Abstract: Objective To construct the eukaryotic expression vector of human PRKCB1 containing enhanced green fluorescence protein gene and transfer into human umbilical vein endothelial cell(HUVEC)，then identify activity of expression protein. Methods The pReceiver-M29-PRKCB1 eukaryotic expression plasmid was constructed by frame amplified from pMD18-T-PRKCB1 plasmid. Then the recombinant plasmids were identified by enzyme analysis and DNA sequencing. According to optimized conditions,the eukaryotic expression plasmids were transfered into HUVEC and observed under fluorescence microscope.After that, transfection efficiency was calculated under random vision.The plasma membrane/cytosol ratio of fluorescence was calculated under confocal microscopy.The translocation was identified. Results The gene sequence was completely consistent with that reported in GenBank. The enhanced green fluorescence protein could be observed in HUVEC after 48 hours.Transfection efficiency was 18.62±0.57%. The translocation was observed. Conclusion The eukaryotic expression plasmid is successfully constructed and transfered into HUVEC, the translocation was identified. It is the molecular instrument for screening HUVEC stably expressing human protein kinase c 2 and isolating protein complex.