovirus vectors carrying angiopoietin-1 gene

Objective To construct and identify recombinant adenovirus vectors carrying mouse angiopoietin-1( Ang-1) gene.Methods The Ang-1 gene sequence was made by chemosynthesis and amplified by PCR,and then subcloned into shuttle plasmid. The recombinant plasmid was transformed into competent cell,the transformants was identified by PCR electrophoresis analysis and DNA sequencing. The adenovirus shuttle plasmid carrying exogenous gene and auxiliary packaging plasmid carrying most of the adenovirus genome were cotransfected into HEK293 cells. The recombinant adenovirus was obtained by repeated freezing and thawing. The recombinant adenovirus titer was detected by endpoint dilution assay. The 293 T cells were transfected with the recombinant adenovirus,and then the supernatants were harvested following the 48-hour transfection for a Western Blot analysis on the expression of Ang-1 protein. Results The PCR electrophoresis analysis showed that there was a characteristic band( approximately 1. 5 kbp in size). The DNA sequencing showed that the Ang-1 gene sequence was in accordance with the Gen Bank sequence. The recombinant adenovirus titer was about 1 × 109 pfu / ml after HEK293 packaging. The Western Blot analysis on the supernatants following 293 T cell transfection showed that there was a characteristic band of 58 k D,indicating the recombinant Ang-1 protein of the correct size was obtained after transfection. Conclusion The recombinant adenovirus vector carrying Ang-1 gene has been constructed successfully,which could provide the experimental evidences for further research on the effect of Ang-1 protein on hematopoietic stem cell mobilization and angiogenesis.