Cloning of P-selectin glycoprotein ligand-1 gene and expression and purification of its Ig fusion protein

Objective To Clone P-selectin glycoprotein ligand-1 (CD162)gene, as well as to express and purify its Ig fusion protein. Methods The CD162 cDNA was cloned by RT-PCR. The CD162 cDNA fragment was then insert into eukaryotic expression vector. The expression vector was then transfected into COS7 cells by DEAD-Dextran method. The supernatant was harvested and the CD162-hIgFc fusion protein was identified by Western Blotting. The biological activity of CD162-hIgFc was assayed by flow cytometry. Results A 801 bp cDNA fragment of CD162 was cloned out. Expression vector pIg-CD162 was therefore constructed and transfected into COS7 cells. The CD162-higFc fusion protein was harvested and purified from the supernatant. Western blotting confirmed the fusion protein identity. Flow cytometry assay showed that the CD162-hIgFc fusion protein was able to identify the CD 62P on the surface of platelets. Conclusion In this study, the CD162-hIgFc fusion protein was expressed and purified,therefore,a solid foundation was built for the further study the interaction of P selectin and its ligand CD162.