Construction of Fusion Plasmid of Human LMO4 Gene and Its Recombinant Protein Expression and localization

Objective To construct the expression plasmid of human LIM domain Only 4(hLMO4) gene and identify its recombinant protein expression and localization.Methods Total RNA was extracted from human breast cancer MCF-7 cells.The hLMO4 coding sequence was amplified by polymerase chain reaction(PCR) method and subcloned into pCDNA3.1-myc-his A vector.After the target region was sequenced,the plasmid was transfected into BGC 823 cell lines.The expression of the recombinant plasmid in BGC 823 cells was proved by Western blot.The localization of pEGFP-LMO4 in breast cancer cell MCF-7 was observed by using laser scanning confocal microscopy.Results hLMO4 had been constructed into expressing vector pCDNA3.1-myc-his A successfully.The length of the fragment was 500bp,identified by restriction enzymes digestion.The expression of myc-LMO4 fusion protein was detected by Western blot,with a molecular weight 23KD.The pEGFP-LMO4 protein was localized more in the nucleus,less in the cytoplasm.Conclusion The recombinant plasmid was successfully cloned into eukaryotic expressing vector,the pEGFP-LMO4 fusion protein was expressed in the nucleus and cytoplasm.