Preparation and identification of the nano antibodies of anti-programmed death receptor-1

Abstract

Objective To prepare camelid-derived nano antibodies with high affinity binding to programmed death receptor-1 (PD-1) antigen, and to provide experimental basis for subsequent functional studies. Methods The PD-1-Fc recombinant protein expressed in eukaryotic expression was used to immunize Xinjiang Bactrian camel 6 times. The peripheral blood was collected and the lymphocytes were isolated. Nested PCR amplification was performed to obtain the genes in variable region of camelid heavy chain antibody (VHH), and to construct a phage display library. The phage display library was screened by solid phase enzyme-linked immunosorbent assay (ELISA). The PD-1 antigen, which was sequentially reduced in mass concentration (5.00、2.50、1.00 μg/ml), was coated in an ELISA plate, and the phage display library was subjected to 3 rounds of affinity selection. Individual clones that bind to PD-1 were further screened by soluble monoclonal ELISA. According to the results of DNA sequencing, three VHH monoclonals with multiple repeats were selected and ligated into pET22b vector, and transformed into E. coli BL21(DE3) competent cells, and then induced by isopropyl-β-D-thiogalactoside. The recombinant VHH antibody protein was purified by nickel column affinity chromatography, and its binding activity and affinity to PD-1 antigen were detected by Western Blot and ELISA. Results After immunization of Bactrian camel 6 times with recombinant protein PD-1-Fc, high titer specific antibody was stimulated, and the immune serum titer reached 1∶32 000. A VHH phage display library with a reservoir size of 2.6×108 cfu/ml was constructed from the immunized camel lymphocytes. After 3 rounds of affinity selection, 46 VHH monoclonals with absorbance (A600) values above 0.6 were obtained by soluble monoclonal ELISA. Among them, three clones of VHH-B7, VHH-H5 and VHH-H12 had higher repeats, indicating that significant enrichment was obtained. The results of Western Blot and ELISA showed that the purified B7, H5 and H12 nanobodies had good binding activity to PD-1 antigen and had high affinity. Their affinity constants were 1.19×1011 and 1.63×1011, 1.59×1011 L/mol, respectively. Conclusion The anti-PD-1 camelid-derived nanobodies were obtained by affinity selection of VHH phage display library, which can bind to the PD-1 antigen with high affinity. This study can provide an experimental basis for subsequent functional studies. Key words: PD-1; Nano antibodies; Heavy chain antibody variable region; Identification; Phage display