Construction of eucaryotic plasmid of human LMO3 Gene and the expression and localization of fusion protein

Objective To construct the expression plasmid of human LIM domain only 3 (hLMO3) gene and identify the expression and localization of its fusion protein in human HEK293 cells.Methods The hLMO3 coding sequence was amplified by polymerase chain reaction by using cDNA library derived from human fetal brain as the template and subcloned into pEGFP vector.After the target region was identified by enzyme digestion and sequencing,the plasmid was transfected into HEK293 cells.The expression of the recombinant plasmid in HEK293 cells was detected by Western blot.The localization of pEGFP-LMO3 in HEK293 cells was observed with laser scanning confocal microscopy.Results hLMO3 was constructed into the expressing vector pEGFP successfully,the length of the fragment identified by restriction enzyme digestion was 440bp.The expression of pEGFP-LMO3 fusion protein with a molecular weight of 42 kDa was detected by Western blot in human HEK293 cells.The pEGFP-LMO3 fusion protein was mostly localized in the nucleus of HEK293 cells.Conclusion The recombinant plasmid of hLMO3 gene was successfully cloned into eukaryotic expressing vector,and the pEGFP-LMO3 fusion protein was mostly localized in the nucleus of HEK293 cells.