Identification of porcine Toll-like receptor2 (TLR2) targeting peptide ligands using cell-based phage display combined with Illumina sequencing

Abstract

Toll-like receptors (TLRs) are the representative PRRs and play an important role in innate and adaptive immunity. Among various types of TLRs, TLR2 could recognize a wide range of bacterial and fungal components and induce innate and adaptive immune responses. In particular, it is known that TLR2-targeting formulations in vaccines showed adjuvants properties and could effectively induce humoral and cellular immune responses. In this study, porcine TLR2-targeting peptide ligands were identified using cell-based phage display combined with high-throughput sequencing. Porcine TLR2-overexpressing cell line was constructed using lentiviral vector and established cell line was confirmed through flow cytometry, western blot and ligand binding assay. Cell-based phage display was performed on the constructed cell line and two types of biopanning were proceeded : subtractive and non-subtractive biopanning. After 3 rounds of biopanning, eluted phage from each round was sequenced using Illumina platform and three candidate peptides were identified: NAGHLSQ, VPSKPGL, RANLDGQ. In the binding assay, NAGHLSQ showed the highest binding affinity to the constructed cell line and binding of NAGHLSQ to TLR2 overexpessing cell line was gradually decreased in response to increasing concentration of TLR2 ligand. Thus, NAGHLSQ peptide could be used in porcine vaccines as TLR 2 targeting formulation.