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. 2020 Dec 2;8(12):562. doi: 10.3390/biomedicines8120562

Figure 1.

Figure 1

Construction of anti-programmed death-1 (PD-1) nanobodies (Nbs) phage-display library. Schematic representation of the generation and selection of PD-1 specific Nbs from an immunized llama. After immunization (1), collection of blood and extraction of mRNA from peripheral lymphocytes was performed (2–4). VHH (Nbs) genes were amplified with specific primers (5) and cloned into pComb3x phagemid vector (6). The library was electroporated into E. coli (7), phages were produced (8) and used for the selection of PD-1 specific Nbs using HEK-293T cells transiently expressing mPD-1 (HEK-293T/mPD-1+ cells) (10), after a preincubation with HEK-293T wild type (wt) cells (9). Eluted phages (11) were used to infect E. coli to perform another round of selection. After three rounds, screening of positive clones was performed in PD-1 binding ELISAs and flow-cytometry assays, using HEK-293T cells expressing mouse or human PD-1 (12). Created with BioRender.com.