Expression And Localization Of Spag6L Gene In Eukaryotic Plasmids And Detection Its Transcriptional Regulation Activity On Promoter Area

Objective: To construct eukaryotic plasmids expressing mouse Spag6 L gene,and investigate its expression and localization in transfected mammalian cells.luciferase reporter recombinant plasmid on promoter are was constructed,and its transcriptional activity was detected.Method: The mouse Spag6 L coding sequence was amplified by ploymerase chain reaction(PCR)and subcloned into the p EGFP-N2 and pc DNA3 vectors,respectively.After sequencing,the constructed plasmids were transfected into CHO and COS-7 cells,the expression and cellular localization of Spag6 L protein were determined by Western blot analysis and immunofluorescence staining.Luciferase reporter recombinant plasmid Spag6L/PGL3 on upstream promoter area was constructed,following with detecting its transcriptional activity.Result: Mouse Spag6 L was successfully cloned into the pEGFP-N2,pcDNA3 and PGL3 vectors.Expression of SPAG6 L and GFP-tagged Spag6 L fusion protein was confirmed,with the relative molecular weight of 56 KD and 82 KD respectively.Spag6 L protein mainly is localized in the cytoplasmic vesicles and microtubules.The transcriptional activity of Spag6 L is significantly stronger than that of Spag6.Conclusion: These recombination plasmids expressing Spag6 L was successfully constructed,which provide a platform to further study its role in vitro.