Construction and expression of eukaryotic expression vector of human SYNOVIOLIN gene

Objective:To construct eukaryotic expression vector containing the EGFP-SYNOVIOLIN gene.Methods:According to the sequence of SYNOVIOLIN gene and the multiple clone sites of the expression vector pIRES2-EGFP plasmid,a specific pair of primers were designed and synthesize.PCR amplification of SYNOVIOLIN gene from the pCDNA3-syno plasmid was performed,and an approximate 1900bp objective fragment was achieved.The fragment was then cloned into T-A vector.The recombined vector confirmed by sequencing was digested by SalⅠ/BamHⅠ to obtain SYNOVIOLIN cDNA fragment,then the fragment was subcloned into the multiple sites of pIRES2-EGFP vector.The recombinant plasmid pIRES2-EGFP-syno was transfected into HEK293 cell by lipofectamine 2000.The result was examined using confocal microscopy.The expression of SYNOVIOLIN was detected by Western blotting.Results:PCR,DNA sequencing and restriction enzyme digestion analysis indicated that the eukaryotic expression vector pIRES2-EGFP-syno was constructed successfully.The expression of EGFP can be seen under confocal microscopy.Western blotting proved the protein expression of SYNOVIOLIN in HEK 293 cells.Conclusions:The SYNOVIOLIN cDNA is acquired and the eukaryotic expression vector,pIRES2-EGFP-syno is successfully constructed with efficient expression in HEK293 cells,which provide a good experimental basis for further study on the gene therapy of anti-tendon adhesion.