The establishment of sex determining region Y box 9 gene adenovirus vectors by AdMax packaging systems and identification of its expression

Objective To build sex determining region Y box 9 (SOX9) gene recombinant adenovirus vectors and carry on the appraisal.Methods First,SOX9 gene was synthesized as a template for polymerase chain reaction (PCR) amplification,then it was restructed into linear pAV-MCMV-green fluorescent protein (GFP)-3 flag,and transfered into DH5a cells.The cloning vectors were obtained.The cloning vetors were identified by technology of PCR.Mter that,the cloning vectors were transfected into HEK293 cells which have been cultivated before by AdMax adenovirus packaging system in which SOX9 gene adenovirus vectors can be packaged,matured and amplified.The SOX9 gene adenovirus vectors were detected by Western blotting.PCR was used to examine the viral DNA and its titer.Results With fragment of 1 565 bp gained,which size is consistent with the SOX9 gene,SOX9 gene was restructed into the adenovirus vectors successfully.Western blotting revealed that there were positive bands from 72 × 103 to 95 × 103,which had the same size with SOX9-GFP-Flag protein (86 × 103),and the titer was 2.3 ×1011 PFU/ml,and the expression of SOX9 protein was detected successfully.Conclusion The safe,stable and effective SOX9 gene recombinant adenovirus vectors were successfully established,and identified correctly. Key words: Intervertebral disc degeneration;  Sex determining region Y box 9 gene;  Adenovirus