Construction of human QKI6 recombinant adenovirus vector and identity of its expression in cardiomyocytes

AIM: To construct recombinant adenovirus vector containing human RNA-binding protein( QKI6) gene and provide an experimental foundation for further research on the protective mechanisms of the QKI6 gene in cardiomyocyte apoptosis and applications of QKI6 in the treatment of diabetic cardiomyopathy. METHODS: Using PCR,QKI6 target gene fragment was amplified from the original plasmid and ligated into recombinant shuttle plasmid( pShuttle-GFP-CMV). pShuttle-GFP-QKI6 was then transferred into pAdxsi vector to gain adenovirus plasmids pAdxsi-GFP-QKI6 containing QKI6 target gene and GFP fluorescence reporter. PCR was performed to identify the recombinant adenovirus following amplification and purification of pAdxsi-GFP-QKI6. After linearization by Pacl,the recombinant adenovirus vector transfected HEK 293 cells for adenovirus packaging. Viral titer was determined and the expression of the target gene was measured. The wrapped adenovirus vector transfected rat neonatal cardiomyocytes.GFP expression was observed to determine the efficiency of viral infection and PCR was performed to detect QKI6 expression in infected cardiomyocytes. RESULTS: The recombinant adenovirus vector pAdxsi-GFP-QKI6 containing QKI6 gene and GFP fluorescence reporter was constructed successfully, which was confirmed by PCR and DNA sequencing. Recombinant adenovirus was reusable to infect HEK 293 cells. GFP and QKI6 expression was detected by fluorescence microscopy and PCR. The packaged recombinant adenovirus efficiently infected neonatal cardiomyocytes and QKI6 expression in cardiomyocytes increased. CONCLUSION: We successfully constructed the adenovirus containing QKI6 gene. QKI6 expression increases after infection of 293 cells and neonatal cardiomyocytes,which establishes the foundation for further research on effects and mechanisms of myocardial protection of QKI6 in diabetic rats.