Abstract:

Abstract:Objective
To construct the rat NKX2.5-pEGFP fusion eukaryotic expression plasmid and screen bone marrow mesenchymal stem cells（MSCs） xpressing rat fusion protein NKX2.5-pEGFP and provide theoretical foundation for the research of cell and gene therapy of myocardial infarction. Methods The total RNAs were extracted from myocardial cells of embryo rats,RT-PCR amplification was performed to produce rat gene of NKX2.5.The recombinant plasmid NKX2.5-pEGFP was obtained
 by inserting the aim gene into pEGFP-N3.The MSCs derived from bone arrow mononuclear cells were obtained by gradient centrifugation and adherent culture.The recombinant plasmids NKX2.5-pEGFP were ransfected into rat MSCs by Lipo2000 and screened with G418 for 2 eeks.The expression of recombinant plasmid in MSCs was detected by Western blotting and fluoroscope analysis.Results The length of NKX2.5 gene obtained by RT-PCR was 981 bp which was consistent with theoretical  numerus.The recombinant plasmid NKX2.5- pEGFP was identified by endonuclease digestion and PCR,which could be digested into two fragments with  BamHⅠ and SalⅠ,about 981  and  4 700 bp,the results were coincident with the anticipated result.The protein with molecular weight of 65 000  was found in MSCs transfected with the recombinant plasmid NKX2.5- pEGFP by Western blotting which was coincident with theoretical numerus. Green fluorescence was positive expression in MSCs transfected with the recombinant plasmid NKX2.5-pEGFP by fluorescence detection.
Conclusion The rat MSCs expressing NKX2.5-pEGFP fusion protein is successfully constructed.

Key words:  mesenchymal stem cells；NKX2.5；enhancement type green fluorescent protein