[Development of recombinant HEK293-16 cell strain expressing chimera receptor EpoR/LR-F3/HAb18GEF with site-specific integration expression system].
OBJECTIVE
To establish recombinant HEK293-16 cell strain expressing chimera receptor EpoR/LR-F3/HAb18GEF via site-specific integration expression system.


METHODS
The HAb18GEF gene fragment was amplified by PCR and digested with Sac I/Not I, and then cloned into the multiple clone site downstream of EpoR/LR-F3 gene of eukaryotic expression vector pCEL2f. The obtained pCEL2f/HAb18GEF vector, checked by partial nucleotide sequencing and restriction endonuclease digestion, was co-transfected with POG44 vector into HEK293-16 cells by mixing with Lipofect AMINE2000 reagent. After selection with hygromycin B for one month, transient and stable expression of EpoR/LR-F3/HAb18GEF were detected by indirect immunofluorescence staining, flow cytometry assay, and Zeocin test.


RESULTS
The recombinant pCEL2f/HAb18GEF vector, containing fused EpoR/LR-F3/HAb18GEF gene with correct open reading frame, was successfully constructed. It was confirmed that both EpoR and HAb18GEF were expressed efficiently on the membrane of co-transfeced HEK293-16 cells. 12 stably-expressed clones, sensitive to Zeocin, were finally obtained, which presented 99.93% EpoR-positive cells with strong immunofluorescence intensity of 1036.39, and simultaneously presented 99.51% HAb18GEF-positive cells with strong immunofluorescence intensity of 652.72.


CONCLUSIONS
HEK293-16 cell strains stably expressing EpoR/LR-F3/HAb18GEF chimera receptor have been developed successfully, which lays foundation for MAPPIT (Mammalian protein-protein interaction trap) screening of binding molecule of hepatoma associated antigen HAb18G/CD147.
Zhao
Pu
P
Department of Cell Biology, Fourth Military Medical University, Xi'an, China. zhaopu6687700@163.com
Zhang
Si-He
SH
Jan
Tavernier
T
Li
Yu
Y
Chen
Zhi-Nan
ZN
D051926:Basigin / Q000235:genetics*; D002460:Cell Line; D004586:Electrophoresis; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017467:Receptors, Erythropoietin / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D051926:Basigin