Expression of recombinant secreted glycoprotein of human CREG/myc-His and the analysis for its bio-activities

Objective:To construct the eukaryotic expressing vector of human cellular repressor of E1A-stimulated gene(hCPEG) and then analyze it expression and bio-activities after transfected into human 293F cells.Methods:The fragments of hCREG ORF with terminator codon mutation were amplificated by RT-PCR and the eukaryotic expressing vector-pcDNA4 myc-His/hCREG was constructed.The human 293F cells were transfected with Lipofectamine 2 000 and stably transfected cell clones were screened.The expression of secreted recombinant hCREG/myc-His protein was induced by serum withdrawal and identified by Western blot.The secreted glycoprotein was detected with PNGaseF digestion and Western blot.The effect of secreted hCREG/myc-His protein on proliferation of human intra-thoracic artery smooth muscle cells(HITASY) was evaluated by Flow cytometry analysis.Results:The cDNA fragments of hCREG ORF with terminator codon mutation was obtained by amplification with RT-PCR and the pcDNA4 myc-His/hCREG eukaryotic expressing vector was constructed by EcoRΙ/BamHΙ digestion.The recombinant eukaryotic expressing vector was confirmed correct by restricted endonuclease digestion and DNA sequencing.The transfected 293F cell clones expressed stably secretory hCREG/myc-His fusion protein in Western blot.The secreted protein of hCREG/myc-His was confirmed to be glycosylated by PNGaseF digestion.Futhermore,more HITASY cells were obviously arrested in G1 phase compared with the control when cultured in supernatant with the secreted glycoprotein by Flow cytometry analysis(58.88% vs 62.89%,P0.005).Conclusion:The eukaryotic expressing vector pcDNA4 myc-His/hCREG is constructed successfully and the secreted glycoprotein of hCREG/myc-His is expressed with biological activity.