Construction, expression and characterization of TfR scFv-SLC fusion protein.

Objective:To construct and express scFv-SLC against transferrin receptor and CCR7,and to identify its cell conjugation activity.Methods:PCR primers were designed according to the complementary sequences of gene SLC in the vector of pDsRed2-N1-CCL21,respectively,which contained inter-linker G4S and the restriction endonuclease EcoRⅠand NotⅠ.SLC fragments was first amplified through PCR and then inserted into plasmid pET28a+scFv,thereby producing a vector which could express a scFv-SLC-His tag(pTfR scFv-SLC).To express scFv-SLC,E.coli.BL21 was cultured in LB broth and was induced by IPTG.The protein was analyzed by SDS-PAGE and Western blot.The cell conjugation activity of scFv-SLC was characterized by FCM.Results:It was demonstrated by digesting and sequencing results of pTfR scFv-SLC that the constructing was successful.Protein was analyzed by SDS-PAGE and Western blot,and the molecular weight was identical with the protein size of TfR scFv-SLC.The results of FCM proved that TfR scFv-SLC had the specificity of conjugating with TfR and CCR7 on the cell surface.Conclusion:TfR scFv-SLC against transferrin receptor and CCR7 have been successfully constructed and expressed.Expression products could specifically bind to TfR and CCR7 on the cell surface of MCF-7 and HepG2 cell.