[Construction and expression of human EpCAM eukaryotic expression vectors and identification of their products].
OBJECTIVE
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.


CONCLUSIONS
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.
Wang
Chun-yan
CY
Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
Xie
Xin
X
Song
Chao-jun
CJ
Jin
Zhen-xun
ZX
Ouyang
Wei-ming
WM
Jin
Bo-quan
BQ
D000818:Animals; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000235:genetics*; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D000071858:Epithelial Cell Adhesion Molecule; D005822:Genetic Vectors; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D011993:Recombinant Fusion Proteins