Mouse E-selectin /human IgG Chimeric Protein: Expression, Purification And Characterization From COS-7 Cell

Adhesion molecules were initially defined as cell surface structuresmediating cell-cell and cell-extracellular matrix (ECM) interactions.Selectins family is very important one of adhesion molecules. E-selectinhas been implicated in the pathogenesis of a number of acute inflammatorydisease states. E-selectin modulate the early adhesion interactions betweencirculating leukocytes and the endothelium .E-selectin can be expressed onthe surface of endothelial cells following stimulation by a number ofinflammatory mediators including the inflammatory cytokines interleukin-1beta (IL-1β), tumor necrosis factor-alpha(TNFα) and endotoxin .E-selectin binding with the ligands including sialyl Lewis(x) and sialylLewis(a) causes leukocytes rolling along the vascular endothelium , somigrating into the place of inflammation . E-selectin not only is specialselectin of the vascular endothelium , but also reacts early stage of acuteinflammation . It has very important significant in the pathogenesis ofischemia-reperfusion (IR) ,atherosclerosis and tumor metastasis. Therefore,using something to block the reaction between E-selectin and his ligandscan inhibit the inflammation and tumor metastasis. Object To express mouse E-selectin and human IgG Fc chimericprotein in mammalian eukaryotic expression system, COS-7 cell, purifyand characterize the expressed protein in vitro. Methods After amplification in Ecoli. HB10/P3 with CaCL2, therecombinated expression plasmid pCDM8-L-selectin/IgG was extracted bya kit to remove endotoxin and was identificated through PCR. Then thepCDM8-L-selectin/IgG was transfected into COS-7 with lipid reagentLipofectAMINE2000TM. The chimeric protein expressed in the cell culturemedia was detected with ELISA and then was purified through protein Aaffinity column chromatography. The protein was further characterized bySDS-PAGE, Dot blot and Western blot analysis and cell adhesion ability. Results The plasmid we got was verified to be mainly superhelixstructure in 0.8 agarose gel and PCR showed that a specially amplifiedband at 342bp. The chimeric protein could be detected in the supernatant ofcell culture media 48 hour after the transfection with ELISA . A proteinband with molecular weight of about 122.4kDa was found in theSDS-PAGE gel after protein A affinity chromatography. And this proteinspecifically bound with mouse E-selectin antibody and human IgGpolyclonal antibody in Dot-blot and Western-blot . After purified chimericprotein hatching with HL-60 cells(having E-selectin ligands sLewisX in thecell surface) , the count was higher than that of purified chimeric proteinhatching with B16 cells(no E-selectin ligands sLewisX in the cell surface)obviously (p<0.01)and was higher than that of L-selectin/IgG chimericprotein (p<0.01) or human IgG (p<0.01) hatching with HL-60 cellobviously too. Conclusion Recombined chimeric protein E-selectin/IgG can besuccessfully expressed in mammalian eukaryotic expression vector COS-7cells and purified through protein A affinity chromatography . we got activechimeric protein .