Abstract:

Abstract: [Objective] To construct the eukaryotic expression vector for enhanced green fluorescent protein (EGFP) fused with Hu Sheep myogenin (MyoG) for investigating the subcellular localization of EGFP-MyoG fusion protein in the transfected NIH-3T3 cells. [Method] MyoG gene was amplified by PCRfrom pMD19-T-MyoG and inserted into the eukaryotic expression vector pEGFP-C1 to construct fusion protein expression vector pEGFP-C1-MyoG. Then NIH-3T3 cell was transfected with the constructed recombinant plasmid pEGFP-C1-MyoG. 48 hours after transfection, the subcellular localization of the fusion protein was monitored by fluorescence microscopy and the expression of MyoG was detected by RT-PCR and western blot. [Results] Enzyme, sequencing analysis indicated that the MyoG gene had been correctly inserted into the pEGFP-C1. RT-PCR detected specific 680bp band, indicating the expression of fusion protein in RNA level. Western blot analysis confirmed that the EGFP-MyoG fusion protein of Mr 53kD was detected in transfected NIH-3T3 cells. Fluorescence observed under fluorescence microscopy showed that EGFP-MyoG protein was positioned in the nucleus.[Conclusion] The fusion protein EGFP-MyoG was successfully expressed in NIH-3T3 cells, and it was positioned in nucleus in single cell level.