Aim: To construct EpCAM eukaryotic expression vectors pIg-EpCAM and pEGFP-EpCAM and to express them in COS7 cells.
Methods: EpCAM cDNA was amplified by PCR and then inserted into the pIg and pEGFP vectors respectively to construct recombinant vectors pIg-EpCAM and pEGFP-EpCAM. The two recombinant vectors were transfected into COS7 cells under the mediation of liposome. The expressed EpCAM-Ig fusion protein was detected by Western blot. The expression of EpCAM-GFP fusion protein was observed under fluorescence microscope.
Results: DNA sequencing demonstrated that EpCAM was correctly cloned into the two vectors. The culture supernatant of the pIg-EpCAM-transfected COS7 cells could bind effectively to mAb against human IgG Fc. Green fluorescence distributed evenly in the cytoplasm and nuclei of pEGFP-transfected COS7 cells, whereas in pEGFP-EpCAM transfected COS7 cells, green fluorescence distributed mainly on the surface of the cells.
Conclusion: Two EpCAM eukaryotic expression vectors have been constructed and expressed successfully, which lays the foundation for functional research of EpCAM and for preparation of mAb to EpCAM.