Development of recombinant HEK293-16 cell strain expressing chimera receptor EpoR/LR-F3/HAb18GEF with site-specific integration expression system

AIM: 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. CONCLUSION: 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.