Construction, Expression And Binding Activity Of Anti-TfR-scFv-EGFP Fusion Protein

Obectives To construct the fusion gene of anti-transferrin receptor single chain variable fragment (anti-TfR-scFv) and enhanced green fluorescent protein (EGFP), study the expression and identify the biologic activity of the fusion protein preliminarily.Methods Two pairs of PCR primers were designed and used to amplify the VH and VL gene of the anti-TfR-scFv from vector Chi7579-pγ-Expr and Chi7579-pκ-Expr respectively. Two restriction endonuclease sites HindⅢand BamHI were introduced. By splicing overlap extension PCR (SOE-PCR),the VH and VL gene fragments were connected with a linker peptide(Gly4Ser)3 and a leader sequence was added at the 5'terminus to be a single chain Fv (scFv: L-VH-Linker-VL). For the convenience of identification,the L-VH-Linker-VL gene was inserted into the pGEM-T vector. The recombinant plasmid, which called T-scFv,was transfomed into E.coli DH5αby CaCl2 method. Positive cell clones were indentified by restriction enzyme digestion and sequenced. Then the scFv gene were digested with HindⅢand BamHI and subcloned into the eukaryotic fusion protein expression vector pEGFP-N1 to obtain pTfRscfv-EGFP. After identification,pTfRscfv-EGFP plasmid was extracted and tansfected into COS-7 cells. The expression of the fusion protein was detected by inverted fluorescence microscopy in 24h after transfection. Fluorescence intensity of the secreting supernatant of the cells was detected by fluorospectrophotometer and the cell-binding activity of the fusion protein was analyzed by flow cytometer (FCM).Results The scFv gene which connected VH and VL with a (G4S)3 linker, was generated by SOE-PCR and identified by sequencing. After transfected with pTfRscfv-EGFP, COS-7 cells could emit green fluorecence detected by inverted fluorecence microscopy. The fluorescence intensity in their supernatant was 2 times higher than that of the control groups. The TfR positive tomor cells could bind with the fusion protein and emit green fluorescence.Conclusions An anti-TfR-scFv-EGFP eukaryotic expression vector was successfully constructed . The expression product had the fluorescent activity of EGFP and binding specificity to TfR as well as anti-TfR-scFv. This vector is potential in future research for application in tumor imaging and treatment.