Construction and identifcation of recombinant adenovirus vector co-expressing HIF-1α and EGFP

Objective To study how to construct the recombinant adenovirus vector of pAdeno-EGFP-HIF-1α. Methods The recombinant pShuttle-EGFP-HIF-1αvector was constructed by amplifying HIF-1αin PCR and cloned into the pShuttle-EGFPHIF-1α vector containing EGFP. The recombinant pAdeno-EGFP-HIF-1αadenoviral vector was constructed by transferring the constructed recombinant pShuttle-EGFP-HIF-1αvector to the pAdeno skeleton vector, amplifed in H293 cells and purifed. Their viral titer was measured and identifed by PCR. Results Agarose gel electrophoresis displayed 2 fragments at 2.5 kb and 5.1 kb in positive clones and 1 fragment at 5.1 kb in negative clones of pShuttle-EGFP-HIF-1αafter KpnI/BamHI enzyme digestion, indicating that the recombinant pShuttle-EGFP-HIF-1α vector was successfully constructed. Agarose gel electrophoresis showed 8 fragments at 4.5 kb, 11.7 kb, 2.66 kb, 2.6 kb, 2.48 kb, 2.47 kb, 1.45 kb, 0.6 kb in positive clones and 6 fragments at 14 kb, 11.8 kb, 4.0 kb, 2.47 kb, 1.45 kb, 0.6 kb in negative clones of recombinant pShuttle-EGFP-HIF-1αvector and pAdeno vector, indicating that the recombinant pAdeno-EGFP-HIF-1α adenovirus vector was successfully constructed. PCR showed that the recombinant pShuttle-EGFP-HIF-1αadenovirus vector was amplifed to a 2.5 kb fragment after package and purifcation, suggesting that the HIF-1αwas integrated in the adenovirus with a titer of 2×10¹⁰ pfu/ml as measured by TCID50. Conclusion The successful construction of recombinant pAdeno-EGFP-HIF-1α adenoviral vector lays a foundation for the further study of its role in spinal cord injury.