Abstract:

　　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.

Key words: SYNOVIOLIN, green fluorescent protein(GFP), eukaryotic expression vector, gene therapy