Construction, Expression And Characterization Of ScFv And BsFv Against Transferrin Receptor

Objective: To construct and express scFv and BsFv against transferrin receptor, and identify its cell conjugation activity and specificity.Methods:1. scFv was constructed by digesting and sub-clone.2.Designed PCR primers from the parent scFv to introduce interlinker G4S and the restriction endonuclease AscI. Two scFv fragments were produced by PCR, SfiI-scFv-linker-AscI and AscI-scFv-NotI respectively. After digested by SfiI, NotI and AscI, the two scFv fragments gene were connected into the secretory expression vector pAB1. Then the connected production was transformed into E. coli. TG1. The recombinant bacterial colonies were identified by extracted plasmid and digested with SfiI/NotI, and one was sent to sequencing.3.The positive clones were cultured and induced by isopropyl-β–D-thiogalactopyanoside(IPTG). The proteins were purified by IMAC.and identified by SDS-PAGE and Western blot.4.The cell conjugation activity and specificity of scFv and BsFv were characterized by FCM.Results:1.Two bands about 750bp and 1500 bp could be observed by agarose gel electrophoresis after the plasmid digested with SfiI/NotI. This results were consistent with expected gene size of scFv and BsFv. The sequencing result of scFv was blast with the squence of parent scFv and primers, and it was demonstrated the sequence of BsFv gene was correct. The genes of scFv and BsFv were coloned into the pAB1 vector successfully. 2.The positive clones were cultured and induced by isopropylβ–D-thiogalactopyanoside(IPTG). The proteins were purified by IMAC and analyzed by SDS-PAGE and Western blot, which molecular weights were 30KD and 60 kD. They were identical with the protein size of scFv and BsFv and proved the expression and purification were successful.3.The biological activities of scFv and BsFv conjugating with K562, HepG2 and static peripheral blood lymphocyte were analyzed by FCM. The values of the BsFv conjugating with K562 and HepG2 were about 60% and 23%, respectively, which was higher than that of scFv. Furthermore, the values of scFv and BsFv combining with static peripheral blood lymphocyte were the same. The results proved the scFv and BsFv had the specificity of conjugating with TfR on the cell surface.The avidity of BsFv was higher than that of the parent scFv.Conclusion: ScFv and BsFv against transferrin receptor have been successfully constructed and expressed. Our study demonstrated expression products could specifically bind to TfR on the cell surface of K562 and HepG2. The avidity of BsFv is higher than that of the parent scFv . The scFv and BsFv will be promising reagents in detecting of TfR on cell surface, sTfR and targeted therapy.