Fig. 2

Schematic diagram of the construction and screening of a single-domain phage display library. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples of a Bactrian camel immunized with PEDV, and total RNA was extracted from the PBMCs and reverse transcribed to cDNA. The VHH gene fragments with restriction enzyme sequences were amplified with nested PCR. In the first round of PCR, the gene fragment (~ 600 bp) containing the VHH, hinge region and CH2 domain was amplified from the cDNA, and then the VHH gene was amplified by a pair of primers specific for VHH gene FR1 to FR4 using the product of the first PCR (600 bp product). SfiI and NotI restriction sites were added on the third round of PCR. The VHH gene was ligated into the pCANTAB 5E plasmid and transformed into E. coli TG1, and after rescue by the M13K07 helper phage, the VHH gene was displayed on the phage surface. The PEDV spike protein-specific phages were enriched by three rounds of biopanning