Abstract: Objective To prepare alpaca-derived anti-human TGF-β1 nanobodies with high specificity and high affinity. Methods The eukaryotic expression plasmid cFUGW-TGF-β1 was constructed and transiently transfected into HEK-293T cells to obtain recombinant TGF-β1 protein. Purified TGF-β1 proteins were mixed with Freund's adjuvant before immunizing alpaca. The peripheral blood mononuclear cells were isolated from immunized alpaca for RNA extraction and cDNA library was prepared by reverse transcription. Nested PCR was performed to obtain heavy chain variable region fragments for a phage display library. ELISA was applied to screen TGF-β1-specific antibody strains. Positive clones were selected and transferred to a prokaryotic expression vector for purification of nanobodies specific against human TGF-β1. Immunoblotting, immunofluorescence and ForteBio methods were performed to identify the specificity and affinity of the antibodies. Results The recombinant plasmid cFUGW-TGF-β1 was successfully constructed and 3 mg TGF-β1 purified proteins were obtained. A TGF-β1-VHH phage display library with a capacity of 1.2×10⁸ was obtained after 5 immunization. After four-rounds selection with ELISA, 7 strains of nanobodies that specifically bound to recombinant TGF-β1 protein were obtained. Both Western blot and immunofluorescence experiments showed that nanobodies B3 and C8 can recognize TGF-β1 in NCI-H520 tumor cells with the affinity as 1.48×10^-9 mol/L and 9×10^-9 mol/L, respectively. Conclusions Two TGF-β1-VHH nanobodies B3 and C8 are obtained, which can specifically bind TGF-β1 with high affinity. These nanobodiesare potential candidates for tumor immunotherapy.

Key words: human transforming growth factor β1, nanobodies, phage display library