Effect of Expressed Product of Lipocalin-2 Gene on Proliferation of Human Embryonic Kidney Cells

Objective To construct the eukaryotic expression vector PL-2 for lipocalin-2(Lcn-2),and observe the effect of PL-2 as well as recombinant Lcn-2 protein previously expressed in prokaryotic cells on the proliferation of human embryonic kidney(HEK)cells.Methods Amplify Lcn-2 gene from murine macrophages RAW264.7 by RT-PCR and clone into eukaryotic expression vector pEGFP-C1.HEK293 cells were transfected with the constructed recombinant plasmid PL-2,and the expressed product was ob-served by fluorescent microscopy and identified by RT-PCR.Treat HEK293 cells with the recombinant plasmid PL-2 and the recom-binant Lcn-2 protein previously expressed in prokaryotic cells,and determine the proliferation of cells by MTT method,and the ex-pression of proliferation cell nuclear antigen(PCNA)by Western blot.Results Restriction analysis and sequencing proved that re-combinant plasmid PL-2 was constructed correctly.Fluorescent microscopy and RT-PCR proved expression of Lcn-2 gene in HEK293 cells transfected with PL-2.After treatment with recombinant Lcn-2 protein,the proliferation level of HEK293 cells and the expres-sion level of PCNA were significantly higher than those of control.However,recombinant plasmid PL-2 showed no significant effect on the proliferation of HEK293 cells and the expression of PCNA.Conclusion The eukaryotic expression vector PL-2 for Lcn-2 was successfully constructed,which showed no significant effect on proliferation of HEK293 cells.However,the recombinant Lcn-2 protein expressed in prokaryotic cells promoted effect on the proliferation of HEK293 cells.It is speculated that the expressed product of Lcn-2 gene may promote the proliferation of HEK293 cells by binding to cell membrane receptor.